U.S. patent application number 15/383598 was filed with the patent office on 2017-04-06 for dipeptide comprising a non-proteogenic amino acid.
The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Caspar Christensen, Jens C. Norrild, Michael Raunkjaer, Rune Severinsen.
Application Number | 20170096450 15/383598 |
Document ID | / |
Family ID | 48696363 |
Filed Date | 2017-04-06 |
United States Patent
Application |
20170096450 |
Kind Code |
A1 |
Christensen; Caspar ; et
al. |
April 6, 2017 |
Dipeptide Comprising a Non-Proteogenic Amino Acid
Abstract
Described is a dipeptide comprising a non-proteogenic amino
acid, methods of making such and methods of using said dipeptide in
a process of making a polypeptide or protein comprising one or more
non-proteogenic amino acids.
Inventors: |
Christensen; Caspar;
(Broenshoej, DK) ; Raunkjaer; Michael; (Bagsvaerd,
DK) ; Severinsen; Rune; (Roskilde, DK) ;
Norrild; Jens C.; (Birkeroed, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Family ID: |
48696363 |
Appl. No.: |
15/383598 |
Filed: |
December 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14368465 |
Jun 24, 2014 |
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PCT/EP2012/076408 |
Dec 20, 2012 |
|
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15383598 |
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61593524 |
Feb 1, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/107 20130101;
C07K 14/605 20130101; C07K 5/06 20130101; C07K 5/06147 20130101;
C07K 4/00 20130101; C07K 14/001 20130101 |
International
Class: |
C07K 5/078 20060101
C07K005/078; C07K 14/605 20060101 C07K014/605; C07K 1/107 20060101
C07K001/107 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 29, 2011 |
EP |
11195998.7 |
Claims
1. A dipeptide of Chem. 1: ##STR00008## wherein R1 is H or an amino
protecting group, and R2 is an amino protecting group; or R1 is a
removable alkyl group, and R2 is H or a removable alkyl group; or
R1 and R2 are jointly forming a ring; R3 is H, or a secondary
ammonium cation, a tertiary ammonium cation or a metal cation
forming a salt with the carbon/late group; and R4 is absent or an
acidic salt.
2. The dipeptide of claim 1, wherein the amino protecting group is
selected from the group consisting of: Boc, Trt, Bpoc, Fmoc, Nsc,
Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps.
3. The dipeptide of claim 1, wherein the removable alkyl group is
selected from the group consisting of: Benzyl and tert-Butyl.
4. The dipeptide of claim 1, wherein, when R1 and R2 are jointly
forming a ring, the jointly formed ring is selected from the group
consisting of: Phatalimide and 1,3,5-dioxazine.
5. The dipeptide of claim 1, wherein R1 is H or an amino protecting
group selected from the group consisting of: Boc, Trt, Bpoc, Fmoc,
Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps, and R2 is an
amino protecting group selected from the group consisting of: Boc,
Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps;
or R1 is a removable alkyl group selected from the group consisting
of: Benzyl and tert-Butyl, and R2 is H or a removable alkyl group
selected from the group consisting of: Benzyl and tert-Butyl; or R1
and R2 are jointly forming a ring selected from the group
consisting of: Phatalimide and 1,3,5-dioxazine; R3 is H, or a
secondary ammonium cation, a tertiary ammonium cation, an alkali
metal cation or an alkaline earth metal cation forming a salt with
the carboxylate group; and R4 is absent or an acidic salt selected
from the group consisting of: A salt of TFA, a salt of HCl, a salt
of HBr and a salt of hydrogensulfate.
6. The dipeptide of claim 1, wherein R4 is absent.
7. The dipeptide of claim 1, wherein R4 is an acidic salt selected
from the group consisting of: A salt of TFA, a salt of HCl, a salt
of HBr and a salt of hydrogensulfate.
8. The dipeptide according to claim 1, which is Fmoc-His-Aib-OH of
Chem. 2 ##STR00009## wherein His is histidine, Aib is the
artificial amino acid 2-aminoisobutyric acid, Fmoc is the
protection group 9-fluorenylmethyloxycarbonyl and R4 is absent or
an acidic salt such as TFA, HCl, HBr or HOAc.
9. The dipeptide according to claim 1, which is the TFA salt of
Fmoc-His-Aib-OH: Fmoc-His-Aib-OH,TFA wherein His is histidine, Aib
is the artificial amino acid 2-aminoisobutyric acid, Fmoc is the
protection group 9-fluorenylmethyloxycarbonyl and TFA is
trifluoroacetic acid.
10. The dipeptide according to claim 1, which is activated by an
activating agent such as a phosphonium based coupling reagent such
as (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP).
11. A method for producing a dipeptide according to claim 1.
12. A method for obtaining a polypeptide or protein comprising one
or more non-proteogenic amino acids, wherein the method comprises a
step of reacting the dipeptide according to claim 1 with a
polypeptide or protein.
13. A method for obtaining a polypeptide or protein according to
claim 12, wherein R1 and/or R2 of said dipeptide is removed in a
deprotection step under basic conditions.
14. A method for obtaining a polypeptide or protein according to
claim 12, wherein pH of the aqueous media is between pH 8.7 and pH
9.4.
15. The method for obtaining a polypeptide or protein according to
claim 12, wherein said dipeptide is reacted with the
.alpha.-N-terminal of the polypeptide or protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/368,465, filed Jun. 24, 2014, which is a 35 U.S.C. .sctn.371
National Stage application of International Application
PCT/EP2012/076408 (WO 2013/098191), filed Dec. 20, 2012, which
claimed priority of European Patent Application 11195998.7, filed
Dec. 29, 2011; this application claims priority under 35 U.S.C.
.sctn.119 of U.S. Provisional Application 61/593,524; filed Feb. 1,
2012; the contents of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention is related to a dipeptide comprising a
non-proteogenic amino acid, methods of making such and methods of
using said dipeptide for producing a polypeptide or protein
comprising one or more non-proteogenic amino acids.
BACKGROUND
[0003] A large number of polypeptides and proteins have been
approved for use in medical practice. The polypeptides and proteins
may be produced in suitable host cells by recombinant DNA
technology or they may be produced synthetically by
well-established peptide synthesis technology. However, native
polypeptides and proteins tend to exhibit high clearance rates
which are unacceptable for many clinical indications where a high
plasma concentration of the polypeptide is required over a
prolonged period of time.
[0004] The native polypeptides and proteins may be altered from the
natural form to analogues and derivatives thereof to change or
enhance certain characteristics. For example non-proteogenic amino
acids (i.e. non-natural amino acids) may be added or substituted
into polypeptides or proteins to e.g. confer a certain protection
against hydrolysis (such as hydrolysis by DPP-IV of GLP-1
peptides).
[0005] Polypeptides containing one or more non-proteogenic amino
acids such as N-terminally modified GLP-1 analogues may be prepared
by introducing the non-proteogenic amino acid(s) via chemical
synthesis in a stepwise manner wherein a coupling step followed by
a deprotection step is applied for each amino acid to be added to
the polypeptide or protein.
[0006] The stepwise synthesis is however time-consuming and
inconvenient, it may lead to the formation of many byproducts,
intermediate purification steps may be needed, and it may result in
a significant amount of racemisation of some amino acid residues
such as histidine residues.
[0007] Alternatively a peptide fragment including the
non-proteogenic amino acid(s) may be coupled to the remaining
polypeptide or protein where the fully protected fragment, such as
e.g. a fragment protected on the N-terminal amino group and the
side chain amino groups, is used in the method.
[0008] Such peptide fragment may, however, not be soluble in
aqueous media limiting its use. Moreover, several deprotection
steps are needed if orthogonal protecting groups are present in the
fragment and if isolated, intermediate purifications may be
necessary between synthetic steps, and problems of intermediate
isolation may occur.
[0009] WO 2009/083549 is related to a method for the preparation of
GLP-1 analogues and derivatives containing non-proteogenic amino
acids. Patent applications WO 2007/147816 A1 and WO 2010/125079 A2
are related to synthetic coupling of peptide fragments. Bourgault,
S. et al. describe in PEPTIDES, vol. 29, no. 6(1), June 2008, pages
919-932 the use of conventional peptide chemistry.
[0010] A peptide fragment for use in an improved method for
obtaining polypeptides containing one or more non-proteogenic amino
acids is still needed.
SUMMARY
[0011] The present invention is related to a dipeptide of Chem.
1:
##STR00001##
wherein [0012] R1 is H or an amino protecting group, and R2 is an
amino protecting group; or [0013] R1 is a removable alkyl group,
and R2 is H or a removable alkyl group; or [0014] R1 and R2 are
jointly forming a ring; [0015] R3 is H, or a secondary ammonium
cation, a tertiary ammonium cation or a metal cation forming a salt
with the carboxylate group; and [0016] R4 is absent or an acidic
salt.
[0017] Also contemplated is a method for producing a dipeptide of
the invention.
[0018] Furthermore, a method for obtaining a polypeptide or protein
comprising one or more non-proteogenic amino acids is described,
wherein the method comprises a step of reacting a dipeptide of the
invention with a polypeptide or protein.
[0019] The invention may also solve further problems that will be
apparent from the disclosure of the exemplary embodiments
DESCRIPTION
[0020] The present invention is related to a dipeptide comprising a
non-proteogenic amino acid, wherein the dipeptide is suitable for
coupling to a polypeptide or protein.
[0021] In one aspect, the dipeptide of the invention has a free
unprotected imidazolyl moiety. In one aspect, the dipeptide of the
invention is in the form of a carboxylic acid salt.
[0022] In one aspect of the invention, the dipeptide is of Chem.
1:
##STR00002##
wherein [0023] R1 is H or an amino protecting group such as, but
not limited to, Boc, Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS,
dNBS, ivDde or Nps, and R2 is an amino protecting group such as,
but not limited to, Boc, Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS,
pNBS, dNBS, ivDde or Nps; or [0024] R1 is a removable alkyl group
such as, but not limited to benzyl or tert-Butyl, and R2 is H or a
removable alkyl group such as, but not limited to, benzyl or
tert-Butyl; or [0025] R1 and R2 are jointly forming a ring such as,
but not limited to, phatalimide or 1,3,5-dioxazine; [0026] R3 is H,
or a secondary ammonium cation, a tertiary ammonium cation or a
metal cation, such as an alkali metal cation or an alkaline earth
metal cation, forming a salt with the carboxylate group; and [0027]
R4 is absent or an acidic salt such as but not limited to a salt of
TFA, HCl, HBr or hydrogensulfate.
[0028] In one aspect of the invention, R1 is H and R2 is an amino
protecting group such as, but not limited to, Boc, Trt, Bpoc, Fmoc,
Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde or Nps; or R1 and R2 are
jointly forming a ring such as, but not limited to, phatalimide or
1,3,5-dioxazine; or R1 is a removable alkyl group such as, but not
limited to benzyl or tert-Butyl and R2 is H or a removable alkyl
group such as, but not limited to benzyl or tert-Butyl.
[0029] In one aspect, R1 is H and R2 is a base sensitive protecting
group such as, but not limited to, Fmoc. In one aspect, R1 is H and
R2 is Fmoc.
[0030] When used herein, the term "amino protecting group" is to be
understood as a protecting group (alternative term: protective
group) known to the person skilled in the art of peptide chemistry
which is introduced into the dipeptide by chemical modification of
an amine (functional) group in order to prevent reaction on the
very same amine during a chemical reaction.
[0031] When used herein, the term "removable alkyl group" is to be
understood as an alkyl group, such as but not limited to a benzyl
group, which can be removed by catalytical hydrogenolysis
methodology. In one aspect of the invention R1 is benzyl and R2 is
H.
[0032] R3 may be hydrogen, a secondary ammonium cation, a tertiary
ammonium cation or a metal cation, wherein the secondary ammonium
cation, the tertiary ammonium cation or the metal cation forms a
salt with the carboxylate group to which it is adjacent. In one
aspect of the invention the metal cation is an alkali metal cation
or an alkaline earth metal cation. In one aspect R3 is selected
from the group consisting of: H, lithium cation, sodium cation,
potassium cation, caesium cation, calcium cation, magnesium cation,
a cation derived from a secondary amine such as but not limited to
N,N-dicyclohexyl ammonium cation, N,N-ditert-butyl amonium cation
or a cation derived from a tertiary amine such as but not limited
to triethylammonium cation.
[0033] In one aspect of the invention R3 is H. In one aspect R3
together with the carboxylate group to which it is adjacent forms a
salt, such as but not limited to, a monovalent salt, a bivalent
salt, or a salt derived from an amine.
[0034] According to an aspect of the invention R3 may be a
secondary ammonium cation, a tertiary ammonium cation or a metal
cation, such as an alkali metal cation or an alkaline earth metal
cation, forming a salt with the carboxylate group to which it is
adjacent. The salt between R3 and the carboxylate group may e.g. be
a monovalent salt, such as but not limited to an alkali salt
including a lithium salt, a sodium salt, a potassium salt or a
caesium salt, a bivalent salt such as but not limited to a calcium
salt or a magnesium salt, a salt derived from a secondary amine
such as, but not limited to, N,N-dicyclohexylamine or
N,N-ditert-butylamine or a salt derived from a tertiary amine such
as, but not limited to, triethylamine.
[0035] It has surprisingly been found by the inventors that the
dipeptide of the invention where R3 is H, or is a secondary
ammonium cation, a tertiary ammonium cation or a metal cation which
forms a salt with the carboxylate group to which it is adjacent,
and R4 is absent or an acidic salt is particularly good in e.g. an
aqueous acylation reaction where the dipeptide is reacted with a
peptide or polypeptide.
[0036] In one aspect of the invention, R4 is absent. In one aspect,
R4 is an acidic component forming a salt with the dipeptide. In one
aspect, R4 is selected from the group consisting of: TFA, HCl, HBr
and hydrogensulfate. In one aspect R4 is TFA.
[0037] In one aspect, the dipeptide of the invention is the
enantiomeric or racemic dipeptide Fmoc-His-Aib-OH of Chem. 2
##STR00003##
wherein * indicates the chiral center of the dipeptide and R4 is
absent or an acidic component such as, but not limited to, TFA,
HCl, HBr or hydrogensulfate, said acidic component forming a salt
with the dipeptide. In one aspect R4 is TFA.
[0038] Herein the term "enantiomeric" in a sample of compounds, is
to be understood as an excess of one enantiomeric form, i.e. either
the L- or the D-form, in the sample. When used herein, the term
"racemic" is to be understood as equivalent amounts of L- and
D-form in a sample of compounds. As a non-limiting example, the
histidine residue of the enantiomeric Fmoc-His-Aib-OH of Chem. 2
may be in the form of L-histidine or D-histidine.
[0039] In one aspect of the invention, the dipeptide of Chem. 1 or
Chem. 2 is activated by an activating agent known by a person
skilled in the art. In one aspect, the dipeptide of Chem. 1 or
Chem. 2 is activated by a phosphonium based coupling reagent. In
one aspect, the phosphonium based coupling reagent is selected from
the group consisting of:
Benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorphosphate (BOP),
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), (7-Azabenzotriazol-1-yloxy) tripyrrolidinophosphonium
hexafluorophosphate (PyAOP),
6-Chloro-benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium
hexafluorophosphate (PyClocK),
O-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl)
phosphonium hexafluorophosphate PyOxP,
O-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl)
phosphonium tetrafluoroborate (PyOxB). In one aspect phosphonium
based coupling reagent is
Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP).
[0040] The term "phosphonium based coupling reagent" is herein to
be understood as a coupling reagent containing a phosphonium salt
which, when reacting in situ with a carboxylic acid, forms an
activated carboxylic acid to be reacted with a polypeptide or
protein.
[0041] The dipeptide of the invention is surprisingly stable and
has a long shelf-life.
[0042] When used herein the term "stabilized" or "stable" when
referring to a dipeptide of the invention refers to a dipeptide
with increased chemical stability, increased physical stability or
increased physical and chemical stability.
[0043] In one aspect a dipeptide of the invention is stable for
more than 6 weeks of usage and for more than 2 years of storage. In
another aspect a dipeptide of the invention is stable for more than
4 weeks of usage and for more than two years of storage. In a
further aspect a dipeptide of the invention is stable for more than
4 weeks of usage and for more than 3 years of storage. In an even
further aspect a dipeptide of the invention is stable for more than
2 weeks of usage and for more than 1 year of storage.
[0044] When used herein the term "ambient temperature" means the
temperature of the surroundings. Under indoors conditions, ambient
temperature is the same as room temperature and may e.g. be
25.degree. C.
[0045] The dipeptide of the invention is easy to handle and the use
thereof in peptide chemistry is easy compared to conventional step
by step solid phase peptide synthesis due to reduced amount of
chemical modification steps, such as deprotection and activation
steps.
[0046] According to an aspect, the dipeptide of the invention may
be used in a method for obtaining a polypeptide or protein
comprising one or more non-proteogenic amino acids.
[0047] In one aspect, the dipeptide according to the invention is
used in a process for coupling covalently the dipeptide to a
polypeptide or protein. In one aspect, the dipeptide is used in a
process for coupling the dipeptide to the N-terminal amine of a
polypeptide or protein. In one aspect, the dipeptide is used in a
process for coupling the dipeptide to nucleophiles in other
molecules not belonging to the chemical group of polypeptides
and/or proteins.
[0048] In one aspect, the polypeptide or protein to which the
dipeptide is coupled consists of proteogenic amino acids, i.e. the
polypeptide or protein to which the dipeptide is coupled does not
comprise any non-proteogenic amino acids.
[0049] In one aspect of the invention, the dipeptide of Chem. 1 or
Chem. 2 is used for coupling said dipeptide to a polypeptide or
protein to form an amide bond between the carboxylate group of the
dipeptide of Chem. 1, i.e. the functional group containing R3, or
the carboxylic acid of Chem. 2 and a free amine of a polypeptide or
protein. In one aspect, the dipeptide of Chem. 1 or Chem. 2 is
reacted to a polypeptide or protein in an aqueous media to form an
amide bond between the carboxylic acid of the dipeptide of Chem. 1
or Chem. 2 and the N-terminal amine of a polypeptide or
protein.
[0050] According to an aspect of the invention, R1 and/or R2 is
removed after completion of the reaction with a polypeptide or
protein. In one aspect, R1 and/or R2 is removed in one chemical
step. In one aspect, R1 and/or R2 is removed in a deprotection step
under basic conditions. In one aspect, R1 and/or R2 is removed in a
deprotection step comprising adding base to the reaction medium. In
one aspect, R1 and/or R2 is removed in a deprotection step
comprising adding an amine to the reaction medium. In one aspect,
R1 and/or R2 is removed in a deprotection step comprising adding
piperidine to the reaction medium.
[0051] In one aspect of the invention, R1 and/or R2 is removed in
situ in one chemical step after completion of the acylation
reaction with a polypeptide or protein.
[0052] According to one aspect, the dipeptide of the invention may
be used in a method for obtaining a polypeptide or protein
comprising one or more non-proteogenic amino acids. In one aspect,
the reaction is carried out in solution. In one aspect, the
activated dipeptide of the invention is reacted with a polypeptide
or protein dissolved in an aqueous media. In one aspect, the
coupling reaction is carried out in a solid phase peptide synthesis
as known by the person skilled in the art. It has by the inventors
been found that by using said method for obtaining a polypeptide or
protein comprising one or more non-proteogenic amino acids and a
histidine N-terminally thereto, a polypeptide or protein product is
obtained in which the histidine residue is not or only slightly
racemized.
[0053] In one aspect the method for obtaining a polypeptide or
protein comprising one or more non-proteogenic amino acids
comprises the following steps:
[0054] 1. activating the dipeptide with a phosphonium based
coupling reagent
[0055] 2. reacting the activated dipeptide with a polypeptide or
protein
[0056] 3. removing the protecting group(s) in situ
whereby the final polypeptide or protein is obtained.
[0057] In one aspect the method for obtaining a polypeptide or
protein comprising one or more non-proteogenic amino acids
comprises the following steps:
[0058] 1. activating the dipeptide of chem. 1 or chem. 2 with a
phosphonium based coupling reagent
[0059] 2. reacting the activated dipeptide with a polypeptide or
protein
[0060] 3. removing the protecting group(s) in situ
whereby the final polypeptide or protein is obtained.
[0061] In one aspect, the activated dipeptide is reacted with a
polypeptide or protein in an aqueous media. As used herein the
terms "aqueous medium" or "aqueous media" include any water based
medium, e.g., water, saline solution, a sugar solution, a
transfusion solution, a buffer, and any other readily available
water-based medium. Further, an aqueous media may contain one or
more water soluble organic solvents such as, but not limited to,
dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP),
dimethylacetamide (DMAC), dimethyl sulfoxide (DMSO), acetonitrile,
dioxane, a water soluble acetal such as e.g. dimethyl acetal,
diethyl acetal or 1,3-dioxalane and a water soluble alcohol such as
e.g. methanol, ethanol, propanol, 2-propanol and
butoxy-2-ethanol.
[0062] In one aspect, the aqueous media in which the activated
dipeptide is reacted with a polypeptide or protein comprises
100-10% water and thus 0-90% further solvent(s), where non-limiting
examples of further solvents e.g. may be selected from the group
consisting of DMF, NMP, DMAC, DMSO, acetonitrile, dioxane, a water
soluble acetal such as e.g. dimethyl acetal, diethyl acetal or
1,3-dioxalane and a water soluble alcohol such as e.g. methanol,
ethanol, propanol, 2-propanol and butoxy-2-ethanol. In one aspect
the aqueous media comprises 80-20% water, such as 60-30% water. In
one aspect the aqueous media comprises 50-30% water. In one aspect
the aqueous media comprises about 50% water. In one aspect the
aqueous media comprises about 40% water. In one aspect the aqueous
media comprises about 30% water.
[0063] In one aspect of the invention the dipeptide is dissolved in
an aprotic organic solvent or a mixture thereof such as, but not
limited to, DMF, NMP, DMAC, DMSO, acetonitrile and dioxane, before
it is added to the aqueous media wherein it is reacted with a
peptide or polypeptide.
[0064] When used herein the term "aprotic" is used for solvents
such as e.g. acetone or dichloromethane which tend to have large
dipole moments, i.e. separation of partial positive and partial
negative charges within the same molecule, and solvate positively
charged species via their negative dipole. Examples of aprotic
solvents include, but is not limited to, dichloromethane (DCM),
tetrahydrofuran (THF), ethyl acetate, acetone, DMF, NMP, DMAC,
DMSO, acetonitrile, dioxane and propylene carbonate.
[0065] In one aspect, the activated dipeptide is reacted with a
polypeptide or protein on solid phase using a procedure known by
the person skilled in the art of peptide chemistry, as e.g.
described in ISBN 0-7167-7009-1 "Synthetic Peptides", ed. Gregory
A. Grant.
[0066] In one aspect the phosphonium based coupling reagent is
PyBOP. In one aspect the protecting group is Fmoc. In one aspect
the final polypeptide or protein is obtained in solution.
[0067] In one aspect the protecting group is removed under basic
conditions. In one aspect the protecting group is removed at a pH
which is at least 7. In one aspect the protecting group is removed
by piperidine, DBU (1,8-diazabicycloundec-7-ene) or
tert-butylamine.
[0068] In one aspect of the invention, the pH of the aqueous
reaction mixture in the acylation step is adjusted to between pH 7
and pH 14. In one aspect pH of the reaction medium is between pH 8
and pH 13. In another aspect the pH is between pH 8 and pH 12. In
another aspect the pH is between pH 8 and pH 10. In another aspect
the pH is between pH 8.3 and pH 9.7.
[0069] The "reaction mixture" is herein to be understood as the
mixture of solvents and reagents used when reacting the dipeptide
of the invention with a polypeptide or protein. The reaction
mixture may be aqueous, i.e. water being present in the reaction
mixture.
[0070] The pH of the reaction mixture may be controlled by means
known to the person skilled in the art. For example a simple pH
paper test (pH stick) or a pH-meter may be used to measure the pH
and acid or base may be added manually to adjust the pH, or a
pH-meter with a feed-back mechanism, which can control the pH of
the solution, may be used.
[0071] Acids suitable for adjusting the pH include but are not
limited to: Hydrochloric acid, sulphuric acid, hydrogen sulphate,
phosphoric acid, citric acid and acetic acid.
[0072] Bases suitable for adjusting the pH include, but are not
limited to: Tertiary amine bases such as, but not limited to,
triethylamine or diisopropylethylamine, N-methylmorpholine,
alkalimetal hydroxides such as, but not limited to, lithium
hydroxide, sodium hydroxide, potassium hydroxide or cesium
hydroxide and alkali carbonates such as, but not limited to,
potassium carbonate, sodium carbonate, lithium carbonate, potassium
hydrogen carbonate, sodium hydrogen carbonate or lithium hydrogen
carbonate.
[0073] In one aspect of the invention the reaction mixture
comprises a buffer. In one aspect of the invention the buffer is
selected from the group consisting of: Phosphate buffer, Sodium
carbonate buffer, Bicine N,N-Bis(2-hydroxyethyl)glycine buffer,
HEPPS buffer (3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic
acid buffer), HEPES buffer
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid buffer), MOPS
buffer (3-(N-Morpholino)propanesulfonic acid buffer) and TEA buffer
(triethylamine buffer). In one aspect of the invention the reaction
mixture comprises a TEA buffer (triethylamine buffer).
[0074] Before addition to the reaction mixture, the dipeptide may
be activated, i.e. the carboxylic acid functionality of the
dipeptide may be converted to an activated ester of said carboxylic
acid. When activating the dipeptide of the invention, the
temperature of the reaction mixture during the activation step may
be between -5.degree. C. and 50.degree. C. such as between
0.degree. C. and 50.degree. C. In one aspect the temperature is
between 5.degree. C. and 40.degree. C. In another aspect the
temperature is between 10.degree. C. and 35.degree. C. In a further
aspect the temperature is between 15 and 25.degree. C. In yet a
further aspect the temperature is about 20.degree. C. during the
activation step.
[0075] The temperature of the reaction mixture during the acylation
step, where the activated dipeptide of the invention is reacted
with a polypeptide or protein, may be between -5.degree. C. and
50.degree. C. such as between 0.degree. C. and 50.degree. C. In one
aspect the temperature is between 5.degree. C. and 40.degree. C. In
another aspect the temperature is between 10.degree. C. and
35.degree. C. In a further aspect the temperature is between 15 and
25.degree. C. In yet a further aspect the temperature is about
20.degree. C.
[0076] The term "polypeptide or protein" as used herein means a
compound composed of at least two constituent amino acids connected
by polypeptide bonds. The constituent amino acids may be chosen
from the group of the amino acids encoded by the genetic code
(proteogenic amino acids) and they may be natural amino acids which
are not encoded by the genetic code, as well as synthetic amino
acids (non-proteogenic amino acids). The 22 proteogenic amino acids
are: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine,
Cystine, Glutamine, Glutamic acid, Glycine, Histidine,
Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine,
Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and
Valine.
[0077] Thus a non-proteogenic amino acid is a moiety which can be
incorporated into a polypeptide or protein via polypeptide bonds
but is not a proteogenic amino acid. Examples are
.gamma.-carboxyglutamate, ornithine, phosphoserine, the D-amino
acids such as D-alanine and D-glutamine. Synthetic non-proteogenic
amino acids comprise amino acids manufactured by chemical
synthesis, i.e. D-isomers of the amino acids encoded by the genetic
code such as D-alanine and D-leucine, Aib (.alpha.-aminoisobutyric
acid), Abu (.alpha.-aminobutyric acid), ornithine, Dap
(2,3-diaminopropionic acid), Dab (2,4-diaminobutanoic acid), Tle
(tert-butylglycine), 3-aminomethyl benzoic acid, anthranilic acid,
des-amino-Histidine, the beta analogs of amino acids such as
.beta.-alanine etc. D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine, (1-aminocyclopropyl) carboxylic acid,
(1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl)
carboxylic acid, (1-aminocyclohexyl) carboxylic acid,
(1-aminocycloheptyl) carboxylic acid or (1-aminocyclooctyl)
carboxylic acid.
[0078] The term "analogue" as used herein referring to a
polypeptide or protein means a modified polypeptide or protein
wherein one or more amino acid residues of the polypeptide or
protein have been substituted by other amino acid residues and/or
wherein one or more amino acid residues have been deleted from the
polypeptide or protein and/or wherein one or more amino acid
residues have been deleted from the polypeptide or protein and or
wherein one or more amino acid residues have been added to the
polypeptide or protein. Such addition or deletion of amino acid
residues can take place at the N-terminal of the polypeptide or
protein and/or at the C-terminal of the polypeptide or protein. A
simple system is often used to describe analogues: For example
[Aib.sup.8, Arg.sup.34]GLP-1(7-37) designates a GLP-1(7-37)
analogue wherein the naturally occurring alanine at position 8 is
substituted with alpha-aminoisobutyric acid and lysine at position
34 has been substituted with arginine. All amino acids for which
the optical isomer is not stated is to be understood to mean the
L-isomer. In aspects of the invention a maximum of 17 amino acids
have been modified. In aspects of the invention a maximum of 15
amino acids have been modified. In aspects of the invention a
maximum of 10 amino acids have been modified. In aspects of the
invention a maximum of 8 amino acids have been modified. In aspects
of the invention a maximum of 7 amino acids have been modified. In
aspects of the invention a maximum of 6 amino acids have been
modified. In aspects of the invention a maximum of 5 amino acids
have been modified. In aspects of the invention a maximum of 4
amino acids have been modified. In aspects of the invention a
maximum of 3 amino acids have been modified. In aspects of the
invention a maximum of 2 amino acids have been modified. In aspects
of the invention 1 amino acid has been modified.
[0079] In one aspect of the invention, the C-terminal of the
derivative according to the invention may be terminated as either
an acid or amide. In one aspect, the C-terminal of the derivative
of the invention is an amide. In another aspect, the C-terminal of
the derivative of the invention is an acid.
[0080] The present invention is especially suitable for making
polypeptides or proteins comprising one or more non-proteogenic
amino acids suitable for treating e.g. diabetes such as
glucagon-like peptides and insulins.
[0081] In one aspect the polypeptide or protein to be reacted with
the dipeptide is a glucagon-like peptide.
[0082] The term "glucagon-like peptide" as used herein means the
glucagon family of polypeptides, exendins and analogues thereof.
The glucagon family of polypeptides are encoded by the
preproglucagon gene and encompasses three small polypeptides with a
high degree of homology, i.e. glucagon (1-29), GLP-1 (1-37) and
GLP-2 (1-33). Exendins are polypeptides expressed in lizards and
like GLP-1, are insulinotropic. Examples of exendins are exendin-3
and exendin-4.
[0083] The terms GLP-1, GLP-2, exendin-3 and exendin-4 are known to
a person skilled in the art. For example "GLP-1 compound" or "GLP-1
polypeptide" as used herein means human GLP-1(7-37), insulinotropic
analogue thereof and insulinotropic derivatives thereof.
Non-limiting examples of GLP-1 analogues are GLP-1(7-36) amide,
Arg.sup.34-GLP-1(7-37), Aib.sup.8Arg.sup.34-GLP-1(7-37),
Gly.sup.8-GLP-1(7-37), Val.sup.8-GLP-1(7-36)-amide and
Val.sup.8Asp.sup.22-GLP-1(7-37). Non-limiting examples of GLP-1
derivatives are desamino-His.sup.7, Arg.sup.26,
Lys.sup.34(N.sup..epsilon.-(.gamma.-Glu(N.sup..alpha.-hexadecanoyl)))-GLP-
-1(7-37), desamino-His.sup.7, Arg.sup.26,
Lys.sup.34(N.sup..epsilon.-octanoyl)-GLP-1(7-37), Arg.sup.26,34,
Lys.sup.38(N.sup..epsilon.-(.omega.-carboxypentadecanoyl))-GLP-1(7-38),
Arg.sup.26,34,
Lys.sup.36(N.sup..epsilon.-(.gamma.-Glu(N.sup..alpha.-hexadecanoyl)))-GLP-
-1(7-36) and Arg.sup.34,
Lys.sup.26(N.sup..epsilon.-(.gamma.-Glu(N.sup..alpha.-hexadecanoyl)))-GLP-
-1(7-37). According to established practice in the art the GLP-1
nomenclature starts at the histidine residue which is referred to
as no. 7, and subsequent amino acid residues are numbered
accordingly, ending with glycine no. 37. Therefore, generally, any
reference herein to an amino acid residue number or a position
number of the GLP-1(7-37) sequence is to the sequence starting with
His at position 7 and ending with Gly at position 37. GLP-1
analogues of the derivatives of the invention may be described by
reference to i) the number of the amino acid residue in native
GLP-1(7-37) which corresponds to the amino acid residue which is
changed (i.e., the corresponding position in native GLP-1), and to
ii) the actual change.
[0084] In one aspect the glucagon-like peptide according to the
invention is dipeptidyl aminopeptidase IV protected. In another
aspect the glucagon-like peptide according to the invention is
dipeptidyl aminopeptidase IV protected.
[0085] The term "dipeptidyl aminopeptidase IV protected" as used
herein means a glucagon-like peptide, e.g. a GLP-1 analogue, which
is more resistant to dipeptidyl aminopeptidase IV (DPP-IV) than the
native compound, e.g. GLP-1(7-37). Such protection may be obtained
by e.g. mutations and/or derivatization of the native compound.
Resistance of a GLP-1 compound towards degradation by dipeptidyl
aminopeptidase IV is determined by the following degradation
assay:
[0086] Aliquots of the GLP-1 compound (5 nmol) are incubated at
37.degree. C. with 1 .mu.L of purified dipeptidyl aminopeptidase IV
corresponding to an enzymatic activity of 5 mU for 10-180 minutes
in 100 .mu.L of 0.1 M triethylamine-HCl buffer, pH 7.4. Enzymatic
reactions are terminated by the addition of 5 .mu.L of 10%
trifluoroacetic acid, and the polypeptide degradation products are
separated and quantified using HPLC analysis. One method for
performing this analysis is: The mixtures are applied onto a Vydac
C18 widepore (30 nm pores, 5 .mu.m particles) 250.times.4.6 mm
column and eluted at a flow rate of 1 ml/min with linear stepwise
gradients of acetonitrile in 0.1% trifluoroacetic acid (0%
acetonitrile for 3 min, 0-24% acetonitrile for 17 min, 24-48%
acetonitrile for 1 min) according to Siegel et al., Regul. Pept.
1999; 79: 93-102 and Mentlein et al. Eur. J. Biochem. 1993; 214:
829-35. Polypeptides and their degradation products may be
monitored by their absorbance at 220 nm (peptide bonds) or 280 nm
(aromatic amino acids), and are quantified by integration of their
peak areas related to those of standards. The rate of hydrolysis of
a GLP-1 compound by dipeptidyl aminopeptidase IV is estimated at
incubation times which result in less than 10% of the GLP-1
compound being hydrolysed.
[0087] The term "insulinotropic" as used herein referring to a
glucagon-like peptide means the ability to stimulate secretion of
insulin in response to an increased plasma glucose level.
Insulinotropic glucagon-like peptides are agonists of the GLP-1
receptor. The insulinotropic property of a compound may be
determined by in vitro or in vivo assays known in the art. The
following in vitro assay may be used to determine the
insulinotropic nature of a compound such as a glucagon-like
peptide. Preferably insulinotropic compounds exhibit an EC.sub.50
value in the below assay of less than 5 nM, even more preferably an
EC.sub.50 value of less than 500 pM.
[0088] Baby hamster kidney (BHK) cells expressing the cloned human
GLP-1 receptor (BHK 467-12A) are grown in DMEM media with the
addition of 100 IU/mL penicillin, 100 .mu.L/mL streptomycin, 10%
foetal calf serum and 1 mg/mL Geneticin G-418 (Life Technologies).
Plasma membranes are prepared by homogenization in buffer (10 mM
Tris-HCl, 30 mM NaCl and 1 mM dithiothreitol, pH 7.4, containing,
in addition, 5 mg/mL leupeptin (Sigma), 5 mg/L pepstatin (Sigma),
100 mg/L bacitracin (Sigma), and 16 mg/L aprotinin
(Calbiochem-Novabiochem, La Jolla, Calif.)). The homogenate is
centrifuged on top of a layer of 41% W/v sucrose. The white band
between the two layers is diluted in buffer and centrifuged. Plasma
membranes are stored at -80.degree. C. until used.
[0089] The functional receptor assay is carried out by measuring
cAMP as a response to stimulation by the insulinotropic polypeptide
or insulinotropic compound. Incubations are carried out in 96-well
microtiter plates in a total volume of 140 mL and with the
following final concentrations: 50 mM Tris-HCl, 1 mM EGTA, 1.5 mM
MgSO.sub.4, 1.7 mM ATP, 20 mM GTP, 2 mM 3-isobutyl-1-methylxanthine
(IBMX), 0.01% w/v Tween-20, pH 7.4. Compounds are dissolved and
diluted in buffer. GTP is freshly prepared for each experiment: 2.5
.mu.g of membrane is added to each well and the mixture is
incubated for 90 min at room temperature in the dark with shaking.
The reaction is stopped by the addition of 25 mL 0.5 M HCl. Formed
cAMP is measured by a scintillation proximity assay (RPA 542,
Amersham, UK). A dose-response curve is plotted for the compound
and the EC.sub.50 value is calculated using GraphPad Prism
software.
[0090] The term "prodrug of an insulinotropic compound" as used
herein means a chemically modified compound which following
administration to the patient is converted to an insulinotropic
compound. Such prodrugs are typically amino acid extended versions
or esters of an insulinotropic compound.
[0091] The term "exendin-4 compound" as used herein is defined as
exendin-4(1-39), insulinotropic fragments thereof, insulinotropic
analogs thereof and insulinotropic derivatives thereof.
Insulinotropic fragments of exendin-4 are insulinotropic
polypeptides for which the entire sequence can be found in the
sequence of exendin-4 and where at least one terminal amino acid
has been deleted. Examples of insulinotropic fragments of
exendin-4(1-39) are exendin-4(1-38) and exendin-4(1-31). The
insulinotropic property of a compound may be determined by in vivo
or in vitro assays well known in the art. For instance, the
compound may be administered to an animal and monitoring the
insulin concentration over time. Insulinotropic analogs of
exendin-4(1-39) refer to the respective molecules wherein one or
more of the amino acids residues have been exchanged with other
amino acid residues and/or from which one or more amino acid
residues have been deleted and/or from which one or more amino acid
residues have been added with the proviso that said analogue either
is insulinotropic or is a prodrug of an insulinotropic compound. An
example of an insulinotropic analog of exendin-4(1-39) is
Ser.sup.2Asp.sup.3-exendin-4(1-39) wherein the amino acid residues
in position 2 and 3 have been replaced with serine and aspartic
acid, respectively (this particular analog also being known in the
art as exendin-3). Insulinotropic derivatives of exendin-4(1-39)
and analogs thereof are what the person skilled in the art
considers to be derivatives of these polypeptides, i.e. having at
least one substituent which is not present in the parent
polypeptide molecule with the proviso that said derivative either
is insulinotropic or is a prodrug of an insulinotropic compound.
Examples of substituents are amides, carbohydrates, alkyl groups,
esters and lipophilic substituents. An example of an insulinotropic
derivative of exendin-4(1-39) and analog thereof is
Tyr.sup.31-exendin-4(1-31)-amide.
[0092] The term "stable exendin-4 compound" as used herein means a
chemically modified exendin-4(1-39), i.e. an analogue or a
derivative which exhibits an in vivo plasma elimination half-life
of at least 10 hours in man, as determined by conventional
methods.
[0093] The term "dipeptidyl aminopeptidase IV protected exendin-4
compound" as used herein means an exendin-4 compound which is more
resistant towards the plasma peptidase dipeptidyl aminopeptidase IV
(DPP-IV) than exendin-4, as determined by the assay described under
the definition of dipeptidyl aminopeptidase IV protected GLP-1
compound.
[0094] The GLP-1 analogues may be such wherein the naturally
occurring Lys at position 34 of GLP-1(7-37) has been substituted
with Arg.
[0095] Also, derivatives of precursors or intermediates of
insulinotropic polypeptides are covered by the invention.
[0096] In one aspect of the invention the glucagon-like peptide is
insulintropic. In a further aspect the insulintropic glucagon-like
peptide is selected from the group consisting of GLP-1, GLP-2,
exendin-4, exendin-3 and analogues and derivatives thereof.
[0097] Conformational stability of protein based drugs is important
for maintaining biological activity and for minimizing irreversible
loss of structure due to denaturation and fibrillation. Especially
large insulinotropic polypeptides and proteins are labile with
respect to conformational change due to complicated refolding
patterns. Also, insulinotropic polypeptides with a known history of
fibrillation, such as GLP-1, are particularly sensitive towards
destabilization of tertiary structure (i.e. formation of a molten
globular state).
[0098] In one aspect, the constituent amino acids of a
glucagon-like peptide according to the invention may be selected
from the group of the amino acids encoded by the genetic code and
they may be natural amino acids which are not encoded by the
genetic code, as well as synthetic amino acids. Natural amino acids
which are not encoded by the genetic code are e.g. hydroxyproline,
.gamma.-carboxyglutamate, ornithine, phosphoserine, D-alanine and
D-glutamine. Synthetic amino acids comprise amino acids
manufactured by chemical synthesis, i.e. D-isomers of the amino
acids encoded by the genetic code such as D-alanine and D-leucine,
Aib (.alpha.-aminoisobutyric acid), Abu (.alpha.-aminobutyric
acid), Tle (tert-butylglycine), .beta.-alanine, 3-aminomethyl
benzoic acid, anthranilic acid.
[0099] In one aspect of the invention, the glucagon-like peptide to
be reacted with the dipeptide according to the invention is a GLP-1
polypeptide. In a further aspect the GLP-1 polypeptide is a GLP-1
peptide having a side chain mentioned in WO 2006/005667, WO
2005/027978, WO 2011/080103 or WO 2006/097537. In one aspect the
GLP-1 polypeptide is GLP-1(9-37); Arg.sup.34-GLP-1(9-37);
Aib.sup.22,Arg.sup.34-GLP-1(9-37); Arg.sup.34,
Pro.sup.37-GLP-1(9-37) or
Aib.sup.22,27,30,35,Arg.sup.34,Pro.sup.37-GLP-1 (9-37)amide having
a side chain mentioned in WO 2006/005667, WO 2005/027978, WO
2011/080103 or WO 2006/097537. In one aspect the GLP-1 polypeptide
is a GLP-1 peptide mentioned in WO 2011/080103 page 84, line 24 to
page 95, line 2, or a GLP-1 analogue mentioned in WO 2006/097537
page 19, line 25 to page 22, line 4.
[0100] In another aspect the glucagon-like peptide to be reacted
with the dipeptide according to the invention is a GLP-1
polypeptide which is selected from the group consisting of: [0101]
Arg.sup.34-GLP-1(9-37); [0102] Aib.sup.22,Arg.sup.34-GLP-1(9-37);
[0103] Arg.sup.34, Pro.sup.37-GLP-1(9-37); [0104]
Aib.sup.22,27,30,35,Arg.sup.34,Pro.sup.37-GLP-1 (9-37)amide; [0105]
N.sup..epsilon.26-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}
ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}eth-
oxy)acetyl]-[Arg.sup.34,Lys.sup.37]GLP-1(9-37)-peptide; [0106]
N.sup..epsilon.26{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy-
)decanoylamino]butyrylamino}-ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acety-
l},
N.sup..epsilon.37-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphe-
noxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}-ethoxy)ethoxy]a-
cetyl}-[Arg.sup.34,Lys.sup.37]GLP-1(9-37)-peptide; [0107]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadeca-
noylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadeca-
noylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}-ethoxy)acetyl][Ar-
g.sup.34,Lys.sup.37]GLP-1(9-37)-peptide amide; [0108]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoy-
lamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoy-
lamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)-acetyl][Arg.s-
up.34,Lys.sup.37]GLP-1(9-37)-peptide amide; [0109]
N.sup..epsilon.26-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamin-
o)butyrylamino]ethoxy}ethoxy)-acetyl],
N.sup..epsilon.37-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamin-
o)butyrylamino]-ethoxy}ethoxy)acetyl][Arg.sup.34,Lys.sup.37]GLP-1(9-37)-pe-
ptide amide; [0110] N.epsilon..sup.26
(17-carboxyheptadecanoyl)-[Arg34]GLP-1-(9-37)-peptide; [0111]
N.sup..epsilon.26-(19-carboxyponadecanoyl)-[Arg34]GLP-1-(9-37)-peptide;
[0112]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyheptadecanoylami-
no)-4(S)-carboxybutyrylamino]-ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acet-
yl][Arg34]GLP-1-(9-37)peptide; and [0113]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(21-Carboxyuneicosanoylamino)-4(S)-
-carboxybutyrylamino]ethoxy)-ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg3-
4]GLP-1-(9-37)peptide.
[0114] In another aspect the glucagon-like peptide to be reacted
with the dipeptide according to the invention is a GLP-1
polypeptide which is selected from the group consisting of: [0115]
Arg.sup.34-GLP-1(9-37); [0116] Aib.sup.22,Arg.sup.34-GLP-1(9-37);
[0117] Arg.sup.34, Pro.sup.37-GLP-1(9-37); and [0118]
Aib.sup.22,27,30,35,Arg.sup.34,Pro.sup.37-GLP-1 (9-37)amide
[0119] In another aspect the glucagon-like peptide to be reacted
with the dipeptide according to the invention is a GLP-1
polypeptide which is selected from the group consisting of: [0120]
N.sup..epsilon.26-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}
ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}eth-
oxy)acetyl]-[Arg.sup.34,Lys.sup.37]GLP-1(9-37)-peptide; [0121]
N.sup..epsilon.26{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[1
0-(4-carboxyphenoxy)decanoylamino]butyrylamino}-ethoxy)ethoxy]acetylamino-
}ethoxy)ethoxy]acetyl},
N.sup..epsilon.37-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphenox-
y)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}-ethoxy)ethoxy]acet-
yl}-[Arg.sup.34,Lys.sup.37]GLP-1(9-37)-peptide; [0122]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadeca-
noylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadeca-
noylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}-ethoxy)acetyl][Ar-
g.sup.34,Lys.sup.37]GLP-1(9-37)-peptide amide; [0123]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(1
1-carboxyundecanoylamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}e-
thoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoy-
lamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)-acetyl][Arg.s-
up.34,Lys.sup.37]GLP-1(9-37)-peptide amide; and [0124]
N.sup..epsilon.26-[2-[2-(2-{2-[(S)-4-Carboxy-4-(1
3-carboxytridecanoylamino)butyrylamino]ethoxy}ethoxy)-acetyl],
N.sup..epsilon.37-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamin-
o)butyrylamino]-ethoxy}ethoxy)acetyl][Arg.sup.34,Lys.sup.37]GLP-1(9-37)-pe-
ptide amide.
[0125] In another aspect the glucagon-like peptide to be reacted
with the dipeptide according to the invention is a GLP-1
polypeptide which is selected from the group consisting of: [0126]
N.sup..epsilon.26
(17-carboxyheptadecanoyl)-[Arg34]GLP-1-(9-37)-peptide; [0127]
N.sup..epsilon.26-(19-carboxyponadecanoyl)-[Arg34]GLP-1-(9-37)-peptide;
[0128]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyheptadecanoylami-
no)-4(S)-carboxybutyrylamino]-ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acet-
yl][Arg34]GLP-1-(9-37)peptide; and [0129]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(21-Carboxyuneicosanoylamino)-4(S)-
-carboxybutyrylamino]ethoxy)-ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg3-
4]GLP-1-(9-37)peptide.
[0130] In one aspect a glucagon-like peptide comprising one or more
non-proteogenic amino acids is obtained by the method of the
invention. In another aspect the glucagon-like peptide comprising
one or more non-proteogenic amino acids obtained by the method of
the invention is a GLP-1 peptide comprising one or more
non-proteogenic amino acids. In a further aspect the GLP-1 peptide
comprising one or more non-proteogenic amino acids is a GLP-1
peptide comprising one or more non-proteogenic amino acids and
having a side chain mentioned in WO 2006/005667, WO 2005/027978, WO
2011/080103 or WO 2006/097537. In one aspect the GLP-1 peptide
comprising one or more non-proteogenic amino acids is
Aib.sup.8,Arg.sup.34-GLP-1(7-37);
Aib.sup.8,22,Arg.sup.34-GLP-1(7-37); Aib.sup.8,Arg.sup.34,
Pro.sup.37-GLP-1(7-37) or
Aib.sup.8,22,27,30,35,Arg.sup.34,Pro.sup.37-GLP-1 (7-37)amide
having a side chain mentioned in WO 2006/005667, WO 2005/027978, WO
2011/080103 or WO 2006/097537. In one aspect the GLP-1 peptide
comprising one or more non-proteogenic amino acids is a GLP-1
peptide mentioned in WO 2011/080103 in the section on page 84, line
24 bridging on to page 95, line 2, or a GLP-1 analogue mentioned in
WO 2006/097537 in the section on page 19, line 25 bridging on to
page 22, line 4.
[0131] In one aspect the GLP-1 peptide comprising one or more
proteogenic amino acids is selected from the group consisting of:
[0132] Aib.sup.8,Arg.sup.34-GLP-1(7-37); [0133]
Aib.sup.8,22,Arg.sup.34-GLP-1(7-37); [0134] Arg.sup.34-GLP-1(7-37);
[0135] Aib.sup.8,Arg.sup.34, Pro.sup.37-GLP-1(7-37); [0136]
Aib.sup.8,22,27,30,35,Arg.sup.34,Pro.sup.37-GLP-1 (7-37)amide;
[0137]
N.sup..epsilon.26-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}
ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}eth-
oxy)acetyl]-[Aib.sup.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37)peptide;
[0138]
N.sup..epsilon.26{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxyphenoxy-
)decanoylamino]butyrylamino}-ethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acety-
l},
N.sup..epsilon.37-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphe-
noxy)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}-ethoxy)ethoxy]a-
cetyl}-[Aib.sup.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37)-peptide; [0139]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadeca-
noylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(15-carboxypentadeca-
noylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}-ethoxy)acetyl][Ai-
b.sup.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37)-peptide amide; [0140]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoy-
lamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoy-
lamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)-acetyl][Aib.s-
up.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37)-peptide amide; [0141]
N.sup..epsilon.26-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamin-
o)butyrylamino]ethoxy}ethoxy)-acetyl],
N.sup..epsilon.37-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamin-
o)butyrylamino]-ethoxy}ethoxy)acetyl][Aib.sup.8,Arg.sup.34,Lys.sup.37]GLP--
1(7-37)-peptide amide; [0142] N.sup..epsilon.26
(17-carboxyheptadecanoyl)-[Aib8,Arg34]GLP-1-(7-37)-peptide; [0143]
N.sup..epsilon.26-(19-carboxyponadecanoyl)-[Aib8,Arg34]GLP-1-(7-37)-pepti-
de; [0144]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyheptadecanoyl-
amino)-4(S)-carboxybutyrylamino]-ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)a-
cetyl][Aib8,Arg34]GLP-1-(7-37)peptide; and [0145]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(21-Carboxyuneicosanoylamino)-4(S)-
-carboxybutyrylamino]ethoxy)-ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Aib8-
,Arg34]GLP-1-(7-37)peptide.
[0146] In one aspect the GLP-1 peptide comprising one or more
proteogenic amino acids is selected from the group consisting of:
[0147] Aib.sup.8,Arg.sup.34-GLP-1(7-37); [0148]
Aib.sup.8,22,Arg.sup.34-GLP-1(7-37); [0149] Arg.sup.34-GLP-1(7-37);
[0150] Aib.sup.8,Arg.sup.34, Pro.sup.37-GLP-1(7-37); and [0151]
Aib.sup.8,22,27,30,35,Arg.sup.34,Pro.sup.37-GLP-1 (7-37)amide.
[0152] In one aspect the GLP-1 peptide comprising one or more
proteogenic amino acids is selected from the group consisting of:
[0153]
N.sup..epsilon.26-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}
ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[10-(4-Carboxyphenoxy)decanoylamino]ethoxy}eth-
oxy)acetyl]-[Aib.sup.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37) peptide;
[0154] N.sup..epsilon.26{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[1
0-(4-carboxyphenoxy)decanoylamino]butyrylamino}-ethoxy)ethoxy]acetylamino-
}ethoxy)ethoxy]acetyl},
N.sup..epsilon.37-{2-[2-(2-{2-[2-(2-{(S)-4-carboxy-4-[10-(4-carboxyphenox-
y)decanoylamino]butyrylamino}ethoxy)ethoxy]acetylamino}-ethoxy)ethoxy]acet-
yl}-[Aib.sup.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37)-peptide; [0155]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(1
5-carboxypentadecanoylamino)butyrylamino]-ethoxy}ethoxy)acetylamino]ethox-
y}ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(1
5-carboxypentadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy-
}-ethoxy)acetyl][Aib.sup.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37)-peptide
amide; [0156]
N.sup..epsilon.26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoy-
lamino)butyrylamino]ethoxy}-ethoxy)acetylamino]ethoxy}ethoxy)acetyl],
N.sup..epsilon.37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(11-carboxyundecanoy-
lamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)-acetyl][Aib.s-
up.8,Arg.sup.34,Lys.sup.37]GLP-1(7-37)-peptide amide; and [0157]
N.sup..epsilon.26-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamin-
o)butyrylamino]ethoxy}ethoxy)-acetyl],
N.sup..epsilon.37-[2-[2-(2-{2-[(S)-4-Carboxy-4-(13-carboxytridecanoylamin-
o)butyrylamino]-ethoxy}ethoxy)acetyl][Aib.sup.8,Arg.sup.34,Lys.sup.37]GLP--
1(7-37)-peptide amide.
[0158] In one aspect the GLP-1 peptide comprising one or more
proteogenic amino acids is selected from the group consisting of:
[0159] N.sup..epsilon.26
(17-carboxyheptadecanoyl)-[Aib8,Arg34]GLP-1-(7-37)-peptide; [0160]
N.sup..epsilon.26-(19-carboxyponadecanoyl)-[Aib8,Arg34]GLP-1-(7-37)-pepti-
de; [0161]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyheptadecanoyl-
amino)-4(S)-carboxybutyrylamino]-ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)a-
cetyl][Aib8,Arg34]GLP-1-(7-37)peptide; and [0162]
N.sup..epsilon.26-[2-(2-[2-(2-[2-(2-[4-(21-Carboxyuneicosanoylamino)-4(S)-
-carboxybutyrylamino]ethoxy)-ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Aib8-
,Arg34]GLP-1-(7-37)peptide.
[0163] The production of peptides and proteins is well known in the
art. Peptides or proteins may for instance be produced by classical
peptide synthesis, e.g. solid phase peptide synthesis using t-Boc
or Fmoc chemistry or other well established techniques, see e.g.
Greene and Wuts, "Protective Groups in Organic Synthesis", John
Wiley & Sons, 1999, "Organic Synthesis on solid Phase",
Florencio Zaragoza Dorwald, Wiley-VCH Verlag GmbH, D-69469
Weinheim, 2000, "Novabiochem Catalog", Merck Biosciences 2006/2007
and "Fmoc Solid Phase Peptide Synthesis", Edited by W. C. Chan and
P. D. White, Oxford University Press, 2000, ISBN 0-19-963724-5. The
peptides or proteins may also be produced by a method which
comprises culturing a host cell containing a DNA sequence encoding
the peptide or protein and capable of expressing the peptide or
protein in a suitable nutrient medium under conditions permitting
the expression of the peptide or protein. For peptides or proteins
comprising non-natural amino acid residues, the recombinant cell
should be modified such that the non-natural amino acids are
incorporated into the peptide or protein, for instance by use of
tRNA mutants.
[0164] The terms "about" or "approximately" as used herein means in
reasonable vicinity of the stated numerical value, such as plus or
minus 10%, or for pH values plus or minus 0.2 or for temperature
plus minus 5 degrees Celsius.
The following is a non-limiting list of embodiments according to
the invention: [0165] 1. A dipeptide of Chem. 1:
##STR00004##
[0165] wherein [0166] R1 is H or an amino protecting group, and R2
is an amino protecting group; or [0167] R1 is a removable alkyl
group, and R2 is H or a removable alkyl group; or [0168] R1 and R2
are jointly forming a ring; [0169] R3 is H, or a secondary ammonium
cation, a tertiary ammonium cation or a metal cation forming a salt
with the carboxylate group; and [0170] R4 is absent or an acidic
salt. [0171] 2. The dipeptide of embodiment 1, wherein the amino
protecting group is selected from the group consisting of: Boc,
Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps.
[0172] 3. The dipeptide of embodiment 1 or 2, wherein the removable
alkyl group is selected from the group consisting of: Benzyl and
tert-Butyl. [0173] 4. The dipeptide of any one of embodiments 1-3,
wherein, when R1 and R2 are jointly forming a ring, the jointly
formed ring is selected from the group consisting of: Phatalimide
and 1,3,5-dioxazine. [0174] 5. The dipeptide of any one of
embodiments 1-4, wherein [0175] R1 is H or an amino protecting
group selected from the group consisting of: Boc, Trt, Bpoc, Fmoc,
Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps, and R2 is an
amino protecting group selected from the group consisting of: Boc,
Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps;
or [0176] R1 is a removable alkyl group selected from the group
consisting of: Benzyl and tert-Butyl, and R2 is H or a removable
alkyl group selected from the group consisting of: Benzyl and
tert-Butyl; or [0177] R1 and R2 are jointly forming a ring selected
from the group consisting of: Phatalimide and 1,3,5-dioxazine;
[0178] R3 is H, or a secondary ammonium cation, a tertiary ammonium
cation, an alkali metal cation or an alkaline earth metal cation
forming a salt with the carboxylate group; and [0179] R4 is absent
or an acidic salt selected from the group consisting of: A salt of
TFA, a salt of HCl, a salt of HBr and a salt of hydrogensulfate.
[0180] 6. The dipeptide of any one of embodiments 1-5, wherein
[0181] R1 is H or an amino protecting group selected from the group
consisting of: Boc, Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS,
dNBS, ivDde and Nps, and [0182] R2 is an amino protecting group
selected from the group consisting of: Boc, Trt, Bpoc, Fmoc, Nsc,
Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps. [0183] 7. The
dipeptide of any one of embodiments 1-5, wherein R1 is H and R2 is
an amino protecting group selected from the group consisting of:
Boc, Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and
Nps. [0184] 8. The dipeptide of any one of embodiments 1-5, wherein
R1 is H and R2 is Fmoc. [0185] 9. The dipeptide of any one of
embodiments 1-5, wherein R1 is an amino protecting group selected
from the group consisting of: Boc, Trt, Bpoc, Fmoc, Nsc, Cbz,
Alloc, oNBS, pNBS, dNBS, ivDde and Nps, and [0186] R2 is an amino
protecting group selected from the group consisting of: Boc, Trt,
Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps.
[0187] 10. The dipeptide of any one of embodiments 1-5, wherein
[0188] R1 is a removable alkyl group selected from the group
consisting of: Benzyl and tert-Butyl, and [0189] R2 is H or a
removable alkyl group selected from the group consisting of: Benzyl
and tert-Butyl. [0190] 11. The dipeptide of any one of embodiments
1-5, wherein [0191] R1 is a removable alkyl group selected from the
group consisting of: Benzyl and tert-Butyl, and [0192] R2 is H.
[0193] 12. The dipeptide of any one of embodiments 1-5, wherein
[0194] R1 is a removable alkyl group selected from the group
consisting of: Benzyl and tert-Butyl, and [0195] R2 is a removable
alkyl group selected from the group consisting of: Benzyl and
tert-Butyl. [0196] 13. The dipeptide of any one of embodiments
1-12, wherein R3 is H. [0197] 14. The dipeptide of any one of
embodiments 1-12, wherein R3 is a secondary ammonium cation forming
a salt with the carboxylate group. [0198] 15. The dipeptide of any
one of embodiments 1-12, wherein R3 is a tertiary ammonium cation
forming a salt with the carboxylate group. [0199] 16. The dipeptide
of any one of embodiments 1-12, wherein R3 is an alkali metal
cation forming a salt with the carboxylate group. [0200] 17. The
dipeptide of any one of embodiments 1-12, wherein R3 is an alkaline
earth metal cation forming a salt with the carboxylate group.
[0201] 18. The dipeptide of any one of embodiments 1-17, wherein R4
is absent. [0202] 19. The dipeptide of any one of embodiments 1-17,
wherein R4 is an acidic salt selected from the group consisting of:
A salt of TFA, a salt of HCl, a salt of HBr and a salt of
hydrogensulfate. [0203] 20. The dipeptide according to any one of
embodiments 1-19, wherein [0204] R1 is H; [0205] R2 is Fmoc; [0206]
R3 is H; and [0207] R4 is absent or an acidic salt such as TFA,
HCl, HBr or HOAc. [0208] 21. The dipeptide according to any one of
embodiments 1-3, which is
[0209] Fmoc-His-Aib-OH of Chem. 2
##STR00005## [0210] wherein His is histidine, Aib is the artificial
amino acid 2-aminoisobutyric acid, Fmoc is the protection group
9-fluorenylmethyloxycarbonyl and R4 is absent or an acidic salt
such as TFA, HCl, HBr or HOAc. [0211] 22. The dipeptide according
to any one of embodiments 1-3, which is the TFA salt of
Fmoc-His-Aib-OH:
[0212] Fmoc-His-Aib-OH,TFA [0213] wherein His is histidine, Aib is
the artificial amino acid 2-aminoisobutyric acid, Fmoc is the
protection group 9-fluorenylmethyloxycarbonyl and TFA is
trifluoroacetic acid. [0214] 23. The dipeptide according to any one
of embodiments 1-22, which is activated by an activating agent such
as a phosphonium based coupling reagent such as
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP). [0215] 24. A method for producing a dipeptide according to
any one of embodiments 1-23. [0216] 25. A method for obtaining a
polypeptide or protein comprising one or more non-proteogenic amino
acids, wherein the method comprises a step of reacting the
dipeptide according to any one of embodiments 1-23 with a
polypeptide or protein. [0217] 26. A method for obtaining a
polypeptide or protein according to embodiment 25, wherein said
dipeptide is reacted with a polypeptide or protein in an aqueous
media. [0218] 27. A method for obtaining a polypeptide or protein
according to embodiment 25, comprising the step of mixing the
dipeptide and the polypeptide or protein in an aqueous media.
[0219] 28. A method for obtaining a polypeptide or protein
according to any one of embodiments 25-27, wherein R1 and/or R2 of
said dipeptide is removed after completion of the reaction with a
polypeptide or protein. [0220] 29. A method for obtaining a
polypeptide or protein according to any one of embodiments 25-28,
wherein R1 and/or R2 of said dipeptide is removed in a deprotection
step under basic conditions. [0221] 30. A method for obtaining a
polypeptide or protein according to any one of embodiments 25-29,
wherein R1 and/or R2 of said dipeptide is removed in a deprotection
step wherein a piperidine is added to the reaction medium. [0222]
31. A method for obtaining a polypeptide or protein according to
any one of embodiments 25-30, wherein R1 and/or R2 of said
dipeptide is removed in situ in one chemical step after completion
of the acylation reaction with a polypeptide or protein. [0223] 32.
A method for obtaining a polypeptide or protein according to any
one of embodiments 25-31 wherein the polypeptide or protein is
N-terminal Fmoc protected, comprising a step wherein the N-terminal
Fmoc protected polypeptide or protein is deprotected in situ.
[0224] 33. A method for obtaining a polypeptide or protein
according to any one of embodiments 25-32, wherein the method
comprises the steps: [0225] 1. activating a dipeptide of Chem. 1 or
Chem 2. with a phosphonium based coupling reagent. [0226] 2.
reacting said activated dipeptide with a polypeptide or protein
[0227] 3. removing the protecting group(s) in situ [0228] whereby
the final polypeptide or protein is obtained. [0229] 34. A method
for obtaining a polypeptide or protein according to embodiment 33,
wherein in step 2 said activated dipeptide is reacted with a
polypeptide or protein in an aqueous media. [0230] 35. A method for
obtaining a polypeptide or protein according to any one of
embodiments 26-34, wherein said aqueous media comprises one or more
water soluble organic solvents selected from the group consisting
of: DMF, NMP, DMAC, DMSO, acetonitrile, dioxane, butoxy-2-ethanol,
a water soluble acetal and a water soluble alcohol. [0231] 36. A
method for obtaining a polypeptide or protein according to
embodiment 35, wherein said one or more water soluble organic
solvents is NMP. [0232] 37. A method for obtaining a polypeptide or
protein according to any one of embodiments 26-36, wherein the
aqueous media in which said activated dipeptide is reacted with a
polypeptide or protein comprises 10-100% water. [0233] 38. A method
for obtaining a polypeptide or protein according to any one of
embodiments 26-36, wherein the aqueous media in which said
activated dipeptide is reacted with a polypeptide or protein
comprises about 40% water. [0234] 39. A method for obtaining a
polypeptide or protein according to any one of embodiments 26-38,
wherein pH of the aqueous media is between pH 7 and pH 14. [0235]
40. A method for obtaining a polypeptide or protein according to
any one of embodiments 26-38, wherein pH of the aqueous media is
between pH 8.7 and pH 9.4. [0236] 41. A method for obtaining a
polypeptide or protein according to any one of embodiments 26-38,
wherein pH of the aqueous media is about pH 9.1. [0237] 42. A
method for obtaining a polypeptide or protein according to any one
of embodiments 26-38, wherein pH of the aqueous media is about pH
9.3. [0238] 43. A method for obtaining a polypeptide or protein
according to any one of embodiments 26-38, wherein the aqueous
media comprises a buffer. [0239] 44. A method for obtaining a
polypeptide or protein according to any one of embodiments 25-38,
wherein the polypeptide or protein comprising one or more
non-proteogenic amino acids is obtained in solution. [0240] 45. A
method for obtaining a polypeptide or protein according to any one
of embodiments 1-23, wherein the polypeptide or protein reacted
with said dipeptide is immobilised on a solid phase. [0241] 46. The
method for obtaining a polypeptide or protein according to any one
of embodiments 1-23, wherein said dipeptide is reacted with the
.alpha.-N-terminal of the polypeptide or protein. [0242] 47. The
method for obtaining a polypeptide or protein according to any one
of embodiments 1-23, wherein the polypeptide or protein to be
reacted with said dipeptide is a GLP-1 peptide.
EXAMPLES
List of Abbreviations
[0242] [0243] ADO: 8-Amino-3,6-dioxaoctanoic acid [0244] Aib:
2-aminoisobutyric acid [0245] Alloc: Allyloxycarbonyl [0246] Boc:
tert-Butoxycarbonyl [0247] Bpoc:
2-(p-biphenylyl)-2-propyloxycarbonyl [0248] Cbz: Benzyloxycarbonyl
[0249] DCM: Dichloromethane [0250] DIC:
N,N'-Diisopropylcarbodiimide [0251] DIPEA:
N,N-Diisopropylethylamine [0252] DME: Dimethyl ether [0253] dNBS:
2,4-Dinitrobenzenesulfonyl [0254] EtOH: Ethanol [0255] Fmoc:
9-fluorenylmethyloxycarbonyl [0256] HBr: Hydrobromic acid [0257]
HCl: Hydrochloric acid [0258] His: Histidine [0259] HOAc: Acetic
acid [0260] HOBt: Hydroxybenzotriazole [0261] ivDde:
1-(4,4-Dimethyl-2,6-dioxocyclo-Hexylidene)-3-methylbutyl [0262]
Lys: Lysine [0263] MeCN: Acetonitrile [0264] Mtt: 4-Methyltrityl
[0265] NMP: N-Methyl-2-pyrrolidone [0266] Nps: o- or
p-Nitrophenylsulfenyl [0267] Nsc:
2-(4-Nitrophenyl)sulfonylethoxycarbonyl [0268] oNBS:
o-Nitrobenzenesulfonyl [0269] OtBu: tert-Butoxy [0270] pNBS:
p-Nitrobenzenesulfonyl [0271] PyBOP:
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
[0272] TBME: tert-Butyl methyl ether [0273] TBTU:
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate [0274] TEA: Trimethylamine [0275] TFA:
Trifluoroacetic acid [0276] TIPS: Triisopropylsilane [0277] Trt:
Triphenylmethyl
Example 1
2-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(1H-imidazol-4-yl)-propan-
oylamino]-2-methyl-propanoic acid trifluoracatate (alternative
name: Fmoc-His-Aib-OH, TFA)
##STR00006##
[0279] Fmoc-His(Trt)-Aib-OH (1 moleq., starting material) was
suspended in DCM (1.5 mL/g) and TIPS (1.7 moleq.) was added. Cooled
(0-10.degree. C.) TFA (3 mL/g) was added and the mixture was
stirred at ambient temperature until the reaction was completed
(High-performance liquid chromatography (HPLC) conversion). DME (1
mL/g starting material) and TBME (11 mL/g starting material) was
added leading to an increase in temperature. The temperature was
slowly allowed to return to room temperature (rt) resulting in
precipitation of a white solid. The mixture was stirred for an
additional 3 hr and filtered. The precipitate was washed twice with
TBME (3 mL/g starting material) and dried overnight in vacuo
affording the de-tritylated dipeptide as TFA-salt in 90% yield.
[0280] Stability studies at freeze (<-15.degree. C..+-.5.degree.
C.), fridge (5.degree. C. .+-.3.degree. C.), and room temperature
(20.degree. C..+-.3.degree. C.) shows more than 24 month
stability.
TABLE-US-00001 NMR data: ##STR00007## Coupling Chemical Shift
Coupling Constants .sup.1H .delta. (ppm) Inte-gral Pattern
.sup.nJ.sub.HH (Hz) H18 1.34 3 H singlet ND H19 1.38 3 H singlet ND
H14 2.93 1 H Double dublet .sup.2J.sub.HH = 14, .sup.3J.sub.HH = 9
H14' 3.08 1 H Double dublet .sup.2J.sub.HH = 14, .sup.3J.sub.HH = 5
H9 4.20 1 H Multiplet ND H10, H10' 4.25 2 H Multiplet ND H13 4.37 1
H multiplet ND H2, H7 7.33 2 H Triplet .sup.3J.sub.HH = 7 H15 7.33
1 H Singlet ND H3, H6 7.42 2 H Triplet .sup.3J.sub.HH = 7 H1, H8
7.68 2 H Dublet .sup.3J.sub.HH = 7 H12 7.71 1 H Dublet
.sup.3J.sub.HH = 8.5 H4, H5 7.90 2 H dublet .sup.3J.sub.HH = 7 H17
8.17 1 H singlet ND H16 8.97 1 H singlet ND H20 12.5 1 H br.singlet
ND
Example 2
N.sup..epsilon.26
[(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazate-
tracontan-1-oyl)] [Aib.sup.8, Arg.sup.34]GLP-1-(7-37) peptide
(alternative name: N.sup..epsilon.26
[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyry-
lamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib.sup.8,
Arg.sup.34]GLP-1-(7-37) peptide)
[0281] Step 1
[0282] In Situ Activation of Dipeptide Fmoc-His-Aib-OH,TFA (Mixture
I):
[0283] To a mixture of Fmoc-His-Aib-OH,TFA (4 moleq.) and
HOBt*H.sub.2O (4 moleq.) was added NMP (4.7 mL/g dipeptide) at
ambient temperature. To the stirred solution TEA was added until pH
8, while the temperature of the solution was kept at ambient
temperature using an ice-bath. A solution of PyBOP (3.9 moleq.) in
NMP (2.4 mL/g dipeptide) was added to the solution containing the
dipeptide at ambient temperature. The pH of the reaction mixture
was adjusted to pH 8 by use of TEA. The mixture was stirred at
ambient temperature for 20 min. prior to the addition to mixture
II.
[0284] Preparation of peptide (N.sup..epsilon.26
[(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazate-
tracontan-1-oyl)] [Arg.sup.34]GLP-1-(9-37) peptide (alternative
name: N.sup..epsilon.26
[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyry-
lamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Arg.sup.34]GL-
P-1-(9-37) peptide for acylation (Mixture II): N.sup..epsilon.26
[(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazate-
tracontan-1-oyl)] [Arg.sup.34]GLP-1-(9-37) was suspended in 40 w/w
% H.sub.2O in NMP (37 g peptide/L solvent mixture). To the cooled
suspension was added TEA until pH 9.3.
[0285] Step 2:
[0286] Acylation of peptide N.sup..epsilon.26
[(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazate-
tracontan-1-oyl)] [Arg.sup.34]GLP-1-(9-37) peptide (Mixture
III):
[0287] Mixture I was added dropwise to mixture II at ambient
temperature. After addition the pH was readjusted to pH 9.3
(pH-meter) by TEA. The mixture was stirred until optimal conversion
(measured by HPLC).
[0288] Step 3:
[0289] Removal of Protecting Group (Fmoc)
[0290] To the mixture III was added piperidine (20
moleq./dipeptide) and the mixture was stirred for 40 min at rt.
[0291] Orbitrap m/z 1028,7 (4+) 1371,4 (3+)
Example 3
Preparation of
N.sup..epsilon.26,N.sup..epsilon.37-bis[(S)-(22-carboxy-33-(4-carboxyphen-
oxy)-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatritriacontan-1-oyl)-
][Aib8,Arg34,Lys37]GLP-1-(7-37) peptide (alternative name:
N.sup..epsilon.26-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphe-
noxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy-
]acetyl]-,
N.sup..epsilon.37-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4--
carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]eth-
oxy]ethoxy]acetyl]-[Aib.sup.8, Arg.sup.34,
Lys.sup.37]-GLP-1-(7-37)-peptide)
[0292] Step 1: Sidechain Acylation
[0293] [Arg.sup.34, Lys.sup.37]-GLP-1-(9-37)-peptide as an
isoprecipitated pellet (15 g, peptide content of approximately 13%
w/w, purity of .about.93%) was suspended in water (50 mL) and NaOH
(aq) (1 M; 1150 .mu.L) was added to dissolve the peptide. The
resulting solution (57 mL) was transferred to a 150 mL reaction
chamber in a titrater setup. The pH of the solution was measured to
10.6. The pH was by the titrator adjusted to 11.3 with dilute NaOH
(aq) (0.5 M, 0.67 mL) and the volume adjusted with water to 60 mL
giving a final peptide concentration of approximately 33 mg/mL.
Assaying the solution to a standard curve of [Arg.sup.34,
Lys.sup.37]-GLP-1-(9-37)-peptide gave a corrected peptide content
of 1.71 g. Using 1.71 g peptide gave a corrected concentration of
the solution of 28.5 mg/mL. Activated sidechain
2,5-dioxopyrrolidin-1-yloxy
(S)-(22-carboxy-33-(4-carboxyphenoxy)-10,19,24-trioxo-3,6,12,15-tetraoxa--
9,18,23-triazatritriacontanate (alternative name:
(4-[9-((S)-1-Carboxy-3-{2-[2-({2-[2-(2-hydroxy-5-oxo-pyrrolidin-1-yloxyca-
rbonylmethoxy)-ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]-ethylcarbamoyl}-pr-
opylcarbamoyl)-nonyloxyybenzoic acid) as 83% active ester, 1.73 g
3.3 eq) was dissolved in NMP (4.8 mL) giving 5.20 mL of solution
(0.63 eq/mL). The NMP solution of the sidechain was added slowly
from a syringe pump at a constant speed keeping pH constant at 11.3
by titrator controlled addition of aq. NaOH (aq) (0.5 M). 3.90 mL
(2.4 eq) of sidechain was added over 1 h and 10 minutes (.about.2
eq/h). Ultra Performance Liquid Chromatography (UPLC) analysis
showed the acylation to be almost complete and addition was
continued to a total of 2.8 eq of sidechain (4.43 mL). During
addition a total of (16.71 mL; 8.4 mmol) 0.5 N NaOH (aq) was added
by the titrator.
[0294] Step 2: Isoprecipitation
[0295] The reaction mixture was transferred with water to
4.times.50 mL centrifuge vials (22 mL in each) and pH in each was
adjusted to 4.8 by addition of conc. acetic acid to give a white
precipitate. EtOH (2.2 mL to a total of app.10% v/v) was added. The
precipitate was centrifuged and used without further purification
in the next step.
[0296] Step 3: Ligation of Dipeptide
[0297] The isoprecipitate was suspended in NMP (48 mL; 50 mg
peptide/mL) and DIPEA (656 .mu.L) was added. pH of the resulting
solution was measured to pH=9.6 in a sample of 100 .mu.L of the
mixture diluted with 900 .mu.L water.
[0298] The water content of the slurry was measured to 1.4% by Karl
Fischer titration. Water (9.12 mL) was added to give a water
content of app. 20%.
[0299] Fmoc-His-Aib-OH.TFA; 1058 mg, 3.5 eq) was activated with
HOBt (248 mg; 3.5 eq), PyBOP (907 mg, 3.325 eq) and triethylamine
(545 .mu.L) in NMP for 15 minutes. pH was 4-5 measured by a wet pH
stick. The mixture was added to the peptide solution and pH was
adjusted with triethylamine to pH 9.3 (by measuring a sample of the
reaction mixture (100 .mu.L) added to water (900 .mu.L). After 1 h
UPLC showed almost complete conversion to the desired product.
[0300] Step 4: Fmoc Deprotection
[0301] To the reaction mixture from the ligation step was added
piperidine (3.35 mL, (5% v/v)) and the mixture was stirred for 25
minutes after which UPLC analysis showed complete conversion to the
product. Water was added to give a 1:1 NMP-water solution and the
pH was adjusted to 8.5 with acetic acid and the product purified by
standard chromatography.
[0302] Analysis:
[0303] RP-UPLC: BEH C18, 1501 mm.COPYRGT.45.degree. C. and 0.1
ml/min; gradient from 30 to 60% 0.04% TFA in MeCN (B eluent) in 30
min then up to 90% B, total run time 38 min. UV@215 nm, 5 Hz Synapt
High Definition Mass Spectrometry (HDMS): positive ES-MS mode from
m/z 200-2500 (1 Hz). V(cap): 3 kV; V(cone): 28V; Desolvation gas
250.degree. C.@750 l/h; cone gas 50 l/h@ 110.degree. C.
[0304] Rt=15.18 min
[0305] Exact mass: 4885,4477 Da; Found: M/4: 1222.35; M/3:
1629.45
Example 4
Preparation of
N.sup..epsilon.26[(S)-(22-carboxy-33-(4-carboxyphenoxy)-10,19,24-trioxo-3-
,6,12,15-tetraoxa-9,18,23-triazatritriacontan-1-oyl)][Aib.sup.8,Arg.sup.34-
,Lys.sup.37]GLP-1-(7-37) peptide (alternative name:
N.sup..epsilon.26-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphe-
noxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy-
]acetyl]-[Aib.sup.8, Arg.sup.34,
Lys.sup.37]-GLP-1-(7-37)-peptide):
[0306] The peptide was synthesised using solid phase peptide
synthesis:
[0307] To the 1.04 g resin Fmoc-Lys(Boc)-Wang-LL (eq. 0.24 mmol/g)
was added in a stepwise manner 4 moleq. Standard Fmoc/OtBu protocol
protected amino acids or Ser-Ser pseudoproline or
Fmoc-L-Lys(Mtt)-OH. The activation amino acid (4 moleq.) in
cartridge was reacted for 7 minutes with 4 moleq DIC and 4 moleq.
HOBt in NMP. The resulting peptide was transferred to a reaction
vessel with resin and reacted for 30 min. DIPEA (4 moleq.) was
added and the reaction was continued for 30 min. The resin was
flowwashed with NMP and subsequently deprotected using 20%
Piperidine (10 ml, 20 min). The resin was again flowwashed with
NMP.
[0308] MTT Deprotection:
[0309] The resin was washed in DCM.
1,1,1,3,3,3-hexafluoro-2-propanol (10 ml for 10 min) was added and
the resin was drained. The deprotection procedure was repeated
overall four times. The resin was washed with DCM, followed by
NMP.
[0310] To a mixture of
(S)-22-(tert-butoxycarbonyl)-33-(4-(tert-butoxycarbonyl)phenoxy)-10,19,24-
-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatritriacontan-1-oic acid,
HOBt (4 moleq.) and DIPEA (4 moleq.) in NMP (10 ml) was added, and
TBTU (3.8 moleq.) was added as a solid, and the mixture was shaken
for 15 min. before added to the resin. After 2. hr. the resin was
drained and flowwashed with NMP and DCM.
[0311] The peptidyl resin was swelled in NMP (10 mL) for 10 min at
ambient temperature. The vessel was drained. 20 vol % piperidine in
NMP (20 mL) was added to the resin. The mixture was swelled for 20
min at ambient temp. The dipeptide Fmoc-L-His-Aib-OH.TFA (580 mg)
and HOBt.H.sub.2O (153 mg) were placed in a 20 mL vial. NMP (4 mL)
was added. To the mixture was added TEA (650 .mu.L) until pH 8
(pH-stick). To the reaction mixture was added a solution of PyBOP
(500 mg) in NMP (4 mL). To the reaction mixture was again added TEA
(200 .mu.L) until pH 8 (pH-stick). The mixture was stirred for 35
min at ambient temp. The vessel was drained. 20 vol % piperidine in
NMP (20 mL) was again added to the resin (double deprotection). The
mixture was swelled for 20 min at ambient temp and the resin was
drained. The resin was flow washed with NMP (50 mL), DCM (50 mL)
and 3 times NMP (50 mL). Finally the resin was flowwashed with DCM
and drained. The peptide was cleaved from the resin by a mixture of
TFA, H.sub.2O and TIPS (95%, 2.5%, 2.5%) for 3 hr. The resulting
cleaved peptide was precipitated in diethylether and isolated by
filtration.
[0312] TOF MS ES+: m/z, found m/4 (1045.54), calculated m/4
(1045.5)
[0313] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit of the invention.
* * * * *