Note: Descriptions are shown in the official language in which they were submitted.
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NON-ENZYMATIC CHEMICAL AMIDATION PROCESS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit ofpriority under 35 U.S.C. ~ 119(e) to U.S.
Provisional Application Ser. No.: 60/089,635 filed June 17, 1998, which is
herein
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
This invention relates to non-enzymatic methods of preparing peptides amidated
at the C-terminal position. Such peptide C-terminal amides are useful in
various
pharmaceutical compositions such as, for example, antimicrobicides.
DESCRIPTION OF THE RELATED ART
Recombinant DNA techniques have been used to produce many naturally
occurring peptides. In addition, recombinant DNA techniques have made possible
the
selection, amplification and modification of such peptides. For example,
alteration of
the DNA coding sequence results in changes in the amino acid sequence of
recombinantly produced peptides, thereby altering their function. However,
some
modifications to a recombinantly produced peptide cannot be accomplished by
altering
the DNA sequence.
The C-terminal carboxyl group in many naturally occurring antimicrobial
peptides, for example, often exists as an amide (R-CONHZ). In this form, C-
terminal
amidated peptides exhibit improved antimicrobial activity. Cuervo, J.H. et
al., Peptide
Res., l, pp. 81-86, 1988; Lee, J.Y. et al., Proc. Natl. Acad. Sci. 86, pp.
9159-9162,
1989. However, it is not possible to produce a C-terminal amidated peptide
using
current recombinant expression techniques. Rather, peptides are converted to
the
amidated form using biological means subsequent to recombinant expression of
the
peptide in a peptidylglycine form.
One such biological means is the in vivo amidation of the C-terminal carboxyl
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2
group of a precursor peptide using a C-terminal a-amidating enzyme. In such a
reaction, the precursor peptide is a peptidylglycine substrate of the general
formula X-
R-Gly, where Gly represents a glycine residue, R represents an amino acid, and
X
represents the remaining part of the peptide. Treatment of the peptidylglycine
substrate
with a C-terminal a-amidating enzyme yields a peptide C-terminal amide of
general
formula X-R-CONH2. Peptidylglycine a-amidating enzymes have been used to
effect
such an amidation. Bradbury, A.F. et al., Nature, 298 (5875), pp. 686-688,
1982.
However, such an enzymatic method is limited to those peptides that possess a
C-
terminal glycine residue. Other a-amidating enzymes have been characterized
and
been used to amidate recombinantly produced peptides in vitro. Eipper, B.A. et
al.,
Peptides, 4(6), pp. 921-8, 1983; Murthy, A.S., J. Biol. Chem. 261, pp. 1815-
1822,
1986; Engels, J.W. et al., Protein Eng. 1, pp. 195-199, 1987.
Disadvantageously,
enzymatic amidation methods are time consuming, costly, unpredictable, and,
oftentimes, substrate specific. In addition, such methods require an
additional step of
1 S purification of the final amidated peptide.
Reductive cleavage of nitrogen-nitrogen bonds with Raney nickel and hydrazine
to form amides has been achieved with non-peptide substrates such as N, N'-
diacylated
hydrazines. Robinson et al., Canadian Journal of Chemistry, Vol. 39, pp. 1171-
1173,
1961. Few non-enzymatic, chemical amidation procedures for production of
peptide C-
terminal amides are known in the art. U.S. Patent 5,589,364 describes the non-
enzymatic chemical amidation of peptide methyl esters in a single step using
ammonia.
However, the use of ammonia can be quite harsh and thus prohibits the
application of
such a non-enzymatic chemical amidation method with relatively non-reactive
esters
(e.g. simple alkyl esters and non-activated aryl esters) and/or with base-
sensitive
substrates under mild conditions without the formation of undesired side
products.
Furthermore, nonoptimal reaction conditions such as elevated pressures must be
used
and slower reaction rates lead to the formation of undesired lactams and
polymeric by-
products.
Accordingly, there still exists a need in the art for a simpler, gentler, more
versatile, non-enzymatic method for the preparation of peptide C-terminal
amides that
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3
can be applied to a variety of peptide substrates.
SUMMARY OF THE INVENTION
The invention answers this need by providing a non-enzymatic method of
preparing a peptide C-terminal amide comprising the steps of: reacting a
peptide C-
terminal carboxylic acid ester with an N-amino or N-oxy amine derivative to
form the
corresponding peptide C-terminal N-amino or N-oxy amide derivative; and
converting
the corresponding peptide C-terminal N-amino or N-oxy amide derivative under
conditions sufficient to the corresponding peptide C-terminal amide.
The invention also provides a non-enzymatic method of preparing a peptide C-
terminal amide comprising the steps of reacting a fusion peptide C-terminal
carboxylic
acid ester with an N-amino or N-oxy amine derivative to form a peptide C-
terminal N-
amino or N-oxy amide derivative; and converting the peptide C-terminal N-amino
or
N-oxy amide derivative under conditions sufficient to the corresponding
peptide C-
terminal amide.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Reaction scheme for conversion of
(a) MSI-344 (GIGKFLKKAKKFGKAFVKILKK corresponding to SEQ ID NO:1 )
to (b) MSI-78: (GIGKFLKKAKKFGKAFVKILKK corresponding to SEQ ID N0:2
which contains a C-terminal amide group).
Figure 2: HPLC analysis following conversion of MSI-344-methyl ester to
MSI-1918 (GIGKFLKKAKKFGKAFVKILKK corresponding to SEQ 1D NO: 5 which
contains a C-terminal hydroxamic acid group) with hydroxylamine.
Figure 3: HPLC analysis following reduction of MST-1918 [SEQ ID NO: 5] to
MSI-78 [SEQ ID N0:2] with hydrazine and Raney nickel (Ni).
Figure 4: HPLC analysis following conversion and cleavage of MSI-1850-
methyl ester to MSI-1918 [SEQ ID NO: 5].
Figure 5: Scheme depicting downstream processing methods for MSI-1922
(MKAIFVLLEHHHHHLKDAQTNSSST>l~NNNNNNNNLGIEGRISEFNGIGKFLKK
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AKKFGKAFVKILKK corresponding to SEQ ID N0:4).
Figure 6: HPLC analysis following conversion and cleavage of MSI-1922-
methyl ester to MSI-1918 [SEQ ID NO: 5].
Figure 7: HPLC analysis following reduction of MSI-1918 [SEQ ID NO: S] to
MSI-78 [SEQ ID N0:2) with hydrazine and Raney nickel as described in Example
3.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a non-enzymatic method of preparing a peptide C-
terminal amide comprising the steps of reacting a peptide C-terminal
carboxylic acid
ester with an N-amino or N-oxy amine derivative to form the corresponding
peptide C-
terminal N-amino or N-oxy amide derivative; and converting the corresponding
peptide
C-terminal N-amino or N-oxy amide derivative under conditions sufficient to
the
corresponding peptide C-terminal amide.
According to the invention, the peptide C-terminal carboxylic acid ester may
be
any synthetic, recombinant, or naturally occurring peptide C-terminal
carboxylic acid
ester, i.e. any synthetic, recombinant, or naturally occurring peptide having
a carboxylic
acid moiety at the C-terminal position. In a preferred embodiment of the
invention, the
peptide C-terminal carboxylic acid ester is of general formula (I):
R,-C(O)ORZ (I).
In formula (I), R, is an amino acid sequence and R2 is a linear or branched,
substituted
or unsubstituted C~-Cio alkyl group, or a substituted or unsubstituted C3-C,
cycloalkyl,
C~-C,o aralkyl, C6-C,2 aryl or heteroaryl group. R, may be any amino acid
sequence. In
a preferred embodiment of the invention, to avoid competing amidation
reactions, the
amino acid sequence of R, does not contain any amino acids having carboxylic
acid
group containing side chains (e.g. glutamic acid, aspartic acid). In addition,
as would
be understood by one of skill in the art, the amino acid sequence may be
chosen to
avoid problems such as, for example, catalyst poisoning by amino acid groups
containing, for example, disulfide linkages. Preferably, R, is a about 2-150
amino acid
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sequence, more preferably, a about 2-110 amino acid sequence, and most
preferably, a
about 2-50 amino acid sequence. Preferably, RZ is a linear or branched,
substituted or
unsubstituted C~-Coo alkyl group, more preferably, a methyl group. Possible
heteroatoms for the heteroaryl group include N, O, and S. Possible
substituents include
those substituents known in the art (e.g. alkyl, hydroxyl, vitro, chloro,
bromo, iodo,
cyano, amino) as long as they do not interfere with the overall C-terminal
amidation
process. In a more preferred embodiment of the invention, the peptide C-
terminal
carboxylic acid ester is a recombinant peptide C-terminal carboxylic acid
ester. In
another more preferred embodiment of the invention, the peptide C-terminal
carboxylic
acid ester is a recombinant peptide C-terminal carboxylic acid ester of
formula (I)
where R, has the following amino acid sequence:
H2N-GIGKFLKKAKKFGKAFVKILKK [SEQ ID NO:1].
The peptide C-terminal carboxylic acid ester for use in a method of the
invention may
be prepared by any means known in the art. For example, the peptide C-terminal
carboxylic acid ester may be prepared from the corresponding peptide C-
terminal
carboxylic acid precursor using esterification methods known in the art.
Bodanszky et
al., Peptide Synthesis, 2nd Edition, John Wiley & Sons, 1976. The peptide C-
terminal
carboxylic acid precursor may be any peptide C-terminal carboxylic acid. As
with the
peptide C-terminal carboxylic acid ester, the peptide C-terminal carboxylic
acid may be
a synthetic peptide, a recombinant peptide or a naturally occurnng peptide, as
understood by one of skill in the art. Preferably, the peptide C-terminal
carboxylic acid
is a recombinant peptide C-terminal carboxylic acid.
The N-amino or N-oxy amine derivative may be any N-amino or N-oxy amine
derivative having at least one free amine hydrogen known in the art, i. e. any
amine
having at least one free amine hydrogen and is N-substituted with an amino or
oxy
group. In a preferred embodiment of the invention, the N-amino or N-oxy amine
derivative is of formula (II):
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~~s)(~s) (II).
In formula (II), Y is NR4 or O and R3, R4, and Rs are, independently,
hydrogen, a linear
or branched, substituted or unsubstituted C~-Coo alkyl group, or a substituted
or
unsubstituted C3-C~ cycloalkyl, C,-C,o aralkyl, C6-C,Z aryl or heteroaryl
group.
Possible heteroatoms of the heteroaryl group and possible substituents are
each as
described above. In a preferred embodiment of the invention, R3 is hydrogen, Y
is O
and Rs is hydrogen, methyl or a benzyl group. In another preferred embodiment
of the
invention, R3 is hydrogen, Y is NR4 and R4 and Rs are each a hydrogen. In a
more
preferred embodiment of the invention, R3 is hydrogen, Y is O and Rs is
hydrogen (i.e.
hydroxylamine).
According to the invention, the high nucleophilicity of the N-amino or N-oxy
amine derivative offers the advantage that it may be used to rapidly convert
relatively
non-reactive peptide C-terminal carboxylic acid esters at about neutral pH to
a peptide
1 S C-terminal N-amino or N-oxy amide derivative, as described below, without
having to
protect the side chain amino or, if present, N-terminal amino groups or
arginine
guanidino side chain groups on other amino acid residues. ~ In addition, the
relatively
low pKa (about 6.0-9.0, preferably, about 6.0-8.0) of the N-amino or N-oxy
amine
derivative allows for reaction under about neutral conditions, permits
reaction with base
sensitive substrates, and prevents competitive amidation reactions.
A peptide C-terminal N-amino or N-oxy amide derivative may be any synthetic,
recombinant or naturally occurring peptide C-terminal N-amino or N-oxy amide
derivative, i. e. any synthetic, recombinant or naturally occurring peptide
having an N-
amino or N-oxy amide moiety at the C-terminal position. Most preferably, a
peptide C-
terminal N-amino or N-oxy amide derivative is any recombinant peptide C-
terminal N-
amino or N-oxy amide derivative. In a preferred embodiment of the invention,
the
peptide C-terminal N-amino or N-oxy amide derivative is of formula (III):
R,-C(O)N(R3)(YRs) (III).
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In formula (III), R,, R3, Y, and RS are each as described above.
According to the invention, a peptide C-terminal N-amino or N-oxy amide
derivative may be prepared by reaction of an N-amino or N-oxy amine derivative
with a
peptide C-terminal carboxylic acid ester in a molar ratio of about 1:1, each
as described
S above. More preferably, an excess of the N-amino or N-oxy amine derivative
is used to
react with a peptide C-terminal carboxylic acid ester. The concentration of
the reactant
N-amino or N-oxy amine derivative ranges between about 2M - neat, preferably,
between about 2-16 M, more preferably, between about 4-10 M. The reaction is
conducted at a pH range of about 7-11, preferably, about 8-9.5, at about
ambient
temperature until formation of the peptide C-terminal N-amino or N-oxy amide
derivative, as described herein, is complete. Completion of formation of the
peptide C-
terminal N-amino or N-oxy amide derivative may be determined by analytical
techniques and methods known in the art (e.g. high performance liquid
chromatography
(HPLC), gas chromatography, capillary electrophoresis). As recognized by one
of skill
in the art, the addition of solvents including, for example, denaturants (e.g.
urea,
guanidine) to solubilize the reactant peptide as necessary is envisioned as
well. The
newly formed peptide C-terminal N-amino or N-oxy amide derivative may also be
further purified by means known in the art such as, for example,
chromatography
techniques (e.g. reverse phase HPLC, ion exchange chromatography).
The peptide C-terminal amide may be a synthetic, recombinant or naturally
occurring peptide C-terminal amide, i.e. any synthetic, recombinant or
naturally
occurring peptide having an amide moiety at the C-terminal position. Most
preferably,
the peptide C-terminal amide is a recombinant peptide C-terminal amide. In a
preferred
embodiment of the invention, the peptide C-terminal amide is of formula (IV):
R,-C(O)NHR3 (IV).
In formula (IV), R, and R3 are each as described above.
According to a method of the invention, conversion of a peptide C-terminal N-
amino or N-oxy amide derivative, as described above, is effected "under
conditions
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sufficient" to the desired corresponding peptide C-terminal amide, as
described above.
"Under conditions sufficient" may include any reaction conditions that effect
conversion of a N-amino or N-oxy amide moiety to an amide moiety. In a
preferred
embodiment of the invention, the peptide C-terminal amide results from the
cleavage of
the N-N or N-O bond of the C-terminal N-amino or N-oxy amide moiety of the
peptide
C-terminal N-amino or N-oxy amide derivative, as described above. In a more
preferred embodiment of the invention, such "under conditions sufficient"
include
reductive reaction conditions. Examples of suitable reductive reaction
conditions
include, but are not limited to, catalytic (e.g. Pd, Pt, Ni, Rh, Ru)
hydrogenation (Babu
et al., Indian J. Technol., Vol. 5, pp. 327-328, 1967; exchange or transfer
hydrogenation (e.g. hydrazine with catalysts containing Ni (e.g. Raney Ni),
Fe, Cu, Sn,
Mg, or Zn) (Robinson et al., Can. .l. Chem. 39, pp. 1171-1173, 1961; Zajac,
W.W. et
al., J. Org. Chem. 36, pp. 3539-3541, 1971); cleavage by sodium or lithium in
ammonia
(Denmark, S.E. et al., J. Org. Chem. 55, pp. 6219-6223, 1990); metal-acid
reduction
(e.g. reductants containing Zn, Sn, or Fe in acid) (Kirk and Othmer,
Encyclopedia of
Chemical Technology, Vol 2., Interscience, New York, 1963); and
electrocatalytic
reductions using electrodes containing Fe, Cu, Sn, or Ni (Cyr, A. et al.,
Electrochim.
Acta, 35, pp. 147-152, 1990). More preferably, the peptide C-terminal amide is
formed
by means of catalytic hydrogenation, exchange or transfer hydrogenation, or
metal-acid
reduction. Most preferably, conversion to the peptide C-terminal amide is
achieved by
exchange or transfer hydrogenation by addition of hydrazine and Raney Ni. In
the
event that the N-amino or N-oxy amine derivative is hydrazine (i.e. in formula
(II), R3
is hydrogen, Y is NR4 and R4 and Rs are each a hydrogen), then conversion of
the
peptide C-terminal carboxylic acid ester to the peptide C-terminal amide may
be
achieved as a one-pot synthesis by the addition of a catalyst such as, for
example,
Raney Ni and, if necessary, additional hydrazine, once formation of the
peptide C-
terminal hydrazide (i.e. the reaction product of a peptide C-terminal
carboxylic acid and
hydrazine) is determined to be complete, as described above. As recognized by
one of
skill in the art, reaction conditions (e.g. reactant amounts, temperature,
time, and pH)
may vary depending upon the type of substrate and reaction methods used. In
general,
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however, the amount of reductant used ranges between about a molar equivalent
to an
excess based on the peptide C-terminal N-amino or N-oxy amide derivative, as
described above. The reaction temperature may range between about 0-
60°C,
preferably, between about ambient temperature to about 45 °C. The pH of
the reaction
mixture may range between about 6-10, preferably, between about 7-9. The
reaction
pH may be adjusted and maintained by the addition of a buffer such as, for
example,
ammonium chloride. As recognized by one of skill in the art, reaction time may
vary
depending on the type and amount of catalyst used but, generally, formation of
the
desired peptide C-terminal amide is complete within about 24 hours.
The invention also provides a non-enzymatic method of preparing a peptide C-
terminal amide comprising the steps of reacting a fusion peptide C-terminal
carboxylic
acid ester with an N-amino or N-oxy amine derivative to form a peptide C-
terminal N-
amino or N-oxy amide derivative; and converting the peptide C-terminal N-amino
or
N-oxy amide derivative under conditions sufficient to the corresponding
peptide C-
1 S terminal amide.
According to the invention, the fusion peptide C-terminal carboxylic acid
ester
may be any synthetic or recombinant fusion peptide C-terminal carboxylic acid
ester
comprising a fusion partner segment and a peptide C-terminal carboxylic acid
ester
segment. In a preferred embodiment of the invention, the fusion peptide C-
terminal
carboxylic acid ester is of formula (V):
R~-R5-C(O)ORZ (V).
In formula (V), RZ is as described above, R6 may be any synthetic,
recombinant, or
naturally occurring amino acid sequence "beginning" (reading from left to
right) (i.e.
the N-terminal position of the amino acid sequence) with an unhindered amino
acid, as
described below, and R, is a fusion partner. Preferably, R6 is a recombinant
amino acid
sequence. Preferably, the amino acid sequence of R6 is a about 5-50 amino acid
sequence, more preferably, a about 8-40 amino acid sequence, and most
preferably, a
about 10-30 amino acid sequence. The fusion partner segment may be any fusion
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partner known in the art and would be understood by one of skill in the art to
be a
peptide. Examples of suitable unhindered amino acids include, but are not
limited to,
glycine, alanine or serine. Preferably, the unhindered amino acid is glycine.
According
to the invention, the junction (i.e. point of connection) of the fusion
partner segment
5 and the peptide segment may be any consecutive amino acid sequence that may
be
cleaved with an N-amino or N-oxy amine derivative, as described herein.
Preferably,
the fusion partner segment "ends" (reading from left to right) (i. e. the C-
terminal
position of the fusion partner segment) with an asparagine or aspartic acid
amino acid
and the peptide segment "begins" (reading from left to right) with a
unhindered amino
10 acid, as described above. In a preferred embodiment of the invention, the
junction of
the fusion partner segment and the peptide segment is a consecutive asparagine
and
glycine amino acid sequence. The peptide C-terminal carboxylic acid ester
segment
may be any peptide C-terminal carboxylic acid ester as described above.
A fusion peptide C-terminal carboxylic acid ester may be prepared by means
known in the art including, for example, esterification of the corresponding
fusion
peptide C-terminal carboxylic acid precursor. Bodanszky et al., Peptide
Synthesis, 2nd
Edition, John Wiley & Sons, 1976. A fusion peptide C-terminal carboxylic acid
precursor may be any fusion peptide C-terminal carboxylic acid precursor
comprising a
fusion partner segment, as described above, and a peptide C-terminal
carboxylic acid
segment (i.e. a peptide C-terminal carboxylic acid ester segment, as described
above,
where the carboxylic acid ester moiety is a carboxylic acid moiety). A fusion
peptide
C-terminal carboxylic acid precursor may be prepared by any means known in the
art.
For example, a fusion peptide C-terminal carboxylic acid precursor may be
prepared by
recombinant means known in the art. In an alternative example, a fusion
peptide C-
terminal carboxylic acid precursor may be prepared by from a fusion partner
and a
synthetic, recombinant or naturally occurnng peptide C-terminal carboxylic
acid
"beginning" with an unhindered amino acid, each as described above.
Reaction of a fusion peptide C-terminal carboxylic acid ester with an N-amino
or N-oxy amine derivative results in the formation of a peptide C-terminal N-
amino or
N-oxy amide derivative, each as described above. Advantageously, according to
a
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method of the invention, both cleavage of the fusion protein at the junction
of the
fusion partner segment and peptide segment, each as described above, and
conversion
of the C-terminal ester moiety of the peptide to a C-terminal N-amino or N-oxy
amide
moiety may be achieved in a single step.
S According to the invention, a peptide C-terminal N-amino or N-oxy amide
derivative may be prepared by reaction of a fusion peptide C-terminal
carboxylic acid
ester with an N-amino or N-oxy amine derivative, each as described above, in a
molar
ratio of about 1:1. More preferably an excess of the N-amino or N-oxy amine
derivative is used to react with the fusion peptide C-terminal carboxylic acid
ester. The
concentration of the reactant N-amino or N-oxy amine derivative ranges between
about
2M - neat, preferably, about 2-16 M, more preferably, about 4-10 M. The
reaction is
conducted at a pH range of about 7-11, preferably, about 8-9.5 at about
ambient
temperature until cleavage and formation of the peptide C-terminal N-amino or
N-oxy
amide derivative, as described above, is complete. Completion of cleavage and
formation of the peptide C-terminal N-amino or N-oxy amide derivative may be
determined by analytical techniques and methods known in the art (e.g. high
performance liquid chromatography (HPLC), gas chromatography, capillary
electrophoresis). As recognized by those of skill in the art, the addition of
solvents
including, for example, denaturants (e.g. urea, guanidine) to solubilize the
reactant
peptide as necessary is envisioned as well. The newly formed peptide C-
terminal N-
amino or N-oxy amide derivative may be further purified by means known in the
art
such as, for example, chromatography techniques (e.g. reverse phase HPLC, ion
exchange chromatography).
According to the invention, the peptide C-terminal N-amino or N-oxy amide
derivative may be converted "under conditions sufficient" to a peptide C-
terminal
amide, each as described above.
Other embodiments of the invention will be apparent to those skilled in the
art
from consideration of the specification and practice of the invention
disclosed. It is
intended that the specifications and examples be considered exemplary only
with the
true scope of the invention being indicated by the claims. Having provided
this detailed
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12
information, applicants now describe preferred aspects of the invention.
EXAMPLES:
EXAMPLE 1.
Per paration of MSI-78 [S~ ID N0~2] from MSI-344 [SEO ID NO~ 11 via a two step
chemical conversion.
MSI-344 [SEQ ID NO:1] is a recombinantly produced peptide with an
unmodified C-terminal carboxyl group, several lysine residues and an
unprotected N-
terminal amino group. MSI-78 [SEQ ID N0:2] is the C'.-terminal amidated form
of
MSI-344 [SEQ ID NO:1 ] and displays significantly greater antimicrobial
activity than
MSI-344 [SEQ ID NO:1 ]. The conversion of MSI-344 [SEQ ID NO:1 ] to MSI-78
[SEQ ID N0:2] is outlined in Figure 1. MSI-344 was first converted to MSI-344-
methyl ester by deprotection of chemically synthesized MSI-344-t-butyl-oxy-
carbonyl
protected methyl ester in a mixture of hydrochloric acid, dioxane and
methanol. The
reaction material contained approximately 90% MSI-344-methyl ester as
determined by
HPLC analysis.
Conversion of MSI-344-methyl ester to MSI-1918 [SEQ ID NO: 5]:
MSI-344-methyl ester hydrochloride salt was added to 8 M hydroxylamine
diluted in deionized water and adjusted to pH 8.2 with acetic acid. The
reaction was
agitated for two hours at 20°C. The reaction was determined to be
complete following
HPLC analysis of sample aliquots from the reaction. The reaction was then
diluted into
5% acetic acid and applied to an amberchrom column. The column was washed with
2% acetic acid to remove excess hydroxylamine. The peptide was eluted from the
column with a 0-70% gradient of acetonitrile in deionized water with a 2%
constant
concentration of acetic acid. All fractions determined to be ninhydrin
positive were
pooled and lyophilized to yield the crude acetate salt of MSI-1918 [SEQ ID NO:
5].
This crude product contained approximately 83% MSI-1918 [SEQ ID NO: S] and 3%
MSI-344 [SEQ ID NO:1 ] along with a number of unidentified impurities
initially
present in the t-butyl-oxy-carbonyl protected methyl ester starting material
as
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demonstrated by the HPLC analysis in Figure 2. The presence of MSI-344 [SEQ ID
NO:1] in the reaction product resulted from hydrolysis of the methyl ester
group to
form the carboxylic acid. More importantly, the reaction products contained no
peptide
lactam side products or polymeric material which would result from reaction of
the
unprotected amine groups present in the peptide. Protection of the E-amino
group of
lysine and the arginine guanidino side chain groups was not necessary to
accomplish
conversion of MSI-344-methyl ester to MSI-1918 [SEQ ID NO: 5].
For the HPLC trace displayed in Figure 2, chromatographic bands were
identified through co-elution with the corresponding synthetic compound.
Control
peak column retention times were as follows (dashed line): MSI-344 [SEQ ID
NO:1]
was 9.160 minutes, MSI-344-methyl ester was 9.673 minutes, MSI-78 [SEQ m N0:2]
was 9.924 minutes and MSI-344-lactam side product was 10.242. Peak column
retention times for the experimental reaction products were as follows (solid
line):
MSI-344 [SEQ ID NO:1] was 9.244 minutes, MSI-1918 [SEQ ID NO: S] was 9.591
minutes while the unidentified impurities ranged from 7.247 to 7.772 minutes.
Reduction of MSI-1918 [SEQ ID NO: SJ to MSI-78 [SEQ ID N0:2J:
The crude MSI-1918 [SEQ ID NO: 5] acetate salt product was converted to the
hydrochloride salt. The crude MSI-1918 [SEQ ID NO: .5] hydrochloride salt was
dissolved in deionized water and methanol. Ammonium chloride was dissolved in
this
solution followed by the addition of Raney nickel. The reaction was vigorously
agitated and warmed to 40°C. A 35% solution of hydrazine was then added
to the
flask, followed by additions at five and eight hours. Progress of the reaction
was
determined by HPLC analysis of sample aliquots following the initial addition
of
hydrazine. At seven hours, the reaction was determined to be approximately SO%
complete. The reaction was allowed to proceed overnight and at twenty-four
hours the
conversion appeared to be almost complete. The resultant material contained
approximately 6.9% MSI-344 [SEQ m NO:1] and 93% MSI-78 [SEQ ID N0:2] as
demonstrated by the HPLC analysis in Figure 3. Again, the reaction products
contained
no peptide lactam side product which would result from reaction of unprotected
amine
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14
groups of lysine residues indicating that protection of E-amino or arginine
guanidine
side chain groups is not necessary to accomplish the reduction.
For the HPLC trace displayed in Figure 3, peak column retention times for the
experimental reaction products were as follows (solid line): MSI-344 [SEQ ID
NO:1 ]
was 9.222 minutes, while MSI-78 [SEQ ID N0:2] was 9.979 minutes.
Mass Spectrum (+ES) for MSI-78 [SEQ ID N0:2]: Calcd. For C,ZZH210N32022~ 2477.
Found: 2477 (M+1 ).
EXAMPLE 2
Cleavage of MSI-1850 jSEO ID N0:3] and conversion to MSI-1918 f~0 ID NO~ S]
in a single step.
MSI-1850 (QPELAPEDPEDEFNGIGKFLKKAKKFGKAFVKILKK
corresponding to SEQ ID N0:3) is a recombinantly produced MSI-344-HSV fusion
peptide with an unmodified C-terminal carboxyl group, several lysine residues
and an
I S unprotected N-terminal amino group. The fusion peptide consists of
fourteen amino
acids derived from Herpes Simplex glycoprotein D on the N-terminal, with the
remainder of the peptide comprising MSI-344 [SEQ ID NO:1 ]. The HSV region of
the
fusion protein contained several aspartic and glutamic acid residues while the
MSI-344
[SEQ ID NO:1] region contained no amino acid residues with carboxylic acid
side
chains. Cleavage of MSI-344 [SEQ ID NO:1 ] from its fusion partner can be
accomplished with hydroxylamine because of the asparagine-glycine cleavage
site
which connects the two peptides. Conversion to the hydroxamic acid
intermediate and
cleavage from MSI-344 [SEQ ID NO:1] fusion partner can therefore be
accomplished
in a single step.
Preparation of the MSI-1850-ester is again necessary prior to cleavage and
conversion to the hydroxamic acid intermediate. For esterfication of MSI-1850
[SEQ
ID N0:3], the fusion peptide was dissolved in 2% hydrochloric acid in methanol
and
agitated under nitrogen for fourteen hours. The solvent was removed under
vacuum
and the residue was subsequently placed under high vacuum for two hours. The
reaction was not forced to completion but instead was processed as the crude
product.
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WO 99/65931 PCT/US99/13626
MSI-344 [SEQ ID NO:1 ) does not contain any carboxylic acid side chains,
therefore
only the C-terminal is converted to the methyl ester.
MSI-1850-methyl ester along with a reference material consisting of
phenylacetic acid was dissolved in deionized water. One volume of the solution
was
S added to one volume of 8 M hydroxylamine and adjusted to pH 8.3 with
hydrochloric
acid and the reaction was allowed to proceed for twenty-four hours at ambient
temperature. The HPLC analysis in Figure 4 demonstrates that the reaction
product
contained MSI-1918 [SEQ ID NO: 5], indicating that both cleavage from the
fusion
partner occurred along with conversion to the hydroxamic acid. The reaction
product
10 also contained MSI-344 [SEQ ID NO:1] which resulted from cleavage of MSI-
1850
[SEQ ID N0:3) which had not been fully converted to the methyl ester in the
previous
esterfication reaction because it was not allowed to proceed to completion.
MSI-1918
[SEQ ID NO: 5] is then converted to MSI-78 [SEQ ID N0:2] according to the
reduction procedure described in Example 1.
EXAMPLE 3
Three step conversion of MSI-1922 [SEQ ID N0~4] to MSI-78 [~FQ ID N0~21
Esterification of MSI-1922 [SEQ ID N0:4J:
The peptide MSI-1922 [SEQ )D N0:4], Figure 5, (1.01 g, 43% active moiety by
HPLC) was added to a 100 mL round bottom flask. Methanol (SO mL) and p-
toluenesulfonic acid (2.0 g) were added to the flask with vigorous magnetic
stirnng.
The flask was equipped with a Soxhlet extractor (125 mL capacity). The thimble
(25 x
80 mm) was filled with 3~ molecular sieves and 25 mL methanol was added to the
extractor. This was done to prevent the volume holdup of the extractor from
concentrating the reaction too much. The reaction was brought to a vigorous
reflux
under N2. The reflux was sufficient to cycle solvent through the extractor at
~ 4-5 min
intervals. The reaction was allowed to proceed for 24 h. The reaction was
worked-up by
chilling the reaction to 0-4°C, adding 1 volume of t-butyl methyl ether
(50 mL), and
allowing it to stand at this temperature for 10 min. The suspension was then
centrifuged
at 1600 x g for 10 min. The pellet was partially dried in vacuo for 30 min @
30 inches
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WO 99/65931 PCT/US99/13626
16
Hg.
Note: The reaction can easily be run at concentrations > SO mg/mL. Dilution in
this case was necessary due to the glassware used. The Soxhlet extractor
procedure
pushes the esterification to completion as evidenced by the ratio of MSI-1918
[SEQ ID
NO:S] to MSI-344 [SEQ ID NO:1 ], in the reaction sample from the cleavage
reaction
described below (see attached chromatogram, Figure 6). Incomplete
esterification will
be detected as the carboxylic acid, MSI-344 [SEQ ID NO:1 ], after cleavage.
Hydroxylamine Conversion to Hydroxamic Acid and Cleavage:
MSI-1922 methyl ester, 2, Figure 5, was suspended in 15 mL 10 M NH20H /
8M urea and the suspension was homogenized with vigorous stirring. The
suspension
was stirred for 2h before dilution with 15 mL of 8 M urea. This solution was
shaken at
rt for 48 h and the cleavage reaction was worked-up by acidification to pH ~
6.0 with
acetic acid. The solution was then diluted and centrifuged for loading onto an
ion
exchange column (SP-Sepharose, elution with a gradient of sodium chloride in
50 riZM
phosphate buffer). The final purified yield of MSI-1918 [SEQ ID NO:S] as the
TFA salt
was 128.8 mg (62% yield, assuming the product is 66% active moiety, based on
elemental analysis).
Mass Spectrum (+ES) for MSI-1918 [SEQ ID NO:S]: Calcd. For C~ZZH2,pN32013~
2493.
Found: 2493 (M+1).
Reduction of MSI-1918 [SEQ ID N0:5] to Amide (MSI-78 [SEQ ID N0:2]):
MSI-1918 [SEQ ID NO:S] (550 mg as the TFA salt) was dissolved in 11 mL of
deionized water in a 50 mL polypropylene centrifuge tube. Raney Ni (Activated
Metals
A-5000, 100 mg) was added to the reaction vessel. The reaction was
magnetically
stirred and warmed to 40°C. The hydrazine solution (17.5% w/v as a
50/50 molar ratio
of hydrazine hydrochloride/hydrazine pH 8.1 ) was loaded into a 10 mL syringe
and
placed on a syringe pump. The pump was set to dispense 6.29 mL of the
hydrazine
buffer over 2h. The evolution of gas was vigorous after several minutes of
hydrazine
addition and foaming of the peptide solution became problematic. At several
time
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17
points during the course of the reaction, n-butanoi was added to reduce the
foaming.
After a total of 700 pL of n-butanol was added, the foaming was controlled.
After 2h of
addition, the reaction was sampled for HPLC analysis and the addition of
hydrazine
was stopped. The reaction was allowed to stir for an additional 1.5 h until
the HPLC
result confirmed that the reaction was akeady completed at the 2 h time point
(see
attached chromatogram, Figure 7). The reaction was worked-up by removal of the
magnetic stir bar and removal of most of the Raney Ni by the magnet. The
solution was
then desalted and converted to the TFA salt by absorbing it on a C,8 column (
50 mL,
30 g, Bakerbond 40 ~.m, prep LC packing) and washing with 0.2% TFA in
deionized
water (500 mL). The peptide was then eluted with 60/40 acetonitrile/water
containing
0.1% TFA. The ninhydrin positive eluant was lyophilized to yield 482.9 mg of
MSI-78
[SEQ )D N0:2] as the TFA salt (88 % yield).
EXAMPLE 4
~3r razinolysis of a peptide C-terminal methyl ester
A peptide C-terminal methyl ester (e.g. MSI-344-OMe) is dissolved in an
aqueous solution containing a hydrazine/hydrazine hydrochloride buffer (pH 8-
10).
The hydrazine solution is prepared by adding the desired acid to a solution of
35%
hydrazine (or a more concentrated solution can be prepared from hydrazine
monohydrate or neat hydrazine if desired). The solution is magnetically
stirred at room
temperature removing an aliquot at regular intervals for HPLC analysis.
Complete
conversion of the peptide C-terminal methyl ester to the peptide C-terminal
hydrazide
is monitored by HPLC. Once the reaction is complete, the reaction mixture is
chilled in
an ice water bath and neutralized by addition of acid. The crude peptide C-
terminal
hydrazide is then desalted and purified by ion exchange or reverse phase
chromatography. The purified peptide C-terminal hydrazide is reduced to the
corresponding peptide C-terminal amide by addition of hydrazine (pH 8) in
water with
Raney Nickel catalysis. Alternatively, in a one-pot procedure, the crude
peptide C-
terminal hydrazide is reduced directly to the amide by the careful addition of
an aliquot
of Raney Ni, or by addition of the reaction mixture to Raney Ni. For saftey
reasons,
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18
this is only practical if the concentration of hydrazine used in the
conversion is
relatively low or the excess hydrazine free base is removed in vacuo.
It should be understood that the foregoing discussion and examples merely
present a detailed description of certain preferred embodiments. It will be
apparent to
those of ordinary skill in the art that various modifications and equivalents
can be made
without departing from the spirit and scope of the invention. All the patents,
journal
articles and other documents discussed or cited above are herein incorporated
by
reference.
CA 02331330 2000-12-12
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SEQUENCE LISTING
<110> Magainin Pharmaceuticals, Inc.
<110> Jones, Stephen R.
Noecker, Lincoln A.
Feibush, Binyamin
<120> Non-enzymatic Chemical Amidation Process
<130> 36870-5075-WO
<140> PCT/US99/13626
<141> 1999-06-17
<150> US 60/089,635
<151> 1998-06-17
<160> 5
<170> PatentIn Ver. 2.0
<210> 1
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide no. MSI-344
<400> 1
Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe
1 5 10 15
Val Lys Ile Leu Lys Lys
<210> 2
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (22)
<223> AMIDATION
<220>
<223> Description of Artificial Sequence: Synthetic
peptide no. MSI-78, containing C-terminal amide
group
<400> 2
Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe
1
CA 02331330 2000-12-12
- WO 99/65931 PCT/US99/13626
1 5 10 15
Val Lys Ile Leu Lys Lys
<210> 3
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide no.
MSI-1850, a recombinantly produced fusion peptide no.
MSI-344-HSV with an unmodified C-terminal carboxyl group
<400> 3
Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp Glu Phe Asn Gly Ile
1 5 10 15
Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val Lys
20 25 30
Ile Leu Lys Lys
<210> 4
<211> 67
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide no. MSI-1922
<400> 4
Met Lys Ala Ile Phe Val Leu Leu Glu His His His His His Leu Lys
1 5 10 15
Asp Ala Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn
20 25 30
Asn Asn Leu Gly Ile Glu Gly Arg Ile Ser Glu Phe Asn Gly Ile Gly
35 40 45
Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val Lys Ile
50 55 60
Leu Lys Lys
<210> 5
<211> 22
<212> PRT
2
CA 02331330 2000-12-12
WO 99/65931 PCT/US99/13626
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (22)
<223> AMIDATION
<220>
<223> Description of Artificial Sequence: Synthetic
peptide no. MSI-1918, containing C-terminal hydroxamic
acid group
<400> 5
Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe
1 5 10 15
Val Lys Ile Leu Lys Lys
3
