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 ie/t-butoxycarbonyl
Bsmoc 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl
Bzl benzyl
Cbz benzyloxycarbonyl
DCC /V,/V-dicyclohexylcarbodiimide
DCE dichloroethane
DCM dichloromethane
DCU L/,/V-dicyclohexylurea
DEA diethylamine
DIPE diisopropyl ether
DIPEA A/./V-diisopropylethylamine
DMA A/./V-dimethylacetamide
DMF L/,/V-dimethylformamide
DOE design of experiments
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 A/-methyl-2-pyrrolidone
OMe methoxy
O Bu fert-butoxy
PG protecting group
PyBOP benzotriazol-1-yloxy-tris(pyrrolidino)-phosphonium hexafluorophosphate
RM reaction mixture
SPPS solid phase peptide synthesis
TAEA tris(2-aminoethyl)amine
TBTU 0-(benzotriazol-1 -yl)-1 ,1,3,3-tetramethyluronium tetrafluoroborate
fBu te -buty
TEA triethylamine
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin layer chromatography
TOTU 0-[cyano(ethoxycarbonyl)methylenamino]-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 AMerminal 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 AMerminal 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), A/./V-dimethylacetamide (DMA) or A/-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 ferf-butoxycarbonyl (Boc), trityl (Trt), tertbutyl
(fBu) and e -butoxy (OiBu) 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 £BuOH/CHCI3.
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 e a/. (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 et al. (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 et al. 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 2-methyltetrahydrofuran,
preferably in combination with an organic solvent selected from the group consisting of
n-heptane, toluene, ethylacetate, isopropylacetate, acetonitrile or tetrahydrofuran (this
group is designated as organic solvent 1). In particular, the solubility of the peptides in
the combination of 2-methyltetrahydrofuran and the organic solvent 1 is generally
higher than in neat 2-methyltetrahydrofuran. Moreover, they found that commonly used
polar aprotic solvents largely partition into the aqueous layer in a biphasic system
comprising water and 2-methyltetrahydrofuran or a combination of 2-
methyltetrahydrofuran and the organic solvent 1.
Therefore, water and neat 2-methyltetrahydrofuran or a combination of 2-
methyltetrahydrofuran 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,
A/,/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 2-methyltetrahydrofuran,
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 : 2-methyltetrahydrofuran 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 A/,A/-dimethylformamide, W,A/-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 2-methyltetrahydrofuran,
component a2) is water,
component a3) is an organic solvent 1, the organic solvent 1 is selected from the group
consisting of n-heptane, toluene, 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 : 2-methyltetrahydrofuran 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
2-methyltetrahydrofuran : organic solvent 1 from 50 : 1 to 1 : 1.
In a preferred embodiment, the biphasic system obtained in step a) is characterised by
the following volume ratios:
polar aprotic solvent : 2-methyltetrahydrofuran 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
2-methyltetrahydrofuran : 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 2-methyltetrahydrofuran,
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 2-methyltetrahydrofuran,
component a2) is water,
component a3) is an organic solvent 1, the organic solvent 1 is selected from the group
consisting of n-heptane, toluene, 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 2-methyltetrahydrofuran, 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 2-methyltetrahydrofuran 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 2-
methyltetrahydrofuran and the organic solvent 1, whereby the addition of 2-
methyltetrahydrofuran 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
0 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 2-methyltetrahydrofuran 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 : 2-methyltetrahydrofuran
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 2-methyltetrahydrofuran and the
organic solvent 1 was shown to be higher than in the neat 2-methyltetrahydrofuran.
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 2-methyltetrahydrofuran : 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 0 : 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 2-methyltetrahydrofuran and a combination of 2-methyltetrahydrofuran
and the organic solvent 1 are particularly suitable for the process for extraction of a
peptide. 2-Methyltetrahydrofuran is an easily recyclable, environmentally friendly
solvent, which can be derived from a variety of agricultural by-products. Accordingly,
the present invention provides an environmentally friendly process for extraction of a
peptide.
The solubility of the peptide in a combination of 2-methyltetrahydrofuran and the
organic solvent 1 is particularly high if the organic solvent 1 is selected from the group
consisting of 7-heptane, toluene, 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 2-methyltetrahydrofuran 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 1. 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 11.
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 /V-terminal 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, 2-methyltetrahydrofuran, 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 A/,A/-dimethylformamide, A/./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-(1H-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-(1H-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 , ,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)]-
methanaminiurri 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 N,N- -C .4 alkylanilines,
such as L/,/V-diethylaniline, 2,4,6-tri-C - alkylpyridines, such as collidine (2,4,6-
trimethylpyridine), or - .4 alkylmorpholines, such as /V-methylmorpholine, with any
C - alkyl being identical or different and independently from each other straight or
branched C -4 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 -aryl substituted triazeno, the triazeno type coupling
additive being preferably a A/-hydroxybenzotriazol derivative (or 1-hydroxybenzotriazol
derivative) or a A/-hydroxybenzotriazine derivative. Such coupling additives have been
described in WO 94/07910 and EP 0 410 182.
Preferred coupling additives are selected from the group consisting of Nhydroxysuccinimide
(HOSu), 6-chloro-1-hydroxybenzotriazole (CI-HOBt), A/-hydroxy-
3,4-dihydro-4-oxo-1 ,2,3-benzotriazine (HOOBt), 1-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/DI PEA, 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,
carboxyiic 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 .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 2-methyltetrahydrofuran or of the mixture of
2-methyltetrahydrofuran 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). 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 2-methyltetrahydrofuran 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 2-methyltetrahydrofuran and the organic solvent 1.
However, if the organic layer containing the peptide comprises more than 60 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 2-methyltetrahydrofuran 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 2-methyltetrahydrofuran 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 2-methyltetrahydrofuran and/or in the
mixture of 2-methyltetrahydrofuran and the organic solvent 1. The organic solvent 2 is
preferably selected from the group consisting of acetonitrile, diethyl ether, diisopropyl
ether, n-heptane and toluene, more preferred from the group consisting of acetonitrile,
diethyl ether, diisopropyl ether and toluene, particularly preferred from the group
consisting of diisopropyl ether, A?-heptane and toluene.
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 -10°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 AMerminal 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 /V-terminal PG of the peptide is fluorenyl-9-
methoxycarbonyl (Fmoc) group.
In one particular embodiment, the peptide after PG cleavage is extracted with MeTHF
or with a mixture of MeTHF 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 MeTHF and,
optionally, the organic solvent .
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 2-methyltetrahydrofuran 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 /V-terminal 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. Wuts "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 DMF, 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 2-methyltetrahydrofuran or a
combination of 2-methyltetrahydrofuran 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 2-
methyltetrahydrofuran 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 2-methyltetrahydrofuran or a combination of 2-
methyltetrahydrofuran 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[PPh3] ,
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+BH ,
NH3BH3, Me2NHBH3, fBu-NH BH3, 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 2-
methyltetrahydrofuran or a combination of 2-methyltetrahydrofuran 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, OfBu 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, A/-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
/V-terminal 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 /V-terminal PG because it can be easily removed under basic conditions.
Furthermore, the Fmoc as a PG of the /V-terminus 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
erminus 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 OfBu, Biz, OMe, NH2, as well as 2-chlorophenyldiphenylmethylester
or /V-methyl-9/-/-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, OfBu 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 OfBu 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-
9H-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 2-methyltetrahydrofuran or/and in the combination of 2-methyltetrahydrofuran 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 /Bu, Trt, Boc,
OrBu 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 AMerminal 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 erminal 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 erminus 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 /V-terminus 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 shows a contour plot illustrating the NMP content (g/L) in the organic layer of
the ternary mixture NMP/MeTHF/water (black circles represent the compositions of the
experimental mixtures, those prepared in duplicates are labelled with "2 x").
Figure 2 shows a contour plot illustrating the volume of the organic layer (mL) of the
ternary mixture NMP/MeTHF/water (black circles represent the compositions of the
experimental mixtures, those prepared in duplicates are labelled with "2 x").
Figure 3 shows a contour plot illustrating the NMP content (g/L) in the organic layer of
the ternary mixture NMP/MeTHF/NaCI solution (black circles represent the
compositions of the experimental mixtures, those prepared in duplicates are labelled
with "2 x").
Figure 4 shows a contour plot illustrating the volume of the organic layer (mL) of the
ternary mixture NMP/MeTHF/NaCI solution (black circles represent the compositions of
the experimental mixtures, those prepared in duplicates are labelled with "2 x").
Figure 5 shows a calculated contour plot of the extraction yield of the pentapeptide HLeu-
Trp(Boc)-Val-Asn(Trt)-Ser(rBu)-NH 2 described in example 6 in water as a function
of the relative composition of the system MeTHF/NMP/water (black circles represent
the compositions of the experimental mixtures, those prepared in triplicate are labelled
with "3 x").
Figure 6 shows a diagram representing the dependency of concentration of NMP in
organic layer as a function of the composition of the system NMP/MeTHF/THF/water.
Figure 7 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.
Test # 1: Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl was isolated
using extraction with DCM.
Test # 3 : Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl was isolated
using extraction with EtOAc.
Test # 5 : Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl was isolated
using extraction with MeTHF.
Figure 8 shows an image of the peptide Boc-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-
Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu(OBzl), which was isolated according to the process
of the present invention.
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 1013±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-009-3TG9
Column: Phenomenex Luna C8(2) 5 m h 250 x 4.6 mm
Oven temperature: 40°C
Flow rate: 1.50 mL/min
Detector wavelength: 215 nm
Gradient run time: 30 min
Gradient composition: 22 to 52 % B in 15 min, 52 to 82 % B in 5 min, 82 to 98 %
B in 5 min, 98 % B in 5 min
Method MIH-009-2TG1
Column: Purospher Star RP18 55 x 4 mm
Oven temperature: 40°C
Flow rate: 2.0 mL/ min
Detector wavelength: 215 nm
Gradient run time: 15 min
Gradient composition: 2 to 78 % B in 5 min, 78 to 98 % B in 10 min
Method MIH-009-RTTG1
Column: Purospher Star RP18 55 x 4 mm
Oven temperature: 40°C
Flow rate: 2.0 mL/min
Detector wavelength: 215 nm
Gradient run time: 15 min
Gradient composition: 2 to 98 % B in 5 min, 98 % B in 5 min
Method MIH-009-025TG3
Column: XBridgeC18 5m 150 x 4.6 mm
Oven temperature: 40°C
Flow rate: 1.5 mL/min
Detector wavelength: 215 nm
Gradient run time: 20 min
Gradient composition: 2 to 98 % B in 5 min, 98 % B in 5 min
Method MIH-009-397TG3
Column: Vydac 214TP54 C4 250 x 4.6 mm
Oven temperature: 40°C
Flow rate: 1.5 mL/min
Detector wavelength: 215 nm
Gradient run time: 25 min
Gradient composition: 43 to 78 % B in 25 min
Method MIH-009-397TG15
Column: Vydac 2 4TP54 5 C4 250 x 4.6 mm
Oven temperature: 40°C
Flow rate: 1.5 mL/min
Detector wavelength: 215 nm
Gradient run time: 2 min
Gradient composition: 33 to 78 % B in 25 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 m
Reagents : Methanol (analytical grade)
2. Sample preparation
Test and reference solution
In a 10 mL volumetric flask, add accurately 400 mI_ 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 pm 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).
Design of Experiments
The Design of experiments (DOE) was performed upon using the DOE software
package Design-Expert ® 8 of Stat-Ease, Inc.
a) Extraction of NMP in the systems MeTHF/water and MeTHF/NaCI solution
The present example demonstrates that the volume of the organic layer and its NMP
content is dependent on the composition of the biphasic systems NMP/MeTHF/water
and NMP/MeTHF/NaCI solution. This dependency was verified with designed biphasic
systems in which the volume fractions of NMP and MeTHF as well as the NaCI content
in water were systematically varied in a quadratic design mode while keeping the
overall volume constant. In order to investigate the influence of the NaCI content in
water, the same set of biphasic systems was prepared with pure water (see Table 1a)
and with a 150 g/L NaCI solution (see Table 1b). After stirring these biphasic systems,
they were left for completion of the phase separation in a metering vessel and the
volume of the organic layer was measured. The NMP content of the organic layer was
measured by gas chromatography. Statistical models were developed to determine the
volume of the organic layer and its NMP content as mathematical functions of the
volume fractions of NMP, MeTHF and water in the overall biphasic system composition
(these volume fractions summing to 1).
a) In the absence of NaCI, the NMP content Ln (g/L) in the organic layer is given by the
quadratic mixture model (with R2 = 0.954):
Ln (NMP in organic layer) = 5.7 * MeTHF volume fraction - 17.1 * NMP volume
fraction - 6.9 * H20 volume fraction + 102.8 * NMP volume fraction * H20
volume fraction
This model of the ternary mixture NMP/MeTHF/water is graphically represented as a
contour plot depicted in Figure 1.
b) The volume of organic layer in the absence of NaCI is given by the linear mixture
model (with R2 = 0.992):
Vol. organic layer (mL) = 22.6 * MeTHF volume fraction - 10.4 * NMP volume
fraction - 4.7 * H20 volume fraction
This model of the ternary mixture NMP/MeTHF/water is graphically represented as a
contour plot depicted in Figure 2 .
Table 1a. Extraction of NMP in the biphasic system NMP/Me HF/water in the absence
of NaCI.
c) In the presence of aqueous solution containing 150 g/L NaCI, the NMP content Ln'
(g/L) in the organic layer is given by the linear mixture model (with R2 = 0.958):
Ln' (NMP in organic layer) = 25.1 * MeTHF volume fraction + 297.3 * NMP
volume fraction - 43.0 * NaCI solution volume fraction
This model of the ternary mixture NMP/MeTHF/NaCI solution is graphically represented
as a contour plot depicted in Figure 3 .
d) The volume of the organic layer in the presence of aqueous solution containing
150 g/L NaCI is given by the linear mixture model (with R2 = 0.991):
Vol. organic layer = 20.5 * MeTHF volume fraction - 1.05 * NMP volume fraction
- .08 * NaCI solution volume fraction
This model of the ternary mixture NMP/MeTHF/NaCI solution is graphically represented
as a contour plot depicted in Figure 4 .
Table 1b. Extraction of NMP in the biphasic system NMP/MeTHF/NaCI solution
(150 g/L NaCI).
In order to achieve an efficient removal of NMP from the organic layer containing the
peptide, it is desirable that the NMP content in the organic layer is sufficiently low,
preferably below 50 g/L and even more preferred below 20 g/L. Such conditions are
found in the lower parts of the ternary mixture diagrams shown in Figures 1-4. If NaCI
is absent, the lowest NMP content in the organic layer can be obtained at a low NMP
volume fraction. On the other hand, a lower MeTHF volume fraction leads to a lower
organic layer volume. Therefore, unless the peptide of interest is highly soluble in
MeTHF, only the conditions corresponding to the bottom left corner of the ternary
diagram are applicable for the process of extraction of the peptide.
In the presence of NaCI, the miscibility of MeTHF and water significantly decreases, so
that the volume of the organic layer is larger. Moreover, the presence of NaCI
increases the density of the aqueous layer, so that the phase separation is quicker.
Therefore, it can be concluded that it is generally preferred to carry out the process for
extraction of a peptide in the presence of NaCI and to repeat the extraction with fresh
NaCI solution to reach very low NMP content in the MeTHF layer.
b) Extraction of H-Leu-Trp(Boc)-Val-Asn(Trt)-Ser(n3u)-NH2 in the system
NMP/MeTHF/NaCI solution
A central composite DoE was performed for the process of extraction of the
pentapeptide H-Leu-Trp(Boc)-Val-Asn(Trt)-Ser(£Bu)-NH 2 described in example 6 below.
The extraction yield of this peptide was measured from a solution in NMP having a
concentration of 200 mg/mL. The relative volumes of MeTHF and of water, as well as
the NaCI content in water, were systematically varied. One experiment was carried out
for each boundary condition and 3 experiments were carried out for the centre point.
The obtained results are shown in Table 2 below.
Mixture MeTHF H20 NaCI in Extraction
# Vx Vx water (g/L) yield (%)
1 2 2 0 90.7
2 3.5 3.5 100 100.0
3 3.5 5 100 100.0
4 5 3.5 100 99.4
5 2 3.5 100 99.7
6 3.5 2 00 96.7
7 5 2 0 57.8
8 5 5 200 100.0
9 3.5 3.5 100 100.0
10 3.5 3.5 100 100.0
11 2 2 200 98.1
12 5 5 0 100.0
13 3.5 3.5 0 97.9
14 5 2 200 98.9
15 2 5 0 99.8
16 2 5 200 100.0
17 3.5 3.5 200 100.0
Table 2 . Extraction yields of H-Leu-Trp(Boc)-Val-Asn(Trt)-Ser(fBu)-NH 2 as a function of
the composition of the organic and of the aqueous layers. MeTHF Vx stands for the
volume ratio MeTHF : reaction mixture (RM). H20 Vx stands for the volume ratio
H20 : reaction mixture (RM).
A good mathematical model (R2 = 0.93) was obtained: the extraction yield of the
peptide can be calculated as a function of the volume ratio water : reaction mixture
(RM) and of the NaCI content in water as:
Extraction yield (%) = 0.77 + 0.089 * (water / RM) + 0.00059 * NaCI (g/L) -
0.00012 * (water / RM) * NaCI (g/L) - 0.0087 (water / RM)2
This model can be represented graphically by the contour plot given in Figure 5 . The
minimum volume ratio water : reaction mixture needs to sufficiently high in order to
reach the extraction yield of over 99%. However, this minimum volume ratio
water : reaction mixture is also dependent on the NaCI content in the aqueous layer.
Indeed, a higher NaCI content in the aqueous layer leads to a lower miscibility of
MeTHF with the aqueous layer. As a consequence, the solubility of the peptide in the
aqueous layer is lower. If NaCI is absent in the aqueous layer, the volume ratio
water : reaction mixture needs to be higher than 4 in order to reach an extraction yield
of over 99%. In the presence of NaCI at 50 g/L in the aqueous layer, a water : reaction
mixture ratio = 2.7 is sufficient.
It can be considered that the necessary volume ratio water : reaction mixture leading to
an extraction yield above 99% is given by:
Water : reaction mixture > 4 - 0.00974 * NaCI (g/L)
On the other hand, the volume ratio MeTHF : reaction mixture = 2 is always sufficient
for an extraction yield to be over 99%.
c) Extraction of NMP in systems DCM/NaCI solution, EtOAc/NaCI solution
and MeTHF/NaCI solution
Extraction properties of MeTHF (according to the present invention) were compared to
those of EtOAc and DCM (comparative). EtOAc and DCM are commonly employed
solvents for extraction of peptides. The experiments were carried out with an aqueous
solution containing 150 g/L NaCI. No peptides were present in the systems of the
present example.
The volume ratios were as follows:
NMP : DCM : NaCI solution = 1 : 3 : 3
NMP : EtOAc : NaCI solution = 1 : 3 : 3
NMP : MeTHF : NaCI solution = 1 : 3 : 3
After the extraction, fraction of NMP in the aqueous layer was determined by GC. The
results of the experiments are summarized in Table 3 below.
Solvent Fraction of NMP in aqueous layer
DCM 0.555
EtOAc 0.935
MeTHF 0.962
Table 3. Extraction of NMP using DCM, EtOAc or MeTHF from an aqueous NaCI
solution (150 g/L NaCI).
As can be noticed from Table 3 above, the extraction with MeTHF leads to a higher
fraction of NMP in the aqueous layer than the extraction using DCM or EtOAc.
Accordingly, the NMP content in the organic layer after the extraction with MeTHF was
lower than after the extraction with DCM or EtOAc.
This result indicates that an extraction of a peptide with MeTHF generally allows a
more efficient separation of polar aprotic solvents such as NMP than a comparative
extraction employing common solvents EtOAc or DCM.
d) Extraction of NMP in the system MeTHF/THF/NaCI solution
The following example relates to mixtures consisting of NMP, MeTHF, THF and an
aqueous solution containing 150 g/L NaCI. In particular, the dependency between the
NMP content (g/L) in the organic layer after the phase separation and the composition
of the mixture was investigated. No peptides were present in the systems of the
present example.
The volume ratio MeTHF : NMP was 3, whereby the volume ratio NaCI solution : NMP
was varied from 2 to 10 and the volume ratio THF : NMP was varied from 0 to 3 . The
objective of these experiments was to illustrate the interactions between these four
components, so these experiments were performed with neat solvents. However, it is
noteworthy that the presence of a peptide may change the NMP distribution. The
obtained results are represented in Figure 6 .
From Figure 6 it can be recognised that if the volume ratio THF : NMP is below 2, the
NMP content in the organic layer ranges from 0 g/L to 20 g/L, even if the volume ratio
water : NMP is low. Typically, if the volume ratio NMP : MeTHF : THF ; NaCI solution =
1 : 3 : 2 : 5 , 90% of NMP is located in the aqueous layer. However, if the volume ratio
THF : NMP is higher than 2 , the extraction yield of NMP is lower.
Nevertheless, already at a volume ratio NMP : MeTHF : THF : NaCI solution =
1 : 3 : 3 : 3 , a high extraction yield of more than 99% can be achieved for many
peptides, whereas 80% of total NMP is removed into the aqueous layer.
e) Extraction of NMP in systems MeTHF/THF/NaCI solution and
EtOAc/THFANaCI solution
Extraction properties of the solvent combination MeTHF/THF (according to the present
invention) were compared to those of the combination EtOAc/THF (comparative). The
experiments were carried out with an aqueous solution containing 150 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
Fraction of NMP in the aqueous layer was determined by GC. The results of the
experiments are summarized in Table 4 .
Table 4 . Extraction of NMP in the biphasic system NMP/solvent combination/NaCI
solution (150 g/L NaCI).
As can be noticed from Table 4 above, the extraction with a solvent combination
MeTHF/THF leads to a higher fraction of NMP in the aqueous layer than the extraction
with EtOAc/THF. Accordingly, the NMP content in the organic layer after the extraction
with MeTHF/THF was lower than after the extraction with EtOAc/THF.
This result indicates that an extraction of a peptide with a solvent combination
containing MeTHF generally allows a more efficient separation of polar aprotic solvents
such as NMP than a comparative extraction employing a solvent combination
containing EtOAc.
Example 1 Use of a continuous LPPS upon usage of Fmoc as a protecting group
for the synthesis of H-Phe-lle-Glu(OiBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-
Pro-Thr(fBu)-Gly-Ser(fBu)-NH
Example 1.1 LPPS
Fmoc-Phe-lle-Glu(OfBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-OH (0.5 g , 0.28 mmol),
H-Pro-Thr(fBu)-Gly-Ser(fBu)-NH 2 (0.15 g, 0.32 mmol) and HOBt (0.044 g , 0.28 mmol)
were combined in NMP (2.5 mL) at 20°C. The mixture was stirred for 10 min at room
temperature until all solids were dissolved, then cooled to 0°C. TBTU (0.093 g ,
0.28 mmol), followed by DIPEA (46 mI_ , 0.28 mmol) was added and the reaction mixture
was stirred at this temperature. After 2 h the reaction was complete as determined by
HPLC. The reaction progress was monitored by the following method: 5 mI_ sample of
the reaction mixture, diluted 50 fold in NMP, were analysed according to method MIH-
009-3TG9 described above.
Example 1.2 Fmoc deprotection
To the solution prepared according to example 1. 1 (4 mL) DEA (0.4 mL, 3.9 mmol) was
added at room temperature. After completion of the Fmoc cleavage, as determined by
HPLC, the volatiles were eliminated by co-evaporations with ACN (3 x 1 mL) at 30°C
and 60 mbar. The reaction progress was monitored by the following method: 5 L
sample of the reaction mixture, diluted 50 fold in NMP, were analysed according to
method MIH-009-3TG9 described above.
Example 1.3 Extraction with MeTHF/THF and isolation
The solution prepared according to example 1.2 (4 mL) was combined with MeTHF
(12 mL), THF (8 mL) and an aqueous solution containing 100 g/L NaCI and 25 g/L
Na2C0 3 (20 mL). After a thorough mixing and phase separation (approx. 4 min), the
lower aqueous layer was removed. The peptide solution was further cleaned up by
addition of THF (8 mL) and of an aqueous solution containing 100 g/L NaCI and 25 g/L
Na2C0 3 (20 mL). After a thorough mixing and a layer separation, the lower layer was
removed. The organic layer was evaporated at 30°C, 60 mbar to a residual volume of
ca. 4 mL. MeTHF and THF were removed by four co-evaporations with ACN
(4 x 10 ml_) to initiate the peptide precipitation. The process of peptide precipitation
was completed by addition of ACN (10 mL) and DIPE (30 mL) to the residue of the
fourth co-evaporation (4 mL). The solid was separated by filtration, washed with DIPE
(3 x 0 mL) and dried under reduced pressure.
The present example demonstrates that the peptide precipitation can take place during
evaporation of the organic layer and the precipitated peptide can be easily separated
by filtration. In the presence of DMF or NMP, formation of such peptide precipitate
would not be possible.
Example 2 Use of a continuous LPPS upon usage of Boc as a protecting group
for the synthesis of Glu(OBzl)-Glu(OBzl)-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-OBzlTos (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 - 0°C until completion of the
reaction as determined by HPLC. The reaction progress was monitored by the
following method: 5 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 50 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 15°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 L sample of the reaction mixture was
diluted 30 fold in methanol and analysed according to method MIH-009-2TG1
described above.
Example 2.3 Boc-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl
DMF (25 mL), DIPEA (2.3 mL, 13.9 mmol), Boc-Glu(OBzl)-OH (4.3 g , 12.7 mmol) and
HOBt (2.0 g , 13.0 mmol) were added to the material prepared according to example
2.2. After the solubilisation was complete, EDC HCI (2.8 g , 14.6 mmol) was added at -
5°C and the peptide coupling reaction was performed at a temperature of from -7°C to -
3°C until completion of the reaction as determined by HPLC. The reaction progress
was monitored by the following method: 5 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.
Solid materials were separated by filtration and subsequently washed with DMF (5 mL).
MeTHF (50 mL) was added to the combined filtrates (20 mL). This resulting mixture
was successively extracted with:
1) aqueous solution containing 20 g/L NaCI (50 mL)
2) aqueous solution containing 20 g/L NaCI and 50 g/L NaHC0 3 (50 mL)
3) aqueous solution containing 20 g/L NaCI and 50 g/L NaHC0 3 (50 mL)
4) aqueous solution containing 20 g/L NaCI (50 mL).
The organic layer was then evaporated at 30°C under reduced pressure.
Example 2.4 H-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl
Boc cleavage was performed at 15°C by addition of toluene (20 mL), phenol (0.25 g)
and TFA (16 mL) to the residue of evaporation. After completion of the Boc cleavage
reaction, as verified by HPLC (5 sample of the reaction mixture diluted 30 fold in
ACN were analysed according to method MIH-009-2TG1 1 described above), the
reaction mixture was evaporated under reduced pressure. The residual TFA was
removed by co-evaporations with toluene (3 x 25 mL).
The peptide precipitated at the end of the evaporation and it was dissolved upon
addition of DMF (10 mL) to the residue of evaporation. Subsequently, MeTHF (50 mL)
was added. This combined organic layer was extracted successively with:
1) aqueous solution containing 150 g/L NaCI (50 mL)
2) aqueous solution containing 150 g/L NaCI (50 mL)
3) aqueous solution containing 20 g/L NaCI and 50 g/L NaHC0 3 (50 mL)
4) aqueous solution containing 20 g/L NaCI and 50 g/L KHS0 (50 mL)
5) aqueous solution containing 150 g/L NaCI (50 mL)
6) aqueous solution containing 150 g/L NaCI (50 mL).
The organic layer was evaporated at 30°C under reduced pressure (60 mbar).
Example 2.5 Boc-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl
DMF (25 mL), DIPEA (2.3 mL, 13.9 mmol), Boc-Glu(OBzl)-OH (4.3 g , 12.7 mmol) and
HOBt (2.0 g, 3.0 mmol) were added to the residue of evaporation obtained according
to example 2.4. After complete solubilisation, the peptide coupling reaction was
performed with EDC HCI (2.8 g , 14.6 mmol) at a reaction temperature of from -7°C to -
3°C. The reaction progress was monitored by the following method: 5 L sample of the
reaction mixture was diluted 50 fold in acetic acid : water (9 : 1) and analysed
according to method MIH-009-2TG1 1 described above.
Solid materials were separated by filtration and washed with DMF (10 mL). The
resulting filtrates were combined.
Subsequently, MeTHF (50 mL) was added to the combined filtrates and the resulting
mixture was extracted successively:
1) twice with aqueous solution containing 20 g/L NaCI (50 mL)
2) once with aqueous solution containing 20 g/L NaCI and 50 g/L NaHC0 3 (50 mL)
3) once with aqueous solution containing 20 g/L NaCI and 50 g/L KHS0 4 (50 mL)
4) three times with aqueous solution containing 20 g/L NaCI (50 mL).
The organic layer was then evaporated at 30°C under reduced pressure and poured in
DIPE (150 mL) for precipitation. After filtration, the collected solid was further washed
three times with DIPE (3 x 50 mL). The resulting solid was finally dried under reduced
pressure.
Example 3 Extraction of H-Ser(fBu)-Lys(Boc)-Gln(Trt)-Met-Glu(fBu)-Glu(iBu)-
Glu(fBu)-Ala-Val-Arg(Pbf)-Leu-Phe-lle-Glu(OfBu)-Trp(Boc)-Leu-Lys(Boc)-
Asn(Trt)-Gly-Gly-Pro-Ser(fBu)-Ser(fBu)-Gly-Ala-Pro-Pro-Pro-Ser(iBu)-NH from
the reaction mixture
Fmoc-Ser(rBu)-Lys(Boc)-Gln(Trt)-Met-Glu(fBu)-Glu(fBu)-Glu(fBu)-Ala-Val-Arg(Pbf)-
Leu-OH ( 1 1.5 g 4 mmol), H-Phe-lle-Glu(OfBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-
Gly-Pro-Ser(fBu)-Ser(fBu)-Gly-Ala-Pro-Pro-Pro-Ser(fBu)-NH 2 (10 g , 4 mmol) and HOBt
( 1 .5 g , 8.8 mmol) were combined in a mixture of NMP (94 mL) and THF (70 mL) at
20°C. The mixture was stirred for 10 min at room temperature until all solids were
dissolved, then cooled to 0°C. TOTU ( 1 .5 g , 4.6 mmol), followed by DIPEA (4 mL,
23 mmol) was added and the reaction mixture was stirred at this temperature. After 2 h
the reaction was complete as determined by HPLC. The reaction progress was
monitored by the following method: 5 sample of the reaction mixture, diluted 50 fold
in NMP, were analysed according to method MIH-009-397TG3.
Consequently, the cleavage of Fmoc protective group was performed by addition of
diethylamine (10 mL) to the reaction mixture according to the procedure described in
example 1.2. After the Fmoc cleavage was complete, as verified by HPLC method
MIH-009-397TG3, the volatiles were removed by four subsequent co-evaporations with
acetonitrile (4 x 50 mL).
The residue of evaporation (120 mL) was divided in 6 equal parts and used for
comparative work up experiments.
The volume ratios were as follows:
b) NMP solution : MeTHF : NaCI solution = 1 : 3 : 3
c) NMP solution :: EtOAc : NaCI solution = 1 : 3 : 3
d) NMP solution : DCM : NaCI solution = 1 : 3 : 3
e) NMP solution : EtOAc : NaCI solution = 1 : 6 : 3
NMP solution : DCM : NaCI solution = 1 : 6 : 3
a) Precipitation in DIPE
The residue of evaporation (20 mL) was co-evaporated twice with THF (20 mL) and the
product was then precipitated by transfer into DiPE (135 mL) at 25°C under stirring.
The product appeared in the form of a gummy solid that could not be filtered in 24
hours.
b) Extraction with MeTHF
MeTHF (60 mL) was added to the residue of evaporation (20 mL). This mixture was
extracted three times with an aqueous solution (60 mL) containing NaCI (15% w/v) and
Na2C0 3 (2.5% w/v). All decantations took less than 2 minutes. The organic layer was
evaporated to a residual volume of 9 mL. MeTHF was exchanged by two coevaporations
with THF (20 mL). The mixture was evaporated to a final volume of 9 mL
and the product was precipitated by transfer into DIPE ( 35 mL) at 25°C under stirring.
The solid could be isolated by filtration in 15 seconds and finally dried. The HPLC
analysis of the mother liquor of precipitation showed that the yield of peptide
precipitation was above 99.9%. The precipitate was not sticky, no product was lost on
the surface of the glassware.
c) Extraction with 3 volumes of EtOAc
EtOAc (60 mL) was added to the residue of evaporation (20 mL). This mixture was
extracted twice with an aqueous solution (60 mL) containing NaCI (15% w/v) and
Na C0 3 (2.5% w/v). On the second extraction, the peptide formed a gel in the form of
an opaque layer which appeared between the organic layer and the aqueous layer.
This intermediate layer did not disappear after 48 hours.
d) Extraction with 3 volumes of DCM
DCM (60 mL) was added to the residue of evaporation (20 mL). This mixture was
extracted three times with an aqueous solution (60 mL) containing NaCI (15% w/v) and
Na2C0 3 (2.5% w/v). All decantations took less than 10 minutes. The organic layer was
evaporated to a residual volume of 8 mL. Solvent exchange was performed by two coevaporations
with THF (20 mL). The mixture was evaporated to a final volume of 8 mL.
This residue of evaporation was transferred into DIPE (135 mL) at 25°C under stirring
but no precipitation was observed. Instead, a phase separation was observed and the
peptide was found in the bottom oily phase.
e) Extraction with 6 volumes of EtOAc
EtOAc (120 mL) was added to the residue of evaporation (20 mL). This mixture was
extracted three times with an aqueous solution (60 mL) containing NaCI (15% w/v) and
Na2C0 3 (2.5% w/v). All decantations took less than 2 minutes. The organic layer was
evaporated to a residual volume of 9 mL. EtOAc was exchanged by two coevaporations
with THF (20 mL). The mixture was evaporated to a final volume of 9 mL
and the product was precipitated by transfer into DIPE (135 mL) at 25°C under stirring.
The solid was isolated by filtration in 17 minutes (instead of 15 seconds in the MeTHF
extraction process). The precipitate was very sticky and more than 15% of the product
was lost on the surface of the glassware.
f) Extraction with 6 volumes of DCM
DCM (120 mL) was added to the residue of evaporation (20 mL). This mixture was
extracted three times with an aqueous solution (60 mL) containing NaCI (15% w/v) and
Na2C0 3 (2.5% w/v). All decantations took less than 10 minutes. The organic layer was
evaporated to a residual volume of 8 mL. Solvent exchange was performed by two coevaporations
with THF (20 mL). The product was precipitated by transfer into DIPE
(135 mL) at 25°C under stirring. The product precipitated as a gum that could not be
isolated by a filtration of 12 hours.
In experiments b) and d)-f) the organic layers were separated and added dropwise to
135 mL DIPE at 25°C. In cases b), d), e) and f), the organic layers were partially
evaporated to a residual volume of 9 ± 1 mL. The products in experiments b), e) and f)
were separated by filtration.
The peptide content in the aqueous layer after the extraction as well as in the filtrate
after the filtration step was determined by analytical HPLC. The isolated product was
dried under reduced pressure at 40°C overnight and, subsequently, the product yield
was determined.
The results of experiments a)-f) are summarized in Table 5 .
No. Extraction Product Filtration Isolated
Solvent Phase Peptide appearanc Filtration Peptide product
sepa¬ content in e after time content in yield
ration the aqueous precipitatio the filtrate
time layer n
a) no no no Gum > 12 h < 0.1% 0%
b) eTHF < 2 < 0.1% Filterable 15 sec NA 84%
(3 vol) min solid
c) EtOAc > 48 h NA NA NA < 0.1 % 0%
(3 vol)
d) DCM < 2 min < 0.1% NA 5 sec 100% 0%
(3 vol)
e) EtOAc < 2 min < 0.1% Filterable 17 min < 0.1% 69%
(6 vol) solid
f) DCM < 2 min < 0.1% Gum > 12h < 0.1% 36%
(6 vol)
Table 5 . Extraction of H-Ser(iBu)-Lys(Boc)-Gln(Trt)-Met-Glu(iBu)-Glu(fBu)-Glu(iBu)-
Ala-Val-Arg(Pbf)-Leu-Phe-lle-Glu(OfBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-
Ser(fBu)-Ser(fBu)-Gly-Ala-Pro-Pro-Pro-Ser(fBu)-NH 2
Results
The direct precipitation in diisopropyl ether (experiment a)) yielded a gum that was
impossible to filter i.e. the filtration time was higher than 12 h. Thus, the product could
not be isolated.
An attempt to carry out the extraction with 3 volumes of EtOAc (experiment c)) resulted
in a stable emulsion i.e. the phase separation time of more than 48 h was observed.
The extraction with 6 volumes of EtOAc (experiment e)) was successful but it required
a higher volume of solvent than with MeTHF (experiment b)). Moreover the filtration
time of the product obtained in experiment e) was rather high.
The extraction with 3 volumes of DCM (experiment d)) was successful but then the
peptide could not be precipitated. The extraction with 6 volumes of DCM (experiment
f)) yielded a gummy precipitate that could not be filtered. These results indicate that the
products after the DCM extractions in both experiments contained considerable
amounts of NMP.
Thus, the best results were achieved in the experiment b) (highlighted in bold), where
MeTHF was employed for the extraction. Moreover, the isolated product yield in the
experiment b) was significantly higher than in other experiments.
Example 4 Extraction of Boc-His(Trt)-Gly-Glu(OiBu)-Gly-Thr(iBu)-Phe-Thr(fBu)-
Ser(iBu)-Asp(OiBu)-Leu-Ser(fBu)-Lys(Boc)-Gln(Trt)-Met-Glu(iBu)-Glu(iBu)-
Glu(iBu)-Ala-Val-Arg(Pbf)-Leu-Phe-lle-Glu(OiBu)-Trp(Boc)-Leu-Lys(Boc)-
Asn(Trt)-Gly-Gly-Pro-Ser(fBu)-Ser(iBu)-Gly-Ala-Pro-Pro-Pro-Ser(iBu)-NH2 from
the reaction mixture
Boc-His(Trt)-Gly-Glu(OfBu)-Gly-Thr(iBu)-Phe-Thr(fBu)-Ser(iBu)-Asp(OfBu)-Leu-OH
(6.94 g , 4.1 1 mmol), H-Ser(fBu)-Lys(Boc)-Gln(Trt)-Met-Glu(fBu)-Glu(fBu)-Glu(fBu)-Ala-
Val-Arg(Pbf)-Leu-Phe-lle-Glu(OiBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-
Ser(fBu)-Ser(fBu)-Gly-Ala-Pro-Pro-Pro-Ser(fBu)-NH 2 (20 g , 4.32 mmol) and HOBt
(0.63 g , 4.1 mmol) were combined in NMP (210 mL) at 20°C. The mixture was stirred
for 10 min at room temperature until all solids were dissolved, then cooled to 0°C.
TOTU (2.7 g , 8.22 mmol), followed by DIPEA (6 mL, 42 mmol) was added and the
reaction mixture was stirred at this temperature. After 2 h the reaction was complete as
determined by HPLC. The reaction progress was monitored by the following method:
5 L sample of the reaction mixture, diluted 50 fold in NMP, were analysed according
to method MIH-009-397TG15.
The obtained reaction mixture was divided in equal samples (sample volume: 5 mL)
and used directly for the extractions tests # 1-17. In each case, a sample of the
reaction mixture (5 mL) was mixed with different organic solvents as summarised in
Table 6 , and then extracted with 15 mL of 20% aqueous solution of NaCI. These
experimental conditions were compared for the phase separation (decantation) time
and the yield of peptide extraction (ratio of the peptide in the organic layer).
In tests # 4, 6 and 11 the peptide was poorly solubilised in the organic layer; it was
found as a gel that slowly settled between the organic layer and the aqueous layer.
In tests # 1, 2 , 7 , 8 , 15-17 a separation between two clear layers was rapidly observed
(in less than 2 minutes). The two layers were separated and the peptide content in
each layer was determined by HPLC.
In tests # 3-6, 9-14 the extraction resulted in an opaque mixture. After several minutes
(more than 60 minutes), the system began to separate but a thick layer of peptide gel
formed between the aqueous layer and the organic layer. A clear separation of the
layers was never observed. However, after 120 minutes, the aqueous layer was
removed from the decantation vessel. The separation of the peptide gel from the
organic layer was practically impossible (the density difference was too low). The
peptide gel and the organic layer were dissolved with NMP and the peptide content
was determined by HPLC. The yield of peptide extraction shown in Table 6 is thus
more indicative of the quality of the decantation between the peptide gel than a real
portioning between the aqueous layer and the organic layer.
The volume ratios of the components employed for the extractions tests # 1-17 and
observations are summarised in Table 6 below.
Decan
Peptide
Test Vol DCM Vol EtOAc Vol MeTHF Vol THF Vol ACN -tation
extraction
# (mL) (mL) (mL) (mL) (mL) time
yield (%)
(min)
1 15 0 0 0 0 < 2 98,8
2 30 0 0 0 0 < 2 98.9
3 0 15 0 0 0 120 70,3*
4 0 30 0 0 0 120 95,2*
5 0 0 15 0 0 120 85,2*
6 0 0 30 0 0 120 97,8*
7 15 15 0 0 0 < 2 52,1
8 15 0 0 0 7,5 < 2 69,0
9 0 15 15 0 0 120 95,3*
10 0 15 0 0 5 120 53,3*
11 0 15 0 0 7,5 120 97,5*
12 0 15 0 0 10 120 96,7*
13 0 15 0 0 15 120 88,5*
14 0 0 15 0 5 120 89,6*
15 0 0 15 0 7,5. < 2 99,3
16 0 0 15 0 10 < 2 98,7
17 0 0 15 0 15 < 2 98,9
*peptide in the gel phase was included in the extraction yield
Table 6 . Extraction of Boc-His(Trt)-Gly-Glu(OfBu)-Gly-Thr(fBu)-Phe-Thr(fBu)-Ser(fBu)-
Asp(OfBu)-Leu-Ser(iBu)-Lys(Boc)-Gln(Trt)-Met-Glu(fBu)-Glu(fBu)-Glu(rBu)-Ala-Val-
Arg(Pbf)-Leu-Phe-lle-Glu(OfBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt)-Gly-Gly-Pro-Ser(iBu)-
Ser(fBu)-Gly-Ala-Pro-Pro-Pro-Ser(fBu)-NH 2
Results
In extractions with neat DCM (tests # 1 and 2) a quick phase separation and a high
peptide extraction yields were observed. However, as shown in example c) (Table 3)
and comparative example 5.1 (Table 7), extractions with neat DCM result in a high
content of the polar aprotic solvent in the organic layer. As a consequence, subsequent
precipitation of the extracted peptide becomes difficult. This disadvantage is illustrated
by Example 3 , tests d) and f). Accordingly, implementation of peptide extractions with
neat DCM suffers from serious drawbacks.
Extractions with neat MeTHF (tests # 5 and 6) showed a higher peptide extraction yield
than extractions with neat EtOAc (tests # 3 and 4).
Extraction properties of mixtures MeTHF/ACN were investigated in tests # 14-17. The
phase separation times observed in tests # 14-17 were shorter and the peptide
extraction yields were higher in comparison to extractions with neat MeTHF (tests # 5
and 6). A comparison between the results of extractions with MeTHF/ACN (tests # 14-
17) and extractions with EtOAc/ACN (tests # 10- 3) reveals that MeTHF/ACN mixtures
have better extraction properties than the corresponding EtOAc/ACN mixtures. In
particular, extractions with MeTHF/ACN mixtures led to shorter phase separation times
and higher peptide extraction yields.
Example 5 Use of continuous LPPS for the coupling of two peptides and Boc
cleavage without precipitation of the intermediates. Preparation of Boc-Gly-Gly-
Gly-Gly-Gly-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl)
Example 5.1 Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl)
Boc-Pro-lle-Leu-Pro-Pro-OH (3.5 g , 5.5 mmol) and H-Glu(OBzl)-Glu(OBzl)-Tyr-
Leu(OBzl) (5.0 g , 5.5 mmol) were dissolved in DMF (25 mL) at 20°C. The resulting
mixture was cooled to -8°C then HOBt H20 (0.88 g , 5.75 mmol), EDC HCI ( 1 .21 g ,
6.31 mmol) were added and the reaction temperature was maintained in the range
from -4°C to -8°C until a complete conversion was confirmed by a HPLC measurement.
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.
To a reaction mixture prepared above, MeTHF (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 20 g/L NaCI (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 KHS0 4 (90 mL)
5) aqueous solution containing 20 g/L NaCI (90 mL).
The organic layer was then evaporated at 30°C under reduced pressure.
Comparative example 5.1 Extraction of Boc-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 1).
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 7 below and were
then extracted with 3 mL of aqueous solution of NaCI (15% w/v) and Na2C0 3 (2.5%
w/v).
A rapid separation between the two clear layers was observed in all extraction tests.
The DMF content in the organic layer was determined by GC.
able 7 . Extraction of Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-OBzl
Results
Extractions with neat MeTHF (tests # 5 and 6) led to a lower DMF content in the
organic layer than extractions with neat DCM (tests # 1 and 2) or neat EtOAc (tests # 3
and 4). Furthermore, extractions with solvent mixtures containing MeTHF (tests # 8 , 9
and 1 ) provided a lower DMF content in the organic layer than extraction with the
mixture EtOAc/DCM (test # 7) or EtOAc/THF (test # 10).
Example 5.2 Removal of the Boc protecting group. H-Pro-lle-Leu-Pro-Pro-
Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl)
Boc cleavage was performed by addition of toluene (20 mL), phenol (0.25 g) and TFA
(16 mL) to the material obtained in example 5.1 at 15°C. After reaction completion, as
determined by HPLC, the reaction mixture was evaporated at 30°C under reduced
pressure. The reaction progress was monitored by the following method: 5 L sample
of the reaction mixture, diluted 20 fold in ACN, were analysed according to method
MIH-009-2TG1 1 described above.
Volatiles were further removed by subsequent co-evaporations with toluene
(2 x 20 mL) at 30°C under reduced pressure. MeTHF (50 mL) was added to the residue
of evaporation and the organic solution was extracted six times with an aqueous
solution containing 20 g/L NaCI (6 x 50 mL). The organic layer was evaporated at 30°C
under reduced pressure.
Comparative example 5.2 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
The products from tests # 1, 3 and 5 of comparative example 5.1 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 8 and graphically presented in Figure 7 .
Conversion (%)
Time (min)
Test # 1 Test # 3 Test # 5
0 0 0 0
60 30,6 95,6 93,3
105 53,7 99,9 99,9
270 8 1,8
330 88,6
450 98
Table 8. Deprotection of Boc-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu-
OBzl
Results
Traces of DMF in the materials obtained in comparative example 5.1 significantly inhibit
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 materials obtained by
extraction with EtOAc and MeTHF. In this particular case no significant difference
between materials obtained by extraction EtOAc and MeTHF was observed.
Example 5.3 Coupling of Boc-Phe-OH with H-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-
Glu(OBzl)-Tyr-Leu(OBzl) by using LPPS without precipitation of the intermediate
Boc-Phe-OH ( 1.53 g , 5.8 mmol) was dissolved in DMF (25 mL) at 20°C and added to
the reaction mixture obtained in example 5.2. 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 mI_ sample of the reaction mixture was diluted 50 fold in acetic acid : water (9 : 1) and
analysed according to method MIH-009-2TG1 1 described above.
Then MeTHF (90 mL) was added and the reaction mixture was successively extracted
with:
1) aqueous solution containing 50 g/L NaCI (90 mL)
2) aqueous solution containing 50 g/L NaCI (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 KHS0 (90 mL)
5) aqueous solution containing 50 g/L NaCI (90 rmL)
6) aqueous solution containing 50 g/L NaCI (90 mL).
The organic layer was then evaporated under reduced pressure at 35°C.
The process of example 5.2 was then applied to the obtained material with the only
difference that the residue was extracted with aqueous NaCI solution seven times
instead of six.
Example 5.4 Coupling of Boc-Ser(Bzl)-OH and H-Phe-Pro-lle-Leu-Pro-Pro-
Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl)
Boc-Ser(Bzl)-OH ( 1.62 g , 5.5 mmol) was coupled to the H-Phe-Pro-lle-Leu-Pro-Pro-
Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl) peptide prepared according to example 5.3, using
the procedure described therein.
a) Extraction and precipitation in DIPE
25 mL of the reaction mixture resulting from example 5.4 and containing 5 g Boc-
Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-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 bar 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 miti
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 8 (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 5.4.
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 5.4, 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 5.4 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.
Example 5.5 Boc cleavage of Boc-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-
Glu(OBzl)-Tyr-Leu(OBzl)
Boc-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu(OBzl) (5 g)
were put in a mixture of toluene (20 mL), phenol (0.2 g) and TFA (16 mL). After
reaction completion as determined by HPLC (5 m of the reaction, diluted 30 fold in
acetonitrile, were analysed according to HPLC method MIH-009-2TG1 1), the reaction
mixture was evaporated under reduced pressure and a residual oil was obtained. The
residual TFA was further removed by two co-evaporations with toluene (2 x 30 mL).
MeTHF (50 mL) was added to the resulting residue of co-evaporations and this mixture
was extracted three times with an aqueous solution containing NaCI at 100 g/L
(3 x 50 mL). The obtained organic layer was separated and evaporated under reduced
pressure at 35°C.
Example 5.6 Coupling of Boc-Gly-Gly-Gly-Gly-OH with H-Ser(Bzl)-Phe-Pro-lle-
Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl) and extraction of the product
Boc-Gly-Gly-Gly-Gly-OH ( 1 .27 g , 2.8 mmol), H-Ser(Bzl)-Phe-Pro-lle-Leu-Pro-Pro-
Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu(OBzl) (5.0 g , 2.7 mmol) and HOBt H20 (0.43 g ,
2.8 mmol) were dissolved in DMF (25 mL) at 20°C and the obtained solution was
added to the reaction mixture obtained in example 5.5 above. The temperature of the
reaction mixture was adjusted to 6±2°C, and EDC HCI (0.6 g , 3.1 mmol) was added
thereto. 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: 3 sample of the reaction mixture, diluted 50 fold in acetic acid : water
(9 : 1), were analysed according to method MIH-009-2TG1 described above.
Then, MeTHF (90 mL) and THF (30 mL) were added and the mixture was successively
extracted with:
1) aqueous solution containing 100 g/L NaCI (100 mL)
2) aqueous solution containing 100 g/L NaCI and 25 g/L NaHC0 3 (100 mL)
3) aqueous solution containing 100 g/L NaCI (100 mL)
4) aqueous solution containing 00 g/L NaCI (100 mL).
The obtained organic layer was then evaporated under reduced pressure at 35°C.
Example 5.7 Boc cleavage
Toluene (20 mL), phenol (0.2 g) and TFA (16 mL) were added to the residue of
evaporation obtained in the example 5.6 above. After reaction completion as
determined by HPLC (5 L of the reaction, diluted 30 fold in acetonitrile, were analysed
according to HPLC method MIH-009-2TG1 1), the reaction mixture was evaporated
under reduced pressure whereby a residual oil was obtained. The residual TFA was
removed by two subsequent co-evaporations with toluene (2 3 mL). MeTHF (60 mL)
and THF (50 mL) were added to the residue of evaporation and the resulting solution
was extracted three times with an aqueous solution containing 100 g/L NaCI
(3 x 100 mL). The obtained organic layer was evaporated under reduced pressure at
35°C.
Example 5.8 Coupling of Boc-Gly-OH with H-Gly-Gly-Gly-Gly-Ser(Bzl)-Phe-Prolle-
Leu-Pro-Pro-Glu(OBzl)-Glu(OBzl)-Tyr-Leu(OBzl)
DMF (25 mL) was added to the residue of evaporation obtained in example 5.7 above.
Boc-Gly-OH (0.5 g , 2.8 mmol), HOBt H20 (0.43 g , 2.8 mmol) and EDC HCI (0.6 g ,
3.1 mmol) were added to the resulting mixture at 6±2°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: 3 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.
Subsequently, MeTHF (90 mL) and THF (30 mL) were added and the resulting mixture
was successively extracted with:
1) aqueous solution containing 100 g/L NaCI ( 00 mL)
2) aqueous solution containing 00 g/L NaCI and 25 g/L NaHC0 3 ( 00 mL)
3) aqueous solution containing 100 g/L NaCI (100 mL)
4) aqueous solution containing 100 g/L NaCI (100 mL)
5) aqueous solution containing 100 g/L NaCI (100 mL).
The resulting organic layer was evaporated under reduced pressure at 35°C.
Example 6 Continuous liquid phase synthesis of H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH2 upon usage of Fmoc as a protecting group
Example 6.1 LPPS of Fmoc-Asn(Trt)-Ser(rBu)-NH
H-Ser(fBu)-NH 2 (2.0 g , 12.5 mmol) and Fmoc-Asn(Trt)-OH (6.77 g , 11.3 mmol) were
added to NMP (30.0 mL) at 20°C. The mixture was stirred for 15 min until the solids
were completely dissolved and cooled to 10°C. PyBOP (6.52 g , 12.5 mmol) was added,
followed by addition of TEA (2.25 mL, 16.0 mmol). The reaction was carried out at
10°C and the conversion was complete after 14 h , as confirmed by HPLC. The reaction
progress was monitored by the following method: 3 sample of the reaction mixture,
diluted 50 fold in NMP, were analysed according to method MIH-009-RTTG1 described
above.
Example 6.2 Removal of the Fmoc protecting group and isolation of Asn(Trt)-
Ser(n3u)-NH2
To a mixture prepared according to example 6.1 , TAEA (8 mL) was added and
completion of the Fmoc cleavage was determined by HPLC, using the same method as
in example 6.1 .
Then MeTHF (140 mL) was added and the reaction mixture was successively
extracted:
1) once with aqueous solution containing 150 g/L NaCI (140 mL)
2) three times with aqueous solution containing 150 g/L NaCI (40 mL)
3) four times with aqueous solution containing 100 g/L KHS0 (25 mL)
4) twice with aqueous solution containing 50 g/L NaHC0 3 (40 mL)
5) twice with aqueous solution containing 150 g/L NaCI (40 mL).
Example 6.3 Coupling of Fmoc-Val-OH with H-Asn(Trt)-Ser(iBu)-NH
NMP (40 mL) was added to the organic layer obtained in example 6.2 and the
combined mixture was evaporated at 30°C under reduced pressure.
To this solution Fmoc-Val-OH (3.85 g, 11.3 mmol), PyBOP (6.5 g, 12.5 mol) and TEA
(2 mL) were added. The reaction was carried out at room temperature until a complete
conversion was detected by HPLC. The reaction progress was monitored by the
following method: 3 L sample of the reaction mixture, diluted 50 fold in DMF, were
analysed according to method MIH-009-RTTG1 described above.
Example 6.4 Removal of Fmoc protecting group. Val-Asn(Trt)-Ser(fBu)-NH 2
Fmoc cleavage was performed by addition of TAEA (5 mL) to the reaction mixture
obtained in example 6.3. After completion of the reaction as determined by HPLC
(same method as above), MeTHF (100 mL) was added to the reaction mixture. The
combined organic layer was extracted:
1) once with aqueous solution containing 150 g/L NaCI (120 mL)
2) three times with aqueous solution containing 150 g/L NaCI (40 mL)
3) five times with aqueous solution containing 100 g/L KHS0 4 (40 mL)
4) twice with aqueous solution containing 50 g/L NaHC0 3 (40 mL)
5) twice with aqueous solution containing 150 g/L NaCI (40 mL).
NMP (45 mL) was added to the organic layer prior to evaporation at 30°C under
reduced pressure.
Example 6.5 Coupling of Fmoc-Trp(Boc)-OH with H-Val-Asn(Trt)-Ser(fBu)-NH2
Fmoc-Trp(Boc)-OH (6.0 g , 1 .3 mmol) and PyBOP (6.5 g , 12.5 mmol) were added to
the peptide solution obtained in example 6.4 at room temperature. The reaction mixture
was neutralised by addition of TEA (2.75 mL) and stirred until completion of the peptide
coupling reaction as confirmed by HPLC (same method as in example 6.3).
Example 6.6 Removal of the Fmoc protecting group. H-Trp(Boc)-Val-Asn(Trt)-
Ser(iBu)-NH 2
Fmoc cleavage was performed by addition of TAEA (5 mL) to the reaction mixture
obtained in example 6.5. After completion of the reaction as determined by HPLC (the
same method as in example 6.3), MeTHF (150 mL) was added to the reaction mixture.
The combined organic layers were extracted:
1) once with aqueous solution containing 150 g/L NaCI (150 mL)
2) three times with aqueous solution containing 150 g/L NaCI (50 mL)
3) four times with aqueous solution containing 100 g/L KHS0 4 (50 mL)
4) twice with aqueous solution containing 50 g/L NaHC0 3 (50 mL)
5) twice with aqueous solution containing 150 g/L NaCI (50 mL).
MP (50 mL) was added to the obtained organic layer prior to evaporation at 30°C
under reduced pressure.
Example 6.7 Coupling of Fmoc-Leu-OH with H-Trp(Boc)-Val-Asn(Trt)-Ser(fBu)-
NH2 by LPPS
Fmoc-Leu-OH (4.0 g , 1.3 mmol) and PyBOP (6.5 g , 12.5 mmol) were added to the
peptide solution obtained in example 6.6. The reaction mixture was neutralised by
addition of TEA (2.75 mL) and stirred at room temperature until completion of the
peptide coupling reaction as determined by HPLC (same method as in example 6.3).
Example 6.8 Removal of the Fmoc protecting group. H-Leu-Trp(Boc)-Val-
Asn(Trt)- Ser(fBu)-NH2
Fmoc cleavage was performed by addition of TAEA (10 mL) to the reaction mixture
obtained in example 6.7. After completion of the reaction as determined by HPLC
(same method as in example 6.3), MeTHF (150 mL) was added to the reaction mixture.
The combined organic layers were extracted:
1) once with aqueous solution containing 150 g/L NaCI (150 mL)
2) three times with aqueous solution containing 50 g/L NaCI (75 mL)
3) four times with aqueous solution containing 100 g/L KHS0 4 (50 mL)
4) twice with aqueous solution containing 50 g/L NaHC0 3 (50 mL)
5) twice with aqueous solution containing 150 g/L NaCI (50 mL).
The organic layer was evaporated at 30°C under reduced pressure and the
composition of the isolated product was determined by HPLC (same method as in
example 6.3).
9.5 g of H-Leu-Trp(Boc)-Val-Asn(Trt)-Ser(fBu)-NH 2 were isolated and the purity of the
product was 79%.
a) Comparative example: direct precipitation of H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH2 in DIPE (70 mL)
The reaction mixture (15 mL) of example 6.8 which was obtained after completion of
the reaction and before addition of MeTHF and containing H-Leu-Trp(Boc)-Val-
Asn(Trt)-Ser(fBu)-NH 2 (3 g) was partially evaporated to reduce its volume to 7 mL. The
obtained residue was subsequently transferred into DIPE (70 mL) for the peptide
precipitation. This resulted in a formation of a gel which was difficult to be transferred to
the filter and was not filterable.
b) Comparative example: direct precipitation of H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(iBu)-NH 2 i DIPE (100 mL)
The reaction mixture (7 mL) of example 6.8 containing H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH 2 (3 g) was partially evaporated to reduce its volume to 6 mL and was then
transferred into DIPE (100 mL) for the peptide precipitation.
The filtration of the precipitated solid on a 2.5 cm diameter, 20 miti pore size filter, took
15 in and yielded a 4.6 cm high cake, corresponding to the K value of .8. The
peptide quantification (3 L filtrate were analysed by HPLC method MIH-009-RTTG1)
indicated that 310 mg peptide, i.e. 10.3% of the crude material, remained in the mother
liquors of the precipitation.
In summary, the method of the direct precipitation of H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH 2 in DIPE suffered from the difficult evaporation of DMF, longer filtration
time and required a larger volume of DIPE for the product precipitation. Moreover, the
isolated yield of the peptide was rather low because the DMF content in the mother
liquors of precipitation was too high, increasing the peptide solubility in the aqueous
layer.
c) Example: precipitation H-Leu-Trp(Boc)-Val-Asn(Trt)- Ser(fBu)-NH2 in DIPE
(70mL) after extraction in MeTHF
The reaction mixture (15 mL) of example 6.8 containing H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH 2 (3 g) was added to MeTHF (50 mL). This mixture was extracted three
times with an aqueous solution containing 20 g/L NaCI (50 mL). The organic layer was
separated and subsequently partially evaporated under reduced pressure to a residual
volume of 12 mL. The partially evaporated organic layer was finally transferred into
DIPE (70 mL).
The filtration of the precipitated solid on a 2.5 cm diameter, 20 pore size filter, took
5.5 minutes and yielded a 4.0 cm high cake, corresponding to a K value of 2 1.6. The
peptide quantification (3 pL filtrate were analysed by HPLC method MIH-009-RTTG1)
indicated that 16 mg peptide, i .e. 0.5% of the crude material, were present
mother liquors of the precipitation.
Example 7 Coupling of Boc-MeLeu-OH and HCI AIa-OMe
HCI AIa-OMe (4.6 g , 33. 1 mmol) was dissolved in DMF (35 mL) at 20°C. The obtained
solution was cooled to -5°C and Boc-MeLeu-OH (7. 1 g , 28.8 mmol), HOBt (3. 9 g,
0.29 mmol) and EDC HCI (5.5 g, 28.8 mmol) were added thereto. The reaction mixture
was kept at -5°C until completion of the reaction as monitored by the following method:
5 sample of the reaction mixture, diluted 10 fold in acetic acid in methanol, were
analysed according to method MIH-009-025TG3 described above.
After completion of the reaction, MeTHF ( 130 mL) was added and the mixture was
extracted:
) once with water ( 130 mL)
2) once with aqueous solution containing 50 g/L NaCI (40 mL)
3) three times with aqueous solution containing 10 g/L KHS0 (40 mL) .
Subsequently, n-heptane ( 10 mL) was added to the organic layer and the combined
layer was extracted:
1) once with aqueous solution containing 50 g/L NaHC0 3 (25 mL)
2) once with water (25 mL) .
The organic layer was then evaporated under reduced pressure. n-Heptane ( 140 mL)
was added to the residue and the mixture was again evaporated under reduced
pressure, whereby the crystallisation of the peptide took place. After 18 hours, the
solids were separated by filtration and rinsed twice with n-heptane.
The collected product was re-dissolved in n-heptane (45 mL) at 40°C and left overnight
for re-crystallisation.
Because HCI H-Ala-OMe is highly hydrolysable, it usually contains some HCI H-Ala-
OH. Therefore, the material isolated after the peptide coupling reaction usually contains
Boc-MeLeu-Ala-Ala-OMe as an impurity. In general, impurities having a double Ala in
the seq uence are known to be difficult to remove by chromatography after the complete
peptide synthesis was carried out.
The re-crystallisation employed in the present example allows decreasing of the
amount of Boc-MeLeu-Ala-Ala-OMe, which is present in the isolated peptide as an
impurity, from 1.2 mol-% to 0.2 mol.-%. This re-crystallisation is only possible in the
absence of DMF.
Example 8 Use of continuous LPPS for a stepwise peptide assembly without
precipitation of the intermediates. Preparation of H-Pro-Ala-Gly-Phe-Ser(fBu)-
xantheneamide
Example 8.1 Coupling Fmoc-Phe-OH to H-Ser(fBu)-xantheneamide
H-Ser(fBu)-xantheneamide (2.5 g, 7.7 mmol) and Fmoc-Phe-OH (3.0 g, 7.7 mmol)
were dissolved in NMP (20 mL) at 20°C. TBTU (2.6 g , 8 .1 mmol) and TEA (2 mL) were
added and the reaction progress was monitored by the following method: 1 \ sample
of the reaction mixture, diluted 50 fold in DMF, was analysed according to method MIH-
009-RTTG 1.
After completion of the reaction, MeTHF (75 mL) and THF (25 mL) were added to the
reaction mixture. The obtained organic layer was extracted with an aqueous solution
(75 mL) containing 100 g/L NaCI. After vigorous stirring of the resulting mixture and
separation of the organic layer, the organic layer was evaporated under reduced
pressure. The peptide was precipitated by addition of acetonitrile ( 100 mL) to the
residue of evaporation. The resulting solid was separated by filtration and dried under
reduced pressure.
Example 8.2 Fmoc cleavage of Fmoc-Phe-Ser(fBu)-xantheneamide
Fmoc-Phe-Ser(fBu)-xantheneamide (2 g) obtained in example 8 .1 was dissolved in a
mixture of NMP ( 15 mL) and TAEA (2 mL). After the reaction completion, as
determined by the method specified in example 6 .1 above, MeTHF ( 100 mL) and THF
( 100 mL) were added to the reaction mixture. It was then extracted :
1) three times with aqueous solution containing 100 g/L NaHC0 3 (30 mL)
2) five times with aqueous solution containing 10 g/L KHS0 (30 mL)
3) five times with aqueous solution containing 20 g/L NaHC0 3 (30 mL)
4) twice with aqueous solution containing 150 g/L NaHC0 3 (30 mL) .
NMP (30 mL) was added and the resulting organic layer was evaporated under
reduced pressure.
Example 8.3 Coupling of Fmoc-Gly-OH with H-Phe-Ser(iBu)-xantheneamide
Fmoc-Gly-OH (0.92 g , 3.1 mmol), TBTU ( 1 .0 g, 3.1 mmol) and TEA (0.9 mL) were
added to the residue of evaporation obtained in example 8.2. The reaction completion
was verified as specified in example 8.1 above.
Example 8.4 Fmoc cleavage of Fmoc-Gly-Phe-Ser(iBu)-xantheneamide
TAEA (3 mL) was added to the reaction mixture obtained in example 8.3. After a
complete conversion was confirmed by the method specified in example 8.1 above,
MeTHF (100 mL) was added to the reaction mixture. It was then extracted:
1) once with aqueous solution containing 100 g/L NaCI (100 mL)
2) four times with mixture of aqueous solution containing 100 g/L NaCI (21 mL) and
NMP (3.7 mL)
3) once with aqueous solution containing 200 g/L NaCI (25 mL).
NMP (30 mL) was added and the resulting organic layer was evaporated under
reduced pressure.
Example 8.5 Coupling of Fmoc-Ala-OH to H-Gly-Phe-Ser(fBu)-xantheneamide
Fmoc-Ala-OH (0.97 g , 3.1 mmol), TBTU ( 1 .0 g , 3.1 mmol) and TEA (0.8 mL) were
added to the residue of evaporation obtained in example 8.4 above. The reaction
completion was verified by the method specified in example 8.1 above.
Example 8.6 Fmoc cleavage of Fmoc-Ala-Gly-Phe-Ser(fBu)-xantheneamide
TAEA (3 mL) was added to the coupling reaction mixture obtained in example 8.5. After
the reaction completion, as determined by the method specified in example 8.1 above,
MeTHF (100 mL) was added to the reaction mixture. It was then extracted:
1) once with aqueous solution containing 100 g/L NaCI (100 mL)
2) four times with mixture of aqueous solution containing 100 g/L NaCI (21 mL) and
NMP (4 mL)
3) once with aqueous solution containing 200 g/L NaCI (25 mL).
NMP (30 mL) was added and the resulting organic layer was evaporated under
reduced pressure.
Example 8.7 Coupling of Fmoc-Pro-OH with H-Ala-Gly-Phe-Ser(fBu)-
xantheneamide
Fmoc-Pro-OH ( 1 .05 g , 3.1 mmol), TBTU ( 1 .0 g , 3.1 mmol) and TEA (0.8 mL) were
added to the residue of evaporation obtained in example 8.6 above. The reaction
completion was verified by the method specified in example 8.1 .
Example 8.8 Fmoc cleavage of Fmoc-Pro-Ala-Gly-Phe-Ser(iBu)-xantheneamide
TAEA (3 mL) was added to the coupling reaction mixture obtained in example 8.7. After
the reaction completion was verified by the method described in example 8.1 above,
MeTHF (100 mL) was added to the reaction mixture. It was then extracted:
1) once with aqueous solution containing 100 g/L NaCI (100 mL)
2) four times with mixture of aqueous solution containing 100 g/L NaCI (42 mL) and
NMP (8 mL)
3) once with aqueous solution containing 200 g/L NaCI (25 mL).
ACN (50 mL) was added to the obtained organic layer and the resulting mixture was
evaporated under reduced pressure to initiate the peptide precipitation. After three
further co-evaporations with ACN (3 x 30 mL), the obtained solid peptide was
separated by filtration and dried under reduced pressure.
Comparative example 1 SPPS of H-Leu-Trp(Boc)-Val-Asn(Trt)-Ser(iBu)-NH upon
usage of Sieber resin and Fmoc and f-Bu as protecting groups
The SPPS was carried out manually on 10 mmol scale upon using Sieber resin (2.3 g)
with loading of 0.61 meq/g. The materials consumed during the peptide synthesis are
listed in the left column of Table 7 below.
The Sieber resin was swollen in DCM (20 mL) for 18 hours and then washed six times
with DMF. The peptide was then assembled onto the resin, using the following
procedure for each amino acid incorporation:
1. Fmoc cleavages: three treatments of 15 min each with mixture of piperidine / DMF
(15 mL, v/v = 2/8).
2 . peptide-resin wash: six times with DMF (10 mL).
3 . amino acid coupling: Fmoc-amino acid (2.1 mmol, 1.5 eq.) with PyBOP (2.1 mmol)
in DMF ( 0 mL) and TEA (0.7 mL). The completeness of the reaction was verified by
the Kaiser test.
4 . peptide-resin wash: six times with DMF (10 mL).
After the final Fmoc cleavage, the resin was washed eight times with DMF ( 0 mL) and
then six times with DCM (10 mL). The peptide was cleaved off the resin with four
successive treatments with DCM / TFA (v/v = 95/5) for 10 min. The resulting solutions
were combined, evaporated under reduced pressure and precipitated in DIPE (20 mL).
The obtained solids were dried under reduced pressure.
605 mg (gross yield = 33%) of H-Leu-Trp(Boc)-Val-Asn(Trt)-Ser(£Bu)-NH 2 were isolated
and the purity of the product was determined to be 57%.
The values in the Table 9 below are given for a 10 mmol synthesis scale carried out by
SPPS vs. LPPS.
Comparative example 1 Example 6
Sieber resin 16.5 g
DCM 142.86 mL MeTHF 540 mL
DMF 8100.00 mL NMP 165 mL
piperidine 257.14 mL TAEA or DEA 28 mL
PyBOP 39.29 g PyBOP 26.02 g
TEA 2 1.43 mL TEA 9.75 mL
Fmoc-Ser(fBu)-OH 5.75 g H-Ser(fBu)-NH 2 2.0 g
Fmoc-Asn(Trt)-OH 8.93 g Fmoc-Asn(Trt)-OH 6.77 g
Fmoc-Val-OH 5.09 g Fmoc-Val-OH 3.85 g
Fmoc-Trp(Boc)-OH 7.86 g Fmoc-Trp(Boc)-OH 6.0 g
Fmoc-Leu-OH 5.36 g Fmoc-Leu-OH 4.0 g
Aq. sol. containing
1565 mL
150 g/L aCI
Aq. sol. containing
700 mL
100 g/L KHS0
Aq. sol. containing
360 mL
50 g/L NaHCOa
Table 9 Materials consumed during the synthesis of H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH according to the methods of example 6 and comparative example 1.
In summary, the time required for the continuous LPPS carried out in example 6 was
nearly the same as the time required for the SPPS carried out in comparative example
1. In addition thereto, the purity and the yield of the target peptide prepared in example
6 were higher while the amounts of consumed solvents and reagents were
considerably lower than in the case of comparative example 1.
Comparative example 2: Continuous LPPS of H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH2 according to Carpino's method
Comparative example 2.1 LPPS of Fmoc-Asn(Trt)-Ser(fBu)-NH
H-Ser(iBu)-NH 2 (2.0 g , 12.5 mmol) and Fmoc-Asn(Trt)-OH (6.8 g , 11.3 mmol) were
added to DCM (50.0 mL) at 20°C. The mixture was stirred for 15 min until the solids
were completely dissolved and further cooled to 10°C. DCC (2.34 g , 11.3 mmol) and
HOBt ( 1.74 g , 1.3 mmol) were added. The reaction was carried out at 10°C and the
conversion was complete after 14 h , as confirmed by HPLC. The reaction progress was
monitored by the following method: 3 m I_ sample of the reaction mixture, diluted 50 fold
in NMP, were analysed according to method MIH-009-RTTG1 described above.
Comparative example 2.2 Removal of the Fmoc protecting group and isolation of
Asn(Trt)-Ser(fBu)-NH 2
To a mixture prepared according to comparative example 2.1 , TAEA (25 mL) was
added and the reaction mixture was stirred at room temperature. The completion of the
Fmoc cleavage was determined by HPLC, using the same method as in example 4.1 .
The DCU was separated by filtration, whereby the filtration process took 6 min. The
resulting filtrate was diluted with DCM to the total volume of 250 mL and subsequently
extracted three times with an aqueous solution containing 100 g/L NaH2P0 4 and
Na2HP0 , pH 5.5 (100 mL).
Comparative example 2.3 Coupling of Fmoc-Val-OH with H-Asn(Trt)-Ser(fBu)-NH2
The organic layer obtained in comparative example 2.2 was evaporated at 30°C under
reduced pressure to a residual volume of 80 mL.
Fmoc-Val-OH (3.85 g , .3 mmol), DCC (2.34 g , 11.3 mmol) and HOBt ( 1 .74 g ,
11.3 mmol) were added. The reaction was carried out at room temperature. After 18 h ,
Fmoc-Val-OH (0.77 g , 2.3 mmol), DCC (0.47 g , 2.3 mmol) and DCM (25 mL) were
added in order to complete the reaction. The reaction progress was monitored by the
following method: 3 L sample of the reaction mixture, diluted 50 fold in DMF, were
analysed according to method MIH-009-RTTG1 described above.
Comparative example 2.4 Removal of Fmoc protecting group. H-Val-Asn(Trt)-
Ser(fBu)-NH2
Fmoc cleavage was performed by addition of TAEA (25 mL) to the reaction mixture
obtained in the comparative example 2.3. The completion of the reaction was verified
by HPLC using the same method as in the comparative example 2.3.
DCU was separated by filtration and rinsed twice with DCM (2 x 25 mL). The obtained
filtrates were combined and diluted to the total volume of 200 mL with DCM. The
solution was extracted three times with an aqueous solution containing 100 g/L
NaH2P0 and Na2HP0 4, pH 5.5 (3 x 100 ml_).
The organic layer was evaporated under reduced pressure at 30°C to a residual
volume of 100 mL.
Comparative example 2.5 Coupling of Fmoc-Trp(Boc)-OH with H-Val-Asn(Trt)-
Ser(fBu)-NH
Fmoc-Trp(Boc)-OH (6.0 g , 1.3 mmol), DCC (2.34 g, 11.3 mmol) and HOBt ( 1 .74 g ,
11.3 mmol) were added to the peptide solution obtained in comparative example 2.4.
The coupling reaction was carried out at room temperature and the reaction time was
18 h . The completion of the reaction was confirmed by HPLC using the same method
as in the comparative example 2.3.
Comparative example 2.6 Removal of the Fmoc protecting group. H-Trp(Boc)-Val-
Asn(Trt)-Ser(fBu)-NH 2
Fmoc cleavage was performed by addition of TAEA (25 mL) to the reaction mixture
obtained in the comparative example 2.5. The completion of the reaction was
determined by HPLC using the same method as in the comparative example 2.3.
DCU was separated by filtration and rinsed twice with DCM (2 x 25 mL). The resulting
filtrates were combined and diluted to the total volume of 200 mL with DCM. The
solution was extracted three times with an aqueous solution containing 100 g/L
NaH2P0 and Na2HP0 4, pH 5.5 (3 x 100 mL).
Since the organic layer became cloudy during the process of extraction, additional
DCM was added to the organic layer, so that its volume was brought to 400 mL.
Nevertheless, some undissolved product was present during the process of extraction.
Therefore, the separation of the layers was difficult and some product was lost in the
aqueous layer.
Comparative example 2.7 Coupling of Fmoc-Leu-OH with H-Trp(Boc)-Val-
Asn(Trt)-Ser(fBu)-NH by LPPS
Fmoc-Leu-OH (4.0 g , 11.3 mmol), DCC (2.34 g , 1 .3 mmol) and HOBt ( 1 .74 g ,
11.3 mmol) were added to the peptide solution obtained in the comparative example
2.6. The coupling reaction was carried out at room temperature and the reaction time
was 18 h . The completion of the reaction was determined by HPLC using the same
method as in the comparative example 2.3.
Comparative example 2.8 Removal of the Fmoc protecting group. H-Leu-
Trp(Boc)-Val-Asn(Trt)-Ser(iBu)-NH 2
Fmoc cleavage was performed by addition of TAEA (25 mL) to the reaction mixture
obtained in the comparative example 2.5. The completion of the reaction was
determined by HPLC using the same method as in the comparative example 2.3.
DCU was separated by filtration and rinsed twice with DCM (2 x 25 mL). The resulting
filtrates were combined and diluted to the total volume of 200 mL with DCM. The
solution was extracted three times with an aqueous solution containing 100 g/L
NaH2P0 and Na2HP0 4, pH 5.5 (3 x 100 mL).
Since the organic layer became cloudy during the process of extraction, additional
DCM was added to the organic layer, so that its volume was brought to 400 mL.
Nevertheless, some undissolved product was present during the process of extraction.
Therefore, the separation of the layers was difficult and some product was lost in the
aqueous layer.
The resulting organic layer was evaporated at 30°C under reduced pressure. The
obtained residual oil was transferred into n-heptane (100 mL) for precipitation. The
resulting solids were isolated by filtration, rinsed three times with n-heptane (3 x 10 mL)
and dried under reduced pressure.
2.6 g of H-Leu-Trp(Boc)-Val-Asn(Trt)-Ser(iBu)-NH 2 (yield = 3 1%) were isolated and the
purity of the final product was 49%.
In summary, the synthetic method described by L . A. Carpino et al. showed several
drawbacks. Because the solubility of the peptides H-Leu-Trp(Boc)-Val-Asn(Trt)-
Ser(fBu)-NH 2 and H-Trp(Boc)-Val-Asn(Trt)-Ser(fBu)-NH 2 in DCM was not sufficient, a
significant amount of these peptides precipitated during the process of extraction in the
interface between the organic layer and the aqueous layer. Despite the volume of the
organic layer was increased to 400 mL, the products were isolated in only a moderate
yield.
In addition thereto, the reaction times of the coupling reaction were longer than in
example 6 . Furthermore, the separation of the resulting DCU by filtration was
demonstrated to be time consuming.
Claims
. 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,
L/,/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 2-methyltetrahydrofuran,
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 : 2-methyltetrahydrofuran 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, toluene, 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 : 2-methyltetrahydrofuran 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 .
3 . The process of claim 2 , whereby the biphasic system obtained in step a) is
characterised by the following volume ratios:
polar aprotic solvent : 2-methyltetrahydrofuran 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 A/./V-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.
7. The process of one or more of claims 1 to 6 , whereby the pH value of the
component a2) ranges from 5 to 8 .
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 /V,/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 0-1 H-benzotriazole
and carbodiimide coupling reagents.
12. The process of claim 10 or 11, whereby a tertiary base is selected from the
group consisting of /V,/V-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 10 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 toluene;
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 iert-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 erminal 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.