A process for extraction of peptides and its application in liquid phase peptide
synthesis
Field of the invention
The present invention relates to a process for extraction of a peptide from a reaction
mixture resulting from a peptide coupling reaction. This process is preferably used in a
method of liquid phase peptide synthesis (LPPS). The process for extraction of a
peptide from a reaction mixture can also be used in other types of peptide synthesis,
for example in a postcleavage isolation of synthetic peptides prepared by a solid phase
peptide synthesis (SPPS). This process is also applicable for hybrid solid and liquid
phase peptide synthesis. Moreover, the process for extraction of a peptide can be
employed for the isolation of peptides from natural sources such as yeast or bacteria,
in particular for the isolation of recombinantly expressed peptides.
Background of the present invention
In the text of the present application, the nomenclature of amino acids and of peptides
is used according to "Nomenclature and symbolism for amino acids and peptides",
Pure & Appl. Chem. 1984, Vol. 56, No. 5 , pp. 595-624, if not otherwise stated.
The following abbreviations have the meaning as given in the following list, if not
otherwise stated:
ACN acetonitrile
Boc ferf-butoxycarbonyl
Bsmoc 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl
Bzl benzyl
Cbz benzyloxycarbonyl
DCC A/./V-dicyclohexylcarbodiimide
DCM dichloromethane
DEA diethylamine
DIPE diisopropyl ether
DIPEA A/,A/-diisopropylethylamine
DMA A/,A/-dimethylacetamide
DMF /V,A/-dimethylformamide
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
eq equivalent(s)
EtOAc ethylacetate
Fmoc fluorenyl-9-methoxycarbonyl
h hour(s)
HOBt 1-hydroxybenzotriazole
HOBt H20 1-hydroxybenzotriazole monohydrate
HPLS high-performance liquid chromatography
LPPS liquid phase peptide synthesis
MeTHF 2-methyltetrahydrofuran
min minute(s)
MS mass spectrometry
NMP /V-methyl-2-pyrrolidone
OMe methoxy
OiBu te/f-butoxy
PG protecting group
PyBOP benzotriazol-1-yloxy-tris(pyrrolidino)-phosphonium hexafluorophosphate
SPPS solid phase peptide synthesis
TAEA tris(2-aminoethyl)amine
TBTU 0-(benzotriazol-1-yl)-1 , 1 ,3,3-tetramethyluronium tetrafluoroborate
fBu ferf-butyl
TEA triethylamine
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin layer chromatography
TOTU 0-[cyano(ethoxycarbonyl)methylenamino]-1 ,1,3,3-tetramethyluronium
tetrafluoroborate
Trt trityl
UV ultraviolet
Processes for extraction of peptides are generally employed in various types of peptide
synthesis, such as liquid phase peptide synthesis (LPPS), solid phase peptide
synthesis (SPPS) as well as hybrid solid and liquid phase peptide synthesis.
LPPS is particularly often used for industrial large-scale preparations of peptides. LPPS
typically involves coupling of two partially protected amino acids or peptides, whereby
one of them bears an unprotected C-terminal carboxylic acid group and the other one
bears an unprotected /V-terminal amino group. After completion of the coupling step,
the /V-terminal amino group or, alternatively, the C-terminal carboxylic acid group of the
resulting peptide can be deprotected by specific cleavage of one of its protecting
groups (PGs), so that a subsequent coupling step can be carried out. LPPS is usually
finalised by a global deprotection step, in which all remaining PGs are removed.
The handling of peptides, in particular of peptides bearing an unprotected C-terminal
carboxylic acid group and/or an unprotected /V-terminal amino group during the LPPS,
is often compromised by the poor solubility of the peptides in common organic
solvents. In general, the solubility of peptides in common organic solvents decreases
with the length of the peptide chain.
Dichloromethane (DCM) is commonly used in LPPS as a suitable reaction solvent.
DCM has good solvent properties, a low boiling point and its limited miscibility with
water allows working-up of the reaction mixtures by extraction with an aqueous
solution. The use of DCM on an industrial scale is, however, problematic for
environmental reasons and generally limited due to its high density, which makes an
extraction of a DCM layer with an aqueous solution time and cost-consuming.
Furthermore, some recently developed and highly efficient coupling reagents such as
benzotriazol-1-yloxy-tris(pyrrolidino)-phosphonium hexafluorophosphate (PyBOP) and
0-(benzotriazol-1-yl)-1 , 1 ,3,3-tetramethyluronium tetrafluoroborate (TBTU) are poorly
soluble in DCM. These coupling reagents are particularly advantageous for a coupling
of two large peptide fragments, which is known to be low-yielding upon usage of other
coupling reagents.
In addition, many peptides show only a poor solubility in DCM under neutral and basic
conditions and are only sufficiently soluble in polar aprotic solvents, such as e.g. N,Ndimethylformamide
(DMF), L/,/V-dimethylacetamide (DMA) or /V-methyl-2-pyrrolidone
(NMP). Therefore, these polar aprotic solvents are traditionally used as reaction
solvents in LPPS, alone or in a mixture with a less polar solvent such as
tetrahydrofuran (THF).
On the other hand, the usage of polar aprotic solvents for LPPS suffers from a number
of drawbacks. Since polar aprotic solvents have a high boiling point, it is difficult to
concentrate the reaction mixture by evaporation. Furthermore, a direct working-up of
the reaction mixture by extraction with an aqueous solution is not possible due to the
miscibility of polar aprotic solvents with water.
When LPPS is carried out on an industrial scale, the intermediate peptide is usually
isolated by a direct precipitation from the reaction mixture after each coupling step, so
that impurities, such as unreacted starting materials, side products as well as an
excess of coupling reagents and bases, etc. can be separated. After the completion of
the peptide coupling reaction, the reaction mixture is typically poured into an antisolvent,
such as e.g. diethyl ether or water, whereby the precipitation of the peptide
takes place. Unfortunately, already the transfer of the reaction mixture into the antisolvent
is known to trigger gel formation issues.
Moreover, polar aprotic solvents commonly interfere with the process of peptide
precipitation, so that the precipitated peptide is obtained as a sticky gum-like solid,
which is difficult to filter and to dry. In some cases, it is not possible to filter the
precipitated peptide or not even possible to transfer the precipitated peptide onto a
filter. Particularly, peptide precipitations carried out on an industrial scale are often
difficult to perform and are very time-consuming, whereby the filtration time determines
the lead time. This problem can be partially overcome by an increase of the volume
ratio anti-solvent : polar aprotic solvent during the precipitation process, so that in
practice a large amount of a suitable anti-solvent is required for obtaining the
precipitated peptide in a filterable form.
In addition, residues of polar aprotic solvents present in the precipitated peptide are
known to interfere with the subsequent deprotection step involving trifluoroacetic acid
(TFA). Therefore, an additional step of removal of the polar aprotic solvent residues by
washing the precipitated peptide with a more volatile solvent is necessary before a
cleavage of acid cleavable type PGs such as terf-butoxycarbonyl (Boc), trityl (Trt), tertbutyl
(fBu) and e -butoxy (OfBu) can be carried out.
Description of related art
WO 2005/08171 1 is directed to drug-linker-ligand conjugates and drug-linker
compounds and to methods for using the same to treat cancer, an autoimmune
disease or an infectious disease. The document discloses inter alia methods for
preparation of peptide based drugs and extractions of peptides using ethylacetate,
dichloromethane and a mixture of fBuOH/CHCI 3.
US 5,869,454 is directed to arginine keto-amide enzyme inhibitors. The document
discloses inter alia synthesis of these inhibitors and extractions with ethylacetate.
US 2005/0165215 relates to methods of synthesizing peptides and methods for the
isolation of peptides during the synthetic process. The document further relates to
improvements for the large scale synthesis of peptides. The document suggests that
suitable solvents for the peptide extractions include halogenated organic solvents, such
as dichloropropane, dichloroethane, dichloromethane, chloroform, chlorofluorocarbons,
chlorofluorohydrocarbons and mixtures thereof. A preferred solvent is dichloromethane.
C. H. Schneider et al. (Int. J. Peptide Protein Res. 1980, 15, pp. 4 1 - 419) describes a
procedure of peptide synthesis in solution based on liquid-liquid extraction for the
purification of intermediates (two-phase method). The peptide extractions employ
dichloromethane as a solvent.
J .W. van Nispen (Pure and Appl. Chem. 1987, Vol. 59, No. 3 , pp. 331 - 344) provides
an overview over synthesis and analysis of (poly)peptides. The document teaches that
a large number of combinations of solvents of widely varying nature is possible in order
to find optimal separation of peptide components. For this purpose so-called Craig
machines are commonly employed, where in the multiplicative distribution, the lower
phase retains its position while the upper phase is mobile.
US 2010/0184952 discloses a method of removing dibenzofulvene and/or a
dibenzofulvene amine adduct from a reaction mixture obtained by reacting an amino
acid compound protected with an Fmoc group with an amine for deprotection, which
comprises stirring and partitioning the reaction mixture in a hydrocarbon solvent having
a carbon number of 5 or above and a polar organic solvent (excluding organic amide
solvents) immiscible with the hydrocarbon solvent, and removing the hydrocarbon
solvent layer in which the dibenzofulvene and/or the dibenzofulvene amine adduct
are/is dissolved. During this method, an amino acid ester or peptide is transferred to a
polar organic solvent. Examples of such polar organic solvents include acetonitrile,
methanol, acetone and the like and a mixed solvent thereof, with preference given to
acetonitrile and methanol.
L . A. Carpino e a/. (Organic Process Research & Development 2003, 7 , pp. 28-37)
describe a rapid, continuous solution-phase peptide synthesis. The methods employing
deprotections of the Fmoc and Bsmoc protective groups of peptide segments in the
presence of tris(2-aminoethyl)amine were shown to be applicable for the gram-scale
rapid, continuous solution synthesis of short peptides as well as for the synthesis of a
relatively long (22-mer) segment (hPTH 13-34). In the latter case, the crude product
was reported to be of a significantly greater purity than a sample obtained via a solidphase
protocol. The Bsmoc methodology was optimised by a new technique involving
filtration of the growing partially deprotected peptide at each coupling deprotection
cycle through a short column of silica gel.
However, the methodology described by L. A. Carpino e a/ has several limitations.
This methodology employs DCM as a reaction solvent and, therefore, cannot be
applied for the preparation of peptides showing a poor solubility in DCM. Moreover, it
employs a high quantity of high-cost tris(2-aminoethyl)amine (TAEA) which further
limits the applicability of this methodology on an industrial scale.
Thus, there is a strong demand for a time- and cost-efficient synthetic methodology for
the preparation of peptides, in particular on an industrial scale. Such methodology must
overcome the drawbacks resulting from the usage of DCM and of polar aprotic solvents
such as DMF, DMA and NMP during LPPS.
Summary of the invention
The authors of the present invention surprisingly found that a broad range of
structurally diverse peptides has an excellent solubility in toluene, preferably in
combination with an organic solvent selected from the group consisting of n-heptane,
2-methyltetrahydrofuran, ethylacetate, isopropylacetate, acetonitrile or tetrahydrofuran
(this group is designated as organic solvent 1). In particular, the solubility of the
peptides in the combination of toluene and the organic solvent 1 is generally higher
than in neat toluene. Moreover, they found that commonly used polar aprotic solvents
largely partition into the aqueous layer in a biphasic system comprising water and
toluene or a combination of toluene and the organic solvent .
Therefore, water and neat toluene or a combination of toluene with the organic solvent
1 are highly suitable for the extraction of a peptide from a mixture containing a polar
aprotic solvent. In one of the embodiments of the present invention, the resulting
organic layer containing the peptide is partially evaporated and the peptide dissolved
therein is precipitated upon addition of a suitable anti-solvent (this group of solvents is
designated as organic solvent 2). Because substantially no polar aprotic solvent is
present during the process of peptide precipitation the resulting peptide can easily be
filtered. By applying the extraction process of the present invention, the time required
for the peptide filtration can be significantly reduced. Thus, by applying such a process
of extraction, the drawbacks resulting from the usage of polar aprotic solvents during
LPPS can be successfully overcome.
The present invention relates to a process for extraction of a peptide from a reaction
mixture resulting from a peptide coupling reaction, the reaction mixture containing the
peptide and a polar aprotic solvent selected from the group consisting of N,Ndimethylformamide,
/V,A/-dimethylacetamide and /V-methyl-2-pyrrolidone, whereby the
process comprises a step a) and a step b):
step a) comprises the addition of a component a1) and a component a2), whereby
component a1) is toluene,
component a2) is water,
to the reaction mixture, so that a biphasic system with an organic layer and an aqueous
layer is obtained;
step b) comprises the separation of the organic layer containing the peptide from the
aqueous layer, whereby
the biphasic system obtained in step a) is characterised by the following volume ratios:
polar aprotic solvent : toluene from 1 : 20 to 1 : 2 ; and
polar aprotic solvent : water from 1 : 20 to 1 : 2 .
One of the preferred embodiments of the present invention relates to a process for
extraction of a peptide from a reaction mixture resulting from a peptide coupling
reaction containing the peptide and a polar aprotic solvent selected from the group
consisting of L/,/V-dimethylformamide, /V,/V-dimethylacetamide and A/-methyl-2-
pyrrolidone, whereby the process comprises a step a) and a step b):
step a) comprises the addition of a component a1), a component a2) and a component
a3), whereby
component a1) is toluene,
component a2) is water,
component a3) is an organic solvent 1, the organic solvent 1 is selected from the group
consisting of n-heptane, 2-methyltetrahydrofuran, ethylacetate, isopropylacetate,
acetonitrile and tetrahydrofuran,
so that a biphasic system with an organic layer and an aqueous layer is obtained;
step b) comprises the separation of the organic layer containing the peptide from the
aqueous layer, whereby
the biphasic system obtained in step a) is characterised by the following volume ratios:
polar aprotic solvent : toluene from 1 : 20 to 1 : 2 ;
polar aprotic solvent : organic solvent 1 from 1 : 5 to 30 : 1;
polar aprotic solvent : water from 1 : 20 to 1 : 2 ; and
toluene : organic solvent 1 from 50 : 1 to 1 : .
In a preferred embodiment, the biphasic system obtained in step a) is characterised by
the following volume ratios:
polar aprotic solvent : toluene from 1 : 6 to 1 : 3 ;
polar aprotic solvent : organic solvent 1 from 1 : 1 to 4 : 1;
polar aprotic solvent : water from 1 : 5 to 1 : 3 ; and
toluene : organic solvent 1 from 10 : 1 to 2 : 1.
In a particularly preferred embodiment, the polar aprotic solvent is N,Ndimethylformamide
or /V-methyl-2-pyrrolidone.
In yet another embodiment of the present invention, the organic solvent 1 is absent in
the biphasic system.
In one of the preferred embodiments of the present invention, the peptide is extracted
but not precipitated. Instead, one or several protecting groups of the peptide are
cleaved and the resulting partially unprotected peptide is extracted and the organic
layer comprising the peptide is employed for the subsequent peptide coupling reaction.
Thus, the present invention provides an efficient synthetic methodology for a
continuous LPPS which is suitable for the preparation of peptides on an industrial
scale.
The continuous LPPS of the present invention is highly suitable for the peptide
synthesis upon usage of Boc, Fmoc and Bzl as protective groups as will be illustrated
by the examples below.
Process for extraction
The present invention relates to a process for extraction of a peptide from a reaction
mixture resulting from a peptide coupling reaction, the reaction mixture containing the
peptide and a polar aprotic solvent, whereby the process comprises a step a) and a
step b):
step a) comprises the addition of a component a1) and a component a2), whereby
component a1) is toluene,
component a2) is water,
to the reaction mixture, so that a biphasic system with an organic layer and an aqueous
layer is obtained;
step b) comprises the subsequent separation of the organic layer containing the
peptide from the aqueous layer.
One of the preferred embodiments of the current invention relates to a process for
extraction of a peptide from a reaction mixture resulting from a peptide coupling
reaction containing the peptide and a polar aprotic solvent selected from the group
consisting of DMF, DMA and NMP, whereby the process comprises a step a) and a
step b):
step a) comprises the addition of a component a1), a component a2) and a component
a3), whereby
component a1) is toluene,
component a2) is water,
component a3) is an organic solvent 1, the organic solvent 1 is selected from the group
consisting of n-heptane, 2-methyltetrahydrofuran, ethylacetate, isopropylacetate,
acetonitrile and tetrahydrofuran,
so that a biphasic system with an organic layer and an aqueous layer is obtained;
step b) comprises the separation of the organic layer containing the peptide from the
aqueous layer.
Optionally, the component a1), the component a2) and the component a3) are mixed
with each other, whereby this can be done in any sequence. The three components
can also be added as premixed mixtures of two or all three components as long as no
precipitation of the peptide takes place during the process for extraction.
The mixture containing the polar aprotic solvent is preferably a crude reaction mixture
resulting from a peptide coupling reaction. Preferably, this mixture does not contain any
compounds, which can act as surfactants and interfere with the phase separation
during the process for extraction. In a particularly preferred embodiment the mixture
does not contain any surfactants known in the prior art, such as cationic tensides and
non-ionic tensides.
The addition of the component a1), the component a2) and the component a3) to the
mixture containing the peptide and a polar aprotic solvent can take place in any order
as long as no precipitation of the peptide takes place during the process for extraction.
For example, it is possible to combine the mixture containing the peptide and a polar
aprotic solvent with toluene, add water thereto and, finally, add the organic solvent 1. It
is also possible that the mixture containing the peptide and a polar aprotic solvent is
transferred into the water and toluene and the organic solvent 1 are added thereto
afterwards.
In the particularly preferred embodiment of the present invention, the mixture
containing the peptide and a polar aprotic solvent is combined with toluene and the
organic solvent 1, whereby the addition of toluene and the organic solvent 1 can take
place in any order. Subsequently, water is added thereto.
It is understood that the added water (component a2)) may contain dissolved
components, such as salts, for instance inorganic salts.
It is preferred that the obtained biphasic system is vigorously stirred. The process of
stirring of the obtained biphasic system can be carried out upon usage of mixing
equipment known in the state of the art and commonly used for extractions. For
example, in the case of batch extractions, jet- or agitator-type mixers can be employed
for the stirring of the biphasic system.
The choice of the suitable equipment for the extraction mainly depends on the scale on
which the process for extraction is being carried out as well as on the extraction
temperature. The process for extraction can be carried out by using batch extractions
or continuous extractions. The process for extraction can also be repeated several
times, if required, so that an optimal extraction of the peptide is achieved.
After the process of stirring has been carried out, it is preferred that a phase separation
is allowed to take place, whereby two liquid layers are formed: an organic layer and an
aqueous layer. The organic layer has a lower density than the aqueous layer. Phase
separation may be accomplished upon usage of settling tanks or by means of
centrifugation. The time required for the phase separation depends on the scale on
which the process for extraction is taking place and on the equipment employed.
Preferably, the phase separation requires less than 1 hour, more preferred less than
10 min, particularly preferred less than 1 min.
After the phase separation has taken place, the peptide is mainly located in the organic
layer, which further contains toluene and, optionally, the organic solvent 1. The upper
organic layer containing the peptide is separated from the aqueous layer. Preferably,
after the process for extraction more than 90 wt.-% of the peptide is located in the
organic layer and less than 10 wt.-% of the peptide is located in the aqueous layer. It is
even more preferred that after the process for extraction more than 98 wt.-% of the
peptide is located in the organic layer and less than 2 wt.-% of the peptide is located in
the aqueous layer. It is particularly preferred that after the process for extraction more
than 99 wt.-% of the peptide is located in the organic layer and less than 1 wt.-% of the
peptide is located in the aqueous layer.
The process for extraction of the present invention allows an efficient extraction of the
peptide from a crude reaction mixture resulting from a peptide coupling reaction. The
solubility of polar aprotic solvents in the organic layer is significantly lower than in the
aqueous layer. Therefore, the organic layer containing the peptide further contains only
a low amount of the polar aprotic solvents after the extraction.
Preferably, after the process for extraction less than 15 vol.-% of the polar aprotic
solvents is located in the organic layer and more than 85 vol.-% of the polar aprotic
solvents is located in the aqueous layer. It is, however, more preferred that after the
process for extraction less than 5 vol.-% of the polar aprotic solvents is located in the
organic layer and more than 95 vol.-% of the polar aprotic solvents is located in the
aqueous layer. It is particularly preferred that after the process for extraction less than
2 vol.-% of the polar aprotic solvents is located in the organic layer and more than
98 vol.-% of the polar aprotic solvents is located in the aqueous layer. This may require
repeated extractions.
Importantly, the process for extraction according to the present invention not only
allows to separate the peptide from a substantial part of the polar aprotic solvent but
also from salts and side products, which originate from the coupling reagents (ureas,
tetrafluoroborates etc.). These salts and side products usually cannot be removed if a
direct precipitation from a crude reaction mixture resulting from a peptide coupling
reaction takes place upon addition of a hydrophobic anti-solvent such as n-heptane or
diethyl ether. However, these salts and side products are known to reduce the capacity
of chromatography columns used for the downstream processing of peptides. Such
additional purification by column chromatography is essential if the prepared peptides
are used as active pharmaceutical ingredients.
Thus, if required, the precipitated peptide can be subsequently purified by column
chromatography. In cases wherein the peptide is used as an active pharmaceutical
ingredient such additional purification steps are used. Therefore, the process for
extraction according to the present invention allows isolating the peptide in a higher
purity than upon usage of the direct precipitation process from the reaction mixture.
The composition of the biphasic system obtained during the process for extraction has
a strong impact on the distribution coefficients of the peptide and of the polar aprotic
solvents between the organic layer and the aqueous layer. In the following the ratios
are given as volume to volume ratios.
It is preferred that the volume ratio polar aprotic solvent : toluene ranges from 1 : 20 to
1 : 2 . Preferably, this volume ratio ranges from 1 : 10 to 1 : 2 . It is particularly preferred
that this volume ratio ranges from 1 : 6 to 1 : 3 .
The solubility of the peptide in a combination of toluene and the organic solvent 1 was
shown to be higher than in the neat toluene. Therefore, the solubility of the peptide in
the organic layer obtained during the process for extraction is particularly high when
the amount of the organic solvent 1 used is sufficiently high. It is preferred that the
volume ratio polar aprotic solvent : organic solvent 1 ranges from 1 : 5 to 30 : 1.
Preferably, this volume ratio ranges from 1 : 3 to 10 : 1. It is particularly preferred that
this volume ratio ranges from 1 : 1 to 4 : 1.
It is preferred that the volume ratio toluene : organic solvent 1 ranges from 50 : 1 to
1 : 1. Preferably, this volume ratio ranges from 20 : 1 to 2 : 1. It is particularly preferred
that this volume ratio ranges from 10 : 1 to 2 : 1.
The volume ratio polar aprotic solvent : water has a significant influence on the
efficiency of the process for extraction and on the solubility of the peptide in the
aqueous layer. In particular, the peptide has a considerably high solubility in the
aqueous layer, if the volume ratio polar aprotic solvent : water in the biphasic system is
higher than 1 : 2 , i.e. if the aqueous layer contains more than 34 vol.-% of the polar
aprotic solvent. It is therefore preferred that the volume ratio polar aprotic
solvent : water ranges from 1 : 20 to 1 : 2 . Preferably, this volume ratio ranges from
1 : 10 to 1 : 3 . It is particularly preferred that this volume ratio ranges from 1 : 5 to 1 : 3 .
Preferably, the polar aprotic solvent present in the mixture containing the peptide is
selected from the group consisting of DMF and NMP.
Thus, both neat toluene and a combination of toluene and the organic solvent 1 are
particularly suitable for the process for extraction of a peptide. Toluene is an easily
recyclable, low-cost solvent which has a relatively low toxicity to humans and aquatic
organisms. Accordingly, the present invention can be advantageously employed on an
industrial scale.
The solubility of the peptide in a combination of toluene and the organic solvent 1 is
particularly high if the organic solvent 1 is selected from the group consisting of nheptane,
2-methyltetrahydrofuran, ethylacetate (EtOAc), isopropylacetate, acetonitrile
(ACN) and tetrahydrofuran (THF), more preferred from the group consisting of EtOAc,
isopropylacetate, ACN and THF, particularly preferred from the group consisting of
ACN and THF. In a particularly preferred embodiment for the process for extraction of
the peptide the organic solvent 1 is selected from the group consisting of ACN and
THF.
The component a2) employed for the process for extraction of the peptide can consist
of water only. However, the miscibility of toluene and of the organic solvent 1 in the
component a2) and, consequently, the solubility of the peptide in the aqueous layer can
be significantly reduced if the component a2) further contains at least one inorganic
salt. In addition, the water content in the organic layer is reduced if the component a2)
contains at least one inorganic salt.
In one of the preferred embodiments the component a2) contains at least one inorganic
salt selected from the group consisting of sodium chloride, sodium hydrogensulfate,
potassium hydrogensulfate, sodium hydrogencarbonate and sodium
hydrogenphosphate. In other embodiments the component a2) can also contain other
compounds such as acids.
In particular, the component a2) can contain inorganic salts which do not act as
buffering agents in the pH range from 2 to 11. An addition of such inorganic salts can
decrease the solubility of the peptide in the aqueous layer and reduce the time required
for the phase separation during the process for extraction. For instance, the component
a2) can contain sodium chloride or sodium sulfate. The concentration of the inorganic
salt present in the component a2) preferably ranges from 1 wt.-% to 20 wt.-%, even
more preferred from 5 wt.-% to 15 wt.-%. A salt like sodium chloride is used to facilitate
the separation of the two phases and a salt that acts as a buffering agent is used to
selectively extract an acid or a base in the aqueous layer.
The pH value of the component a2) can have a strong influence on the solubility of the
peptide as well as on the solubility of some impurities in the aqueous layer. In addition,
the choice of the pH value of the component a2) depends on the chemical stability of
the peptide as well as on the chemical stability of its PGs. It is preferred that the pH
value of the component a2) ranges from 2 to 11, particularly preferred from 5 to 8 , so
that the tertiary bases used for the peptide coupling reaction predominantly remain in
the aqueous layer during the process for extraction. The pH value of the component
a2) can be adjusted by an addition of an acid or a base and/or upon using a buffering
agent.
The choice of the acid which can be used for the adjustment of the pH value of the
component a2) is not particularly limited as long as the acid present in the component
a2) does not interfere with the process for extraction of the peptide and does not cause
the degradation of the peptide. For example, Bransted acids such as sulphuric acid,
hydrochloric acid, phosphoric acid, trifluoroacetic acid or citric acid can be employed for
this purpose.
The choice of the base which can be used for the adjustment of the pH value of the
component a2) is not particularly limited as long as the base present in the component
a2) does not interfere with the process for extraction of the peptide and does not cause
the degradation of the peptide. For example, hydroxides of alkali metals such as
sodium hydroxide, potassium hydroxide and lithium hydroxide are suitable for the
adjustment of the pH value of the component a2).
It is preferred that the component a2) contains the buffering agent, so that the pH value
of the aqueous layer is kept within the desired range during the process for extraction.
Preferably, the buffering agent is selected from the group consisting of ammonium
chloride, sodium hydrogensulfate, potassium hydrogensulfate, sodium
hydrogencarbonate, sodium carbonate, sodium hydrogenphosphate, sodium
dihydrogenphosphate and sodium phosphate. The concentration of the buffering agent
present in the component a2) preferably ranges from 1 wt.-% to 10 wt.-%, even more
preferred from 3 wt.-% to 8 wt.-%.
Optionally, the obtained organic layer containing the peptide can be additionally
washed at least one time with an aqueous solution. Preferably, the pH value of the
aqueous solution used for this purpose ranges from 2 to 1.
Depending on the conditions of the peptide coupling reaction and the reagents used,
the organic layer can contain compounds with free primary, secondary or tertiary amino
groups as impurities, for instance, peptides with unprotected AMerminal amino groups
or tertiary bases. In such cases, it is preferred that the organic layer is washed with an
aqueous solution having a pH value of from 2 to 7.
In other cases, the organic layer can contain compounds having a free carboxylic acid
group, for instance, peptides with unprotected C-terminal carboxylic acid groups. In
these cases, it is preferred that the organic layer is washed with an aqueous solution
having a pH value of from 7 to 11.
The temperature at which the process for extraction of the peptide is preferably carried
out (hereinafter designated as extraction temperature) depends on the choice of the
solvents employed as well as on the properties of the peptide. The extraction
temperature has a strong influence on the miscibility of the solvents employed and on
the solubility of the peptide in the organic layer and in the aqueous layer. The
extraction temperature is therefore chosen in such a way that a biphasic system is
formed during the process for extraction and the solubility of the peptide in the organic
layer is sufficiently high. Preferably, the process for extraction of the peptide is carried
out at the extraction temperature of from 0°C to 60°C. It is particularly preferred that the
extraction temperature ranges from 20°C to 30°C.
Depending on the conditions of the peptide coupling reaction and on the coupling
reagents employed, a formation of solids can take place before and/or during the
process for extraction. This can be, for instance, the case, if carbodiimides are used as
coupling reagents. For this reason, it may be required that a filtration of the biphasic
system obtained after combining the mixture containing the peptide, a polar aprotic
solvent, toluene, optionally, the organic solvent 1 and the component a2) is carried out.
Therefore, in one of the embodiments of the present invention, a filtration of the
biphasic system is carried out before the organic layer containing the peptide is
separated.
The peptide extracted by the process for extraction of the present invention may be any
peptide. Preferably, the peptide extracted by the process for extraction comprises 100
or less amino acid residues, more preferably 50 or less amino acid residues, most
preferably 20 or less amino acid residues. The amino acids of the peptide can be Dand/
or L-a-amino acids, b-amino acids as well as other organic compounds containing
at least one primary and/or secondary amino group and at least one carboxylic acid
group. Preferably, the amino acids are a-amino acids, even more preferably L-a-amino
acids, whereby proteinogenic amino acids are particularly preferred.
Preparation of the peptide
Another aspect of the present invention relates to a process for preparation of a
peptide in liquid phase comprising a step aa), a step bb) and a step cc):
in step aa) a peptide coupling reaction is carried out in the polar aprotic solvent
selected from the group consisting of L/,/V-dimethylformamide, L/,/V-dimethylacetamide
and A/-methyl-2-pyrrolidone in the presence of a coupling reagent and, optionally, a
tertiary base;
in step bb) the resulting peptide is extracted according to a process described above;
and
in step cc) at least a part of the organic layer obtained in step bb) is evaporated.
As starting materials for the peptide coupling reaction according to step aa) a
combination of two partially protected amino acids, of two partially protected peptides
or a combination of a partially protected amino acid and a partially protected peptide is
employed.
The process for preparation of a peptide in liquid phase according to the present
invention is highly suitable in a liquid phase peptide synthesis (LPPS). In one of the
embodiments of the present invention, the peptide coupling reaction according to step
aa) employs a combination of two partially protected peptides prepared by SPPS.
Thus, the process of the present invention allows coupling of peptide fragments and
can be used in combination with SPPS.
The peptide coupling reaction according to step aa) is carried out using conventional
process parameters and reagents typical for peptide coupling reactions.
The peptide coupling reaction is conventionally carried out in a polar aprotic solvent
and upon using one or more coupling reagents, preferably in the presence of one or
more coupling additives, and preferably in the presence of one or more tertiary bases.
The coupling reagents used for the peptide coupling reaction are chosen in such a way
that they do not react with the polar aprotic solvent under the conditions of the peptide
coupling reaction and no substantial epimerisation of the stereogenic centre adjacent to
the activated carboxylic acid group takes place. Preferred coupling reagents are
therefore phosphonium or uronium salts of 0-1H-benzotriazole and carbodiimide
coupling reagents.
Phosphonium and uronium salts are preferably selected from the group consisting of
BOP (benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate),
PyBOP (benzotriazol-1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate),
HBTU (0-(1 H-benzotriazole-1 -yl)-1 ,1,3,3-tetramethyluronium hexafluorophosphate),
HCTU (0-(1/-/-6-chloro-benzotriazole-1-yl)-1 , 1 ,3,3-tetramethyluronium
hexafluorophosphate),
TCTU (0-(1 H-6-chlorobenzotriazole-1 -yl)-1 ,1,3,3-tetramethyluronium
tetrafluoroborate),
HATU (0-(7-azabenzotriazol-1-yl)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate),
TATU (0-(7-azabenzotriazol-l-yl)-1 , 1 ,3,3-tetramethyluronium tetrafluoroborate),
TBTU (0-(benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate),
TOTU (0-[cyano(ethoxycarbonyl)methyleneamino]-1 ,1,3,3-tetramethyluronium
tetrafluoroborate),
HAPyU (0-(benzotriazol-1 -yl)oxybis-(pyrrolidino)-uronium hexafluorophosphate),
PyAOP (benzotriazole-1 -yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate),
COMU ( 1-[(1 -(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylaminomorpholinomethylene)]-
methanaminium hexafluorophosphate),
PyClock (6-chloro-benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium
hexafluorophosphate), PyOxP (0-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-
oxytri(pyrrolidin-1-yl)-phosphonium hexafluorophosphate) and
PyOxB (0-[(1 -cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1 -yl)-
phosphonium tetrafluoroborate).
Preferred coupling reagents selected from phosphonium or uronium coupling reagents
are TBTU, TOTU and PyBOP.
Carbodiimide coupling reagents are preferably selected from the group consisting of
diisopropyl-carbodiimide (DIC), dicyclohexyl-carbodiimide (DCC) and water-soluble
carbodiimides (WSCDI) such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC).
Water-soluble carbodiimides are particularly preferred as carbodiimide coupling
reagents, whereby EDC is mostly preferred.
The tertiary base employed in the peptide coupling reaction is preferably compatible
with the peptide and with the coupling reagent and does not interfere with the process
for extraction by acting as a surfactant.
Preferably, the conjugated acid of said tertiary base used in the peptide coupling
reaction has a pKa value from 7.5 to 15, more preferably from 7.5 to 10. Said tertiary
base is preferably selected from the group consisting of trialkylamines, such as N,Ndiisopropylethylamine
(DIPEA) or triethylamine (TEA), further V, -di-C1 _4 alkylanilines,
such as A/,A/-diethylaniline, 2,4,6-tri-C 1-4 alkylpyridines, such as collidine (2,4,6-
trimethylpyridine), or -C1-4 alkylmorpholines, such as A/-methylmorpholine, with any
C -4 alkyl being identical or different and independently from each other straight or
branched C alkyl. DIPEA, TEA and /V-methylmorpholine are particularly preferred as
tertiary bases for the peptide coupling reaction.
A coupling additive is preferably a nucleophilic hydroxy compound capable of forming
activated esters, more preferably having an acidic, nucleophilic A/-hydroxy function
wherein N is imide or is /V-acyl or A/-aryl substituted triazeno, the triazeno type coupling
additive being preferably a /V-hydroxybenzotriazol derivative (or 1-hydroxybenzotriazol
derivative) or a A/-hydroxybenzotriazine derivative. Such coupling additives have been
described in WO 94/0791 0 and EP 0 4 10 82.
Preferred coupling additives are selected from the group consisting of Nhydroxysuccinimide
(HOSu), 6-chloro-1-hydroxybenzotriazole (CI-HOBt), /V-hydroxy-
3,4-dihydro-4-oxo-1 ,2,3-benzotriazine (HOOBt), l-hydroxy-7-azabenzotriazole (HOAt),
1-hydroxybenzotriazole (HOBt) and ethyl-2-cyano-2-hydroxyiminoacetate (CHA). CHA
is available under trade name OXYMAPURE ®. CHA has proved to be an effective
coupling additive as epimerisation of the stereogenic centre of the activated carboxylic
acid is suppressed to a higher degree in comparison to benzotriazole-based coupling
additives. In addition, CHA is less explosive than e.g. HOBt or CI-HOBt, so that its
handling is advantageous and, as a further advantage, the coupling progress can be
visually monitored by a colour change of the reaction mixture. Preferably, HOBt is used
as coupling additive for the peptide coupling reaction.
In the preferred embodiment of the present invention, the combination of reagents in
the peptide coupling reaction is selected from the group consisting of
TBTU/HOBt/DIPEA, PyBOP/TEA, EDC/HOBt and EDC/HOBt/DIPEA.
The reaction solvent for the peptide coupling reaction is selected from the group
consisting of DMF, DMA, NMP or mixtures thereof. The particularly preferred reaction
solvent for the peptide coupling reaction is selected from the group consisting of DMF
and NMP.
Preferably, the reaction solvent is substantially water-free. Preferably, the reaction
solvent contains less than 1 wt.-% water, more preferred less than 0.1 wt.-% water,
even more preferred less than 0.01 wt.-% water and particularly preferred less than
0.001 wt.-% water. The water content in a solvent can be determined by Karl Fischer
titration according to the standard test method ASTM E203-8 as known in the prior art.
Preferably, the reaction solvent for the peptide coupling reaction is substantially free of
impurities selected from the group consisting of primary and secondary amines,
carboxylic acids and aliphatic alcohols. The reaction solvent for the peptide coupling
reaction is considered to be substantially free of these impurities if less than 1 mol.-%
of any of the starting materials used in substoichiometric or stoichiometric amount
undergoes an undesired reaction with these impurities during the peptide coupling
reaction.
The choice of the appropriate reaction temperature depends on the employed coupling
reagent as well as on the stability of the peptide. Preferably, the peptide coupling
reaction is carried out at a reaction temperature of from -15°C to 50°C, more preferably
from -10°C to 30°C, even more preferably from 0°C to 25°C.
Preferably, the peptide coupling reaction is carried out at the atmospheric pressure.
However, it is also possible to carry out the peptide coupling reaction at a pressure
which is higher or slightly lower than the atmospheric pressure.
Preferably, the peptide coupling reaction is carried out under an ambient atmosphere.
However, an atmosphere of a protective gas such as nitrogen or argon is also
preferable.
In the present application, the term "reaction time" refers to the time required until the
conversion of the reaction is substantially complete. The conversion of the reaction is
considered to be substantially complete, once the amount of the starting material used
in substoichiometric or stoichiometric amount decreases to less than 5 mol.-% of its
initial amount, preferably to less than 2 mol.-% of its initial amount. The progress of the
reaction can be monitored by analytical methods known in the art, for instance, by
analytical high-performance liquid chromatography (HPLC), thin layer chromatography
(TLC), mass spectrometry (MS) or HPLC-MS, whereby HPLC is particularly preferred
for this purpose.
Preferably, the reaction time for the peptide coupling reaction ranges from 15 min to
20 h, more preferably from 30 min to 5 h, even more preferably from 30 min to 2 h .
The term "part" in this description of reaction conditions of the peptide coupling reaction
is meant to be a factor of the parts by weight of the total weight of the peptides and/or
amino acids employed as starting materials for the peptide coupling reaction.
Preferably, from 1 to 30 parts, more preferably from 5 to 10 parts of the reaction
solvent are used.
Preferably, from 0.9 to 5 mol equivalents, more preferably from 1 to 1.5 mol equivalents
of coupling reagent is used, the mol equivalent being based on the mol of reactive Cterminal
carboxylic acid groups.
Preferably, from 0.1 to 5 mol equivalents, more preferably from 0.5 to 1.5 mol
equivalents of coupling additive is used, the mol equivalent being based on the mol of
coupling reagent.
Preferably, from 1 to 10 mol equivalents, more preferably from 2 to 3 mol equivalents,
of tertiary base is used, the mol equivalent being based on the mol of coupling reagent.
Any peptide is obtainable by the process for preparation of a peptide in liquid phase of
the present invention.
Preferably, the peptide obtained by the process for preparation of a peptide in liquid
phase of the present invention comprises 100 or less amino acid residues, more
preferably 50 or less amino acid residues, most preferably 20 or less amino acid
residues. The amino acids of the peptide can be D- and L-a-amino acids, b-amino
acids as well as other organic compounds containing at least one primary and/or
secondary amino group and at least one carboxylic acid group. Preferably, the amino
acids of the peptide obtained by the process for preparation of a peptide in liquid phase
of the present invention are a-amino acids, even more preferably L-a-amino acids,
whereby proteinogenic amino acids are particularly preferred.
Preferably, after the process for extraction, the organic layer containing the peptide is
partially evaporated. In the present application, the obtained layer is thus designated as
"partially evaporated organic layer". The temperature at which the partial evaporation
takes place is not particularly limited and is chosen according to the thermal stability of
the peptide as well as to the properties of toluene or of the mixture of toluene with the
organic solvent 1. It is preferred that the partial evaporation of the organic layer is
carried out at a temperature of from 30°C to 50°C. If required, the partial evaporation of
the organic layer is carried out under reduced pressure of from 20 mbar to 1000 mbar
(20 hPa to 1000 hPa), preferably under reduced pressure of from 50 mbar to 200 mbar
(50 hPa to 200 hPa). A person skilled in the art is aware that the pressure at which the
partial evaporation of the organic layer takes place is preferably adjusted according to
the desired evaporation temperature.
Since toluene and the organic solvent 1 are sufficiently volatile, the partial evaporation
of the organic layer containing the peptide can be easily carried out.
In one of the embodiments of the present invention, the organic layer containing the
peptide is directly evaporated until dryness and the remaining residue is dissolved in a
solvent which is distinct from toluene and the organic solvent 1. However, if the organic
layer containing the peptide comprises more than 30 vol.-% of a solvent selected from
the group consisting of MeTHF, and THF, the complete evaporation until dryness is
preferably avoided for safety reasons. Instead, the partial evaporation of the organic
layer containing the peptide can be carried out, followed by an addition of toluene and
a subsequent evaporation until dryness.
Because toluene present in the organic layer forms an azeotrope with water, the traces
of water in the organic layer containing the peptide are efficiently removed during the
process of partial evaporation.
In one of the preferred embodiments, the substantial part of the peptide is precipitated
upon combining the partially evaporated organic layer with an organic solvent 2 .
In another preferred embodiment of the present invention, the organic layer containing
the peptide is evaporated until dryness and the remaining residue is dissolved in a
solvent which is distinct from toluene and the organic solvent 1. The obtained solution
is subsequently combined with the organic solvent 2 , whereby the peptide precipitation
takes place.
The volume ratio partially evaporated organic layer : organic solvent 2 employed during
the process for precipitation of the peptide has a strong impact on the completeness of
the process for precipitation and on the properties of the precipitated peptide. In the
following the ratios are given as volume to volume ratios.
It is preferred that the volume ratio partially evaporated organic layer : organic solvent 2
ranges from 1 : 20 to 1 : 1. Preferably, this volume ratio ranges from 1 : 12 to 1 : 2 . It is
particularly preferred that this volume ratio ranges from 1 : 6 to 1 : 3 .
The organic solvent 2 is preferably selected from organic solvents having a boiling
point of less than 160°C at the atmospheric pressure. Preferably, the solubility of the
peptide in the organic solvent 2 is lower than in toluene and/or in the mixture of toluene
and the organic solvent 1. The organic solvent 2 is preferably selected from the group
consisting of acetonitrile, diethyl ether, diisopropyl ether and n-heptane, more preferred
from the group consisting of acetonitrile, diethyl ether and diisopropyl ether, particularly
preferred from the group consisting of diisopropyl ether and n-heptane.
Because the partially evaporated organic layer containing the peptide is substantially
free of the polar aprotic solvent, the amount of the organic solvent 2 required for the
precipitation of the peptide is significantly lower than in the precipitation processes of
the prior art, which use crude reaction mixtures resulting from the peptide coupling
reaction. In addition, contrary to the precipitation processes of the prior art, the
precipitated peptide is a non-sticky solid material.
Preferably, during the precipitation process at least 80 wt.-% of the peptide present in
the partially evaporated organic layer precipitates as a solid material. It is even more
preferred that at least 90 wt.-% of the peptide present in the partially evaporated
organic layer precipitates as a solid material. It is yet even more preferred that at least
95 wt.-% of the peptide present in the partially evaporated organic layer precipitates as
a solid material. It is particularly preferred that at least 98 wt.-% of the peptide present
in the partially evaporated organic layer precipitates as a solid material.
The temperature at which the precipitation process is carried out (this temperature is
hereinafter designated as precipitation temperature) depends on the composition of the
partially evaporated organic layer, choice of the organic solvent 2 and on the properties
of the peptide.
The precipitation temperature has a strong influence on the completeness of the
precipitation of the peptide and on the physical properties of the precipitated peptide.
Preferably, the precipitation process is carried out at the precipitation temperature of
from - 0°C to 60°C, whereby the precipitation temperature of from -10°C to 30°C is
even more preferred. It is, however, particularly preferred that the precipitation
temperature ranges from -10°C to 0°C.
Since the partially evaporated organic layer containing the peptide is substantially free
of the polar aprotic solvent, the precipitated peptide can be easily separated by
filtration. Therefore, the time required for the filtration process is significantly shortened.
Preferably, the precipitated peptide is separated by filtration and dried under reduced
pressure.
It is also possible, however, to separate the precipitated peptide by centrifugation.
If desired, the filtrate collected during the filtration can be subjected again to a partial
evaporation and to a subsequent precipitation, so that a second batch of the
precipitated peptide can be collected.
In another embodiment of the present invention, the partially evaporated organic layer
containing the peptide is directly treated with a reagent cleaving one or several PGs of
the peptide. Because the partially evaporated organic layer containing the peptide is
substantially free of the polar aprotic solvent, the choice of the reagents for the
cleavage of one or several PGs of the peptide is not particularly limited. For instance,
the partially evaporated organic layer containing the peptide can be treated with an
acidolytic reagent, whereby no undesired reactions between the acidolytic reagent and
polar aprotic solvent or inhibition of the cleavage take place. This embodiment of the
present invention is particularly preferable if the /V-terminal PG of the peptide is tertbutoxycarbonyl
(Boc) group.
In other embodiments of the present invention, the partially evaporated organic layer is
used for carrying out other reactions such as disulphide bridge formation.
In another embodiment of the present invention, the reagent cleaving one or several
PGs of the peptide is added directly to the reaction mixture resulting from a peptide
coupling reaction. After the cleavage of the targeted PG is complete, the resulting
peptide is extracted from the reaction mixture. This embodiment of the present
invention is particularly suitable if the erminal PG of the peptide is fluorenyl-9-
methoxycarbonyl (Fmoc) group.
In one particular embodiment, the peptide after PG cleavage is extracted with toluene
or with a mixture of toluene and the organic solvent 1. This is typically the case with
Fmoc protected peptides that are difficult to keep in solution without NMP or DMF. After
Fmoc cleavage these can be extracted in an organic layer containing toluene and,
optionally, the organic solvent 1.
With Boc protected peptides, it is the opposite, NMP and DMF have to be removed
before the Boc cleavage, but these peptides are usually soluble in the presence of TFA
> 5 vol-% in toluene, ethylacetate or, eventually, heptanes.
In yet another embodiment of the present invention, the organic layer containing the
peptide is evaporated until dryness as described above, the remaining residue is
dissolved in a solvent distinct from toluene and the organic solvent 1 and the reagent
cleaving one or several PGs of the peptide is added thereto afterwards.
Protecting groups
Protecting groups (PGs), be it for protecting functional groups in side chains of amino
acids or peptides or for the protection of AMerminal amino groups or C-terminal
carboxylic acid groups of amino acids or peptides, are for the purpose of the present
invention classified into four different groups:
1. PGs cleavable under basic cleaving conditions, in the following called "basic type
PGs",
2 . PGs cleavable under strongly acidic cleaving conditions but not cleavable under
mildly acidic cleaving conditions, in the following called "strong type PGs",
3 . PGs cleavable under mildly acidic cleaving conditions, in the following called "weak
type PGs",
4. PGs cleavable under reductive cleaving conditions, in the following called "reductive
type PGs", and
5 . PGs cleavable under saponification cleaving conditions, in the following called
"saponification type PGs".
PGs and typical reaction conditions, parameters and reagents for cleaving PGs, which
are conventionally used in the process for preparation of a peptide in liquid phase of
the present invention, are known in the art, e.g. T.W. Greene, P. G. M. uts "Greene's
Protective Groups in Organic Synthesis" John Wiley & Sons, Inc., 2006; or P. Lloyd-
Williams, F. Albericio, E. Giralt, "Chemical Approaches to the Synthesis of Peptides
and Proteins" CRC: Boca Raton, Florida, 1997.
Basic cleaving conditions involve treatment of the peptide with a basic cleaving
solution. Preferably, the basic cleaving solution consists of a basic reagent and a
solvent. Basic reagents used in the present invention are preferably secondary amines,
more preferably the basic reagent is selected from the group consisting of diethylamine
(DEA), piperidine, 4-(aminomethyl)piperidine, tris(2-aminoethyl)amine (TAEA),
morpholine, dicyclohexylamine, 1,3-cyclohexanebis(methylamine)-piperazine, 1,8-
diazabicyclo[5.4.0]undec-7-ene and mixtures thereof. Even more preferably, the basic
reagent used in the process for preparation of a peptide in liquid phase of the present
invention is selected from the group consisting of DEA, TAEA and piperidine.
The basic cleaving solution can also comprise an additive, preferably selected from the
group consisting of 6-chloro-1-hydroxy-benzotriazole, 1-hydroxy-7-azabenzotriazole,
1-hydroxybenzotriazole and ethyl-2-cyano-2-hydroxyiminoacetate and mixtures thereof.
Preferably, the solvent of the basic cleaving solution is identical to the polar aprotic
solvent employed for the peptide coupling reaction. Thus, the solvent for the basic
cleaving solution is preferably selected from the group consisting of D F, DMA and
NMP. Alternatively, the peptide containing organic layer which is obtained by the
process for extraction of a peptide from a reaction mixture resulting from a peptide
coupling reaction can be evaporated until dryness as described above. The remaining
residue can be dissolved in one of the solvents selected from the group consisting of
DMF, DMA, pyridine, NMP, acetonitrile or a mixture thereof and subsequently treated
with a basic cleaving solution. DMF or NMP may be necessary to keep the peptide in
solution in Fmoc cleavage reaction mixture as shown in example 1.
The terms "part" and "wt.-%" in the description of basic, strongly acidic, mildly acidic
and reductive cleaving conditions are meant to be a factor of the parts by weight of the
peptide carrying the corresponding groups PG(s) which are being cleaved. For
instance, the expression "5 parts of basic cleaving solution are used" means that 5 g of
basic cleaving solution are used for the treatment of each 1 g of the peptide carrying a
basic type PG.
Preferably, from 5 to 20 parts, more preferably from 5 to 15 parts of basic cleaving
solution are used. Preferably, the amount of basic reagent ranges from 1 to 30 wt.-%,
more preferably from 10 to 25 wt.-%, even more preferably from 15 to 20 wt.-%, with
the wt.-% being based on the total weight of the basic cleaving solution.
Strongly acidic cleaving conditions, as defined in the present invention, involve
treatment of the peptide with a strongly acidic cleaving solution. The strongly acidic
cleaving solution comprises an acidolytic reagent. Acidolytic reagents are preferably
selected from the group consisting of Bronsted acids, such as TFA, hydrochloric acid
(HCI), aqueous hydrochloric acid (HCI), liquid hydrofluoric acid (HF) or
trifluoromethanesulfonic acid, Lewis acids, such as trifluoroborate diethyl ether adduct
or trimethylsilylbromid, and mixtures thereof.
The strongly acidic cleaving solution preferably comprises one or more scavengers,
selected from the group consisting of dithiothreitol, ethanedithiol, dimethylsulfide,
triisopropylsilane, triethylsilane, 1,3-dimethoxybenzene, phenol, anisole, p-cresol and
mixtures thereof. The strongly acidic cleaving solution can also comprise water, a
solvent or a mixture thereof, the solvent being stable under strong cleaving conditions.
Preferably, the solvent of the strongly acidic cleaving solution is identical to the solvent
present in the partially evaporated organic layer containing the peptide. Thus, the
solvent for the strongly acidic cleaving solution is toluene or a combination of toluene
and the organic solvent 1. Alternatively, the organic layer containing the peptide can be
evaporated until dryness as described above and the remaining residue can be
dissolved in one of the solvents selected from the group consisting of ACN, toluene,
DCM, TFA and mixtures thereof. Because toluene and the organic solvent 1 are
sufficiently volatile, the evaporation of the organic layer can be easily carried out.
Preferably, from 10 to 30 parts, more preferably from 15 to 25 parts, even more
preferably from 19 to 2 1 parts of strongly acidic cleaving solution are used. Preferably,
the amount of acidolytic reagent ranges from 30 to 350 wt.-%, more preferably from 50
to 300 wt.-%, even more preferably from 70 to 250 wt.-%, especially from 100 to
200 wt.-%, with the wt.-% being based on the total weight of the strongly acidic
cleaving solution. Preferably, from 1 to 25 wt.-% of total amount of scavenger is used,
more preferably from 5 to 15 wt.-%, with the wt.-% being based on the total weight of
the strongly acidic cleaving solution.
Mildly acidic cleaving conditions according to the present invention involve treatment of
the peptide with a weakly acidic cleaving solution. The weakly acidic cleaving solution
comprises an acidolytic reagent. The acidolytic reagent is preferably selected from the
group consisting of Bronsted acids, such as TFA, trifluoroethanol, hydrochloric acid
(HCI), acetic acid (AcOH), mixtures thereof and/or with water.
The weakly acidic cleaving solution can also comprise water, a solvent or a mixture
thereof, the solvent being stable under weak cleaving conditions. Preferably, the
solvent of the weakly acidic cleaving solution is identical to the solvent present in the
partially evaporated organic layer containing the peptide. Thus, the solvent for the
weakly acidic cleaving solution is toluene or a combination of toluene and the organic
solvent 1. Alternatively, the organic layer containing the peptide can be evaporated
until dryness as described above and the remaining residue can be dissolved in one of
the solvents selected from the group consisting of ACN, toluene, DCM, TFA, and
mixtures thereof.
Preferably, from 4 to 20 parts, more preferably from 5 to 10 parts, of weakly acidic
cleaving solution are used. Preferably, the amount of acidolytic reagent ranges from
0.01 to 5 wt.-%, more preferably from 0.1 to 5 wt.-%, even more preferably from 0.15 to
3 wt.-%, with the wt.-% being based on the total weight of the weakly acidic cleaving
solution.
Reductive cleaving conditions employed in one of the embodiments of the present
invention involve treatment of the peptide with a reductive cleaving mixture. The
reductive cleaving mixture comprises a catalyst, a reducing agent and a solvent.
The catalysts employed for the reductive cleaving conditions are selected from the
group consisting of derivatives of Pd(0), derivates of Pd(ll) and catalysts containing
metallic palladium, more preferably selected from the group consisting of Pd[PPh ]4,
PdCI2[PPh3]2, Pd(OAc) 2 and palladium on carbon (Pd/C). Pd/C is particularly preferred.
The reducing agent is preferably selected from the group consisting of Bu N+BH4 ,
NH3BH3, Me2NHBH3, £Bu-NH2BH3, Me3NBH3, HCOOH/DIPEA, sulfinic acids comprising
PhS0 2H, tolS0 Na and /-BuS0 2Na and mixtures thereof as well as molecular
hydrogen; more preferably the reducing agent is tolS0 2Na or molecular hydrogen.
Preferably, the solvent employed under reductive cleaving conditions is identical to the
solvent present in the partially evaporated organic layer containing the peptide.
Accordingly, the solvent employed under reductive cleaving conditions is preferably
toluene or a combination of toluene and the organic solvent 1. Alternatively, the organic
layer containing the peptide can be evaporated until dryness as described above and
the remaining residue can be dissolved in one of the solvents selected from the group
consisting of NMP, DMF, DMA, pyridine, ACN and mixtures thereof; more preferably
the solvent is NMP, DMF or a mixture thereof. Preferably, the peptide is soluble and
dissolved in the solvent employed under reductive cleaving conditions.
Preferably, from 4 to 20 parts, more preferably from 5 to 10 parts, of reductive cleaving
solution are used.
Saponification cleaving conditions involve treatment of the peptide with a saponification
cleaving solution. Preferably, the saponification cleaving solution consists of a
saponification reagent and a solvent. Saponification reagents used in the present
invention are preferably hydroxides of alkaline and earth alkaline metals, more
preferably the saponification reagent is selected from the group consisting of sodium
hydroxide, lithium hydroxide and potassium hydroxide. Even more preferably, the
saponification reagent used in the process for preparation of a peptide in liquid phase
of the present invention is sodium hydroxide.
Preferably, the solvent of the saponification cleaving solution comprises a mixture of
water with a solvent selected from the group consisting of THF, MeTHF, ethanol,
methanol and dioxane.
According to the present invention, the basic type PGs are not cleavable under strongly
acidic or mildly acidic cleaving conditions. Preferably, the basic type PGs are not
cleavable under strongly acidic, weak or reductive cleaving conditions.
Under the term "strong type PGs" are protecting groups understood which are not
cleavable under mildly acidic or basic cleaving conditions. Preferably, the strong type
PGs are not cleavable under mildly acidic, basic or reductive cleaving conditions.
Usually strong acidic PGs like Bzl are cleaved by hydrogenation. Typically, the global
deprotection of a peptide is carried out by hydrogenation under very mild conditions.
The weak type PGs are not cleavable under basic cleaving conditions, but they are
cleavable under strongly acidic cleaving conditions. Preferably, the weak type PGs are
not cleavable under basic or reductive cleaving conditions, but they are cleavable
under strongly acidic cleaving conditions.
According to one of the embodiments of the present invention, the basic type PG is
preferably Fmoc. Preferably, the strong type PGs are selected from the group
consisting of Boc, fBu, O Bu and Cbz. Preferably, the weak type PGs are selected from
the group consisting of Trt and 2-chlorophenyldiphenylmethyl group. Preferably, the
reductive type PGs are selected from the group consisting of Bzl, /V-methyl-9Hxanthen-
9-amino group and Cbz. Preferably, the saponification type PG is OMe.
In the process for preparation of a peptide in liquid phase of the present invention, the
A/-temninal PG of the peptide is removed in a deprotection reaction before the
subsequent peptide coupling reaction is carried out. According to the present invention,
the /V-terminal PGs are preferably Fmoc, and Boc.
In one of the embodiments of the present invention, Fmoc is highly preferred for the
LPPS as an A/-terminal PG because it can be easily removed under basic conditions.
Furthermore, the Fmoc as a PG of the erminus of the peptide is compatible with the
side chain PGs in order to represent an orthogonal system. The term "orthogonal
system" is defined in G. Baranay and R. B. Merrifield (JACS, 1977, 99, 22, pp. 7363-
7365).
In yet another embodiment of the present invention, Boc is highly preferred as an Nterminal
PG of the peptide for process for the preparation of a peptide in liquid phase.
Its removal can be carried out under strongly acidic conditions. Usage of Boc PG of the
/V-terminus is also compatible with the side chain PGs in order to represent an
orthogonal system.
According to the present invention, the C-terminal PG of the peptide is removed in the
final deprotection step.
Preferred C-terminal PGs are OiBu, Biz, OMe, NH2, as well as 2-chlorophenyldiphenylmethylester
or A/-rmethyl-9H-xanthen-9-amide.
In one of the embodiments of the present invention, Bzl is highly preferred for the
process for preparation of a peptide in liquid phase as a C-terminal PG because it can
be easily removed under reductive cleaving conditions described above. Furthermore,
the Bzl PGs of the C-terminus is compatible with the side chain PGs in order to
represent an orthogonal system.
In another embodiment of the present invention, OiBu as a C-terminal PG is used for
the process for preparation of a peptide in liquid phase. Its removal can be carried out
under strongly acidic cleaving conditions as described above. Usage of OiBu PG of the
C-terminus is also compatible with the side chain PGs in order to represent an
orthogonal system.
In another embodiment of the present invention, OMe as a C-terminal PG is used for
the process for preparation of a peptide in liquid phase. OMe can be easily cleaved by
saponification and is particularly useful if the /V-terminal PG of the peptide is Boc.
In yet another embodiment of the present invention, the solubility of the peptide in the
organic layer can be additionally increased by using a hydrophobic PG for the Cterminus
of the peptide. For this purpose, the C-terminal carboxylic acid group of the
peptide can be protected with a weak type PGs, which are cleavable in mildly acidic
conditions, such as a 2-chlorophenyldiphenylmethylester or A/-methyl-9H-xanthen-9-
amide. These PGs are particularly useful for the synthesis of peptide fragments, which,
in turn can be employed in a convergent peptide synthesis. These C-terminal
carboxylic acid protecting groups have another important advantage: they are cleaved
under mildly acidic conditions, allowing for the liquid phase synthesis of protected
peptides, as an alternative to SPPS, that are used as peptide fragments in a
convergent synthesis strategy. Actually, 2-chlorophenyldiphenylmethylester and Nmethyl-
9/-/-xanthen-9-amide are chemical functions that are used as linkers on SPPS
resins for the synthesis of protected peptide fragments.
According to the present invention, it is desirable that the hydroxy-, amino-, thio- and
carboxylic acid groups of the amino acids side chains of the peptide obtained by the
process for preparation of a peptide in liquid phase are protected with suitable PGs, so
that undesired side reactions are avoided. In addition, usage of the side chain PGs
generally improves the solubility of the peptide in the polar aprotic solvents as well as
in toluene or/and in the combination of toluene and the organic solvent 1.
Generally, side chain PGs are chosen in such a way that they are not removed during
the deprotection of the /V-terminal amino groups during the process for preparation of a
peptide in liquid phase. Therefore, the PG of the /V-terminal amino groups or C-terminal
carboxylic acid groups and any side chain PG are typically different, preferably they
represent an orthogonal system.
According to the present invention, the preferred side chain groups are iBu, Trt, Boc,
OfBu and Cbz.
Once the amino acid sequence of the peptide obtained by the process for preparation
of a peptide in liquid phase is identical to the amino acid sequence of the target
peptide, preferably the /V-terminal PG, the C-terminal PG and any side chain PG are
removed so that the unprotected target peptide is obtained. This step is called global
deprotection. Preferably, the PGs used during the process for preparation of a peptide
in liquid phase are selected to allow global deprotection under mildly acidic, strongly
acidic or reductive cleaving conditions, as defined above, depending on the nature of
PGs.
Any side chain PGs are typically retained until the end of the LPPS. Global
deprotection can be carried out under conditions applicable to the various side chain
PGs, which have been used. In case that different types of side chain PGs are chosen,
they may be cleaved successively; e.g. this is the case for the synthesis of a branched
peptide. Advantageously, the side chain PGs are chosen in such a way so that they are
cleavable simultaneously and more advantageously concomitantly with /V-terminal PG
or with C-terminal PG of the peptide prepared by LPPS.
In one of the embodiments of the present invention, it is possible that the /V-terminal
PG of the peptide in the partially evaporated organic layer is directly removed. Thus, in
this case, the precipitation of the peptide upon usage of the organic solvent 2 is not
required and LPPS of the present invention can be carried out without an isolation of
the intermediate peptides, e.g. as a continuous LPPS.
Depending on the nature of the /V-terminal PG of the peptide, appropriate cleaving
conditions can be chosen for this step.
If the /V-terminal PG of the peptide is a strong type PG or a weak type PG, as defined
above, the organic layer containing the peptide is preferably treated with TFA or HCI.
Because the organic layer containing the peptide is substantially free from the polar
aprotic solvents, the removal of the /V-terminal PG of the peptide is not inhibited by an
undesired reaction between TFA or HCI and the polar aprotic solvent. In one of the
embodiments of the present invention, the /V-terminal PG of the peptide is Boc group.
If the /V-terminal PG of the peptide is a basic type PG, as defined above, the peptide
can be deprotected upon usage of an organic base, as known in the prior art.
Preferably, for this purpose the reaction mixture resulting from a peptide coupling
reaction is directly treated with a basic reagent selected from the group consisting of
DEA, TAEA and piperidine and the peptide with an unprotected /V-terminus is extracted
from this reaction mixture. Alternatively, the organic layer containing the peptide is
treated with the basic reagent. Alternatively, the organic layer containing the peptide
can be evaporated until dryness as described above and the remaining residue can be
dissolved in one of the solvents selected from the group consisting of DMF, DMA,
pyridine, NMP or a mixture thereof and subsequently treated with the basic reagent.
In one of the preferred embodiments of the present invention, the /V-terminal PG of the
peptide is fluorenyl-9-methoxycarbonyl (Fmoc) group. Cleavage of the Fmoc group of
the peptide is accompanied by formation of dibenzofulvene. If DEA or piperidine is
used as a basic reagent and the solvent of the basic cleaving solution is acetonitrile,
the resulting solution containing the peptide with an unprotected A/-terminus is
subsequently washed with a hydrocarbon such as e.g. n-heptane so that
dibenzofulvene is substantially removed. If TAEA is used as a basic reagent for the
cleavage of the Fmoc group, the resulting solution is subsequently subjected to the
extraction process of the present invention. Thus, the solution containing the peptide
with an unprotected erminus is substantially free of dibenzofulvene before a
subsequent peptide coupling reaction is carried out.
After the cleavage of the /V-terminal PG of the peptide, the solution containing the
peptide with an unprotected /v-terminus can be at least partially evaporated and
employed for the subsequent peptide coupling reaction or, alternatively, to the global
deprotection step.
Thus, the present invention provides continuous LPPS methodology, which has a
number of advantages over commonly used SPPS methodology.
Concentrations of reagents present in the reaction mixture during the peptide coupling
reactions and deprotection reactions in the case of the continuous LPPS of the present
invention are higher than in the case of SPPS. As a consequence, the corresponding
reaction times are shorter and batch reactors with a lower capacity can be used for the
synthesis of a given amount of target peptide. The total time required for the synthesis
of a peptide carried out by the continuous LPPS of the present invention is nearly the
same as the total time required for its synthesis if SPPS is used. Thus, use of the
continuous LPPS of the present invention leads to reduced operating costs.
A peptide coupling reaction in the LPPS of the present invention requires a lower
excess of an amino acid or a peptide having an unprotected C-terminal carboxylic acid
group ( 1 .1-1 .2 equivalents) than the corresponding peptide coupling reaction in SPPS
( 1 .5 equivalents or more). Moreover, SPPS further requires a high amount of solvents
for rinsing the resin after each peptide coupling step. Thus, the amount of solvents
required in the case of SPPS is significantly higher than in the case of the continuous
LPPS of the present invention. Hence, use of continuous LPPS of the present invention
leads to a significant reduction of material costs in comparison to use of SPPS.
In addition thereto, the scaling up of the continuous LPPS process of the present
invention is known to be easier than the scaling up of the corresponding SPPS
process, and the target peptide prepared by the continuous LPPS of the present
invention has a higher purity than the corresponding peptide prepared by SPPS.
In summary, the continuous LPPS of the present invention provides a number of
advantages over other methodologies for peptide synthesis, known in the prior art, and
is particularly useful for the preparation of peptides on an industrial scale.
Description of the drawings
Figure 1 illustrates the influence of residual DMF on the rate of removal of the Boc
protecting group of peptide Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-
OBzl.
Figure 2 shows an image of peptide Boc-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-
Glu(OBzl)-Tyr(Bzl)-Leu(OBzl) precipitated in the absence of DMF.
EXAMPLES
The following non-limiting examples will illustrate representative embodiments of the
invention in detail.
All experiments were carried out at room temperature of 20±3°C and atmospheric
pressure of 10 3±50 kPa if not specified otherwise.
Methods Description
A) HPLC analysis
Detection in HPLC method A was done with a UV photodiode array detector.
Step 1 Sample preparation:
Mobile Phase A: 0.1 Vol.-% TFA in water
Mobile Phase B: 0.085 Vol.-% TFA in ACN
Step 2 Chromatography conditions:
Method MIH-009-2TG1 1
Column: Purospher Star RP1 8 55 x 4 mm
Oven temperature: 40°C
Flow rate: 2.0 mL/ min
Detector wavelength: 2 15 nm
Gradient run time: 15 min
Gradient composition: 2 to 78 % B in 5 min, 78 to 98 % B in 10 min
Step 3 Chromatographic profile analysis:
The composition of the isolated products was determined by the measurement of the
areas of all chromatography peaks. The determined purity of the expected products
corresponds to the area-% of the corresponding product peaks.
1. Apparatus and equipment
Gas chromatograph : GC equipped with a flame ionization detector and an
automatic injector system coupled with acquisition
software
Analytical GC column Fused silica column, length 50 m; 0.53 mm internal
diameter; stationary phase : CP SIL 8CB DF = 5.0 mhh
Reagents Methanol (analytical grade)
2. Sample preparation
Test and reference solution
In a 10 mL volumetric flask, add accurately 400 i of sample and make up to volume
with methanol.
3. Chromatographic conditions
Carrier Gas: Helium 30 kPa
Oven temperature: 35°C, 14 minutes 5°C/min 55°C, 3 minutes 5°C/min
110°C, 5 minutes 10°C/min 225°C, 5 minutes
Injector temperature: 225°C
Detector temperature: 260°C
Injected volume: 1 L
Injection mode: Split
Split flow: 85 mL/min
Ratio: 24
Filterability measurements
The mixtures containing precipitated peptides were transferred into a 2.7 cm diameter
filtration column equipped with a 20 m pore size filter. Filtrations were carried out at
20°C under a pressure of 50 mbar. The flow rate and the cake heights were measured
and the filterability coefficient K was calculated as:
K = volume of mother liquor (mL) x cake heights (cm) / filter surface (cm2) / pressure
(bar) / filtration time (min).
Example 1 Extraction of N P in systems eTHF THF/NaCI solution,
EtOAc/THF/NaCI solution and toluene/THF/NaCI solution
Extraction properties of the solvent combination toluene/THF (according to the present
invention) were compared to those of the combination MeTHF/THF and EtOAc/THF
(comparative). The experiments were carried out with an aqueous solution containing
50 g/L NaCI. No peptides were present in the systems of the present example.
The volume ratios were as follows:
NMP : EtOAc : THF : NaCI solution = 1 : 3 : 3 : 3
NMP : MeTHF : THF : NaCI solution = 1 : 3 : 3 : 3
NMP : toluene : THF : NaCI solution = 1 : 3 : 3 : 3
Fraction of NMP in the aqueous layer was determined by GC. The results of the
experiments are summarized in Table 1 below.
Table 1. Extraction of NMP in the biphasic system NMP/solvent 1/solvent 2/NaCI
solution (150 g/L NaCI).
As can be noticed from Table 1 above, the extraction with the combination toluene/THF
led to a higher fraction of NMP in the aqueous layer than the extractions using
EtOAc/THF and MeTHF/THF. Accordingly, the NMP content in the organic layer after
the extraction with toluene/THF was lower than after an extraction with MeTHF/THF or
EtOAc/THF.
Example 2 Synthesis of H-Tyr(Bzl)-Leu-OBzl
Example 2.1 LPPS of Boc-Tyr(Bzl)-Leu-OBzl
Boc-Tyr(Bzl)-OH (4.7 g , 12.7 mmol) and H-Leu-OBzl Tos (5.0 g , 12.7 mmol) were
dissolved in DMF (25 mL) at 20°C. The reaction mixture was cooled to -8°C then
HOBt H20 (2.0 g , 13.1 mmol, 1.0 eq) and EDC HCI (2.8 g , 14.6 mmol) were added.
The reaction temperature was kept in the range of -5°C to -10°C until completion of the
reaction as determined by HPLC. The reaction progress was monitored by the
following method: 5 L sample of the reaction mixture, diluted 50 fold in acetic
acid : water (9 : 1), were analysed according to method MIH-009-2TG1 1 described
above.
Example 2.2 Boc cleavage: H-Tyr(Bzl)-Leu-OBzl
To the mixture prepared according to example 2.1 , toluene (90 mL) was added and the
reaction mixture was successively extracted with:
1) aqueous solution containing 20 g/L NaCI (90 mL)
2) aqueous solution containing 150 g/L NaCI and 50 g/L NaHC0 3 (90 mL)
3) aqueous solution containing 20 g/L NaCI and 50 g/L NaHC0 3 (90 mL)
4) aqueous solution containing 20 g/L NaCI and 50 g/L NaHC0 3 (90 mL)
5) aqueous solution containing 150 g/L NaCI (90 mL).
The combined organic layers were then concentrated under reduced pressure at 35°C,
so that the volume of the combined organic layer was reduced to 20 mL.
The removal of the Boc protecting group was performed by addition of phenol (0.25 g ,
2.6 mmol) and TFA (20 mL) at 5°C. After completion of the reaction, as determined by
HPLC, the reaction mixture was evaporated under reduced pressure at 35°C. Residual
TFA was removed by co-evaporations with toluene (3 x 25 mL). The reaction progress
was monitored by the following method: 5 pL sample of the reaction mixture was
diluted 30 fold in methanol and analysed according to method MIH-009-2TG1 1
described above.
Example 3 (comparative) Influence of residual DMF on the removal of the Boc
protecting group. H-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl
Boc-Pro-lle-Leu-Pro-Pro-OH (3.5 g), H-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl (5.0 g)
and HOBt (0.88 g) were dissolved in DMF (20 mL). The coupling reaction was
performed overnight under stirring at -6°C to 0°C with EDC HCI ( 1 .2 g) and TEA
( 1 .5 mL). Completion of the reaction was verified by HPLC (method MIH-009-2TG1 ) .
The reaction mixture was filtered to remove insoluble salts. Samples of 1 mL of
reaction mixture were mixed with organic solvents as shown in Table 2 below and were
then extracted with 3 mL of aqueous solution of NaCI (15% w/v) and Na2C0 3 (2.5%
w/v). The DMF content in the organic layer was determined by GC.
Table 2 . Extraction of Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bz )-Leu-OBzl
The extraction with EtOAc (tests # 2) led to a lower DMF content in the organic layer
than the extraction with DCM (tests # 1)
The obtained products from tests # 1 and 2 were further processed. The organic layers
were separated and the solvents were exchanged by three co-evaporations with
toluene (bath temperature = 40°C, pressure = 50 mbar). After the volatile solvents were
completely evaporated, toluene (4 mL) and phenol (0.05 g) were added to the residues
of evaporation. Boc cleavages were performed at 0°C by addition of 3.5 mL TFA. The
reactions were monitored by HPLC (method MIH-009-2TG1 1).
The obtained results are summarised in Table 3 and graphically presented in Figure 1
Table 3 . Deprotection of Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-
OBzl
Results
Traces of DMF in the materials significantly inhibited the removal of Boc protective
group. Thus, Boc cleavage of the material obtained by extraction with DCM was
significantly slower than in the case of material obtained by extraction with EtOAc.
The process for extraction of the present invention allows an efficient separation of
polar aprotic solvents such as DMF from the isolated peptide. Accordingly, acidolytic
cleavage of the peptide material isolated by the process for extraction according to the
present invention can be expected to proceed smoothly.
Example 4 (comparative) Coupling of Boc-Ser(OBzl)-OH with H-Phe-Pro-lle-Leu-
Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl)
Boc-Ser(OBzl)-OH ( 1.62 g , 5.5 mmol) was dissolved in DMF (25 mL) at 20°C and
added to crude H-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl).
HOBt H20 (0.89 g , 5.8 mmol) and EDC HCI ( 1 .2 g, 6.3 mmol) were added thereto, and
the reaction mixture was cooled to 5°C. The reaction mixture was kept at this
temperature until a complete conversion was confirmed by HPLC. The reaction
progress was monitored by the following method: 5 m I_ sample of the reaction mixture
was diluted 50 fold in acetic acid : water (9 : 1) and analysed according to method MIH-
009-2TG1 described above.
a) Extraction with MeTHF and precipitation in DIPE
25 mL of the reaction mixture containing 5 g Boc-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-
Glu(OBzl)-Glu(OBzi)-Tyr(Bzl)-Leu(OBzl) were combined with MeTHF (75 mL) and an
aqueous solution containing 100 g/L NaCI (75 mL). After a thorough mixing and phase
separation (approx. 4 min) the lower aqueous layer was removed. The upper organic
layer was further extracted three times with an aqueous solution containing 100 g/L
NaCI (3 x 75 mL). The organic layer was finally isolated and partially evaporated at
30°C, 60 mbar to a residual volume of 10 mL. The partially evaporated organic layer
was added dropwise under stirring into DIPE (250 mL) at 0°C whereby the precipitation
of the peptide took place. The resulting mixture was transferred into a 2.7 cm diameter
filtration column equipped with a 20 m h pore size filter. The filtration was carried out
under a pressure of 50 mbar. The total mother liquor of precipitation (260 mL) was
filtered in 3 minutes and 45 seconds. The cake heights after filtration was 3.5 cm giving
a filterability coefficient K = 848. The solids were collected and dried under reduced
pressure. 4.5 g of the peptide was isolated as a solid material.
An image of the isolated peptide is shown as Figure 2 (40x enlargement).
The aqueous layer resulting from the extraction process and the mother liquors of
precipitation were analysed by HPLC. The amount of the peptide detected therein was
below 0.5 wt.-% of the total amount of the peptide present in 25 mL of the reaction
mixture resulting from example 4.
b) Comparative example: Influence of DMF addition to the mother liquors of
precipitation
The procedure of extraction and precipitation was performed as described under a)
above but DMF (2.5 mL) was added to the precipitation mixture before the filtration of
the peptide was carried out. The solid precipitate immediately turned into a gum-like
solid that was not filterable.
c) Comparative example: Direct precipitation in DIPE
25 mL of the reaction mixture obtained in example 4 , containing 5 g Boc-Ser(Bzl)-Phe-
Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu(OBzl) were added dropwise into
DIPE (250 mL) under stirring at 0°C for precipitation. The peptide precipitated in the
form of a sticky gum-like solid. After decantation the supernatant was pumped off and
replaced with a second batch of DIPE (250 mL). The resulting mixture was stirred for
one hour in order to de-aggregate the sticky gum-like solid. After decantation the
supernatant was replaced again with a third batch of DIPE (250 mL). The mixture was
stirred again for one hour and it was finally transferred into the filtration column.
However, a large part of the solid was still in the form of a sticky gum-like solid that was
left stuck onto the precipitation vessel and therefore could not be transferred. The
mother liquors were filtered in 2 min 30 sec, yielding a 1.75 cm high cake. This gave a
filtration coefficient K = 636. The collected solids were dried under reduced pressure.
2.45 g of the peptide were isolated.
d) Comparative example: Direct precipitation in water.
25 mL of the reaction mixture resulting from example 4 and containing 5 g Boc-
Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu(OBzl) were added
dropwise into water (250 mL) under stirring at 0°C for precipitation. This yielded a very
thin precipitate that was subsequently transferred into the filtration column. The
filtration rate was very low (< 3 mL/h), a considerable amount of precipitate went
through the filter in the beginning of the filtration and the filter was definitely clogged
after about 65 min. Moreover, there was no clear decantation of the precipitate. Thus, it
was not possible to collect the obtained precipitate.
Results
Precipitation of the peptide Boc-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-
Glu(OBzl)-Tyr(Bzl)-Leu(OBzl) in the presence of DMF (Examples b)-d)) led to gum-like
solids, which were difficult to handle. In Example a) the precipitation of the peptide Boc-
Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu(OBzl) was carried
out after the traces of DMF had been substantially removed by an extraction. The
resulting product could be isolated in a good yield and was easily filterable.
The process for extraction of the present invention allows an efficient separation of
polar aprotic solvents such as DMF from the peptide. Accordingly, the undesired
interference of polar aprotic solvents with the process of peptide precipitation can be
excluded.
Claims
1. A process for extraction of a peptide from a reaction mixture resulting from a
peptide coupling reaction, the reaction mixture containing the peptide and a
polar aprotic solvent selected from the group consisting of N,Ndimethylformamide,
/V./V-dimethylacetamide and /V-methyl-2-pyrrolidone,
whereby the process comprises a step a) and a step b),
step a) comprises the addition of a component a1) and a component a2),
whereby
component a1) is toluene,
component a2) is water,
to the reaction mixture, so that a biphasic system with an organic layer and an
aqueous layer is obtained;
step b) comprises the separation of the organic layer containing the peptide
from the aqueous layer,
whereby
the biphasic system obtained in step a) is characterised by the following volume
ratios:
polar aprotic solvent : toluene from 1 : 20 to 1 : 2 ; and
polar aprotic solvent : water from 1 : 20 to 1 : 2 .
2 . The process of claim 1, wherein in step a) a further component a3) is added to
the reaction mixture,
component a3) is an organic solvent 1, the organic solvent 1 is selected from
the group consisting of n-heptane, 2-methyltetrahydrofuran, ethylacetate,
isopropylacetate, acetonitrile and tetrahydrofuran,
so that a biphasic system with an organic layer and an aqueous layer is
obtained;
whereby
the biphasic system obtained in step a) is characterised by the following volume
ratios:
polar aprotic solvent : toluene from 1 : 20 to 1 : 2 ;
polar aprotic solvent : organic solvent 1 from 1 : 5 to 30 : 1; and
polar aprotic solvent : water from 1 : 20 to 1 : 2 .
The process of claim 2 , whereby the biphasic system obtained in step a) is
characterised by the following volume ratios:
polar aprotic solvent : toluene from 1 : 6 to 1 : 3 ;
polar aprotic solvent : organic solvent 1 from 1 : 1 to 4 : 1; and
polar aprotic solvent ; water from 1 : 5 to 1 : 3 .
The process of one or more of claims 1 to 3 , whereby the polar aprotic solvent
is selected from the group consisting of L, -dimethylformamide and /V-methyl-
2-pyrrolidone.
The process of one or more of claims 2 to 4 , whereby the organic solvent 1 is
selected from the group consisting of acetonitrile and tetrahydrofuran.
The process of one or more of claims 1 to 5 , whereby the component a2)
contains at least one inorganic salt selected from the group consisting of
sodium chloride, sodium hydrogensulfate, potassium hydrogensulfate, sodium
hydrogencarbonate and sodium hydrogenphosphate.
The process of one or more of claims 1 to 6 , whereby the pH value of the
component a2) ranges from 5 to 8 .
The process of one or more of claims 1 to 7 , whereby a filtration of the biphasic
system obtained in step a) is carried out before step b).
The process of one or more of claims 1 to 8 , whereby step a) and step b) are
carried out at a temperature of from 20°C to 30°C.
10. A process for preparation of a peptide in liquid phase comprising a step aa), a
step bb) and a step cc):
in step aa) a peptide coupling reaction is carried out in the polar aprotic solvent
selected from the group consisting of A/,/V-dimethylformamide, N,Ndimethylacetamide
and A/-methyl-2-pyrrolidone, and in the presence of a
coupling reagent;
in step bb) the resulting peptide is extracted according to a process according
to one or more of claims 1 to 10; and
in step cc) at least a part of the organic layer obtained in step bb) is evaporated.
11. The process of claim 10, whereby the coupling reagent is selected from the
group consisting of uronium salts, phosphonium salts of 0-1H-benzotriazole
and carbodiimide coupling reagents.
12. The process of claim 10 or 11, whereby a tertiary base is selected from the
group consisting of A/,A/-diisopropylethylamine, triethylamine and Nmethylmorpholine,
and said tertiary base is present in the peptide coupling
reaction of step aa).
13. The process of one or more of claims 0 to 12 comprising further a further step
dd), a step ee) and a step ff), wherein
in step dd) the organic layer obtained in step cc) is combined with an organic
solvent 2 selected from the group consisting of acetonitrile, diethyl ether,
diisopropyl ether and n-heptane;
in step ee) at least a substantial part of the peptide is precipitated; and
in step ff) the precipitated peptide is separated by filtration.
14. The process of one or more of claims 10 to 12, whereby the organic layer
obtained in step cc) is treated with trifluoroacetic acid in the case that a Nterminal
protecting group of the peptide is a ferf-butyloxycarbonyl protecting
group, said terf-butyloxycarbonyl protecting group is removed by said treatment
with trifluoroacetic acid.
15. The process of one or more of claims 10 to 12, whereby the reaction mixture
resulting from the peptide coupling reaction and obtained in step aa) is treated
with piperidine in the case that a /V-terminal protecting group of the peptide is a
fluorenyl-9-methoxycarbonyl protecting group, said fluorenyl-9-methoxycarbonyl
protecting group is removed by said treatment with piperidine.
16. The process of one or more of claims 10 to 15, whereby the C-terminal
carboxylic acid group of the peptide is protected as a 2-
chlorophenyldiphenylmethylester or A/-methyl-9H-xanthen-9-amide.