Note: Descriptions are shown in the official language in which they were submitted.
De~cription 13 3 S ~ g 3
A process for the preparation of tripeptides
Tripeptides are important intermediates in the synthesis
of bioactive peptides such as, for example, the hypo-
thalamus hormone gonadorelin and its analogs. For this
purpose the tripeptides must be available in the most
~traightforward manner possible, on the one hand in good
yields and, on the other hand, in high purity. The
processes hitherto disclosed for the preparation of
tripeptides do not meet these requirements in an optimal
manner and are associated with disadvantages, some of
which are serious. Thus, for example, even the products
prepared by the process described in EP-A 156,280 are
contaminated with byproducts which become disadvanta-
geously evident in the subsequent synthetic steps. Thus
the object of the present invention is to provide a
process for the preparation of tripeptides which does not
have the said disadvantages and provides, in a straight-
forward manner, products of high purity in good yields.
Accordingly, the invention relates to a process for thepreparation of tripeptides of the general formula I
U - A - B - C - OH
in which
U denotes hydrogen or a urethane protective group
A denotes a natural ~-amino acid or derivatives
thereof
B denotes a natural ~-amino acid or derivatives
thereof and
C denotes an aromatic ~-amino acid,
which comprises reacting a compound of the general
formula II
U' - B - OH
~ II
- 2 - 1 33~93
in which U' is a urethane protective group which can be
eliminated by hydrogenolysis, and B has the above-
mentioned meaning, with a compound of the general formula
III
H - C - OR
III
in which R represents alkyl having 1 to 4 carbon atoms,
and C has the abovementioned meaning, in the presence of
propylphosphonic anhydride, eliminating the protective
group U' by hydrogenolysis, reacting the resulting
compound of the general formula IV
H - B - C - OR IV
with a compound of the general formula V
U - A - OH V
in the presence of propylphosphonic anhydride, and
finally eliminating R enzymatically.
The urethane protective groups representing U are prefer-
ably the urethane protective groups customary in peptide
chemistry, as are described, for example, in Kontakte
Merck 3/79, page 14.
The benzyloxycarbonyl and the tert.-butyloxycarbonyl
groups are particularly preferred.
A urethane protective group U' which can be eliminated by
hydrogenolysis is preferably the benzyloxycarbonyl group.
Natural ~-amino acids or their derivatives representing
A and/or B are preferably Gly, Ala, Ser, Thr, Val, Leu,
Ile, Glu, Gln, p-Glu, Tyr, Phe, Trp and His. Ser, Thr,
Trp and Phe are particularly preferred.
An aromatic Q-amino acid representing C is preferably Tyr
or Phe.
_ 3 _ 133549~
R in the general formula IV preferably denotes methyl.
A process in which U and U' denote benzyloxycarbonyl, A
denotes Trp, B denotes Ser, C denotes Tyr and R denotes
methyl is very particularly preferred.
S The formation of a peptide linkage in the presence of
propylphosphonic anhydride is known as the PPA method
(Angew. Chem. Int. Ed. 19, 133 (1980)). This reaction i8
preferably carried out in polar solvents ~uch as, for
example, dimethylacetamide, dimethylformamide, dimethyl
sulfoxide, phosphoric tris(dimethylamide), N-methyl-
pyrrolidone or water. However, chloroform, methylene
chloride or ethyl acetste are also employed.
It is also po~sible in an advantageous manner to use
mixtures of the said solvents with water. An ethyl
acetate/water mixture is particularly preferred. The
~ynthesis can be carried out between -10C and room
temperature. It is preferable to start at about 0C and
subse~uently to raise to room temperature.
The elim~nation of the U' protective group by hydrogeno-
lysis is advantageou~ly carried out in a known manner
with hydrogen on a Pd/C catalyst.
The enzymatic e~terolysis in the last reaction step is
prefersbly carried out with trypsin and/or ~-chymotrypsin
(Hoppe-Seylers Zeit~chrift f. physiol. Chemie, 336, 248
(1964)). Trypsin is particularly preferred. Where ap-
propriate, enzymes which are immobilized by known methods
on ~ support are also used, such as de~cribed, for
example, in Canadian Patent 1,265,083. In this case, the
enzymes are advantageously employed in amounts of 0.01 to 20%
by weight relative to the amount of substrate. An amount of
2~ by weight of enzyme is particularly preferred.
Examples of solvents which can be employed sre water,
dimethylformamide, methanol, ethanol, isopropanol,
butanol, ethyl acetate, butyl acetate, toluene or
J~
133S493
methylene chloride.
An ethyl acetate/water mixture is preferred. The tempera-
tures are advantageously between O and 60C. A temperature
ranqe from 20 to 35C is preferred. The pH of the reaction
medium is preferably in the range between 4 and 10,
particularly preferably between 4 and 8.
The process according to the invention can be carried out
in ~uch a way that each intermediate is isolated. How-
ever, it i~ preferably carried out in a one-pot process,
that i8 to say without isolation of the intermediates.
The starting compounds of the general formulae II, III
and V are known and can be obtained by the customary
methods.
The process according to the invention surpri~ingly
provides products of high chemical and optical purity,
which can be employed- without difficulty in further
syntheses. The yields are likewise excellent and are
between 40 and 50 % based on the amount of the compound
of the general formula III employed.
It has to be regarded as particularly surprising that the
process according to the invention is distinctly su-
perior, in terms both of purity and of yield, to the
proces~ of Canadian Patent 1,278,650, which ha~ only three
stages.
B~ample
Z-Trp-Ser-Tyr-OH
a) 350 ml of water are placed in a 2 1 stirred
apparatus, and 47.8 g (0.200 mol) of Z-Ser-OH, 46.4
g (O.200 mol) of H-Tyr-O~ex~Cl and 150 g of sodium
chloride are introduced. Also added are 700 ml of
ethyl acetate and, after everything has dissolved,
the pH of the mixture is ad~usted to 5.0 by addition
of about 25 ml of N-ethylmorpholine. Durinq the
addition of about 220 ml (0.42 mol) of PPA solution
.,; .
~ 5 ~ 1335~93
(w(PPA) in % = 50) in about 30 minutes at a maximum
of 30C (cool somewhat at the end), about 110 ml
(0.86 mol) of N-ethylmorpholine are added via a pH-
stat pump at pH 5Ø The PPA addition is terminated
when a precipitate forms in the reaction mixture.
The precipitate is redissolved by subsequent add-
ition of 350 ml of water. The aqueous phase i8
separated off in a separating funnel and then the
ester phase is washed with 700 ml of potassium
bisulfate solution (w(RHSO4) in % = 10) and 700 ml of
sodium bicarbonate solution (w(NaHCO3) in % = 5). The
aqueous phase from the reaction and the wash phases
are discarded.
b) About 700 ml of ester phase from the 1st coupling,
200 ml of water and 3.3 g of palladium on carbon
w(Pd) in % = 2.5 are placed in a 2 1 stirred ap-
paratus and a stream of hydrogen is passed in at 25-
30C. During the reaction the pH is maintA i n~ at
4.0 with a pH-stat pump and addition of about 160 ml
(0.16 mol) of hydrochloric acid c(HCl) = 1 mol/l.
After the reaction is complete, when no more hydro-
chloric acid is consumed, (about 30 minutes) the
reaction mixture is filtered through a suction
funnel, and the aqueous phase is separated from the
ester phase in a separating funnel. The ester phase
is discarded.
c) About 430 ml of aqueous phase from the hydrogeno-
lysis and 700 ml of ethyl acetate are placed in a
2 1 stirred apparatus and 50.7 g (0.15 mol) of
Z-Trp-OH and 125 g of sodium chloride are sdded.
After everything has dissolved, the pH i8 ad~usted
to 5.0 with about 19 ml of N-ethylmorpholine. During
the addition of about 220 ml (0.42 mol) of PPA
solution (w(PPA) in % = 50) in about 30 minutes at
a maximum of 30C (cool somewhat at the end), about
110 ml (0.86 mol) of N-ethylmorpholine are added via
a pH-stat pump at pH 5Ø The PPA addition is
- 6 - 1335493
terminated when a precipitate forms in the reaction
mixture. The precipitate is redissolved by
subsequent addition of 350 ml of water. The aqueous
phase is separated off in a separating funnel, and
then the ester phase is washed with 700 ml of
potassium bisulfate solution (w(RHSO4) in % = 10) and
several times with 700 ml portions of sodium bicar-
bonate solution (w(N-~CO~) in ~ = 5) until Z-Trp-OH
has been completely removed (according to TLC
analysis). The aqueous phase from the reaction and
the wash phases are discarded.
d) About 700 ml of ester phase from the 2nd coupling
and 700 ml of water are placed in a 2 1 stirred
apparatus and heated to 35-40C, and 1 g of trypsin
is initially added. The reaction starts immediately
and, during it, the pH is maintAineA constant at
pH 7.0 with about 110 ml (0.11 mol) of sodium
hydroxide solution (c(NaOH) = 1 mol/l). The reaction
lasts about 7 hours and, during this, the rate is
increased now and again by further addition of 0.5 g
of trypsin. It is complete when trypsin addition now
brings about only a slight increase in the rate of
absorption of sodium hydroxide solution, or TLC
analysis shows hardly any starting material remain-
ing. The reaction solution is clarified through a
suction funnel, and the ester phase is separated
from the aqueous phase in a separating funnel. The
ester phase is discarded.
The aqueous phase is initially extracted by shaking
twice at pH 5.8-6.0, by addition and dissolution of
4.0 g of potassium dihydrogen phosphate, with 700 ml
of ethyl acetate each time. The ester phases are
discarded. The aqueous phase is then extracted by
shAking three times at pH 5.0, ad~usted by addition
of about 5 ml of glacial acetic acid, with 700 ml of
ethyl acetate each time. The aqueous phase is
discarded. The ester phases contain the tripeptide
~ 7 ~ 1335493
which, on evaparation to dryness in vacuo, remains
in the form of loosely packed crystals. The product
is dried in a vacuum oven at 40C.
Weight: 51.2 g
Yield: 42.0 % based on H-Tyr-OM~Y~Cl
Purity: 98.2 % (determined with HPLC LiChrosorb Si
60/peptide buffer)
Comparison ExEmple
Z-Trp-Ser-Tyr-OH was prepared by the process specified in
EP-A 156,280.
Yield: 30 %
Purity: 78.8 % (determined with HPLC LiChrosorb Si
60/peptide buffer)
