Process for the selective formation of disulfide bridges in polypeptides and therapeutic compositions

A process for the selective formation of at least two disulfide bridges in a polypeptide which process comprises treating a polypeptide starting material carrying at least two SH-groups to mask at least one of said SH-groups with a p-methoxybenzyl protective group and to mask at least one of the SH-groups with an acetamido methyl protective group, then splitting-off said p-methoxybenzyl protective group with pyridine-polyhydrogen fluoride in the presence of anisol, and then treating with iodine in an acid solution to produce said disulfide bridge-containing polypeptide.

The invention relates to a process for the selective formation of two or 
more disulfide bridges in synthetic and/or natural polypeptides and more 
specifically in the production of insulin derivatives. In further aspect, 
the invention relates to therapeutic compositions which contain, as active 
ingredients, the insulin derivatives obtained in this manner. 
Recently, the biological insulin synthesis by means of Escherichia 
coli-mutants has caused considerable sensation (see D. V. Goeddel, D. G. 
Kleid, F. Bolivar, H. L. Heynecher, D. G. Yansura, R. Crea, T. Hirose, A 
Krazewski, K. Itakura, and A. D. Riggs, Proc. Natl. Acad. Sci. USA (76) 
[in press]). Because of the danger of infection in men with 
insulin-producing bacteria, in the process, the insulin A- and B-chains 
are built up separately from each other in different cultures and are 
obtained as free polypeptide chains. They are then linked to the insulin 
molecule by a bridge by means of statistical disulfide-oxidation, as is 
also the case in the earlier total synthesis of insulin (R. E. Humbel, H. 
R. Bosshard, and H. Zahn in D. F. Steiner and N. Freinkel, Handbook of 
Physiology, Vol. 1 (Baltimore: Williams & Wilkins, 1972), pp. 11 ff.). In 
this statistical combination of the two chains, however, insulin is formed 
as only one product amony many possible ones (3 monomeric bi-disulfides of 
the A-chain, 1 disulfide of the B-chain, 12 isomeric insulins, and many 
oligomers and polymers of the individual and of both chains), so that thus 
far, it has not been possible to combine the A-chain and the B-chain into 
insulin by means of disulfide synthesis in yields higher than 10 to 20%. 
Even recombination attempts with natural A- and B-chains in the proportion 
of 1:1 produced insulin yields of only about 10%. 
B. Kamber et al. have already reported on a total synthesis of human 
insulin in which the three disulfide bridges were, for the first time, 
aimed at different steps of a fragment-like synthesis (Helvetica Chimica 
Acta, Vol. 57 (1974), Fasc. 8, pp. 2617-2621 and Vol. 60 (1977), Fasc. 1, 
pp. 27-37, and the places in the literature cited there). Even in this 
case, two of three disulfide bridges of peptide chains A and B of the 
insulin were not formed first by linking these two complete chains, but in 
preliminary steps of the synthesis. 
Along with the size and complexity of the peptide chains A and B that form 
the hormone insulin, the problem of insulin synthesis, therefore, is 
characterized, first of all, in that the three disulfide bridges linking 
the molecule intrachenarically and interchenarically should be formed by 
targeting, and not statistically, in order to increase the insulin yields. 
In so doing, one should be able to proceed, as much as possible, from 
biologically formed A-chains and B-chains of insulin. 
Human insulin corresponds to the following formula I: 
##STR1## 
In Formula I above, the upper amino acid sequence (1 to 21) corresponds to 
the A-chain, while the lower amino acid sequence (1 to 30) represents the 
B-chain of human insulin. 
The aim of the present invention consists in indicating a process that 
succeeds in selectively forming two or more disulfide bridges in natural 
and/or synthetic polypeptides, and does this, if necessary, in the 
presence of one or several already existing disulfide bridges, and at the 
same time, this process is said to be especially suited for the production 
of synthetic or semisynthetic human insulin by combining a synthetic 
A-chain of insulin with a synthetic or natural B-chain of insulin. 
It was now found that one is successful in selectively forming disulfide 
bridges, if specific amino acid-protective groups are used, and these are 
split off again in a specific manner while simultaneously forming 
disulfide bridges. Thus, it has become evident in a surprising manner that 
if one or both SH-groups that are supposed to be converted into a first 
disulfide bridge are masked with a p-methoxybenzyl protective group, and 
one or both or additional SH-groups, which are supposed to be converted 
into a second or additional disulfide bridges, are masked with an 
acetamido methyl protective group, and the p-methoxybenzyl protective 
group is then split off with pyridine-polyhydrogen fluoride (HF/pyridine 
in the presence of anisol, and then treated with iodine in acid solution, 
by means of which the first disulfide bridge is formed, after which the 
acetamido methyl protective groups are split off, and the SH-groups set 
free in this manner are oxidized to the second or additional disulfide 
bridges. 
The subject of the present invention, therefore, is a process for the 
selective formation of two or more disulfide bridges, if necessary in the 
presence of one or several already existing additional disulfide bridges, 
in synthetic and/or natural polypeptides, a process that is characterized 
in that at least one of the two SH-groups that are to be converted to a 
first disulfide bridge is masked with a p-methoxybenzyl protective group, 
and at least one of the two or of several SH-groups that are to be 
converted into a second or several disulfide groups is masked with an 
acetamido methyl protective group, and the p-methoxybenzyl protective 
group or groups are then split off with pyridine-polyhydrogen fluoride 
(HF/pyridine) in the presence of anisol, and then treated with iodine in 
acid solution. 
In accordance with a preferred form of carrying out the present invention, 
the process is applied to the preparation of active human insulin, for 
which a synthetic insulin A-chain that exhibits an acetamido methyl 
protective group in the A.sup.7 -position and a p-methoxybenzyl protective 
group in the A.sup.20 -position is caused to react with an equimolar 
quantity of synthetic or natural, reduced insulin B-chain. 
In so doing, it is advantageous to use an insulin B-chain that exhibits an 
acetamido methyl protective group in the B.sup.7 -position and a 
p-methoxybenzyl protective group in the B.sup.19 -position, since in this 
way, the formation of insulin isomers can be completely suppressed. 
In this manner, it is possible to selectively link the four SH-groups that 
are to be combined with the correct reaction partner in each case, with 
the formation of two disulfide bridges, without, in so doing, endangering 
the already existing disulfide linkages, like the little intrachenaric 
disulfide ring [A.sup.6 -A.sup.11 ] that is already present in the 
A-chain. 
In the process for the preparation of semisynthetic insulin according to 
the present invention that is preferred, one proceeds from a completely 
synthetic A-chain that exhibits four SH-groups, two of which are to be 
bound to the small intrachenaric disulfide ring, and are masked with 
tertiary-butyl mercapto protective groups, while the other two SH-groups 
exhibit a p-methoxybenzyl group or an acetamido methyl group, namely in 
the A.sup.20 - or A.sup.7 -positions. In so doing, the tertiary-butyl 
mercapto groups are first selectively split off by treatment with tributyl 
phosphane under nitrogen, after which the disulfide bridge, by means of 
selective oxidation with air, is closed intrachenarically, with the 
formation of the small ring. With this method of treatment, the acetamido 
methyl protective group and the p-methoxybenzyl protective group remain 
unaffected. Then, in accordance with the process that is according to the 
present invention, the p-methoxybenzyl protective group is split off by 
means of treatment with pyridine-polyhydrogen fluoride (HF/pyridine), and 
in so doing the acetamido methyl protective group is preserved. Then 
treatment is carried out with iodine in 30% acetic acid solution, in the 
presence of a natural B-chain of insulin in which the two SH-groups are 
unprotected. In so doing, the following steps are carried out in 
succession. First, the second disulfide bridge is linked (A.sup.20 
-B.sup.19), then the acetamido methyl protective group is split off, and 
the SH-group liberated in the process, along with the additional free 
SH-group of the B-chain that still remains, is oxidized to the third 
disulfide bridge (A.sup.7 -B.sup.7). In so doing, it has turned out that, 
in a surprising way, about 30% of the quantity of of free B-chain employed 
reacts selectively with the correct SH-group of the B-chain. 
In order to control the disulfide-bridging reaction even more exactly, 
however, it is undoubtedly possible to proceed from a natural or synthetic 
insulin B-chain which exhibits an acetamido methyl protective group in the 
B.sup.7 -position and a p-methoxybenzyl protective group in the B.sup.19 
-position, so that the corresponding SH-groups of the A- or B-chain of the 
insulin are correspondingly masked, and then in carrying out the required 
procedure in the indicated reaction sequence, only one SH-group in each of 
the two chains is available for the reaction. 
In accordance with this principle of the process according to the present 
invention, it is also possible, naturally, to form two different rings 
within a single chain via disulfide bridges, or to build two or several 
disulfide bridges in one or several chains. In this case, too, the 
SH-groups to be brought together for reaction with each other are 
protected with masking groups of the same kind, according to the present 
invention. 
The process according to the present invention is of special significance 
in the synthesis of human insulin. In the various insulins, as a rule and 
according to their origin, the B-chain, aside from the C-terminus, is 
identical with the B-chain of human insulin, while the A-chains are 
clearly different. Since the C-terminus of the B-chain can easily be 
exchanged, it is sufficient to synthesize the A-chain and insert a B-chain 
of natural origin after changing the C-terminus, in order to then form 
human insulin, proceeding from these starting materials in accordance with 
the procedure according to the present invention. 
The procedure according to the present invention is, however, also suitable 
for the preparation of insulin analogs, as they are made clear, for 
example, in the schematic of FIG. 6. 
As shown in FIG. 6, formula I to IV represent, in a schematized manner, the 
insulin derivatives which are accessible with the aid of the process 
according to the present invention. Thus, the general formula I stands for 
natural insulin, while formulas II and III represent insulin derivatives 
with an unnatural intrachenaric disulfide ring, in which the disulfide 
ring is present in the A.sup.7 -A.sup.11 -position or in the A.sup.6 
-A.sup.7 -position. Formula IV to VI make clear the antiparallel variants 
of the compounds of the formulae I to III in which the insulin B-chain is 
bound antiparallel to the corresponding insulin A-chain. 
These latter antiparallel variants of insulin with natural or unnatural 
arrangement of the intrachenaric disulfide bridge are of special 
significance, since, under certain pH conditions, they gradually 
reconstruct the natural form of bridging, so that they represent 
long-lived, inactive, synthetically produced storage forms of insulin 
which, under physiological conditions, are very slowly converted into 
active insulin, as is desirable in diabetes therapy. 
For the production of the insulin analog of formula II ([A.sup.7 -A.sup.11, 
A.sup.6 -B.sup.7 -cystine]-insulin), a synthetic [A.sup.7 -A.sup.11 
]-insulin A-chain is used as the starting material, which has an acetamido 
methyl protective group in the A.sup.6 -position and a p-methoxybenzyl 
protective group in the A.sup.20 -position. 
For the production of the insulin analog of formula III above ([A.sup.6 
-A.sup.7, A.sup.11 -B.sup.7, A.sup.20 -B.sup.19 -cystine]-insulin), an 
[A.sup.6 -A.sup.7 ]-insulin A-chain is used as the starting material, 
which exhibits an acetamido methyl protective group in the A.sup.11 
-position and a p-methoxybenzyl protective group in the A.sup.20 
-position. 
For the production of the antiparallel insulin analog of formula IV above 
([A.sup.6 -A.sup.11, A.sup.7 -B.sup.19, A.sup.20 -B.sup.7 
-cystine]-insulin), a natural or synthetic [A.sup.6 -A.sup.11 ]-insulin 
A-chain is used as the starting material, which exhibits an acetamido 
methyl protective group in the A.sup.7 - or A.sup.20 -position and a 
p-methoxybenzyl protective group in the A.sup.20 - or A.sup.7 -position, 
which is converted with a natural or synthetic insulin B-chain that is 
present in reduced form, or more preferably exhibits an acetamido methyl 
protective group in the B.sup.19 - or B.sup.7 -position and a 
p-methoxybenzyl protective group in the B.sup.7 - or B.sup.19 -position. 
For the production of the antiparallel insulin analog of formula V above 
([A.sup.7 -A.sup.11, A.sup.6 -B.sup.19, A.sup.20 -B.sup.7 
-cystine]-insulin), a synthetic [A.sup.7 -A.sup.11 ]-insulin A-chain, 
which exhibits an acetamido methyl protective group in the A.sup.6 - or 
A.sup.20 -position and a p-methoxybenzyl protective group in the A.sup.20 
- or A.sup.6 -position, is converted with a natural or a synthetic insulin 
B-chain that is present in reduced form, or more preferably, exhibits an 
acetamido methyl protective group in the B.sup.19 -position or B.sup.7 
-position and a p-methoxybenzyl protective group in the B.sup.7 - or 
B.sup.19 -position. 
For the production of the antiparallel insulin analog of formula VI above 
([A.sup.6 -A.sup.7, A.sup.11 -B.sup.19, A.sup.20 -B.sup.7 
-cystine]-insulin), a synthetic [A.sup.6 -A.sup.7 ]-insulin A-chain, which 
exhibits an acetamido methyl protective group in the A.sup.11 - or 
A.sup.20 -position and a p-methoxybenzyl protective group in the A.sup.20 
- or A.sup.11 -position, is converted with a natural or synthetic insulin 
B-chain that is present in reduced form, or more preferably, exhibits an 
acetamido methyl protective group in the B.sup.19 - or B.sup.7 -position 
and a p-methoxybenzyl protective group in the B.sup.7 - or B.sup.19 
-position. 
The object of the present invention is, furthermore, a process 
characterized in that a natural or synthetic insulin A-chain, with the 
intrachenaric disulfide ring in the A.sup.6 -A.sup.11 -position, the 
A.sup.7 -A.sup.11 -position, or in the A.sup.6 -A.sup.7 -position, in a 
parallel or antiparallel manner connected to a natural or synthetic 
insulin B-chain that is present in reduced form or exhibits acetamido 
methyl protective groups or p-methoxybenzyl protective groups in the 
positions defined above is linked by means of a reactive anchor bond to a 
polymeric carrier. In the process, an especially long-lived, 
unphysiological storage form of insulin is obtained that is of great 
advantage for purposes of therapeutic application. 
In carrying out the process according to the present invention, the 
formation of the disulfide bridge is effected by means of oxidation with 
iodine, preferably in 30% aqueous acetic acid. 
The splitting off of the p-methoxybenzyl protective group or groups is 
advantageously effected in such a way that the reagent 
pyridine/polyhydrogen fluoride (HF/pyridine) is used as the solvent. 
The use of pyridine-polyhydrogen fluoride (HF/pyridine) as an agent for 
splitting off protective groups in peptide chemistry is already known (see 
J. C. S. Chem. Comm. (1976), pp. 451 and 452). It could, however, not be 
expected that the splitting off of the p-methoxybenzyl protective group in 
the presence of anisol as a cation-catcher succeeds especially smoothly, 
and this is without the influence of the acetamido methyl protective group 
which is still available, and without the influence of the disulfide 
bridges which may, perhaps, already be available. 
In what follows below, the present invention is explained in detail with 
respect to the synthesis of bovine insulin. In so doing, first, the 
fragment synthesis, which is known in itself, of an insulin A-chain, with 
sulfur protective groups that can be selectively split off, to a polymeric 
carrier, as well as the conversion, according to the present invention, of 
this synthetic A-chain, under the gradual, oxidative, disulfide bridge 
formation with a natural B-chain, into a fully active, crystalline bovine 
insulin is explained. The concept applied in doing this is a further 
development of the principles that led to the synthesis of the MCD-peptide 
(C. Birr and W. Wengert-Muller, Angewandte Chemie 91 (1979), p. 156 and 
German Published Patent 28 30 442. 
In so doing, the following are used as sulfur protective groups: the 
disulfide with tertiary butyl mercaptan (StBu or SBU) on cysteine in the 
A.sup.6 - and A.sup.11 -positions, the acetamido methyl protective group 
(Acm or ACM) in the A.sup.7 -position, and the p-methoxybenzyl protective 
group (Mbzl or MBZL) in the A.sup.20 -position. 
In the following, the present invention is explained in detail, with 
reference to the attached drawings.

The abbreviations indicated in FIGS. 1 to 4 have the following meanings: 
DDZ or Ddz=.alpha.,.alpha.-dimethyl-3,5-dimethoxy-benzyloxycarbonyl group, 
OME=methoxy group, 
OBU or OBu.sup.t =tertiary-butoxy group, 
OBZL or OBzl=benzyloxy group, 
SBU or SBu.sup.t =butyl mercapto group, tef. 
ACM or Acm=acetamido methyl group, 
BU or Bu.sup.t =butyl group, tef. 
MBZL or Mbzl=p-methoxybenzyl group. 
The preparation of the individual Fragments I, II, III, and IV was reported 
by C. Birr in Danzig in 1978. 
Fragment I (1-3) is formed from Ddz-amino acids in solution, with a yield 
of 84%, according to a method that was recently published (C. Birr et al., 
Int. J. Peptide Protein Res. 13 (1979), pp. 287-295). Fragments II (4-14) 
and IV (15-21) [see the models represented in FIGS. 2 and 3] are built up 
on a polystyrene gel cross-linked with 0.5% divinyl benzene. Referred to 
the initial charge, yields of 90% (Fragment II) or 93% (Fragment IV) are 
obtained. All the synthesizing measures in the production of the fragment 
and their condensation on the polymeric carrier are continually controlled 
with a recording instrument, photometrically, by means of the 
spectroscopic properties of the acid-labile, temporarily N-terminal 
Ddz-protective group (C. Birr in Y. Wolman, Peptides 1974 New York; 
Halsted Press, 1975), pp. 117 ff. 
Asp (OBzl) or Glu (OBzl) are inserted in the 5-, 15-, and 18-positions in 
place of Asn and Gln, in order to avoid a nitrile formation at the 
amide-side function by the dicyclohexylcarbodiimide-condensation reagent. 
In C-terminal Fragment IV, Asp (OH)OBu.sup.t with its .beta.-carboxyl 
function is bound to the aromatic substances of the polystyrene carrier 
via an electrophile 2-oxoethyl-ester linkage. In this manner, all the 
future amide-side functions can be introduced simultaneously at the end of 
the synthesis by means of an ammonolysis of the designated benzyl- and 
2-oxoethyl ester linkage in the separation of Fragments IV or V from the 
polymeric carrier (see FIG. 4). 
All the other side functions of the insulin A-chain are safely protected 
during the synthesis of the fragments by tertiarybutyl protective groups. 
By means of the condensation of Fragment I in a fifteen-fold excess, with 
the aid of the condensation reagent's dicyclo hexyl carbodiimide in 
dimethyl formamide with the carrier-bound Fragment II, Fragment III (1-14) 
is formed on the carrier in a yield of 86% (referred to the II-gel 
polymer). By basic splitting of the 2-oxoethyl ester anchor bond of 
Fragment III on the polystyrol carrier (0.5 n-triethylamine in 
methanol/dioxane (1/1, volume/volume)+0.5% by volume 1 n aqueous sodium 
hydroxide solution)), followed by chromatographic purification of the 
separated, fully protected peptic acid, 1 g of the pure Fragment III is 
obtained (yield=59% [referred to the III gel polymer]). The stable 
existence of the benzyl ester in the A.sup.5 -position under the cleavage 
conditions was proved via the mass spectrum. C-terminal Fragment IV is 
split off from the carrier with dioxane saturated with ammonia, followed 
by a liquid ammonia/methanol mixture (4/1, volume/volume) under pressure 
at 20.degree. C. After the chromatographic purification (DEAE A 25 
Sephadex/methanol-0.5 n acetic acid (4/1, volume/volume) and Sephadex LG 
20/methanol)), 1.45 g of the pure Fragment IV is obtained (yield=86%, 
referred to the carrier-linked preliminary stage). In model experiments, 
it has turned out that, under the conditions mentioned, at the C-terminal 
the .beta.-amide of aspartic acid-.alpha.-tertiary-butyl ester is formed, 
and not the succinimide derivative. 
The condensation of 0.5 mMol of Fragment III with 1 mMol of Fragment IV to 
the fully protected Fragment V (1-21), with the aid of the 
carbodiimide-condensation reagent in dimethyl formamide (for a period of 4 
days), results, after chromatographic purification, in 1.3 g of Fragment V 
(which corresponds to a yield of 78%, referred to Fragment III). A similar 
condensation of Fragment III in a 2.4-fold excess with Fragment IV on the 
carrier, with the use of carbonyl diimidazole, results in a yield of 
Fragment V of 12%. At the same time, 50% of the excess of Fragment III can 
be recovered. 
In Fragment V, the benzyl ester in the A.sup.5 -position is converted into 
the amide with ammonia in methanol under pressure. 
Then the tertiary-butyl disulfides in the A.sup.6 - and A.sup.11 -positions 
are split by reduction with tributyl phosphane, according to the model 
represented in FIG. 4, and the intrachenaric disulfide ring A.sup.6 
-A.sup.11 is formed selectively by oxidation in air with a yield of 84% 
(referred to the fully protected Fragment V). Then all the tertiary-butyl 
ester and ether protective groups are split off with trifluoroacetic acid. 
In this stage, the synthetic insulin A-chain still has the sulfur 
protective groups only in the A.sup.7 (Acm)- and A.sup.20 
(Mbzl)-positions. The p-methoxybenzyl protective group (Mbzl) is 
selectively split off with hydrogen fluoride in pyridine in the presence 
of anisol (with a yield of about 80%). 
The insulin A-chain with a single cysteinyl-mercapto function (A.sup.20) 
that has been obtained in this way is immediately dissolved in 30% acetic 
acid, an equimolar quantity of reduced, natural B-chain from bovine 
insulin (2 free SH-funtions) in an acetic acid solution is mixed with it, 
and it is oxidized with 0.1 n iodine solution. Along with the 
interchenaric formation of the disulfide bridge exclusively between the 
A.sup.20 -B.sup.19 -positions that has been aimed at, as a secondary step 
in so doing, the last still remaining acetamido methyl protective group 
(Acm) in the A.sup.7 -position is removed by iodolysis, and the second 
interchenaric disulfide bridge (A.sup.7 -B.sup.7) is then also formed by 
oxidation. 
Then chromatography is immediately carried out in 10% acetic acid with 
Sephadex G 50. In the process, bovine insulin is obtained by 
electrophoresis and thin-layer chromatography which migrates in a manner 
identical with that of the authentic comparison material, with a yield of 
25%. The freeze-dried raw material shows an activity of 52% in fat cells 
in the biological lipogenesis test, and an activity of 65% in the 
competitive receptor binding test. 
The material can be crystallized as a zinc complex, and shows the 
characteristic crystalline form of insulin (FIG. 5). After repeated gel 
chromatography with Biogel P6 in 1% acetic acid and ion-exchange 
chromatographie with carboxy methyl cellulose, the semisynthetic bovine 
insulin formed according to the present invention shows full biological 
activity in both test systems mentioned above. 
It is immediately evident that in using the selectively cleavable sulfur 
protective groups only on the insulin A-chain, the yield of the 
combination when using the chains in the proportion of 1:1, as compared 
with the previously known statistical disulfide bridge formation, can 
already be improved by more than 100%. This method can even be 
significantly improved, if the B.sup.7 -positions are also protected with 
an acetamido methyl protective group and the B.sup.19 -positions with a 
p-methoxybenzyl protective group, and can then be selectively set free in 
the process according to the present invention. 
The following example will serve for further explanation of the invention. 
EXAMPLE 
The individual steps of the peptide synthesis are carried out in an 
automatic synthesizing machine made by the Schwarz Bio Research Company, 
with the aid of a centrifugal reactor. Conversion control is carried out 
by measuring the UV-absorption at 280 nm and 230 nm, and is registered 
with a two-channel recording instrument. 
(a) Synthesis of the Fragment Insulin 1-3 (Fragment I) in Solution 
(a)1 Synthesis of ValOMe 
C.sub.6 H.sub.13 NO.sub.2 (131,173) 
15.8 ml of thionyl chloride is added to 70 ml of methanol which is cooled 
in an ice/NaCl mixture to -5.degree. to -10.degree. C. Then 23.4 g of 
valine is added, during which the temperature should not rise above 
-5.degree. C. The temperature is then slowly raised to 40.degree. C., and 
stirring is carried out at this temperature for 2 hours. The mixture is 
then concentrated in a vacuum, and taken up in 5% NaHCO.sub.3. It is then 
extracted with acetic ester, and the acetic ester phase is washed neutral 
with H.sub.2 O, and dried with Na.sub.2 SO.sub.4. After evaporating in a 
vacuum at 40.degree. C., the white residue is dried in a desiccator over 
P.sub.2 O.sub.5. 
Yield: 19 g: 80%. 
Thin-layer chromatogram (n-butanol/glacial acetic acid/water; 4/1/1): 
Rf=0.46. 
(a)2 Ddz-Ile-ValOMe (Mixed anhydride method) 
C.sub.24 H.sub.38 N.sub.2 O.sub.7 (466,577) 
(1) Preparation of the amine component 2.6 g (20 mMol) of ValOMe is 
dissolved in 50 ml of CH.sub.2 Cl.sub.2 and cooled to -15.degree. C. 
(2) Preparation of the carboxyl component and coupling 
7.3 g (20 mMol) of Ddz-Ile is dissolved in 50 ml of CH.sub.2 Cl.sub.2 and 
treated with 2.21 ml (20 mMol) of N-methyl morpholine. The mixture is then 
cooled to -15.degree. C., activated by the addition of 2.24 ml (20 mMol) 
of chloroformic acid isopropyl ester (activation period 8 minutes), and 
the amine component is added all at one time. 
(3) Work-up 
The reaction mixture is extracted five times with ice-cold 0.1 n KHSO.sub.4 
-solution, once with water saturated with NaCl, and five times with 
KHCO.sub.3 solution. The solvent is dried with Na.sub.2 SO.sub.4 and 
subjected to vacuum distillation. The remaining residue is dried over 
P.sub.2 O.sub.5. 
Yield: 7.4 g: 80%. 
Thin-layer chromatogram (benzol/glacial acetic acid; 7/1: Rf=0.39 
(a)3 Ddz-Gly-Ile-ValOMe 
C.sub.26 H.sub.41 N.sub.3 O.sub.8 (523,630) 
(1) Preparation of the amine component 
7.4 g (16 mMol) of Ddz-Ile-ValOMe is dissolved in 100 ml of 5% 
trifluoroacetic acid in CH.sub.2 Cl.sub.2. (The protective group is split 
off in 15 minutes.) The reaction solution is then neutralized with 
N-methyl morpholine (with pH-paper to pH 7-7.5), and cooled to -15.degree. 
C. 
(2) Preparation of the carboxyl component and coupling 
5.6 g (20 mMol) of Ddz-Gly is dissolved in 50 ml of CH.sub.2 Cl.sub.2, and 
treated with 2.21 ml (20 mMol) of N-methyl morpholine. The reaction 
mixture is then cooled to -15.degree. C., activated by the addition of 
2.24 ml (20 mMol) of chloroformic acid isopropyl ester (activation period 
8 minutes), and the amine component is added all at one time. 
(3) Work-up 
The reaction mixture is extracted five times with ice-cold 0.1 n KHSO.sub.4 
solution, once with water saturated with NaCl, and five times with 
KHCO.sub.3 solution. The solvent is dried with Na.sub.2 SO.sub.4, and 
subjected to vacuum distillation. The remaining residue is dried over 
P.sub.2 O.sub.5. 
Yield: 7.1 g: 88%. 
Melting point 147.degree. C. 
Amino acid analysis: HCl/propionic acid 15 minutes 160.degree. C. 
______________________________________ 
Gly Val Ile 
______________________________________ 
Calculated 1 1 1 
Found 1.10 1.00 1.01 
______________________________________ 
Mass spectrum: m/e=523. 
(a)4 Ddz-Gly-Ile-Val 
C.sub.25 H.sub.39 N.sub.3 O.sub.8 (509,604) 
7.1 g (14 mMol) of Ddz-Gly-Ile-ValOMe is dissolved in 20 ml of dioxane, and 
treated with 2 equiv. 1 n NaOH. The reaction mixture is stirred at room 
temperature, neutralized with 1 n acetic acid, concentrated in a vacuum, 
and purified by means of gel chromatography (Sephadex LG-20/methanol). 
The saponification is carried on with thin-film chromatography: complete 
saponification. 
Thin-layer chromatogram (chloroform/methanol/pyridine; 95/5/3): Rf=0.10. 
O.R.D. value: [.alpha.].sub.D.sup.25 =+1.5 (C=1.4) methanol. 
(b) Automatic solid phase synthesis of insulin A-chain fragments 15-21 and 
4-14 (IV or II) 
______________________________________ 
(1) 1 .times. 
2 min Splitting off Ddz* with trifluoro- 
acetic acid in CH.sub.2 Cl.sub.2 
(2) 1 .times. 
15 min 
(3) 6 .times. 
2 min each Washing with CH.sub.2 Cl.sub.2 
(4) 1 .times. 
2 min Repetition of the Ddz-cleavage with 
(5) 1 .times. 
15 min 5% trifluoroacetic acid 
(6) 6 .times. 
2 min each Washing with CH.sub.2 Cl.sub.2 
(1-6) The filtrate of the cleavage product and the wash 
liquid are collected for photometric determina- 
tion of the Ddz-cleavage product; 
conversion control 
(7) 4 .times. 
2 min each Reaction with triethyl amine 
[CH.sub.2 Cl.sub.2 /dimethylformamide (1/1)] 
1:9 
(8) 12 .times. 
2 min each Washing with CH.sub.2 Cl.sub.2 
(9) 1 .times. 
addition of 20 ml of Ddz-amino acid solution 
(calculated on the basis of the pre- 
ceding Ddz-values) in CH.sub.2 Cl.sub.2 
(10) 1 .times. 
addition of 20 ml of dicyclo hexyl carbodiimide 
solution (same equivalents as Ddz-amino acid) in 
dimethyl formamide 
(11) 1 .times. 
addition of 20 ml dimethyl formamide/CH.sub.2 Cl.sub.2 (1/1) 
(12) 1 .times. 
60 min Reaction time 
(13) 3 .times. 
2 min Washing out with CH.sub.2 Cl.sub.2 
(14) 5 .times. 
2 min Washing out with CH.sub.2 Cl.sub.2 /methanol 
(15) 4 .times. 
2 min Washing out with CH.sub.2 Cl.sub.2 
(16) Repetition of steps 9-15 twice 
(17) 1 .times. 
addition of 40 ml 0.1 M 3-nitrophthalic acid an- 
hydride in pyridine 
(18) 1 .times. 
addition of 20 ml dimethyl formamide/CH.sub.2 Cl.sub.2 
/(1/1) 
(19) 1 .times. 
10 min Reaction time 
(20) 8 .times. 
2 min Washing out CH.sub.2 Cl.sub.2 
(21) 5 .times. 
2 min Washing out with CH.sub.2 Cl.sub.2 /methanol 
(4/1) 
(22) 4 .times. 
2 min CH.sub.2 Cl.sub.2 
(23) 5 .times. 
2 min Washing out with CH.sub.2 Cl.sub.2 /methanol 
(4/1) 
(24) 4 .times. 
2 min Washing out with CH.sub.2 Cl.sub.2 
______________________________________ 
*Ddz = .alpha.,dimethyl-3,5-dimethoxy-benzyloxy-carbonyl group 
The synthesizing program described above is used for the synthesis of 
insulin A 15-21 (Fragment IV) and A 4-14 (Fragment II). The carboxyl 
component (Ddz-amino acid) in each case is used in each cycle in fourfold 
excess, referred to the previous Ddz-value. Methylene chloride that has 
been purified over an Al.sub.2 O.sub.3 -column serves as a solvent and 
wash liquid for steps 1 to 9. The synthesis is started with 3 g of 
Ddz-AspOBu.sup.t -polystyrene (cross-linked with 0.5% divinyl benzene) and 
3 g of Ddz-Tyr(Bu.sup.t) -polystyrene (cross-linked with 0.5% divinyl 
benzene) in a centrifugal reactor. 
Charges: 
0.526 mMol Ddz-AspOBu.sup.t /g polymer 
0.664 mMol Ddz-AspOBu.sup.t /g polymer 
0.900 mMol Ddz-Tyr(Bu.sup.t)/g polymer 
0.857 mMol Ddz-Tyr(Bu.sup.t)/g polymer 
If no volume is indicated in the program, a volume of 60 ml is measured out 
at this point by the automatic synthesizing machine. 
The conversions, referred to the initial charge, are found on the basis of 
photometric measurement of the Ddz-cleavage product as follows: 
Conversion results insulin 15-21. 
______________________________________ 
Con- 
First Synthesis ver- Second Synthesis 
Charge sion Charge Conver- 
mMol/g carrier % mMol/g carrier 
sion % 
______________________________________ 
AspOBu.sup.t 
0.526 -- 0.664 -- 
Cys(Mbzl) 
0.529 100 0.714 100 
Tyr(Bu.sup.t) 
0.412 78 0.660 100 
Asp(OBzl) 
0.358 92 0.670 100 
Glu(OBu.sup.t) 
0.308 86 0.593 90 
Leu 0.250 81 0.563 95 
Glu(OBzl) 
0.240 96 0.600 100 
______________________________________ 
See FIG. 2. The average yield of both syntheses amounts to 93%. 
Conversion rresults insulin 4-14. 
______________________________________ 
Con- 
First Synthesis ver- Second Synthesis 
Charge sion Charge Conver- 
mMol/g carrier % mMol/g carrier 
sion % 
______________________________________ 
Tyr(Bu.sup.t) 
0.900 -- 0.857 -- 
Leu 0.523 58 0.600 70 
Ser(Bu.sup.t) 
0.502 96 0.591 97 
Cys(SBu.sup.t) 
0.491 98 0.571 98 
Val 0.470 96 0.442 77 
Ser(Bu.sup.t) 
0.463 99 0.400 90 
Ala 0.470 100 0.414 100 
Cys(Acm) 
0.466 99 0.407 98 
Cys(SBu.sup.t) 
0.500 100 0.392 96 
Glu(OBzl) 
0.433 86 0.352 89 
Glu(OBu.sup.t) 
0.300 69 0.300 86 
______________________________________ 
See FIG. 3. The average yield of both syntheses is 90%. 
The second synthesis is not interrupted after the tenth cycle, instead the 
conversion with insulin A 1-3 follows. In each case, the tripeptide is 
used in fivefold excess in 3 conversions. For this reaction, the synthesis 
program is changed as follows: 
(9) 1.times.addition of 20 ml of Ddz-tripeptide solution (CH.sub.2 
Cl.sub.2) 
(10) 1.times.addition of 20 ml 1-hydroxybenzotriazole solution (in dimethyl 
formamide) 
(11) 1.times.addition of 10 ml of dicyclohexylcarbodiimide solution in 
dimethyl formamide/CH.sub.2 Cl.sub.2 (1/1) 
(12) 1.times.addition of 20 ml of CH.sub.2 Cl.sub.2 dimethyl formamide 
(1/1) 
(13) 1.times.4 hours reaction time 
(14) 3.times.2 min washing out with CH.sub.2 Cl.sub.2 
(15) 5.times.2 min washing out with CH.sub.2 Cl.sub.2 /methanol (4/1) 
(16) 4.times.2 min washing out with CH.sub.2 Cl.sub.2 
(17) Repetition of steps 9-16 twice. 
In the condensation of the fragment, 15.75 mMol tripeptide 1 equivalent 
dicyclo hexyl carbodiimide, and 1.5 equivalents of 1-hydroxybenzotriazole 
are used in each cycle. 
Conversion result insulin 1-14 (Fragment III). 
______________________________________ 
Charge Conversion 
mMol/g carrier 
% 
______________________________________ 
Insulin A 4-14 
0.300 -- 
Insulin A 1-14 
0.258 86 
______________________________________ 
(c) Synthesis of the insulin A-chain to the polymeric carrier 
(c)1 Automated cleavage of the insulin Fragments II (4-14), III (1-14), and 
IV (15-21) from the polymeric carrier 
The cleavage takes place with modified Beyerman reagent (1 n 
triethylamine/methanol+1% 1 n aqueous NaOH)/dioxane (1:1), with the 
following synthesis program: 
(1) 1.times.2 min peptide cleavage 
(2) 3.times.4 min peptide cleavage 
(3) 20.times.30 min peptide cleavage 
The whole cleavage reaction is continuously recorded by means of the 
photometric control device. The end of the cleavage reaction can be read 
from the recording. Most of the time, the cleavage is finished after about 
3-6 hours. The filtrate is collected in a flask in which dry ice has been 
placed to neutralize the basic peptide solution. Since 3-nitrophthalic 
acid anhydride has been used in the synthesis as a blocker, the core 
sequences can be separated by means of a first fractionation with DEAE-ion 
exchanger A-25, using a mixture of methanol/0.5 n acetic acid (4/1). After 
this, a purification is carried out with column chromatography, using 
Sephadex LH-20 (2.5.times.200) and methanol as a vehicle. 
(c)2 
Ddz-Glu(OBu.sup.t)-Glu(OBzl)-Cys(SBu.sup.t)-Cys(Acm)-Ala-Ser(Bu.sup.t)-Val 
-Cys(SBu.sup.t)-Ser(Bu.sup.t)-Leu-Tyr(Bu.sup.t) [1] 
C.sub.94 H.sub.148 N.sub.11 O.sub.24 (1816,285) 
After the separation and purification described above, the mixture of 
methyl ester and free acid of Fragment II (4-14) is subjected to 
saponification. 
About 1.1 g of Fragment II (4-14) is dissolved in 3 ml of dioxane, and 
treated with 2 equivalents of 1 n NaOH. The pH of the solution is kept at 
11.5 with a pH-stat. After 4 hours at room temperature, the reaction is 
concluded. Neutralization is then carried out with 1 n acetic acid, and 
concentration takes place in a vacuum. The removal of salt then follows by 
means of gel chromatography with Sephadex LH-20/methanol. Finally, drying 
is carried out over P.sub.2 O.sub.5. The yield is 73% (1.1 g), referred to 
the last Ddz-value of the N-terminal amino acid of the heptapeptide bound 
to the carrier. 
Thin layer chromatogram (n-butanol/glacial acetic acid/water; 4/1/1): 
Rf=0.83. 
Mass spectroscopic evidence of the protective groups: 
______________________________________ 
Cleavage product of 
Ddz Acm OBzl 
______________________________________ 
m/e 178 72 107 
______________________________________ 
Amino acid analysis: (HCl/propionic acid, 15 minutes, 160.degree. C.). 
______________________________________ 
Cys 
CysSO.sub.3 H Ser Glu Ala Val Leu Tyr 
______________________________________ 
Calculated 
3 2 2 1 1 1 1 
Found 2.38 1.82 1.64 1.10 1.13 1.00.sup.a 
0.95 
______________________________________ 
.sup.a Reference amino acid 
O.R.D. value: [.alpha.].sub.D.sup.25 =-38.2 (C=0.5)MeOH. 
(c)3 
Ddz-Gly-Ile-Val-Glu(OBu.sup.t)-Glu(OBzl)-Cys(SBu.sup.t)-Cys(Acm)-Ala-Ser(B 
u.sup.t)-Val-Cys(SBu.sup.t)-Ser(Bu.sup.t)-Leu-Tyr(Bu.sup.t). [2] 
C.sub.107 H.sub.171 N.sub.14 O.sub.27 (2085,634) 
After the separation described, in general, above, and the purification 
operations, a separation of Fragment III (1-14) [2] takes place with 
sephadex LH-60/methanol. The mixture of methyl ester and free acid that is 
also not separated by this process is saponified in a manner analogous to 
that for Fragment II [1] again. 
The yield is 59% (1.1 g), referred to the last Ddz-value of the N-terminal 
amino acid of the heptapeptide bound to the carrier. 
Thin-film chromatogram (chloroform/methanol/glacial acetic acid; 85/15/5): 
Rf=0.63 (uniformly). 
Mass spectroscopic evidence of the protective groups: 
______________________________________ 
Cleavage product of 
Ddz Acm OBzl Bu.sup.t 
______________________________________ 
m/e 178 72 107 57 
______________________________________ 
Amino acid analysis: 6 n HCl, 28 hours, 110.degree. C. 
__________________________________________________________________________ 
Cys 
CysSO.sub.3 H 
Ser 
Glu 
Gly 
Ala 
Val 
Ile 
Leu 
Tyr 
__________________________________________________________________________ 
Calculated 
3 2 2 1 1 2 1 1 1 
Found 2.86 2.08 
1.78 
1.05 
1.18 
2.04 
1.03 
1.00.sup.a 
0.86 
__________________________________________________________________________ 
.sup.a Reference amino acid 
Rotation value: [.alpha.].sub.D.sup.25 =-32.3 (C=0.6 methanol). 
This fragment is later put into solution for the condensation. 
(c)4 
Ddz-Glu(OBzl)-Leu-Glu(OBu.sup.t)-Asp(OBzl)-Tyr(Bu.sup.t)-Cys(MBzl)-AspOBu. 
sup.t. [3] 
C.sub.81 H.sub.109 B.sub.7 O.sub.22 (1532,803) 
After the separation and purification of Fragment IV (15-21) [3], the 
saponification is carried out in a manner analogous to that for Fragment 
II [1]. 
The yield is 51% (0.5 g), referred to the last Ddz-value of the N-terminal 
amino acid of the heptapeptide bound to the carrier. 
Thin-film chromatogram (n-butanol/glacial acetic acid/water; 4/1/1): 
Rf=0.80. 
Mass spectrometric evidence of the protective groups: 
______________________________________ 
Cleavage product of 
Ddz OBzl MBzl Bu.sup.t 
______________________________________ 
m/e 178 107 121 57 
______________________________________ 
Amino acid analysis: HCl/propionic acid, 15 minutes, 160.degree. C. 
______________________________________ 
Asp Glu Leu Tyr Cys 
______________________________________ 
Calculated 2 2 1 1 1 
Found 1.67 1.70 1.00.sup.a 
0.87 0.83 
______________________________________ 
.sup.a Reference amino acid 
Rotation value: [.alpha.].sub.D.sup.25 =-41.2.degree. (C=2) methanol. 
(c)5 Recoupling Fragment IV (Insulin 15-21) to the polymeric carrier 
##STR2## 
400 mg (0.26 mMol) insulin A 15-21 [3] is dissolved in 5 ml of methanol. 
Then 38.4 mg (0.23 mMol) of CsOH solution is added (10% shortage). The 
solvent is distilled off in a vacuum, and the residue is dried over 
P.sub.2 O.sub.5. 
For resin esterification, the dried cesium salt is dissolved in absolute 
dimethyl formamide, and added in fivefold excess to the resin (1.89 mMol 
Br/g resin) (634 mg). The charge is agitated for 3 days at 40.degree. C. 
The resin is filtered off through a glass frit, and thoroughly washed with 
the following solvents: dimethyl formamide, chloroform, dioxane/methanol 
(3/1), methanol, dioxane and methanol. After that, it is dried in a 
desiccator over P.sub.2 O.sub.5. The charge of the resin is determined by 
means of elementary analysis for nitrogen, by photometric analysis of the 
cleavage of the Ddz-protective group, and by quantitative amino-acid 
analysis. A quantitative amino-acid analysis showed a charge of 0.11 
mMol/g of resin. 
(c)6 Recovery of Fragment IV (15-21] 
The various wash solutions are combined, and concentrated by evaporation on 
a rotary evaporator. The residue is dissolved in acetic ester, and 
converted into the free acid by extraction with 0.5 n KHSO.sub.4 solution. 
The organic phase is then washed neutral with H.sub.2 O and dried over 
Na.sub.2 SO.sub.4. Further concentration then takes place by means of 
evaporation under vacuum on a rotary evaporator, and drying is carried out 
over P.sub.2 O.sub.5. 200 mg of Fraction IV (insulin 15-21), which has 
been purified by means of thin layer chromatography, is recovered. 
(c)7 Fragment condensation to Fragment III (insulin 1-14) in solution 
Ddz-Gly-Ile-Val-Glu(OBu.sup.t)-Glu(OBzl)-Cys(SBu.sup.t)-Cys(Acm)-Ala-Ser(Bu 
.sup.t)-Val-Cys(SBu.sup.t)-Ser(Bu.sup.t)-Leu-Tyr(Bu.sup.t) [5] 
C.sub.107 H.sub.171 N.sub.14 O.sub.27 (2085,634) 
763 mg (1.5 mMol) of Fraction I (insulin A 1-3), 270 mg (2 mMol) of 
1-hydroxybenzotriazole and 0.22 ml (2 mMol) of N-methyl morpholine are 
dissolved in 20 ml of absolute dimethyl formamide. The solution is cooled 
to 0.degree. C., and treated with 206 mg (1.0 mMol) of dicyclohexyl 
carbodiimide. The mixture is agitated for 1 hour at 0.degree. C., and then 
600 mg (0.35 mMol) of Fragment II (insulin A 4-14 [1])(adjusted to pH 7.5 
with N-methyl morpholine) is added. Agitation is then carried out for 12 
hours overnight, concentration then follows in a vacuum, and purification 
takes place by means of gel chromatography (Sephadex LH-20/methanol). 
The yield amounts to 413 mg of Fragment III (insulin A 1-14), which 
corresponds to a conversion of 52%. Amino acid analysis: HCl/propionic 
acid, 20 minutes, 160.degree. C. 
__________________________________________________________________________ 
Cys 
CysSO.sub.3 H 
Ser 
Glu Gly 
Ala Val 
Ile Leu 
Tyr 
__________________________________________________________________________ 
Calcul. 
3 2 2 1 1 2 1 1 1 
Found 
1.66 1.64 
1.99 
1.02 
1.00.sup.a 
2.21 
0.93 
1.04 
0.65 
__________________________________________________________________________ 
.sup.a Reference amino acid 
O.R.D. value: [.alpha.].sub.D.sup.25 =-32.1 C=0.1 methanol. 
(c)8 Fragment condensation to Fragment V (insulin 1-21) on the polymeric 
carrier 
##STR3## 
700 mg of insulin 15-21-phenacetyl resin [4] (charge 0.074 mMol) is placed 
in a shaking frit, and treated with 5 percent trifluoroacetic acid in 
CH.sub.2 Cl.sub.2. Agitation takes place for half an hour, and then 
thoroughly washing out with CH.sub.2 Cl.sub.2. This procedure is repeated 
twice. The shaking frit is then dried over P.sub.2 O.sub.5. The peptide is 
deprotonized in the shaking frit with triethyl amine [CH.sub.2 Cl.sub.2 
/dimethyl formamide (1:2)](1:3), and thoroughly washed with dimethyl 
formamide/CH.sub.2 Cl.sub.2 and dimethyl formamide. 
400 mg (0.18 mMol) of Fragment III (insulin 1-14 [5]) and 28 mg (0.17 mMol) 
of carbonyl diimidazole are dissolved in 5 ml of absolute dimethyl 
formamide at 0.degree. C., and the solution is added to the above Fragment 
IV (15-21) which is bound to the carrier. Agitation is carried out for 4 
days, the reaction solution is filtered off, and the resin is dried over 
P.sub.2 O.sub.5. Fragment III (insulin 1-14) can be recovered, as 
described further below. A quantitative amino acid analysis, referred to 
alanin in Fragment III (insulin 1-14), shows a charge of 0.0086 mMol, that 
is 30 mg of insulin 1-21. This corresponds to a conversion of 11.6%. The 
conversion is repeated, but an increase in the conversion can not be 
attained. 
Recovery of Fragment III (insulin 1-14) 
The various wash solutions are combined, and evaporated under vacuum in a 
rotary evaporator. By adding water, excess imidazolide is converted into 
the free acid. Evaporation is carried out again (in the rotary 
evaporator), and drying follows. The residue is purified by gel 
chromatography with LH-20. 210 mg of Fragment III (insulin 1-14) is 
recovered. 
(c)9 Separating the insulin A-chain from the polymeric carrier 
Ddz-Gly-Ile-Val-Glu(OBu.sup.t)-Gln-Cys(SBu.sup.t)-Cys(Acm)-Ala-Ser(Bu.sup.t 
)-Val-Cys(SBu.sup.t)-Ser(Bu.sup.t)-Leu-Tyr(Bu.sup.t)-Gln-Leu-Glu(OBu.sup.t) 
-Asn-Tyr(Bu.sup.t)-Cys(MBzl)-AsnOBu.sup.t. [1] 
The polymeric carrier [6] is put into suspension with 10 ml of methanol in 
a steel cylinder. With the exclusion of humidity, the steel cylinder is 
cooled, and treated with 80 ml of liquid ammonia. In this way, the 
.beta.-carboxyl function should be split off from its linkage to the 
phenacetyl carrier, and the .beta.-benzyl ester groupings of the aspartic 
acid in position 18 should also be split, and the .gamma.-benzyl ester 
grouping of glutamic acid in positions 5 and 15, and they should be 
converted in one step to the corresponding amide. The cylinder is sealed, 
and it is agitated for 3 days at room temperature. 
After the cleavage reaction, the resin is filtered off, and thoroughly 
washed with methanol. An amino acid analysis of a resin sample in 6 n HCl 
at 160.degree. C. for 20 minutes shows that the cleavage has proceeded 
completely. The cleavage solution is evaporated under a vacuum at 
40.degree. C., and re-dissolved in methanol. According to the thin-layer 
chromatographic control, the cleavage solution contains, along with the 
insulin A-chain that was aimed for, the fraction of Fragment IV (15-21 21) 
that was not converted. Besides, it remains an insoluble residue, which 
can not be dissolved even in acetic ester. An amino acid analysis is 
carried out on this solid residue. The analysis shows that the insoluble 
constituent consists of insulin A-chain. Since a mercaptan odor can be 
established, however, it can be assumed that the S-tertiary-butyl 
protective groups have been split off, in part, by the basic conditions, 
and the solid residue consists of oligomeric insulin A-chain (20 mg). The 
solid residue and the methanol solution are combined, and used for further 
reaction. The mixture is dried over P.sub.2 O.sub.5. 
Crude yield (methanol phase) about 80 mg. 
Insoluble residue about 20 mg. 
Thin-film chromatogram (n-butanol/glacial acetic acid/water; 4/1/1): Rf=0.9 
(fragment 15-21), 0.2 (insulin A-chain) 
(c)10 Formation of the intrachenaric disulfide bridge A.sup.6 -A.sup.11 
##STR4## 
The peptide mixture [1] from the last cleavage (c)9) is used for the 
reduction of Cys 6 and Cys 20. The reduction is carried out with tributyl 
phosphane. Then the closing of the disulfide linkages takes place by 
oxidation in air. 
30 mg of Fraction V (insulin 1-21) about 100 mg of mixture [1] are 
dissolved in 2.0 ml of propanol/water, and nitrogen is allowed to flow 
through. Then 4 .mu.l of tributylphosphane is added. The reaction solution 
is allowed to react for 48 hours under nitrogen at room temperature. 
Then all the reaction solution is poured into 1 l of water that has been 
adjusted to pH 8 with ammonia. Air is passed through the solution for 48 
hours. It is then concentrated under a vacuum, and separation takes place 
by means of gel chromatography with LH-20. 20 mg of pure, fully protected 
insulin A-chain is obtained [8]. 
The yield of the closing of the inner ring amounts to 66%. 
Amino acid analysis: HCl/propionic acid, 35 minutes, 160.degree. C. 
__________________________________________________________________________ 
Cys 
CysSO.sub.3 H 
Asp 
Ser 
Glu 
Ala 
Val 
Ile 
Leu 
Tyr 
Gly 
__________________________________________________________________________ 
Calc. 
4 2 2 4 1 2 1 2 2 1 
Found 
3.05 1.86 
2.12 
4.10 
1.03 
2.00.sup.a 
0.87 
2.24 
1.87 
0.97 
__________________________________________________________________________ 
.sup.a Reference amino acid 
O.R.D. value: [.alpha.].sub.D.sup.25 =-42.degree. (C=0.1) methanol 
(d) The synthesis of insulin A-chain in solution 
(d)1 The separation of Fragment IV (insulin A 15-21) from the polymeric 
carrier 
Ddz-Gln-Leu-Glu(OBu.sup.t)-Asn-Tyr(Bu.sup.t)-Cys(MBzl)-AsnOBu.sup.t. [9] 
C.sub.67 H.sub.100 N.sub.10 O.sub.19 (1349,560) 
The cleavage, in which both the .beta.-carboxyl function is converted from 
its linkage to the phenacetyl carrier to the amide and the .omega.-benzyl 
ester groupings of aspartic acid in position 18 as well as of the glutamic 
acid residue in position 15 to the amide, takes place with 
dioxane/NH.sub.3. The whole cleavage is continuously controlled with an 
UV-photometer, and registered by a recording instrument. The end is 
indicated after two days. A quantitative amino acid analysis shows that 
only 50% of the peptide has been split off from the carrier. 
The polymeric carrier is then put into suspension with 10 ml of methanol in 
a steel cylinder. With the exclusion of humidity, the cylinder is cooled 
to -70.degree. C., and treated with 80 ml of liquid ammonia. The cylinder 
is sealed, and agitated for 3 days at room temperature. After the cleavage 
reaction, the resin is filtered off and thoroughly washed with methanol. 
An amino acid analysis in 6 n HCl at 160 C/20 minutes shows that the 
cleavage has completely run its course. The various cleavage reactions are 
worked up as follows. 
A fractionation is then carried out with a mixture of methanol/0.5 n acetic 
acid (4/1), using a DEAE-A 25 ion exchanger. Purification follows, using 
column chromatography with Sephadex LH-20-column (2.5.times.200) cm, with 
methanol as a vehicle. The product of the cleavage with liquid ammonia 
contains two spots that run differently in the electrophoresis and on the 
DC-plate. It can be shown by dansylation that the Ddz protective group has 
been split off from a part of the peptide. 
The yield in the cleavage with dioxan/ammonia is 700 mg (42%), referred to 
the last Ddz-value of the N-terminal amino acid of the heptapeptide bound 
to the carrier. 
Thin layer chromatogram (chloroform/methanol/glacial acetic acid; 85/15/5): 
Rf=0.7. 
Thin-layer chromatogram (n-butanol/glacial acetic acid/water; 4/1/1): 
Rf=0.9. 
The yield in the cleavage with liquid ammonia under pressure amounts to 
0.75 g (44%), referred to the last Ddz-value of the N-terminal amino acid 
of the heptapeptide bound to the carrier. 
Thin-layer chromatogram (chloroform/methanol, glacial acetic acid; 
85/15/5): Rf=0.7 and 0.5. 
Thin-layer chromatogram (n-butanol/glacial acetic acid/water; 4/1/1: Rf=0.9 
and 0.8 
The total yield amounts to 86%) (1.45 g), referred to the last Ddz-value of 
N-terminal amino acid of the heptapeptide bound to the carrier. 
Amino acid analysis: HCl/propionic acid, 15 minutes, 160.degree. C. 
______________________________________ 
Asp Glu Leu Tyr Cys 
______________________________________ 
Calc. 2 2 1 1 1 
Found 1.91 1.84 1.00.sup.a 
0.76 0.58 
______________________________________ 
.sup.a Reference amino acid 
The pherogram at pH 1.9 l h 1000V shows a broad band at the start and about 
2 cm from the starting line on the anode side, and both of them give a 
ninhydrin positive reaction. 
(d)2 Condensation of the fragment to an insulin A-chain in solution 
Ddz-Gly-Ile-Val-Glu(OBu.sup.t)-Glu(OBzl-Cys(SBu.sup.t)-Cys(Acm)-Ala-Ser(Bu. 
sup.t)-Val-Cys(SBu.sup.t)-Ser(Bu.sup.t)-Leu-Tyr(Bu.sup.t)-Gln-Leu-Glu(OBu.s 
up.t)-Asn-Tyr(Bu.sup.t)-Cys(MBzl)-AsnOBu.sup.t. [10] 
1.1 g (0.5 mMOl) of the insulin A 1-14 [2] described in Section (c)3 is 
dissolved in 20 ml of absolute dimethyl formamide. The reaction solution 
is cooled down to -10.degree. C., and treated with 1 equivalent (103 mg) 
of dicyclohexyl carbodiimide. The mixture is agitated for 10 minutes at 
-10.degree. C. Then 2 equivalents of 1-hydroxybenzotriazole (135 mg) and 2 
equivalents of N-methyl morpholine (0.11 ml) are added, and the 
temperature is allowed to rise to 0.degree. C. (about 10 minutes). At 
0.degree. C., 2 equivalents (1.16 g) of the amine component [9] and 2 
equivalents of N-methyl morpholine (0.11 ml) are added, the mixture is 
heated to room temperature and agitated for 4 days. 
Then the reaction solution is concentrated in a vacuum, and separated by 
means of gel chromatography with methanol, using LH-20, and with a 
chloroform/methanol gradient, using silica gel K-60 (ready-made column). 
The yield amounts to 1.3 g of fully protected A-chain, which corresponds to 
a conversion of 78%, referred to Fragment III (1-14). 
Thin-layer chromatogram (n-butanol/glacial acetic acid/water; 4/1/1): 
Rf=0.2. 
The amino acid analysis (6n HCl, 64 hours, 110.degree. C.) produced the 
following results: 
__________________________________________________________________________ 
Cys 
CysSO.sub.3 H 
Asp 
Ser 
Glu 
Gly 
Ala 
Val 
Ile 
Leu 
Tyr 
__________________________________________________________________________ 
Calc. 
4 2 2 4 1 1 2 1 2 2 
Found 
4.12 2.21 
1.86 
3.94 
1.01 
1.02 
1.00.sup.a 
0.96 
2.24 
1.86 
__________________________________________________________________________ 
.sup.a Reference amino acid 
O.R.D. value: [ ].sub.D.sup.25 =37.3 (C=0.05) MeOH. 
(d)3 Conversion of Glu(OBzl) into Gln in Position 5 
Ddz-Gly-Ile-Val-Glu(OBu.sup.t)-Gln-Cys(SBu.sup.t)-Cys(Acm)-Ala-Ser(Bu.sup. 
t)-Val-Cys(SBu.sup.t)-Ser(Bu.sup.t)-Leu-Tyr(Bu.sup.t)-Gln-Leu-Glu(OBu.sup.t 
)-Asn-Tyr(Bu.sup.t)-Cys(MBzl)-AsnOBu.sup.t. [11] 
The peptide is dissolved in methanol, and placed in a steel cylinder. The 
steel cylinder is cooled, and with the exclusion of humidity, liquid 
ammonia is added. The cylinder is agitated for 4 days at room temperature. 
In this way, the .gamma.-benzyl ester is split off, with the simultaneous 
formation of amide. The cleavage solution is concentrated by evaporation 
in a vacuum at 40.degree. C., and is dissolved in methanol. An insoluble 
portion remains as a residue. 
Crude yield is about 1.1 g. 
The insoluble residue is about 0.2 g. 
The soluble portion and the solid residue was used together and for further 
reaction. 
(d)4 Formation of the intrachenaric disulfide bridge A.sup.6 -A.sup.11 
##STR5## 
The reduction of the disulfide bonds is carried out with tributyl 
phosphane; the closing of the disulfide bond takes place by air oxidation. 
1.3 g of the insulin A-chain [11] is dissolved in 20 ml of propanol/H.sub.2 
O (1.2 l), and N.sub.2 is made to flow through the solution. Then 155.6 
.mu.l of tributyl phosphane is added. The solution is allowed to react 
under nitrogen for 48 hours at room temperature. After that the whole 
reaction solution is poured into 6 l of H.sub.2 O, which has been adjusted 
to pH 8 with NH.sub.3. Air is passed through the solution for 48 hours. 
Then concentration is carried out in a vacuum, and LH-20 is separated by 
gel chromatography. 
1.09 g of the material [12] is obtained, which corresponds to a yield of 
84%. 
Amino acid analysis (110.degree. C. 6 n HCl, 24 hours). 
__________________________________________________________________________ 
Cys 
CysSO.sub.3 H 
Asp 
Ser 
Glu 
Gly 
Ala 
Val 
Ile 
Leu 
Tyr 
__________________________________________________________________________ 
Calc. 
4 2 2 4 1 1 2 1 2 2 
Found 
3.22 1.91 
2.02 
3.93 
1.05 
1.00.sup.a 
1.58 
0.67 
2.11 
2.21 
__________________________________________________________________________ 
.sup.a Reference amino acid 
O.R.D. value: [.beta.].sub.D.sup.25 =-42.0 (C=0.1) methanol. 
(d)5 Splitting off the protective groups of the combined insulin A-chain 
##STR6## 
The fully protected insulin A-chains [8] and [12] are combined. In order to 
split off the protective groups, concentrated trifluoroacetic acid is 
added to the peptide, and the mixture is allowed to stand for an hour at 
room temperature. Then the reaction solution is concentrated in a vacuum. 
The deblocked insulin A-chain is purified again by gel chromatography with 
Sephadex LH-20/methanol. Finally, the insulin A-chain is freeze-dried, and 
by so doing 750 mg of pure insulin A-chain is obtained. 
Previous oxidation: Performic acid.sup.106, 0.degree. C., 12 hours. 
Amino acid analysis: 6 n hydrochloric acid, 110.degree. C., 42 hours. 
__________________________________________________________________________ 
CysSO.sub.3 H 
Asp 
Ser 
Glu 
Gly 
Ala 
Val 
Ile 
Leu 
Tyr 
__________________________________________________________________________ 
Calc. 
4 2 2 4 1 1 2 1 2 2 
Found 
3.95 1.89 
1.70 
3.91 
1.00.sup.a 
1.08 
1.96 
0.69 
2.10 
1.67 
__________________________________________________________________________ 
.sup.a Reference amino acid 
O.R.D. value: [.alpha.].sub.25.sup.D =-76.2 C=0.5 methanol. 
At pH 1.9 l h 1000 V, the pherogram shows a broad band on the anode side 
about 3.5 cm from the start line, which reacts Cl.sub.I/II -positively. 
(e) The combination of purified synthetic bovine insulin A-chain with 
natural bovine insulin B-chain 
(e)1 The splitting off of the MBzl-protective group with HF/pyridine 
40.8 mg of the synthetic bovine insulin A-chain, formed in the above 
manner, is dissolved in 2 ml of HF/pyridine. The cleavage reagent serves, 
at the same time, as a solvent. 0.25 ml of anisol is added as a cation 
trap. After 60 minutes of reaction time at room temperature, the peptide, 
which has been reduced to Cys 20, is precipitated with ether. A sticky 
white precipitate is formed, which is thoroughly washed with ether several 
times. 
The deblocked A-chain is then immediately dissolved in 3 ml of 30 percent 
acetic acid. 
53.4 mg of reduced B-chain has already been dissolved previously in 2 ml of 
30 percent acetic acid. 
The prepared solution is then added to the reaction solvent and titrated 
with 0.1 n iodine solution until the color of the iodine remains constant 
after about 4 minutes. The excess iodine is back titrated with ascorbic 
acid until the reaction solution is colorless. 
(e)2 Purification of semisynthetic bovine insulin 
The reaction solution is immediately fractionated with the use of Sephadex 
G-50 in acetic acid (column 1.50.times.60 cm). Fractions 3, 4, 5, and 6 
are individually freeze-dried, and compared by electrophoresis with 
authentic bovine insulin. Fractions 3, 4, and 5 contain bovine insulin 
which has been identified by electrophoresis and thin layer 
chromatography. The crude yield amounts to 24.2 mg (25%). Biological tests 
are carried out with the crude product. 
The various fractions are again individually passed through, in 1 percent 
acetic acid, a biogel-P-6-column (column 2 m.times.0.8 cm). The fractions 
that contain insulin after this second separation are combined, and again 
passed through the same column. The main fraction now contains 18.2 mg of 
insulin (yield 20%). 
With this, a biological test was carried out again. Previous oxidation: 
performic acid .sup.106, 0.degree. C., 12 hours Amino acid analysis: 6 n 
HCl, 110.degree. C., 42 hours 
______________________________________ 
CysSO.sub.3 H 
Asp Thr Ser Glu Pro Gly Ala 
______________________________________ 
Calc. 6 3 1 3 7 1 4 3 
Found 5.91 3.70 0.86 2.71 6.75 0.91 4.03 3.23 
______________________________________ 
______________________________________ 
Val Ile Leu Tyr Phe His Lys Arg 
______________________________________ 
Calc. 5 1 6 4 3 2 1 1 
Found 5.12 0.86 6.00.sup.a 
3.73 3.38 1.89 1.11 1.06 
______________________________________ 
.sup.a Reference amino acid 
FIG. 5 represents a photomicrograph of the zinc complex of the fully active 
bovine insulin obtained. 
With the aid of the process according to the present invention, insulin and 
insulin analogs can be produced with precision, which have the 
intrachenaric and/or the interchenaric disulfide rings in natural or 
unnatural arrangement, and which are of great importance for therapeutic 
purposes. Thus, the antiparallel variants of insulin, especially, with a 
natural or unnatural arrangement of the intrachenaric small disulfide 
ring, represents interesting storage forms of insulin, since under 
physiological conditions, they are very slowly converted into active 
insulin. The insulin that is built up on and bound to the polymeric 
carrier, with a natural or unnatural arrangement of the intrachenaric 
and/or interchenaric disulfide rings, also represents such an especially 
long-lived, unphysiological insulin storage form. 
The object of the present invention, therefore, is also drugs which contain 
the insulin products obtained with the aid of process according to the 
present invention, in accordance with claims 2 to 18, with a natural or 
unnatural arrangement of the intra- and/or interchenaric disulfide 
bridges, if necessary in combination with the usual, pharmaceutically 
acceptable binders, carriers, and/or adjuvants. 
It will be understood that the specification and examples are illustrative 
but not limitative of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art.