PROCEDE DE PREPARATION D'ESTERS DE L'INSULINE

PROCESS FOR PREPARING INSULIN ESTERS

Abrégé anglais

ABSTRACT
Insulin and insulin-like compounds from
certain species can in the presence of an L- threonine
ester, water, a water miscible organic solvent, and
trypsin be transpeptidized into a threonineB30 ester of
human insulin in which the protecting group(s)
can be removed whereby human insulin is formed.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing threonineB30 esters of
human insulin or a salt or complex thereof, characterized by
transpeptidizing at position B30 an insulin compound or a
salt or complex thereof convertible thereinto with an
L-threonine ester or a salt thereof in a mixture of water, a
water miscible organic solvent, and trypsin, the content of
water in the reaction mixture being less than 50 per cent
(volume/volume), and the reaction temperature being below
50°C, and in the optional presence of an-acid.
2. A process according to claim 1, wherein the
concentration of L-threonine ester in the reaction mixture
exceeds 0.1 molar, that the reaction temperature is above
the freezing point of the reaction mixture, and that the
reaction mixture contains from 0 (zero) to 10 equivalents of
an acid per equivalent of the L-threonine ester.
3. A process according to claim 1 or claim 2, wherein
the insulin compound is a compound selected from the group
consisting of porcine, dog, finwhale, sperm-whale, and
rabbit insulin, porcine diarginine insulin, porcine split
proinsulin, porcine desdipeptide proinsulin, porcine, dog,
monkey, and human proinsulin, and mixtures thereof.
4. A process according to claim 1, wherein the
insulin compound is of porcine origin.
5. A process according to claim 4, wherein relatively
crude insulin is used as the insulin compound.
6. A process according to claim 2, wherein the
reaction temperature is from 0-37°C.
7. A process according to claim 6, wherein the
reaction temperature is below ambient.
31

8. A process according to claim 1, wherein the
content of water in the reaction mixture is between 40 and
10 per cent (volume/volume).
9. A process according to claim 2, wherein the molar
ratio of the L-threonine ester to the insulin compound is
above 5:1.
10. A process according to claim 2, wherein the molar
ratio of the L-threonine ester to the insulin compound is
from about 5:1 to about 1000:1.
11. A process according to claim 4, claim 6 or claim
8, wherein the molar ratio of the L-threonine ester to the
insulin compound is from 20:1 to 250:1.
12. A process according to claim 1, wherein the
organic solvent is a polar solvent.
13. A process according to claim 12, wherein the
solvent is methanol, ethanol, 2-propanol, 1,2-ethanediol,
acetone, dioxane, tetrahydrofuran, formamide, N,N-dimethyl-
formamide, N,N-dimethylacetamide, N-methylpyrrolidone,
hexamethylphosphortriamide, or acetonitrile.
14. A process according to claim 2, wherein the acid
is an organic acid.
15. A process according to claim 4, claim 6 or claim
12, wherein the acid is formic acid, acetic acid, propionic
acid, and butyric acid.
16. A process according to claim 14, wherein the
amount of acid in the reaction mixture is between 0.5 and 5
equivalent(s) per equivalent of the L-threonine ester.
32

17. A process according to claim 16, wherein the
weight ratio between trypsin and the insulin compound in the
reaction mixture is above 1:200.
18. A process according to claim 17, wherein said
weight ratio is above 1:50 and below 1:1.
19. A process according to claim 1, wherein the
transpeptidation is carried out in the presence of calcium
ions.
20. A process according to claim 1, wherein the
transpeptidation is carried out in a buffer system.
21. A process according to claim 1, wherein the
L-threonine ester is added as an acetate, propionate,
butyrate, or hydrohalide.
22. A process according to claim 21, wherein the
L-threonine ester is added as a hydrochloride.
23. A process according to claim 2, wherein the
reaction temperature and the content of water and acid in
the reaction mixture are chosen so that the yield of
threonineB30 ester of human insulin is higher than 60 per
cent.
24. A process according to claim 2, wherein the
reaction temperature and the content of water and acid in
the reaction mixture are chosen so that the yield of
threonineB30 ester of human insulin is higher than 80 per
cent.
33

25. A process according to claim 2, wherein the
reaction temperature and the content of water and acid in
the reaction mixture are chosen so that the yield of
threonineB30 ester of human insulin is higher than 90 per
cent.
26. A process according to claim 2, wherein the
insulin compound is a compound having the formula I
<IMG> (I)
wherein <IMG>
represents the human des(ThrB30)-insulin moiety wherein
GlyA1 is connected to the substituent designated R1 and
LysB29 is connected to the substituent designated R2,
R2 represents an amino acid or a peptide chain containing
not more than 36 amino acids, and R1 represents hydrogen
or a group of the general formula R3-X-, wherein X
represents arginine or lysine, and R3 represents a peptide
chain containing not more than 35 amino acids, or R2
together with R3 represents a peptide chain containing not
more than 35 amino acids, with the proviso that the number
of amino acids present in R1 plus R2 is less than 37.

27. A process according to claim 26, wherein R is
hydrogen, and R2 is -Ala, -Ser, <IMG>, or
<IMG>, R3 is <IMG>
, and R2 is <IMG>
, or R3 together with R2
is <IMG>
, wherein the
terminal alanyl is connected to LysB29,
<IMG>,
wherein the terminal alanyl is connected to
<IMG>,
wherein the terminal threonyl is connected to LysB29,
or
<IMG>, wherein
the terminal threonyl is connected to LysB29.
28. A process according to claim 27, wherein R1 is
hydrogen, and R2 represents alanine.
29. A process according to claim 1, wherein the
L-threonine ester has the general formula II
Thr(R5)-OR (II)
wherein R4 represents a carboxyl protecting group, and
R5 represents hydrogen or a hydroxyl protecting group.

30. A process according to claim 29, wherein R4 is
lower alkyl, substituted benzyl or diphenylmethyl or a group
of the general formula -CH2CH2SO2R6 , wherein R6
represents lower alkyl.
31. A process according to claim 30, wherein R4 is
methyl, ethyl, tert-butyl, p-methoxy-benzyl,
2,4,6- trimethylbenzyl, or a group of the formula
-CH2CH2SO2R6, wherein R6 is methyl, ethyl,
propyl, or n-butyl.
32. A process according to claim 1, wherein the
insulin compound is porcine insulin or porcine proinsulin,
that the L-threonine ester is Thr-OMe, Thr-OBut or
Thr-OTmb, that the water miscible organic solvent is
N,N-dimethylformamide, N,N-dimethylacetamide,
N-methylpyrrolidone, hexamethylphosphortriamide, dioxane or
acetonitrile, that the reaction temperature is in the range
from about 32 to 37°C, that the content of water in the
reaction mixture is in the range from about 20 to 43 per
cent, that the weight ratio between trypsin and the insulin
compound is in the range from about 1:8 to 1:27, that the
molar ratio between the L-threonine ester and the insulin
compound is in the range from about 60:1 to 180:1, and that
there is added between about 1.25 and 3.0 equivalents of
acetic acid per equivalent of the L-threonine ester.
36

33. A process according to claim 32, wherein the
insulin compound is porcine insulin, that the L-threonine
ester is Thr-OMe or Thr-OBut, that the water miscible
organic solvent is N,N-dimethylformamide or
N,N-dimethylacetamide, that the reaction temperature is
about 37°C, that the content of water in the reaction
mixture is between 41 and 43%, that the weight ratio between
trypsin and the insulin compound is about 1:8, that the
molar ratio between the L-threonine ester and the insulin
compound is about 120:1, and that there is added between 1.2
and 1.5 equivalents of acetic acid per equivalent of the
L-threonine ester.
34. A process according to claim 1, claim 2 or claim
3, wherein the content of water in the reaction mixture is
in the range from 43 to 20 per cent (volume/volume).
35. A process for preparing human insulin according to
claim 1, wherein as a subsequent step, the resulting
threonineB30 ester of human insulin is deblocked.
36. A process according to claim 35, wherein the
deblocking is performed in an aqueous medium in the presence
of a base.
37. A process according to claim 35 or claim 36,
wherein the deblocking is performed at a pH value of from 8
to 12.
38. A process according to claim 35, wherein the
deblocking is performed by acidolysis.
39. A process according to claim 35, wherein the
deblocking is performed by trifluoroacetic acid.
37

Description

~L~62~
This invention relates to the conversion o
insulin and insulin-like compounds
into human insulin.
In the treatment of diabetes mellitus insulin
preparations derived from porcine or bovlne insulin have
generally been used. Bovine, poxcine, and human insulins
exhibit minor differences with respect to their amino
acid composition, the difference between human and porcine
insulin being confined to a single amino acid in that the
B30 amino acid of human insul'in is threonine whereas
that of porcine insUlinisalanine However, it could be argued
that the ideal insulin preparation for treatment of
human beings would be an insul'in having exactly the same
chemical structure as that of human insulin.
For the production of natural human insulin
- the necessary amount of human pancreas glands i~ not
; available.
'Synthetic human insulin has been prepared ~n
a small scale at great expense, vide Helv.Chi~.Acta
57, 2617, and 60, 27. Semisynthetic human insulin has
been prepared from porcine insulin by tedious path-
ways, vide Hoppe-Seyler's Z.~hysiol.Chem. 357, 759,
and Nature 280, 412. However, the present invention
relates to a process which can be used for preparing human
insulin on an industrial scaleO ~
.,. . '''~
'

2 i~28~
US p~tent specification ~o. 3,276,961 purports to
relate to a process for preparin~ semisynthetic human insu-
lin. l1cwever, the ~ield of human insulin is poor because
the process is performed in water, under which conditions
trypsin causes a splitting of the Arg - Gl~ bond,
vide J. Biol.Chem. 236, 743.
A known semisynthetic process for preparing
human insulin comprises the following three steps:
First, porcine insulin is converted into porcine
des-(AlaB30)-insulin by treatment with carboxypeptidase
A, vide Hoppe-S~yler's A.Physiol.Chem. 3S9, 799. In
the second step porcine des-(t~laB3C)-insulin is subjec
ted to a trypsin-catalysed coupling with Thr-OBut,
whereby human insulin ThrB30-tert-butyl ester is formed.
Finally, said ester is treated~with triflouroacetic
acid yielding human insulin, vide Nature 280, 412. The
first step, however, results in a partial removal of
Asn , yielding des-(~la ~AsnA21)-insulinO This
derivative gives, after the two subsequent reactions,
rise to a contamination of des-(AsnA21)-insulin in the
semisyntetic human insulin prepared, a contamination which
cannot easily be removed with known preparative methods.
Des-(AsnA2~ sulin L~ossesses low biological activity
~about 5%), vide Amer.J.Med. 40, 750.
One object of this invention is to provide
a process which converts a non-human insulir.
in~o human insulin.
.
A second object of this invention is to
provide a process which converts porcine insulin
and certain impurities therein into human insulin
via a threonineB30 ester o~ human insulin.
Further objects and the advantages of this
invention will be apparent in this description.

~L6~
This invention is based upon the discovery
that the amino acid or peptide chain bound to the carbo-
nyl group of LysB29 in the insulin compound can be inter-
changed with a threonine ester. Said interchange is
herein referred to as a transpeptidation.
The term "insulin compounds" as used herein
encompasses insulins and insulin-like compounds con~
taining the human des~ThrB30)insulin moiety, the B30
amino acid of-the insulin being alanine (in insulin ~rom,
e.g., hog, dog, and fin and sperm whale) or serine
(rabbit~. The term "insulin-like compounds" as used
herein encompasses proinsulin derived from any of the
above species and primates, together with intermediates
from the conversion of proinsulin into insulin. As
examples of such intermediates can be mentioned split
proinsulin, desdipeptide proinsulins, desnonapeptide pro-
insulin, and diarginine insulins, vide R. Chance: In
Proceedings of the Seventh Congress of IDF, Buenos Aires
1970, 292 - 305, Editors: R.R. Rodrig~es & J.Y.-Owen,
Excerpta Medica, Amsterdam.
The process according to this invention
comprises transpeptidizing an insulin compound or a
salt or complex thereof with an L-threonine ester or
a salt thereof in a mixture of water, a water miscible
organic solvent, and trypsin, the content of water
in the reaction mixture being less than about 50 per cent
(volume/volume) and the reaction temperature being
below about 50 C;
~. ~

Although trypsin is best known for its
proteolytic properties, workers in the art
have recognized that trypsin is capable of
catalyzing the coupling of des-(AI.aB3a) -insulin and
a threonine~tert-butyl ester, vide Nature 280, 41~.
In the present process the trypsin is used to catalyze
transpeptidation.
!
The transpeptidation can be performed by
dissolving 1~ the insulin compound, 2) an L-threonine
ester, and 31 trypsin in a mixture of water and at
least one wa~er miscible organic solvent, optionally in
the ~resence of an acid.
Preferred water miscible orqanic solvents are
polar solvents. As specific examples of such solvents
can be mentioned methanol, ethanol, 2-propanol/ 1,2-ethan-
~ diol, acetone, di.oxane, tetrahydrofuran, N,N-dimethylfor-
mamide, formamide, N,N-dimethylacetamide, N-methylpyrro-
lidone, hexamethylphosphortriamide, and acetonitrile.

Depending on which water miscible organic solvent is
used, on the chosen reaction temperature, and on the
presence oE an acid in the reaction mixture, the content of
water in the reaction mixture should be less than about 50
per cent (v/v), preferably less than about 40 per cent
(v/v), and more than 10 per cent (v/v).
On advantage of decreasing the amount of water in
the reaction mixture is that the formation of byproducts is
thereby decreased. Similarly, by increasing the amount of
acid in the reaction mixture it is possible to decrease the
byproduct formation. The increase in yield is positive
correlated to a high percentage of organic solvent.
The type of trypsin used is not material to the
practice of this invention. Trypsin is a well
characterized enzyme which is commercially available in
high purity, notably of bovine and porcine source.
Furthermore, trypsin of microbial origin may be used.
Moreover, the trypsin form, e.g., crystalline trypsin
(soluble form), immobilized trypsin or even trypsin
derivatives (so long as the trypsin activity is retained)
is not material to practice of this invention. The term
trypsin as employed herein is intended to include trypsins
from all sources and all forms of trypsin that retain the
transpeptidating activity, including proteases with
trypsin-like specificity, e.g. Acromobacter lyticus
protease, vide Agric.Biol.Chem. 42, 1443.
1~

62~8
As examples of active trypsin derivatives can
be mentioned acetylated trypsin, succ:inylated trypsin,
glutaraldehyde treated trypsin, and immo~ilized
trypsin derivatives.
If an immobilized trypsin is used it is
suspended in the medium.
Organic or inorganic acids such as hydrochloric acid,
forric aci~, acetic aci~, propionic acid, and
butyric acid, or bases such as pyridine, TRIS,
N-methylmorpholine, and N-ethylmorpholine may be
added to bring about a suitab]e buffer system. Oryanic acids
are preferred. However, the reaction can be conducted without
such additions. The amount of acid added is usually less
than about 10 equivalents per equivalent of the L-threonine
ester. Preferably, the amount of acid is between 0.5 and
5 equivalents per equivalent of the L-threonine ester. Ions,
which stabilize trypsin such as calcium ions, may be added.
The process may be performed at a tempera-
ture in the range between 50 C and the freezing
point of the reaction mixture.
Enzymatic reactions with trypsin are usually performed at
- about 37 C in order to give a sufficient reaction rate,
However, in order to avoid inactivation of trypsin
- it is advantageous to perform the process according to the
present invention at a temperature below ambient. In
practice reaction temperatures above about 0 C are
preferred.
. .. .. .

7 1~2~6~
The reaction time is usually between several
hours and several days, depending upon the reaction tempera-
ture, upon the amount of trypsin added, and upon other
reaction conditions.
The weight ratio between trypsin and the insu-
lin compound in the react:ion mixture is normally above
about 1:200, preferably ahove about 1:50, and below about
1 : 1 .
The L-threonine esters contemplated for
practice of this invention can be depicted by the
general formula
Thr(R5)-OR4 II
wherein R represents a carboxyl protecting group,
and R represents hydrogen or a hydroxyl protecting
group, or a salt thereof.
The threonineB30 esters of human insulin
resulting from the transpeptidation can be depicted
by the general formula
; (Thr(R5)-oR4)B3o-h-In III
wherein h-In represents the human des-(Thr 30~.insulin
moiety, and R4 and R5 are as defined above.
... .

~1~ii2~
Applicable L-threonine esters of formula II
are those in which R~ is a carboxyl protectinq group
which can be removed from the threonineB30 ester of ~uman insulin
-(formula III) under conditions which do not cause substantia
irreversible alteration in the insulin molecule. As
examples of such carboxyl pxotecting groups can be
mentioned lower aLkyl, e.g., methyl, ethyl, and tert-butyl,
substituted benzyl such as
p-methoxybenzyl and 2,4,6-trimethylbenzyl and diphenyl-
methyl, and groups of the general formula - CH2CH2SO2R6,
where~n R represents lower al~yl such as methyl, ethyl,
propyl, and n-butyl. Cuitable hydroxyl Drotecting grouPs
R5 are thoSe which can be removed from the threonineB30
ester of human insulin (formula III) under conditions
which do not cause substantial irreversible alteration in
the insulin molecule. As an example of such a group (R5)
can be mentioned tert-butyl.
Lower al~yl groups contain less than 7
carbon atoms, preferably less than S carbon atoms.
Further protection groups commonly used are
described by Wunsch Methoden der Organischen Chemie
(Houben-Weyl), Vol. XV/l, editor: Eugen Muller, Georg
Thieme Verlag, Stuttgart 1974.

~2~
Some L-threonine esters (formula II) are known
compounds and the remaining L-threonine esters (formula II)
can be prepared in analogy with the preparation of known
compounds.
The L-threonine esters of formula II may be the
free bases or suitable organic or inorganic salts thereof
preferably acetates, propionates, butyrates, and
hydrohalides such as hydrochlorides.
It is desirable to use the reactants, i.e. the
insulin compound and the L-threonine ester (formula II), in
high concentrations. The molar ratio between the
L-threonine ester and the insulin compound is preferably
above about 5:1, most preferably from about 5:1 to about
1000:1, and optimally from about 20:1 to about 250:1.
It is desirable that the concentration of
L-threonine ester (formula II) in the reaction mixture exeed
0.1 molar.
Human insulin can be obtain from the
threonine esters of human insulin (formula III) by
removal of the protecting group R and any protecting
group R by known methods known ~ se. In case

R4 is methyl, ethyl, or a group -C~H2S02R6, wherein R6
is as defined above, the said protecting group can be
- removed at gentle basic conditions in an aqueous medium,
preferably at a pH value of about 8 - 121 e.q. at
about 9.5. As the base can be used ammonia, triethylamine,
or hydroxides of alkali metals such as sodium hydroxide.
In case R~ is tert-butyl, substituted benzyl such as
p-metho~ybenzyl or 2,4,6--trimethylbenzyl or diphenyl-
methyl the said group can be removed by acidolysis,
preferably with trifluoroacetic acid. The trifluoro-
acetic acid may be non-aqueous or may contain some water,
- or it may be diluted with an organic solvent such as
dichloromethane. In case R5 is tert-butyl said group
can be removed by acidolysis, vide above.
Preferred threonineB30 esters of human
insulin of the formula III are compounds, wherein R5
is hydrogen, and these are prepared from
L-threonine esters of the formula II, wherein R5
is hydrogen.
The transpeptidation of insulin compounds
~into threonine esters of human insulin can be
described in more detail on the basis of the following
formula assigned to the insulin compounds:
- Rl-tl-----A-----21)
11
(l___B~__2g)-R2 (I)
wherein
. ............................ ~

~L~L6%53~
11
A---21)
(l---B---29)~
represents the human des(ThrB30)insulin moiety
wherein GlyAl is connected to the substituent designated
~ and LysB29 is connected to the substituent designated
R2, R2 represents an amino acid or a peptide chain
containing not more than 36 amino acids, and Rl repre-
sents hydrogen or a ~roup of the general formula
R -X-, wherein X represents arginine or lysine, and
R3 represents a peptide chain containing not more
than 35 amino acids, or R2 together with R3 represent
a peptide chain containing not more than 35 amino
acids, with the proviso that the number of amino acids
present in Rl plus R2 is less than 37.
Thus, the transpeptidation of this invention
converts any of the above insulin compounds into
threonineB30 esters of human insulin (formula III),
which then can be deblocked to form human insulin.
:

8~
12
A further advantage of this inventiol- is
that insulin-lik~ compounds present in crude
insulin and present in some commercial insulin
preparations and covered by the formula I by the
transpeptidation of this invention are convert~d
into threonineB30 esters of human insulin, which
then can be deblocked to form human insulin.
Examples of insulin-like compounds of formula I
appear from the following:
Porcine diar~inine insulin ;Rl is hydro~en, and
R is -Ala-Arg-Arg), porcine proinsulin(R3 toaether.with
R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-
-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-~ila-
-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, wherein the terminal alanyl
is connected to LysB29~ do~.proinsulin ~3 together with
R2 is -Ala-Arg-Arg-Asp-Val-~lu-Leu-Ala-Gly-Ala-Pro-Gly-
-Glu-Gly-Gly-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ala-Leu-Gln-
-Lys-, wherein the terminal alanyl is connected to LysB29~,
porcine split proinsulin
(R is Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, and R is -Ala-
-Arg-Ar~-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-
-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu), porcine
desdipeptide proinsulin (Rl is hydro~en, and R2 is
-Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-
-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-
-Leu-Glu-Gly-Pro-Pro-Gln), human proinsulin (R to~ather
with R is -Thr-Arg-Arg-Glu-Ala-Glu-Asp~Leu~Gln-Val-Gly-
-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-
-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, wherein
the terminal threonyl is connected to LysB29), and
monkey proinsulin (R3 toqether with R2 is -Thr-Ar~-Ar~-
-Glu-Ala-Glu-As~-Pro-Gln-Val-Glv-Gln-Val-Glu-Leu-Gly-
-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-
-Glu-Gly-Ser-Leu-Gln-Lys-~ wherein the ter~inal threonyl
is connected to ~ysB29.

2B~
: 13
Hence, in all these impurities covered by formula I
the R substituent designated R3-X- is exchanged with
hydrogen.
A preferred embodiment of the present invention
comprises reacting crude porcine insulin containing
insulin-like compounds or a salt or complex thereof with an
L-threonine ester (formula II) or a salt thereof under the
above conditions whereafter the protecting group R4 and
any protecting group R5 is removed. By this process the
porcine insulin together with insulin-like compounds
therein is converted into human insulin.
As examples of a complex or a salt of an insulin
compound (formula I) can be mentioned a zinc complex or
zinc salt.
When selecting the reaction conditions according to
the above explanation and considering the results obtained
in the following examples it is possible to obtain a yield
of threonineB30 ester of human insulin which is higher
than 60 per cent, and even higher than 80 per cent, and
under certain preferred conditions higher than 90 per cent.

3Li~2~
-14-
The process according to the present
invention has, therefore, the followincJ advantagcs
over the prior art:
a) The enzymatic hydrolysis to remove
Alas30 e.g., with carboxypeptidase A, ls
omitted.
b) The isolation of an intermediate
compound, such as porcine des-(AlaB30)-insulin, is
unnecessary.
c) Contamination with des(Asn l)-insulin
deriv~tives is avoided.
d) Proinsulin and other insulin-like im-
purities present in crude insulin
are - via the threonineB30 ester of human insulin -
converted into human insulin by the process of
this invention, whereby the yield is increased.
. .
el Antigenic insulin-like compounds, vide
British Patent No. 1,285,023, are converted into numan
ir.sulin.
A preferred procedure for preparing human
insulin is as follows:
1) The starting material used for the
transpeptidation is crude porcine insulin,
e.g., crystalline insulin obtained by the use o~ a
citrate buffer , vi~e Patent Specification No. U.S.A.
2,626,228.
2) If there is any trypsin activity left
after the transpeptidation, it is pre~erred to remove
it, e.g., under conditions where trypsin is inactive,
e.g., in acid medium below pH 3. Trypsin can be
removed by separation according to molecular weight,
e.g., by gel filtration on "Sephadex G-50~ or "~io-gel P-3U~
in lM acetic acid, vide Nature 280, 412.
* Trade Mark
B

3~ Other impurities
such as unreacted porcine insulin may be removed
by the use o anion and/or cation exchange chromato-
graphy, vide Examples 1 and 2.
4) Thereafter, thle threonineB30 ester of
human insulin is debloc~ed and human insulin is isolated,
e.g., crystalli~ed,in a manmer known ~ se.
By this process human insulin of an accep-
table pharmaceutical purity can be obtained and
be further ~purified, if desired.
Furthermore, the present invention relates to novel
threonineB30 esters of human insulin wherein the ester
moiety is different from tert-butyl and methyl.
Abbreviations- used are in accordance with
the rules approved (1974) by the IUPAC-IUB Commission
on Biochemical Nomenclature, vide Collected Tentative
Rules & Recommendations of the Commission on Bio-
chemical Nomenclature IUPAC-IUB, 2nd ed., Maryland
1975.

~L~62~
16
Analytical tests:
The conversion of porcine in.sulin and porcine
proinsulin into human insulin esters can be demonstrated by
DISC PAGE electrophoresis in 7.5% polyacrylamide gel in a
buffer consisting of 0.375 M Tris, 0.06 M HCl, and 8 M
urea. The pH of the buffer is 8.7. ESters of the formula
III migrate with a speed of 75~ of that of porcine
insulin. Porcine proinsulin migrating with 55% of the
speed of porcine insulin is by the process according to the
present invention converted into the same product.
Identification of the conversion product as compounds of
the formula III is due to the following criteria:
(a) The electrophoretic migration of the human
insulin esters of formula III in relation to porcine
insulin corresponds to the loss of one negative charge.
(b) The amino acid composition of the stained
protein bonds in the gel representing compounds of the
formula III is identical with that of human insulin, i.e. 3
mol threonine and l mol alanine per mol insulin, and the
composition of porcine insulin is 2 mol threonine and 2 mol
alanine per mol insulin. The technique for analyzing amino
acid compositions of protein bonds in bolyacrylamide gels
has been described in Eur.J.Biochem. 25, 147.
(c) The proof that the incorporated threonine is
placed as C-terminal amino acid in the B chain, is proved
by oxidative sulfitolysis of the S-S bridges o~
insulin in 6 M guanidinium hydrochloride followed by
separation of ~ and B chains by ion exchange
chromatography on "SP Sephadex". DigeStion of the B
chain S-sulfonate with carboxypeptidase A liberates
.~
!~

only the C-terminal amino acid. The technique has been
described by Markussen in Proceedings of the Symposium
on Proinsulin, Insulin and C-peptide, Tokushima,
12 - 14 July, 1978, (Editor: saba, Kaneko & Y~niahara)
Int.Congress Series No. 468, Excerpta Medica, Amsterdam-
Oxford. The analysis is performed after the ester group
has been split from compounds of the formula III.
Those three analyses prove unambiguously that
the conversion into human insulin has taken place.
The conversion of poxcine insulin and porcine
proinsulin into human insulin esters can be followed
quantitatively by E~PLC (high pressure liquid chromato-
graphy) on reverse phase. ~ 4 x 300 mm~/u Bondapak
C18 column" (Waters Ass.) was used and the elution was
performed with a buffer comprising 0.2 M ammonium
sulphate (adjusted to a pH value of 3.5 with sulphuric
acid) and containing 26 - 50% acetonitrile. The
optimal acetonitrile concentration depends on which
ester of the formula III one desires to separate from
porcine insulin. In case R4 is methyl, and R5 is
hydrogen, separation is achieved in 26~ (v/v) of
acetonitrile. Porcine insulin and (Thr-0~4e)B30-h-In
(Me is methyl) elute after 4.5 and 5.9 column volumes,
respectivily, as well separated symmetrical peaks.
Before the application on the HPLC column the proteins
in the reaction mixture were precipitated by addition
of 10 volumes of acetone. The precipitate was isolated
by centrifugation, dried in vacuo, and dissolved in
0.02 M sulph~ric acid.
The process for preparing human insulin
esters and human insulin is illustrated by the following
examples which, however, are not to be construed as
limiting. The examples illustrate some preferred em-
bodiments of the process according to the invention.
.
.... . ........ . . . . . . ....... ... .... . .. _ .... _ .. _ _ _ _ __ _ _ _ . . _.. .... . ... ... .

18
Example 1
200 mg crude porcine insulin, crystallized
once, was dissolved in 1.8 ml 3.33 M acetic acid. 2 ml
of a 2 M solution of Thr-OMe (Me is methyl) in N,N-di~.ethyl-
acetamide, and 20 mg of trypsin dissolved in 0.2 ~nl of
water were added. After storage for 18 hours at 37C
the proteins were precipitated by the addition OI 40 ml
of acetone, and the precipitate was isolated by centri--
fugation. The supernatent was discarded. Analysis of
the precipitate by ~IPLC using 26~ acetonitrile (vide
Analytical tests) showed a 60% conversion of porcine
insulin into(Thr-~e)B30-h-In . The precipitate was dis-
solved in 8 ml freshly deionized 8 M urea, the pH value
was adjusted to 8.0 with 1 M ammonia, and the solution
was applied to a 2.5 x 25 cm column packed with "PAE A-25
Sephadex",-equilibrated with a 0.1 M ~nium chloride buf~er, which
contained 60% (v/v) ethanol, the pH value of which was adjusted to 8.0
with ammonia. Elution ~7as carried out with the same
buffer, and fractions of 15 ml were collected.
(Thr-CMe) -h-In was found in the fractions Nos. 26 - 46,
and unreacted porcine insulin in the fractions Nos.
90 - 120. The fractions Nos. 26 - 46 were pooled,
the ethanol evaporated _ vacuo, and the (Thr-OMe)B30-
-h-In was crystallized in a citrate buffer as described
by Schlichtkrull et al., Handbuch der inneren Medizin,
7/2A, 96, Berlin, Heidelberg, New York 1975. The yield
was 95 mg of crystals having the same rhombic shape as
porcine insulin crystallized in the same manner. The
amino acid composition was found to be identical with
that of human insulin. Further analytical tests descri-
bed in the above section: "Analytical Tests", prooved
that the resulting product was ~Thr-OMe)B30-h-In.
~xample 2
100 mg porcine insulin fulfilling the puri-
ty requirements stated in British Patent No. 1,285,023
was dissolved in 0.9 ml 3.33 M acetic acid and, there-
.
. . ~ .
. ~ ,___,,~ _, ___,.. ... . _ . .. . . . . .. ... _ .. ~ ......... ... _ _ .. _.. _. _ .. __ ... __.. _ .. _ . ___.__ ___._.___._.. _____.____
.. ____.___._.. --- -- -- - ~a~ ~ ~ -

~2~
19
after, L ml of a 2 M solution of threonine methyl ester
in N,N-dimethylfon~ude and 12 mg TFCK (tosylphenylaL~L~hlorcmeth
ketone) treated trypsin dissolved in 0.1 ml water were
added. After an incubation for 24 hours at 37C the
reaction was stopped by the addition of 4 ml 1 M
phosphoric acid. The(Thr-oMe)B3o-h~In obtained was
separated from non-reacted porcine insulin by lon ex-
change chromatography on a 2.5 x 25 cm column of ~SP-
Sephadex~ with an eluent comprising 0.09 ~ sodium
chloride and 0.02 M sodium dihydrogen phosphate(pH value
of the buffer: 5.5) in 60~ ethanol. Frac~ions containing
(Thr-o~e)B30-h-In were collected, the ethanol was removed
Ln vacuo, and the product was crystallized as described
in Example 1. The yield was 50 mg of ~Thr-OMe3B30-h-In.
Example 3
100 mg porcine proinsuiin was dissolved in 0.9 ml
of 3.33 M acetic acid and converted into 5Thr-OMe)B30-h-
-In and purified as described for porcine insulin in
Example 1. The conversion of proinsulin into (Tpr-OMe) -
-h-In was found to be 73% by HPLC analysis of the
acetone precipitate. The yield of crystalline (Thr-O~e)B30-
-h-In was 54 mg.
Example 4
100 mg porcine insulin was dissolved in 0.9 ml of
2.77 M:acetic acid in water and reacted analogously ~ the
process described in Example 2. After completion o~ the
reaction the proteins were precipitated by the addition
of 10 volumes of acetone. Analysis by DISC PAGE showed a
conversion into (Thr-OMe)B30-h-In of 70%.
Example 5
1~0 mg porclne insulin was dissolved in 0.9 ml of
~,

1~6~
3.33 ~. acetic acid and 1 ml 2 M Thr-OMe in N-methylpyrro-
lidone was added. The reaction was performed in a mar.ner
~loyous ~o that described in Example 4 and the conversion
into (Thr-OMe)B30-h-In was 20%.
Example 6
100 mg porcine insulin was dissolved in 0.9 ml of
2.77M acetic acid in~water and 1 ml 2 M Thr-OMe in
HMPA (hexamethylphosphortriamide) was added. The reaction
was performed in a m~n~er analogous to'that described in
Example 4. The convers~on into (Thr-OMe)B30-h-In was 80~.
Example 7
100 mg porcine insulin was dissolved in 0.9 ml of
3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-
acetamide was added. The reaction was performed in a manner
ana,logous to that described in Ex,ample 4. The conversion
into (Thr~OMe)B30-h-In was 80%
Example 8
100 mg porcine insulin was dissolved in 0.9 ml
of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-
dimethylacetamide was added. Thereafter, 200 U trypsin
activity (measured against the substrate BAEE) immobi-
lized on 1 g of glass beads was added and after incubation
at 37C during 24 hours the trypsin bound to the glass
was filtred off. After completion of the reaction the
proteins were precipitated by the addition of 10 volumes
of acetone. Analysis by DISC PAGE showed a conversion
into (Thr-OMe) 3,-h-In of 40~.
Example 9
100 mg porcine insulin was dissolved in 0.9 ml of
3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-
; ~ . .,
~ -,, acetamide was added. Thereafter, 300 U trypsin

21
(activity measured with the substrate BAEE) immobilized
on 200 mg CNBr activated "Sephadex G-150" was added.
~fter incubation at 37C during 24 hours the trypsin
bound to "S~phadex" was filtred off. After completion
of the reaction the proteins were precipitated by the
addition of 10 volumes of acetone. Analysis by DISC PAGE
showed a conversion into (Thr-OMe) 30-h-In of 70~.
Example 10
The process described in Example 7 was repeted,
provided that the ester used was 2 M Thr-OBut (But is
tert-butyl) in N,N-dimethylacetamide. The conversion into
(Thr oBu!t)B30 h In was 80%
Example 11
The process described in Example 8 was repeted,
provided that the ester used ~as 2 M Thr-OBut in N,N-di-
methylacetamide. The convers~on into (Thr-OBut)B30-h-In was
30%.
Example 12
The process described in Example 9 was repeted,
provided that the ester used was 2 M Thr-OBut in N,N-di-
methylacetamide. The convers~on into (Thr-OBut) 3 -h-In
was 70~.
Example 13
100 mg porcine proinsulin was dissolved in 0.9 ml
:of 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-
acetamide was added. The reaction was perfonm~d in a manner
analogous to that described in Example 4. The convers'ion
into (Thr OMe)B30 h In was 80~
s ~.~

6~3
Example 14
100 mg porcine proinsulin was dissolved in 0.9ml
of 3.33M ace~c acid and 1 ml 2 M Thr-OMe in N,N-dimethyl-
acetamide was added. Themixture was treated with immobi-
lized trypsin analogically to the ~rocess described in
Example B. The convexs\~on into ~Thr-OMe) -h-In was 40~.
Example 15
100 mg porcine proinsulin was dissolved in 0.9ml
O~ 3.33 M acetic acid and 1 ml 2 M Thr-OMe in N,N-di-
methylacetamide was added. The mixture was treated
analogically to the process described in Example 9 with
immobilized trypsin. The convers~on into (Thr-OMe)R30-h-In
was 70%.
Example 16
100 mg porcine proinsulin was dissol~ed in 0.9 ml
of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in N,N-di-
methylacetamide was added. The reaction was performed in a
manner anahogous to that described in Example 4.
The conversion into (Thr-OBut)B30-h-In was 80%
.
Example 17
100 mg porcine proinsulin was dissolved in 0.9 ml
Of 3.33 M acetic acid and 1 ml 2 M Thr-OBu in N,N-di-
methylacetamide was added. The mixture was treated
with trypsin immobilized to glass beads analogically
to process described in Example 8. The conver~ion into
(Thr-OBut)B3 -h-In was 40%.
Example 18
100 mg poxcine proinsulin was dissolved in 0.9 ml
of 3.33 M acetic acid and 1 ml 2 M Thr-OBut in N,N-di-
methylacetamide was added. The mixture was treated

~Z8~
23
wit,. trypsin immob~lized to CNBr activated "Sephadex
G-150" analogically with the process described in
Example 9. The conversion into ~Thr-OBu )B30-h-In was
70%.
Example 19
100 mg porcine insulin was dissolved in 0.5 mlof
6 M acetic acid and 1 ml 1 M Thr-OTmb (Tmb is 2,4,6-tri~
methylbenzyl) in N,N-dimethylac~tamide was added.
Furthermore, 0.5 ml N,N-dimethylacetamide and 5 mg TPCK
treated trypsin in 0.1 ml water were added. The reaction
mixture was stored at 32C for 44 hours. After completion
of the reaction the proteins were precipitated by the
addition of 10 volumes of acetone. Analysis by DISC
PAGE showed a conversion into (Thr-OTmb)B30-h-In of 50%.
Example 20
100 mg porcine insulin was dissolved in 0.9 ml of
3 M acetic acid and 1 ml 2 M Thr-OMe in dioxane was added.
The reaction was performed in a manner analogous to that
descri~ed in Example 4 and ~he conversion i~to (Thr-OMe)B30_
-h-In was 10%.
Example 21
100 mg porcine insulin was dissolved in 0.9 ml of
3 M acetic acid and 1 ml 2 M Thr-OMe in acetonitrile was
added. The reaction was performed in a manner analogous to
that described in Example 4 and the conversion into
(Th~-OMe)B30--h-In was 10~.
'~' ''I

24 ~ i~
Example 22
250 mg of crystallin~(Thr-oMe)B3o-h-In was dis-
persed in 25 ml of water and dissolved by the addition of
1 N sodium hydroxide solution to a pH value of 10Ø The
pH value was kept constant at 10.0 for 24 hours at 25 C.
The human insulin formed was crystallized by the addition
of 2 g of sodium chloride, 350 mg of sodium acetate tri-
hydrate and 2.5 mg of zinc acetate dihydrate followed by
the addition of 1 N hydrochloric acid to obtain a pH value
of 5.52. After storage for 24 hours at 4 C the
rhombohedral crystals were isolated by centrifugation,
washed with 3 ml of water, isolated by centrifugation, and
dried in vacuo. Yield: 220 mg of human insulin.
Example 23
10Q mg (Thr-OTmb~B30-h-In was dissolved in 1 ml
of ice cold trifluoroacetic acid and the solution was
stored for 2 hours at 0 C. The human insulin formed was
precipitated by the addition of 10 ml of tetrahydro-
furan and 0.97 ml o~ 1.03 M hydrochloric acid in tetra-
hydrofuran. The precipitate formed was isolated by centri-
fugation, washed with 10 ml of tetrahydrofuran, isolated
by centrifugation, and dried in vacuo. The precipitate
was dissolved in 10 ml of water and the pH value of the
solution was adjusted to 2.5 with 1 N sodium ~ydroxide
solution. The human insulin was precipitated by the
addition of 1.5 g of sodium chloride and isolated by
centrifugation. The precipitate was dissolved in 10 ml
of water, and the human insulin was precipitated by the
addition of 0.8 g of sodium chloride, 3.7 mg of zinc
acetate dih~drate and a.l4 g of
sodium acetate trihydrate followed by the addition of 1 N
sodium hydroxide solution to obtain a p~ value of 5.52.
After storage for 24 hours at 4 C, the precipitate was
isolated by centrifugation, washed with 0.9 ml of water,
isolated by centrifugation and dried in vacuo. Yield:
90 mg of human insulin.

2~
Example 24
100 mg of ~orcine insulin was dissolved in 0.5 ml
of 10 M acetic acid and 1.3 ml of 1.54 M Thr-OMe in N,N-
dimethylacetamide was added. The mixture was cooled to
12C. 10 mg of trypsin dissolved in 0.2 ml of 0.05 M
calcium acetate was added. After 48 hours at 12C the
proteins were precipitated by addition of 20 ml of
acetone. The conversion of porcine insulin into (Thr-
OMe) -h-In was 97% by HPLC.
Example 25
20 mg of porcine insulin was dissolved in a mix-
ture of 0.08 ml of 10 M acetic acid and 0.14 ml of water.
0.2 ml of 2 M Thr-OMe in N,N-dimethylacetamide was added
and the mixture was cooled to -10C. 2 mg of trypsin dis-
solved in 0.025 ml of 0.05 M calcium acetate was added.
After 72 hours at -10C the proteins were precipitated by
addi~ion of 5 ml of~acetone. The conversion of porcine
insulin into (Thr-OMe) -h-In was 64% by HPLC.
Example 26
20 mg of porcine insulin was dispensed in 0.1
ml of water. Addition of 0.6 ml of 2 M Thr-O~e in N,N-
dimethylacetamide caused the insulin to go into solution.
The mixtu~e was cooled to 7C. 2 mg of trypsin dissolved
in 0.025 ml of 0.05 M calcium acetate was added. After
24 hours at 7C the proteins were precipitated by addition
of 5 ml of acetone. The conversion of porcine insulin into
(Thr-OMe)B30-h-In was 62~ by HPLC.
.1 ~

25a ~162~
Example 27
20 mg of porcine insulin was dissolved in 0.135 ml
of 4.45M propionic acid. 0.24 ml of 1.67 M Thr-OMe in N,N-
dimethylacetamide was added. 2 mg of trypsin in 0.025 ml of
~..,

~L6;2 ~
o.05 M calcium acetate was added and the mixture was kept
at 37C for 24 hours. The proteins were precipitated by
addition of 10 vol~nes of 2-propanol. The conversion of
porcine insulin into (Thr-OMe)B30-h-In was 75~ by HPLC.
Example 28
20 mg of porcine insulin was dispersed in 0.1 ml
of water. 0~4 ml 2 M T~Me in N,N-dimethylace~de was added
follcwed by 0.04 ml of 10 N hydro~hLoric acid caused the insulLn to go
in~4 solution. 2mg ~f trypsin dissolved in 0.025 ml of 0.05 M
calcium acetate was added and the mixture was ~ept at 37C
for 4 hours. Analysis by HPLC showed a 46% conversion of
porcine insulin into (Thr-OMe)B30-h-In.
Example 29
20 mg of porcine insulin was dissolved in 0.175
ml of 0.57 M acetic acid. 0.2 ml of 2 M Thr-OMe in N,N-
dimethylacetamide was added followed by the addition of 0.025
ml of 0.05 M calcium acetate. 10 mg of a crude preparation
of AchromObacter lyticus protease was added and the mixture
was kept for 22 hours at 37C. The proteins were precipi-
tated by the addition of 10 volumes of acetone. The conver-
sion of porcine insulin into (Thr-OMe)B30-h-In waS 12% ~EP~C.
.
Example 30
20 mg of porcine insulin was dispersed in 0.1 ml
of 0.5 M acetic acid. Addition of 0.2 ml of 0.1 M Thr-OMe
in N,N-dlmethylacetamide dissolved the insulin. The mix-
ture was cooled to 12C and 2 mg of trypsin in 0.025 ml of
0.05 M caLcium acetate was added. After 24 hours a~ 12C
the analysis by HPLC showed a 42% conversion of porcine
insulin into (Thr-OMe)B30-h-In
~.
. ~;

~z~
27
Example 31
2 mg of rabbit insulin was dissolved in 0.135 ml
of 4.45 M acetic acid. 0.24 ml of 1.67 M Thr-OMe in N,N-
dimethylacetamide was added followed by the addition of
1.25 mg trypsin in 0.025 ml of 0.05 M calcium acetate.
The mixture was kept at 37C for 4 hours. Analysis by
HPLC showed an 88% conversion of rabbit insulin into
(Th~OMe)B30-h-In. Rabbit insulin elutes before porcine
insulin from the HPLC column, the ratio between the elu-
tion volumes of rabbit insulin to (Thr-OMe)B30-h-In being
0.72.
Example 32
2 mg of porcine diarginine insulin (Arg 31-ArgB32-
insulin) was dissolved in 0.135 ml of 4.45 M acetic acid.
0.24 ml of 1.67 M Thr-OMe in N,N-dimethylacetamide was
added followed by the addition of 1.25 mg of trypsin in
0.025 ml of 0.05 M calcium acetate. The mixture was kept
at 37C for 4 hours. Analysis by HPLC showed a 91% conver-
sion of diarginine insulin into (Thr-OMe) -h-In. Diarginine
insulin elutes before procine insulin from the HPLC column,
the ratio between the elution volumes of diarginine insulin
to (Thr-OMe)B30-h-In being 0.50.
Example 33
2 mg of porcine intermediates (i.e. a mixture of des-
dipeptide (Lys62-Arg63)proinsulin and desdipeptide(Arg31-
Arg32)proinsulin) was reacted analogously to the reaction
described in Example 32. Analysis by HPLC showed 88% (Thr-
OMe)B30-h-In
.

Z8~i~
28
Example 34
20 mg of porcine insulin was dissolved in 0.1 ml
of 2 M acetic acid. 0.2 ml of 2 M Thr-OMe in N,N-dimethyla-
cetamide was added and the mixture was cooled to -18C. 2 mg
of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate
was added and the mixture was kept for 120 hours at -18C.
Analysis by HPLC showed that the conversion into (Thr-
OMe)B30-h-In was 83%.
Example 35
The process described in Example 34 was repeted
with the proviso that the reaction was carried out at 50C
for 4 hours. Analysis by HPLC showed that the conversion
into (Thr-OMe) -h-In was 23%.
Example 36
20 mg of porcine insulin was dissolved in 0.1 ml
of 3 M acetic acid. 0.3 ml of 0.33 M Thr-O(CH2)2-SO2-CH3,
CH3COOH (threonine 2-(methylsulfonyl)ethylester, hydroace-
tate) in N,N-dimethylacetamide was added. The mixture was
cooled to 12C and 2 mg of trypsin in 0.025 ml of 0.05 M
calcium acetate was added. HPLC showed after 24 hours at
12 C a 77% conversion into (Thr-O(~H2)2-SO2-CH3) ~ -h-In- me
product elutes approximately at the position of (Thr-OMe)
h-In.
Example 37
20 mg of porcine insulin was dissolved in 0.1 ml
of 10 M acetic acid. 0.2 ml of 2 M Thr-OEt (Et is ethyl) in
N,N-dimethylacetamide was added and the mixture was cooled
to 12 C. 2 mg of trypsin in 0.025 ml of 0.05 M calcium
acetate was added. HPLC showed after 24 hours at 12C a
.

~1~%8~
29
75% conversion into (Thr-O~t)B30-h-In. The product
eluted in a position slightly after that o (Thr-OMe)B30-
h-In.
ExampLe 38
20 mg of porcine insulin was dissolved in 0.1 ml
of 6 M acetic acid. 0.3 ml of 0.67 M Thr(Bu )-OBu (Bu is
ter~iary ~utyl) in N,N-dimethylacetamide was added. The
mixture was cooled to 12C and 2 mg of trypsin in 0.025 ml
of 0.05 M calcium acetate was added. After 24 hours at
12C the conversion into (Thr(But)-OBu~B30-h-In was 77~l
The product was eluted from the HPLC column by applying a
gradient in acetonitrile from 27% to 40%.
Example 39
20 mg of porcine insulin was dissolved in 0.1 ml
of 4 M acetic acid. 0.2 ml of 1.5 M Thr-OMe dissolved in
tetrahydrofuran was added and the mixture was cooled to
12C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M
calcium acetate was added. After 4 hours at 12C the
analysis by HPLC showed a conversion o~ 75% into (Thr-
OMe)B30-h-In~
Example 40
20 mg of porcine insulin was dissolved in 0.1 ml
of 4 M acetic acid. 0.8 ml o~ 2 M Thr-OMe dissolved in
1,2-ethanediol was added and the mixture was cooled to 12C.
2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium
acetate wa's added. After 4 hours a~ 12C the analysis by
HPLC showed a conversion of 48% into (Thr-OMe)B30-h-In~

~z~
Example 41
20 mg of porcine insulin was dissolved in 0.1 ml
of 4 M acetic acid. 0.2 ml of 2 M Thr-OMe dissolved in
ethanol was added and the mixture was cooled to 12C~ 2 mg
of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate
was added and the reaction was carried out for 4 hours at
12 C. Analysis by HPLC showed a 46% conversion into (Thr-
OMe)B30-h-In
- Example 42
20 mg of porcine insulin was dissolved in 0.1 ml of
4 M acetic acid. 0.2 ml of 2 M Thr-OMe in acetone was added
and the mixture ~as cooled to 12C. 2 mg of trypsin dis-
solved in 0.025 ml of 0.05 M calcium acetate was added.
After 4 hours at 12C the analysis by HPLC showed a con-
version of 48% into (Thr-OMe) -h-In.
~ . . .
..
I