Note: Descriptions are shown in the official language in which they were submitted.
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WO 98/02192 PCT/US97112084
RADIOMETAL-BINDING PEPTIDE ANALOGUES
Background of the invention
This invention provides derivatives of biologically
useful cyclic and acyclic peptides in which one or more
amino acid side chains or a segment attached to the
peptide chain contain chelating moieties that can tightly
bind metal ions, including radionuclides. The labeled
peptides carry the metal to specific in vivo targets such
as receptors and antigens, and are useful for
radiodiagnostic imaging, therapy and radiotherapy. New
methods for preparing the peptides are also provided.
Radiolabeled peptides are useful in the diagnosis and
therapy of a variety of human disease states that are
characterized by overexpression of peptide hormone
receptors. Thus, for example, it has been shown that
radiolabeled analogues of LHRH (luteinizing hormone
releasing hormone) and somatostatin selectively bind to
hormone-sensitive tumors characterized by cell-surface
overexpression of LHRH hormone receptors. Similarly,
peptide hormone analogues such as 'z3I-vasoactive
intestinal peptide (VIP) , 99"'Tc-P829, "'In-DTPA Octreotide
and "'In-bisMSH-DTPA have been used to image human tumors
that over express VIP, somatostatin, somatostatin and
melanocyte stimulating hormone (MSH) receptors
respectively. See: Virgolini et al. Engl. J. Med.
169:1116 (1994); Virgolini et al.~J. Nucl. Med. 36:1732,
(1995); Lister-James et al. Nuc?. Med., 36, 91P, #370,
1995 meeting abstract; Pearson et al. J. Med. Chem.
39:1361, (1996); Krenning et al. J. Nucl. Med., 33:652
(1992); and Wraight et al. Brit. J. Radiol. 65:112
(1992).
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Many tyrosine-containing peptides may be labeled with
izsl by well known methods and used for receptor binding
studies. For example, the incidence of VIP receptor
upregulation has been studied in vitro in a wide range of
cancer types using "~I- [Tyr'°] -VIP as the radioligand.
See Reubi, Nucl. Med. 36:1846 (1995). The VIP receptor
was detected in a wide variety of cancer types, including
breast, prostate, ovarian, pancreatic, endometrial,
bladder, colon, esophageal, SCLC, astrocytoma,
l0 glioblastoma, meningioma, pheochromocytoma, lymphoma,
neuroblastoma adenoma, and GEP tumors. An iodinated VIP
analogue '''I- [Tyr'°] -VIP has also been used to image VIP
receptor-rich tumors in humans. See Virgolini et al,
supra.
The use of radioiodine for in vivv diagnostic and
therapeutic uses has distinct disadvantages, however.
1231 ~ the most useful isotope in vivo, is very expensive
($45.30/mCi) and must be produced in a cyclotron. This
isotope, furthermore, has a half-life of only 13.2 hours,
thereby requiring that it be produced in a geographic
location close to where any radioiodinated imaging agent
must be used. Other radioisotopes, such as ~'°'T'c and '88Re
are preferred for diagnostic and therapeutic uses,
respectively. 99°'Tc, for example, is inexpensive
($0.50/mCi), is readily available (produced in a
generator from 9~Mo, a reactor product), and has an ideal
gamma emission energy for imaging with a gamma camera.
Some peptides either directly contain, or are
amenable to the introduction of, residues that allow
direct binding of radiometals such as 99'"Tc and '$8Re to the
peptide. For example, somatostat~n contains a disulfide
bond that, upon reduction, provides two sulfhydryl-
containing cysteine side chains that can directly bind
99mTC. See U.S. Patent no. 5,225,180. See also WO
94/28942, WO 93/21962 and WO 94/23758. Complexes of this
type tend, however, to be heterogeneous and unstable,
which limits their clinical utility. Moreover, the use
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of free sulfhydryis in this manner limits the radiometals
which can be used to label the peptide to those that
tightly bind free S-H groups. This method further
suffers from the problem that direct binding of the metal
to an amino acid side chain can greatly influence the
peptide conformation, thereby deleteriously altering the
receptor binding properties of the compound.
Most peptides either do not contain a metal-binding
amino acid sequence motif or, for various reasons such as
those described supra, are not amenable to suitable
sequence modifications that would permit introduction of
such a motif. Some means of rendering the peptide
capable of binding radiometals must therefore be
introduced into the peptide . A preferred approach is to
attach a metal binding ligand to a specified site within
the peptide so that a single defined, stable, complex is
formed. The ligands used to bind metals often contain a
variety of heteroatoms such as nitrogen, sulfur,
phosphorous, and oxygen that have a high affinity for
metals.
Chelates have conventionally been attached via
covalent linkages to the N-terminus of a peptide or
peptide analogue, following independent synthesis of the
peptide and chelate moieties. For example, Maina et al.
have described the coupling of a tetra-amine chelator to
the N-terminus of a somatostatin analogue, allowing ~9"'Tc
labeling of the peptide. See J. Nucl. Biol. Med. 38:452
(1994). Coupling in this manner is, however, undesirable
when the N-terminus of the peptide plays an important
role in its receptor binding properties. Accordingly,
application of this method is limited by the requirement
that the N-terminus of the peptide accommodate the
presence of a (usually sterically bulky) chelator without
deleteriously affecting the binding properties of the
peptide.
Alternatively, chelating agents have been introduced
into peptide side chains by means of site-selective
reactions involving particular amino acid residues. For
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example, the lysine residue at position 6 of LHRH has
been directly acylated with a chelating group. See
Bajusz, S. et al. Proc. Natl. Acad. Sci. USA 86:6313
(1989). This method is inherently limited by the lack of
chemical selectivity available when more than one side
chain can potentially react with the chelator, or when
the peptide sequence does not contain an amino acid that
can be derivatized in this way. A further limitation of
this approach can arise when multidentate ligands are
used. A single ligand molecule can react with multiple
peptide molecules resulting in the formation of
significant amounts of cross-linked products.
Chelating agents have been introduced on the side
chain of a peptide through tris amino acids as described
by Dunn T.J. et al. WO 94/26294. This method does not
provide a method for cyclizing the peptides. The side
chain protecting groups used to introduce the ligand
described in this work are the same as those typically
used for peptide amide cyclization. See Felix et al.
Int. J. Peptide Protein Res. 32:441 (1988).
A fully protected BAT (bisaminothiol) chelating agent
has been synthesized and coupled to the side chain of a
lysine residue, which could then be incorporated into a
peptide. See Dean et a1. WO 93/25244. These fully
protected precursors are very time consuming, expensive
and cumbersome to prepare. The difficulty and expense of
preparing such precursors make this method untenable for
preparing a diverse array of ligands attached to the
variety of linkers that is needed to design a metal
carrying targeting agent.
One potential solution to this problem is to use a
protecting group strategy that allows selective coupling
of a chelator moiety to specified positions within a
peptide chain. The diversity of chemical reactivities
present within the amino acid side chains of a peptide
has, however, led to difficulties in achieving sufficient
selectivity in site-specific deprotection of protecting
groups. This lack of selectivity has also heretofore
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hampered efforts to selectively deprotect two or more
different functional groups within a peptide to allow
coupling of these groups in, for example, a cyclic
peptide.
Edwards et a1. J. Med. Chem. 37:3749 (1994) have
disclosed a fragment method of assembling a cyclic
disulfide on a resin with a subsequent attachment of an
intact ligand (DTPA). This approach afforded the known
somatostatin targeting agent DTPA-Octreotide. This
l0 approach was specifically designed for the preparation of
a known compound. A more typical situation, however,
requires that a variety of labeled peptides to optimize
binding to a particular target. Such a situation
requires, therefore, a broader approach allowing the I
assembly of multiple ligands, best assembled in
fragments, placed at any point desired in a sequence
which can also be cyclized at a variety of positions in
the peptide sequence.
Additional considerations for the synthesis of
peptides that can selectively bind metals include the
effect of the chelate on the conformation of the peptide.
Most peptides are highly conformationally flexible,
whereas efficient receptor binding usually requires that
a peptide adopt a specific conformation. Whether or not
the peptide can adopt this specific conformation is
greatly influenced by charge and hydrophilic/hydrophobic
interactions, including the effects of a covalently
attached metal chelating moiety. It is possible to
enhance peptide receptor affinity and selectivity by
restricting the conformations that the peptide can adopt,
preferably locking the peptide into an active
conformation. This is often most readily achieved by
preparing cyclic peptides. Cyclic peptides have the
added advantage of enhanced resistance to proteases, and
therefore frequently demonstrate a longer biological
half-life than a corresponding linear peptide.
Peptides can be cyclized 'by a variety of methods such
as formation of disulfides, sulfides and, especially,
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lactam formation between carboxyl and amino functions of
the N- and C-termini or amino acid side chains. However,
the plethora of functionality within a peptide chain
typically means that, for all but the shortest peptides,
selective coupling between two desired functional groups
within a peptide is very difficult to achieve.
It is apparent, therefore, that cyclic peptides that
can chelate metals ions while retaining the ability to
specifically bind with high affinity to a receptor are
greatly to be desired. It is also desirable to have a
means of attaching a chelating moiety to any
predetermined position within a peptide, and to have a
means of selectively forming cyclic peptides between any
two preselected positions within a peptide chain.
Additionally, it is desirable to have access to a method
that would allow a chelating moiety to be coupled to a
peptide at any desired stage during peptide synthesis.
Summary of the Invention
It is therefore an object of the present invention
to provide peptides that can bind radionuclides while
retaining the ability to specifically bind to the peptide
receptor. It is a further object of the invention to
provide methods of preparing and radiolabeling peptides
that can bind radionuclides while retaining the ability
to specifically bind to the peptide receptor. It is a
still further object of the invention to provide
diagnostic and therapeutic methods of using, the
radiolabeled peptides to image or treat a tumor, an
infectious lesion, a myocardial infarction, a clot,
atherosclerotic plaque, or a normal organ or tissue.
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_ 'J _
In accomplishing the foregoing objects of the
invention, there has been provided, in accordance with
one aspect of the current invention, a peptide comprising
a radiometal-binding moiety, wherein said binding moiety
comprises the structure I:
.Rto
N
R2Rt 'peptide
n
where R'. R2, and R' independently are selected from
the group consisting of H, lower alkyl, substituted lower
alkyl, cycloalkyl, substituted cycloalkyl,
heterocycloalkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, alkaryl, and a protecting group
that can be removed under the conditions of peptide
synthesis, provided that at least one of R', R=, or R3 is
H. R5, R~, R8, R~ and R10 independently are selected from
the group consisting of H, lower alkyl, substituted lower
alkyl, aryl, and substituted aryl, or R8 and R9 together,
or R? and R9 together may form a cycloalkyl or substituted
cycloalkyl ring: R4 and R6 together form a direct bond or
are independently selected from the group consisting of
lower alkyl, substituted lower alkyl, aryl, and substituted
aryl; and wherein NR10 is located at the N-terminus of said
peptide, or is located on an amino acid side chain of said
peptide.
In preferred embodiments of the invention, R' is ~I,
R' is H, R, is' H, or R'' and R° together form a direct bond.
In other preferred embodiments, Ri is lower alkyl or
3o s~stituted or tinsubstituted phenyl, or more preferably
methyl or pheayl. In other prefe;red embodiments. R° and
R9 are methyl.
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_ g _
(Chel)yAbuVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID N0:4),
(Chel)yAbuYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID N0: 5),
HSDAVFTDNYTRLRK(Chel)QMAVKKYLNSILN-NH2 (SEQ ID N0:2),
<GHWSYK(Chel)LRPG-NH2 (SEQ ID N0:6),
<GHYSLK(Chel)WKPG-NH2 (SEQ ID N0:7),
AcNaldCpadWdSRKd(Chel)LRPAd-NH2 (SEQ ID N0:8),
(Chel)yAbuSYSNIeDHFdRWK-NH2 (SEQ ID NO: 9),
(Chel)yAbuNleDHF~RWK-NH2 (SEQ ID N0:1),
(Chel)NIeDHFdRWK-NH2 (SEQ ID NO: 1),
Ac-HSDAVFTENYTKLRK(Chel)QNIeAAKKYLNDLKKGGT-NH2 (SEQ ID N0: 10),
(Chel)yAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2, (SEQ ID N0: 2),
(Chel)yAbuVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID NO: 4),
(Chel)yAbuNleDHF~RWK-NH2c (SEQ ID NO:l),
<GHWSYK(Chel)LRPG-NH2 (SEQ ID N0: 6),
<GHYSLK(Chel)WKPG-NH2 (SEQ ID N0: 7),
AcNaldCpadWdSRKd(Chel)LRPAd-NH2(SEQ ID N0:8),
<GHYSYLK(Chel)WKPG-NH2 (SEQ ID N0: 11),
<GHYSLK(Chel)WKPG-NH2 (SEQ ID NO: 7),
NaldCpadWdSRKd(Chel)WKPG-NH2 (SEQ ID N0: 12),
<GHWSYKd(Chel)LRPG-NH2 (SEQ ID NO: 13),
AcNaldCpadWdSRKd(Chel)LRPAd-NH2 (SEQ ID N0: 8),
<GHWSYK(Chel)LRPG-NH2 (SEQ ID NO: 6),
AcK(Chel)FdCFW Ka TCT-OH (SEQ ID N0: 14),
AcK(Chel)DFdCFW Kd TCT-OH (SEQ ID NO: 15),
AcK(Chel)FdCFW Kd TCT-of (SEQ ID N0: 14),
AcK(Chel)DFdCFWdKTCT-of (SEQ ID NO: 15),
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(Chel)DFdCFWdKTCT-OH (SEQ ID N0: 16),
K(Chel)DFdCFWdKTCT-of (SEQ ID NO: 15),
K(Chel)KKFdCFWdKTCT-of (SEQ ID N0: 17),
K(Chel)KDFdCFWdKTCT-OH (SEQ ID N0: 18),
K(Chel)DSFdCFWdKTCT-OH, SEQ ID N0: 19),
K(Chel)DFdCFWdKTCT-OH, (SEQ ID N0: 15),
K(Chel)DFdCFWdKTCD-NH2 (SEQ ID N0: 20),
K(Chel)DFdCFWdKTCT-NH2 (SEQ ID N0:15),
K(Chel)KDFdCFWHKTCT-NHNH2 (SEQ N0: 18),
AcK(Chel)FdCFWdKTCT-NHNH2 (SEQ ID N0: 14),
K(Chel)FdCFWdKTCT-of (SEQ ID N0: 14), and
FdCFWdKTCTK(Chel)-NH2 (SEQ ID N0: 21),
wherein (Chel) is a radiometal-binding moiety having the
structure set forth above.
Other objects, features and advantages of the present
invention' will become apparent from the following
detailed description. It should be understood, however,
that the detailed description and the specific examples,
while indicating preferred embodiments of the invention,
are given by way of illustration only, since various
changes and modifications within the spirit and scope of
the invention will become apparent to those skilled in
the art from this detailed description.
Detailed Description
The present invention provides new chelating moieties
that can be covalently linked to peptides, cyclic
peptides and peptide analogues. The chelating moieties
allow the peptide , cyclic peptides and peptide analogues
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(Chel)yAbuSYSNIeDHFdRWK-NHZ, (Chel)yAbuNleDHF~RWK-NH, ,
(Chel)NIeDHF~RWK-NHz ,
Ac-HSDAVFTENYTKLRK(Chel)QNIeAAKKYLNDLKKGGT-NH,,
{Chel)yAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH~,
(Chel)yAbuVFTDNYTRLRKQMAVKKYLNSILN-NHZ,
(Chel)yAbuNleDHFdRWK-NH,', <GHWSYK(Chel)LRPG-NH,,
<GHYSLK { Chel ) WKPG-NH2, AcNaldCpadWdSRICd ( Chel ) LRPAd-NH,,
<GHYSYLK (Chel) WKPG-NH2, <GHYSLK (Chel) WKPG-NHz,
NaldCpadWdSRKd {Chel) WKPG-NH2, <GHWSYIC~ (Chel) LRPG-NH,,
AcNaldCpadWdSRK~ (Chel) LRPA~-NHz,
AcNaldCpadWdSRItd ( Chel ) LRPA,a-NH2,
AcNaldCpadWdSRF~ ( Chel ) LRPA.~-NH2, <GHWSYK ( Chel ) LRPG-NH,,
AcK (Chel ) FdCFW,,KTCT-OH, AcK (Chel ) DFdCFW,~KTCT-OH,
AcK { Chel ) F,dCFW~KTCT-of , AcK (Chel ) DFdCFW~KTCT-of ,
(Chel) DF~CFW~KTCT-OH, K (Chef) DFdCFWaKTCT-ol,
K(Chel)KKF~,CFW,RTCT-ol, K(Chel)KDF~CFW~KTCT-OH,
K (Chel ) DSF,dCFW,3KTCT-OH, K (Chel ) DFdCFWdKTCT-OH,
K ( Chel ) DF~CFW~KTCD-NH2, K ( Chel ) DF~CFW~KTCT-NH2,
K(Chel)KDF~CFW~KTCT-NHNHz, AcK(Chel)FdCFW,~KTCT-NHNH,,
2 0 K ( Chel ) F CFWdKTCT-of , and FdCFW,,KTCTK ( Chel ) -NHz ,
wherein (Chel) is a radiometal-binding moiety having the
structure set forth above.
Other objects, features and advantages of the present
invention will become apparent from the following
detailed description. It should be understood, however,
that the detailed description and the specific examples,
while indicating preferred embodiments of the invention,
are given by way of illustration only, since various
changes and modifications within the spirit and scope of
the invention will become apparent to those skilled in
the art from this detailed description.
Detailed Description
The present invention provides new chelating moieties
that can be covalently linked to peptides, cyclic
peptides and peptide analogues. The chelating moieties
allow the peptides, cyclic peptides and peptide analogues
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to stably bind metals, especially radiometals. Methods
of preparing these chelators, peptides and peptide
analogues are also provided. The peptides and peptide
analogues are prepared by site-specifically introducing
the metal-chelating moieties into peptides that are
synthesized by solid-phase or solution phase methods.
The chelating moieties may be attached to an amine-
bearing side-chain of an amino acid within the peptide
chain, or may be attached to the N-terminus of the
l0 peptide. Peptides according to the invention include,
but are not limited to, cyclic metal-binding analogues of
LHRH, vasoactive intestinal peptide (VIP), heregulins
( erbB binding peptides ) c~, (31, X32 , and /33 , melanotropin
(a-MSH), somatostatin, calcitonin, epidermal growth
factor, gonadotrophin releasing hormone, heregulins
growth hormone releasing hormone, dynorphin, calcitonin
gene-related peptide, vasotocin, mesotonin,
adrenocorticotropic hormone,corticotropin, gonadotropin,
prolactin, vasopressin, oxytocin, substance P, substance
K, and angiotensin.
The chelating moieties may be represented by the
general formula I:
n
g N-~rd~
K
I R~ (~Y' 0 NS
where R', Rz, and R3 independently are selected from
the group consisting of H, lower alkyl, substituted lower
alkyl, cycloalkyl, substituted cycloalkyl,
heterocycloalkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, alkaryl, and a protecting group
that can be removed under the conditions of peptide
synthesis. At least one of R', R2, or R3 must be H. R4,
R5, RG, R', R8, R9 and R'° independently are selected from
the group consisting of H, lower alkyl, substituted lower
alkyl, aryl, and substituted aryl. R4 and R~ together
also may optionally form a direct bond, and R8 and R''
together or R' and R9 together also may form a cycloalkyl
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or substituted cycloalkyl ring. NR'° is located at the N-
terminus of the peptide to which the chelator is
attached, or is located on an amino acid side chain of
that peptide. When R', R2, R5, or R~ bears a heteroatom
substituted function, the heteroatom also may be used to
carry out additional peptide coupling reactions.
Examples of lower alkyl include, but are not limited
to, straight or branched chain C,-C~ alkyl groups, such as
a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s
butyl, t-butyl, n-pentyl, i-pentyl, and n-hexyl.
Cycloalkyl includes C3-C6 cycloalkyl, such as cyclohexyl.
Heterocycloalkyl includes tetrahydrofuran,
tetrahydropyran, pyrrolidine, and piperidine. Heteroaryl
includes pyrrolyl, furanyl, thienyl, imidazolyl,
oxazolyl, oxazolylthio, thiazolyl, pyrazolyl,
pyrrolidinyl, pyridinyl, pyrimidinyl, morpholinyl, and
piperizinyl. Aryl includes C6-C,= aryl such as phenyl, a-
naphthyl, or ~3-naphthyl.
Alkaryl includes : C6-C,ZarylC,-Cbalkyl, such as
phenylC,-Cbalkyl, or a- or ~3-naphthylC,-Cbalkyl, such as
benzyl, phenylethyl, phenylpropyl, phenylbutyl,
phenylpentyl, a- or /3-naphthylmethyl, napthylethyl,
naphthylpropyl, naphthylbutyi, or naphthylpentyl.
Examples of substituent groups include : C,-C~, alkoxy,
for example, methoxy, ethoxy, propoxy; C,-C~ alkylthio,
for example methylthio, ethylthio, propylthio; C~
C,=arylC,-Cbalkoxy, for example phenylC,-C6 alkoxy such as
benzyloxy; aralkylthio, for example phenylC,-Cbalkylthio
such as benzylthio; amino, substituted amino, for example
C,-Cbalkylamino such as methyl amino, ethylamino; C~,-
C,,arylC,-Csalkyl, such as phenyl,-C~ alkyl for example
benzyl ; C6-C,,aryl such as phenyl ; C,-C8 cycloalkyl such as
cyclohexyl; and C3-C$ cycloalkylC,-Cbalkyl such as
cyclohexylmethyl.
Preferred embodiments of the invention include
compounds where Rz is H, methyl, or phenyl, and where Rg
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and R9 are methyl. Other preferred embodiments are where
R', R3, R5, R' and R'° are H.
The peptides may be synthesized using differentially
protected bis-amino acid derivatives in which either
amino function can be selectively deprotected. These
derivatives are introduced into a growing peptide chain
during peptide synthesis by conventional peptide coupling
methodology. One of the amino functions is then
selectively deprotected, allowing subsequent coupling of
either all or a part of a chelating molecule, or addition
of further amino acid residues to continue the peptide
synthesis. Peptide synthesis can be continued by
coupling at the a-amino group, leading to a peptide with
a conventional amide backbone, or at the side-chain amino
group to produce a peptide whose amide backbone is
interrupted by the side chain structure. Alternatively,
the free amino function can be used to cyclize onto a
reactive functionality located elsewhere in the peptide,
thereby producing a cyclic peptide. Suitable bis-amino
acids will be readily apparent to the skilled
practitioner, and include lysine, ornithine, and 2,3-
diaminopropionic acid (amino-serine). Alternatively, the
chelating moiety may be introduced at the end of peptide
synthesis by coupling the chelating moiety to the
deprotected N-terminus of the resin-bound peptide. The
chelating moiety may be added as a complete unit, in
protected or unprotected form, or may be synthesized in
stepwise fashion to construct the complete chelating
structure.
Bis-amino acids used in the present invention may be
generally represented by the formula : ZHN-CH ( -R-NHY) -COZH
where R is (CH,) ~ or (CHz) ~-X- (CH~) n where X is a heteroatom
such as O,S,or N and n=1-20. Alternatively the hydrogen ,
atoms of the CH, groups can be replaced with lower alkyl,
substituted lower alkyl, or alkenyl groups, or cyclic or
heterocyclic rings such as cyclohexane, benzene, and
piperidine, or other groups well known to the skilled
artisan. The substituents Z and Y independently can be
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H, N, lower alkyl, substituted lower alkyl, aryl, or
substituted aryl.
If peptide synthesis is continued, selective
deprotection of the second amino group of the bis-amino
acid can be accomplished at any point during the peptide
synthesis to introduce the chelating moiety. The
complete chelating moiety can be synthesized prior to
coupling to the peptide, or it can be synthesized by
sequentially coupling segments to the peptide. Once
assembly of the entire peptide/chelator structure is
complete, cleavage, deprotection, and purification
affords the desired peptide derivative. This derivative
is then labeled with a radiometal for use in
radiodiagnostic and radiotherapeutic applications.
Alternatively, if all or part of the chelating
molecule is coupled to the deprotected amino group first,
the second step is to deprotect the other amino group and
continue with the peptide synthesis. If only part of the
chelator moiety is coupled to the peptide at this stage,
the synthesis of the chelator can be finished at any
point during or after synthesis of the peptide chain by
appropriate deprotection and coupling reactions. Final
cleavage, deprotection and purification steps once again
yield the pure peptide derivative, which is then
radiolabeled as before.
Attachment of the chelator to the peptide prior to
cleavage from the resin results in reduced formation of
cross-linked products even when multidentate activated
chelators such as DTPA-dianhydride are used.
Preparation of cyclic peptides is achieved by
selective deprotection of two compatible functional
moieties at specified positions of the peptide sequence,
followed by cyclization between the compatible moieties.
Cyclization can be achieved between any two points of the
peptide sequence, including between the N- and C-termini,
between a terminus and an internal functional group
within the peptide sequence, or between two internal
functional groups. Cyclization can be achieved using
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either solution-phase or solid-phase peptide syntheses,
but is preferably carried out using solid-phase
techniques.
The deprotection and cyclization can be carried out
at any point during the synthesis of the peptide prior to
the final deprotection reactions. For example, the
entire protected peptide sequence can be prepared prior
to the cyclization, or the cyclization can be carried out
on a protected peptide intermediate, followed by
completion of the synthesis. Similarly, the cyclization
can be carried out either before or after all or part of
the metal chelating moiety is coupled to the peptide.
Use of a photocleavable or other resin known to those
skilled in the art on a solid phase peptide synthesizer
also allows release of a protected peptide from a solid
support, with subsequent solution phase selective
deprotection and cyclization. Alternatively, the side
chains to be cyclized can be selectively deprotected
prior to cleavage from the resin, and the cyclization
carried out in solution phase.
Reactions involving the C-terminus of the peptide,
including but not limited to cyclization reactions, may
be accomplished through the release of a protected
peptide from the resin in the manner described above.
Alternatively, the growing peptide chain may be attached
to the resin via the side chain of a residue and the C-
terminal carboxyl group suitably protected. When a
reaction with the C-terminal carboxyl group is desired,
it is selectively deprotected and the reaction allowed to
proceed. In the case of cyclization reactions, the
deprotection of the C-terminus can be accomplished
before, during, or after the selective deprotection of
the compatible reactive group.
The radiometal chelating peptides of the present
invention stably retain radionuclide in blood and other
bodily fluids and tissues. Both the reagents and the
conditions in the present method are greatly simplified
over those in the prior art, and the labeled peptides are
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particularly suitable for radiodiagnostic and
radiotherapy applications using technetium or rhenium
labeling.
The approach outlined above allows the placement of
a radiometal-binding moiety anywhere in a peptide
sequence. Placing the chelating moiety on an amino acid
side-chain, either directly or via a spacer group, rather
than on the N-terminus of a peptide, has the added
advantage of spatially distancing the metal complex from
the peptide backbone, thereby minimizing the effect of
the metal complex on the peptide conformation. This also
allows the N-terminus of the peptide to be used for
cyclizing the peptide, if necessary.
It is known that peptide conformation is greatly
influenced by charge and hydrophilic/hydrophobic
interactions, and it is therefore important to consider
these variables when designing a chelating ligand to be
used in peptides. It is preferred that a variety of
chelating complexes of varying charge and hydrophilicity
and containing spacer groups of various lengths are
prepared and tested to select the metal-complexed peptide
that displays the optimum combination of target
selectivity, pharmacokinetics, and chelate stability.
The skilled artisan will appreciate that such testing is
routine in the art.
The radiolabeled peptides of the present invention
bind specifically to a diseased cell or tissue that
exhibits both a high receptor density and high affinity
for the peptide. The radioactivity of the radionuclide
allows diagnosis and/or treatment of the tumor or
diseased tissue. The invention also includes
pharmaceutical compositions comprising an effective
amount of at least one of the radiolabeled peptides of
the invention, in combination with a pharmaceutically
acceptable sterile vehicle, as described, for example, in
Remington~s Pharmaceutical Sciences; Drug Receptors and
Receptor Theory, 18th ed., Mack Publishing Co., Easton,
PA (1990) . The invention also includes kits for labeling
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peptides which are convenient and easy to use in a
clinical environment.
A. Design and Synthesis of Linear Peptides
Incorporating Chelating Moieties
(i) In General
The peptides of the invention contain radiometal-
chelating amino acid derivatives that are characterized
by the presence of at least one thiol or thiocarbonyl
l0 group, and at least one nitrogen present as either a
tertiary amine, a hydrazone, or a secondary amide or
hydrazide. The sulfur and nitrogen atoms are suitably
disposed to form a multidentate ligand capable of tightly
and preferentially binding a metal ion. The multidentate
ligand may also contain a spacer group that serves to
separate the chelated metal from the rest of the peptide.
The metal ion is preferably a reduced radionuclide, and
in a preferred embodiment is ~''"Tc, 'g6Re, or 'BgRe.
The invention also provides a method for placing
ligands for other metals at any point in a peptide
sequence. These ligands can be introduced intact, for
example DTPA, or as fragments. In this way ligands for
other metals of medical interest including, but not
limited to, In, Ga, Y, Cu, Pt, Mn, Gd, Au, Ag, Hg, and Lu
can be placed in a peptide targeting sequence.
The method allows the introduction of any metal
chelate or chelate~fragment that is suitably protected
for peptide synthesis. The method also provides a method
for the introduction of base-sensitive ligand derivatives
that can be placed at any point in the peptide sequence
as long as it is introduced at the end of the synthesis.
An example is the synthesis of a'ligand attached to the
side chain of lysine using the following ligand fragment
Trityl-S-COCH,N(Boc)CH,COZH. The S-Trityl ester will be
sensitive towards base so it would not be possible to
place this ligand fragment on the peptide at an earlier
point in the synthesis.
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Each of the chelating moieties of the invention can
be prepared by methods well known to the skilled
practitioner in the art of organic synthesis. The
chelating moieties are constructed from subunits that are
linked together by simple coupling or condensation
reactions, such as the condensation of an amino,
hydrazino, or hydrazido function with an activated
carboxyl group, coupling of hydrazines with aldehydes, or
reductive amination reactions between amines and
l0 aldehydes. As used herein the term "condensation" is
intended to encompass reactions that couple together
subunits of the chelating moiety, and thus encompasses
reactions such as reductive amination in addition to
reactions that conform to the classical definition of a ,/
condensation reaction.
Following a condensation reaction, additional
functional groups on the subunit may be deprotected to
allow additional condensation reactions. For example, a
second subunit carrying a free carboxyl group and a
protected amino function can be condensed with an amino,
hydrazino, or hydrazido function on a first subunit to
produce a larger, suitably protected fragment of the
metal binding ligand. The amino function on the second
subunit moiety can then be deprotected and further
coupled to a third subunit. As used herein, the term
"fragment" is intended to encompass a subunit or assembly
of subunits comprising all or part of the metal binding
ligand.
Methods of activating carboxyl groups for such
condensation reactions are well known to those of skill
in the art of organic synthesis and peptide synthesis,
and include the use of active esters and of carbodiimide
and phosphoryl azide coupling agents. Suitable
protecting groups are used for protecting functions on
the subunits when the reactivity of the functions is
incompatible with a reaction used to j oin the subunits or
with reactions used for synthesis of the peptide chain.
Protecting groups for mercapto, amino and carboxylic acid
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functions are well known in the art. See, for example,
Greene, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley
Interscience, NY, 1981). The subunits used to construct
the chelate are either readily prepared by methods well
known in the art, or are commercially available from
suppliers such as Advanced ChemTech (Lexington, KY),
Milligen (Burlington, MA), Applied Biosystems (Foster
City, CA), or Aldrich Chemical Corp. (Milwaukee, WI).
The condensation reactions used to link together the
chelator subunits can either be carried out prior to
peptide synthesis, or during synthesis of the peptide
sequence. When the amino acid derivative is assembled
from its subunits prior to peptide synthesis, a-amino and
a-carboxyl functions must be suitably protected in a
manner that is subsequently compatible with selective
deprotection and activation of these functionalities for
peptide synthesis. Examples of such protecting groups
are well known in the art, and include the
fluorenemethyloxycarbonyl (Fmoc), benzyloxycarbonyl
(Cbz), ~butoxycarbonyl (Boc), allyloxycarbonyl (aloc), 4
methoxytrityl (mtt), and 1-(4,4-dimethyl-2,6
dioxocyclohex-1-ylidene)ethyl (Dde) groups for amino
protection. Groups for carboxyl protection include the
methyl (Me), benzyl (Bn), 'butyl ('Bu), and allyl esters,
respectively.
The amino and carboxyl protecting groups must be
selected such that each group can be selectively
deprotected in the presence of the other. Such
protecting moieties are said to be orthogonal. The
requirement that orthogonal protecting groups be used
precludes, for example, use of the Cbz group for
protection of the amino function in the presence of a
carboxyl group protected as a benzyl ester. See Greene,
supra. In a preferred embodiment the cx-amino group is
protected as an Fmoc group, and the a-carboxyl group is
a methyl ester. The thiol protecting group used in the
compounds of the invention can be any organic or
inorganic group which is readily removed under mild
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conditions to regenerate the tree sulfhydryl in the
presence of the peptide without substantially altering
the activity of the protein. Suitable protecting groups
are listed in Greene, supra, pp. 193-217. Examples of
suitable protecting groups include substituted and
unsubstituted trityl groups, thiol esters, thiocarbamates
and disulfides. In a preferred embodiment the thiol
protecting group is a trityl group or a 4-methoxytrityl
group. Those skilled in the art are familiar with the
l0 procedures of protecting and deprotecting thiol groups.
For example, benzoate thioesters may be deprotected under
mild and selective conditions using hydroxylamine. Once
assembly of the protected chelating moiety is complete,
the a-carboxy function is deprotected and coupled to the
amino terminus of the peptide chain using conventional
methods of peptide synthesis. See Bodanszky et al., THE
PRACTICE OF PEPTIDE SYNTHESIS (Springer verlag,
Heidelberg, 1984).
When the metal-chelating amino acid derivative is
assembled from its subunits during peptide synthesis, the
peptide chain is assembled by conventional solution phase
or, preferably, solid phase synthesis until the point
where the derivative is to be incorporated. The
differentially protected bis-amino acid is then coupled
to the amino terminus of the peptide chain. Subsequent
selective deprotection of one of the amino groups of the
derivative allows either peptide synthesis or chelator
synthesis to continue.
If the a-amino function is deprotected first, all or
part of the remaining amino acid residues are then
coupled to the peptide chain in the conventional manner.
The side chain amino function of the derivative is then
deprotected, and the chelating moiety is assembled as
described above. The complete peptide can then be
deprotected and purified by standard methods.
If the side chain amino function is deprotected
first, all or part of the chelating moiety is then
assembled as described above, followed by deprotection of
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the a-amino group. Peptide synthesis is completed in the
conventional manner as described above.
Once peptide synthesis is complete the fully
protected peptide is deprotected and purified. Methods
for deprotection and purification of synthetic peptides
are well known in the art. See, for example, Bodanszky,
supra. If the peptide was synthesized by solid phase
techniques the peptide must also be cleaved from the
resin used as the solid support for the synthesis.
Methods for achieving this cleavage also are well known
in the art. Methods for purifying synthetic peptides
such as those of the present invention also are well
known to those of skill in the art. Such methods
include, for example, ion exchange, gel filtration
chromatography, and reversed phase high pressure liquid
chromatography (RP-HPLC). In a preferred embodiment of
the invention the peptide is purified by RP-HPLC using a
preparative-scale octadecylsilane (C18) silica column
packing, eluting with a gradient of acetonitrile in 0.1%
trifluoroacetic acid (TFA). The purity of the peptide
can be confirmed by standard methods such as analytical
RP-HPLC or capillary electrophoresis. The identity of
the peptide can be confirmed by NMR spectroscopy or, in
a preferred embodiment of the invention, by mass
spectrometry.
As noted above, it is important that the chelating
moiety does not interfere with peptide binding to the
appropriate receptor. Determining the residues within
the peptide that can be replaced without deleteriously
affecting receptor binding can be carried out in a
systematic and straightforward way by preparing a series
of peptides in which each successive residue is replaced
with, for example, alanine, (an "alanine scan"). The
alanine-substituted peptides then are screened for
biological activity. Modern peptide synthesizers make
synthesis of peptides in this way quite straightforward,
and screening of a large number of peptides is routine
for the skilled artisan. Retention of high receptor
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binding affinity in a peptide containing such an alanine
substitution denotes that the substituted amino acid is
less important for receptor binding, and indicates a
position where the metal-binding residue may be placed.
Synthesis of peptides wherein a chelator is placed in
each of these positions is then straightforward, and
routine screening establishes the optimal position for
the chelator.
As set forth above, a wide variety of peptides
containing metal binding ligands may be prepared using
the methods of the present invention. Additional methods
of preparing metal chelating peptides using the methods
of the claimed invention will be apparent to the skilled
artisan. Specific applications using these methods are
set forth below to further exemplify the invention, but
it will be appreciated that these examples are merely
illustrative and are not meant to limit the scope of
application of the invention.
(ii) Preparation of chelating moieties
In a preferred embodiment, the chelator contains a
thiol group together with a thiosemicarbazide or
thiosemicarbazone group, and can be represented by the
general formula I:
Z ~~ ~y N !~~ -,~ .~N ,~ N - '~d.9
0
HS
where R', R2, and R3 independently are selected from
the group consisting of H, lower alkyl, substituted lower
alkyl, cycloalkyl, substituted cycloalkyl,
heterocycloalkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, alkaryl, and a protecting group
that can be removed under the conditions of peptide
synthesis. At least one of R~, RZ, or R3 must be H. R4,
R5, R°, R', R8, R~ and R'° independently are selected from
the group consisting of H, lower alkyl, substituted lower
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alkyl, aryl, and substituted aryl. R4 and R6 together
also may optionally form a direct bond, and R$ and R~
together or R' and R9 together also may form a cycloalkyl
or substituted cycloalkyl ring. NR~° is located at the N-
terminus of the peptide to which the chelator is
attached, or is located on an amino acid side chain of
that peptide. When R~, RZ, R5, or R6 bears a heteroatom
substituted function, the heteroatom also may be used to
carry out additional peptide coupling reactions.
l0 Although an understanding of the mechanism of metal
binding by the chelating moieties is not necessary for
practicing the invention, and without wishing to be bound
by any theory, it is believed that the metal is bound to
the chelator via the two sulfur atoms plus two nitrogen
atoms. It is hypothesized that the metal-binding
nitrogens are the a-nitrogen of the (3-thiol-containing
amino acid and the thiocarbazide/thiocarbazone nitrogen
distal to the thiocarbonyl group. When the metal is
reduced radioperrhenate or reduced radiopertechnetate,
the two sulfur and two nitrogen atoms provide four
coordination positions on the metal.
These compounds may be prepared by methods that are
well known in the art of organic synthesis. Thus, for
example, compounds having formula I may be prepared by
amide coupling between an a-carboxyl-protected amino acid
moiety having a protected or unprotected ~3-thiol-
containing side chain (a "cysteine-type amino acid°), and
a carboxyl-containing thiosemicarbazone or
thiosemicarbazide moiety. This coupling can be carried
out using well-known methods, such as carbodiimide-
mediated coupling. Deprotection of the a-carboxyl group
of the amino acid allows amide coupling of the chelating
moiety to an amino side chain, or the amino terminus, of
the peptide. Alternatively, an N- and S-protected
cysteine-type amino acid having a /3-thiol containing side
chain may first be coupled to the peptide, followed by N-
deprotection and amide coupling to a carboxyl-containing
thiosemicarbazone or thiosemicarbazide moiety.
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Cysteine-type amino acids may be prepared by standard
methods of amino acid synthesis. The configuration at
the a-carbon may be (R) or (S), or the amino acid may be
racemic. Similarly, the configuration at the ~i-carbon,
when asymmetrically substituted may be (R), (S), or
(R/S). The amino acid is protected for subsequent
coupling reactions using standard methods.
Thiosemicarbazones may be prepared by the
condensation of semicarbazides with carbonyl compounds.
Reduction of the thiosemicarbazones with, for example,
sodium borohydride, provides substituted
thiosemicarbazides. In a preferred embodiment, a
thiosemicarbazide is reacted with glyoxylic acid to form
the corresponding thiosemicarbazone, which optionally may
be reduced to form a substituted thiosemicarbazide.
Many thiosemicarbazides are commercially available
from, for example, Aldrich Chemical Company, Milwaukee,
WI. Other thiosemicarbazides may be prepared by the
reaction of a hydrazine with, for example, an
isothiocyanate. Asymmetrically substituted hydrazines
also are commercially available, or may be prepared by
nitrosation of amines to the nitrosamine, followed by
reduction to the hydrazine. Isothiocyanates may be
prepared by reaction of an amine with thiophosgene.
Other methods of preparing thiosemicarbazides are well
known to the skilled artisan.
In some instances, it is found that
thiosemicarbazides have low solubility in the solvents
used for coupling to the cysteine-like amino acid. In
such instances, the coupling can be carried out using the
thiosemicarbazone, followed by coupling to the peptide.
The thiosemicarbazone then may be reduced to the
thiosemicarbazide using sodium borohydride or another
suitable reducing agent. When the thiosemicarbazide is
sufficiently soluble to be used directly, the a-nitrogen
must first be protected prior to coupling. Suitable
protecting groups are well known in the art and include
Boc and Cbz groups.
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(iii) Lin~ar VIP Receptor Targeting Agents
Naturally occurring VIP has the sequence:
HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID N0: 2)
An alanine scan has revealed several residues whose
replacement with alanine does not greatly affect receptor
binding. These residues include Lys-15, Gln-16, Val-19,
Lys-21, Asn-24, Ser-25 and the N- and C-termini. These
locations are possible sites for the attachment of a
metal binding ligand according to the present invention.
Chelating derivatives based on attachment of the
metal binding ligand at these positions include, but are
not limited to, those with a metal binding moiety
attached, either directly or via a spacer group, to the
pharmacophore via the side chain amine of a lysine or
other bis-amino acid residue. Specif is chelating
derivatives of this general structure include, but are
not limited to:
MaGCyAbuHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID NO :2)
AcCGCHSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID N0: 22)
KPRRPYTDNYTRLRK(PtscGC)QMAVKKYLNSILN-NH2 (SEQ ID NO: 3)
MaGCyAbuVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID NO: 4)
AcCGCVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID N0: 23)
MaGCyAbuYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID N0: 5)
HSDAVFTDNYTRLRK(PtscGC)QMAVKKYLNSILN-NH2 (SEQ ID NO: 2)
HSDAVFTDNYTRLRK(Dtpa)QMAVKKYLNSILN-NH2 (SEQ ID NO: 2)
HSDAVFTDNYTRLRK(AGC)QMAVKKYLNSILN-NH2 (SEQ ID N0: 2)
where Ma is mercaptoacetic acid,
PtscG is 2-(4-phenyl-3-thiosemicarbazidyl)acetic
acid or PhNFiCSNHI~tFICH~CO,H,
yAbu is 7-aminobutyric acid,, and
in K(PtscGC), the parenthese$ denote that enclosed
amino acids are attached to the a amine of
lysine and the first amino acid attached is C
followed by PtscG.
t
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In each of the compounds described above, the
chelating moiety may be replaced by a chelator of the
general formula I, as described above.
(iv) Linaar hHRH ~acaptor Targetiag Ageats
Naturally occurring LFiRH has the. sequence:
<GHWSYGLRPG-NHi (SEQ ID NO: 24)
where <G is pyroglutamic acid. It is further known that
the bicyclic peptide
AcNaldCpadWd (cyclo4-10 ) D (cyclo5-8 ) ERdLKPDap-NH2 (S~ m t~: 25)
(where Wd indicates that the D isomer of the amino acid
was used, Nal is 2-naphthylalanine, Cpa is 4-
chlorophenylalanine and Dap is 2,3-diaminopropionic acid)
binds to the LHRH receptor. See Hienstock et al. J.~ Med.
Chem. 36:3265 (1993). It is also known that the side
chain of position 6 of LFiRFi is very bulk tolerant . See
Barbacci et al. J. 8iol. Chem. 270:9585' (1995) . This
location is a possible site for the attachment of a metal
binding ligand according to the-present invention.
Linear chelating derivatives based on attachment of
the metal binding ligand at this position include, but
are not limited to, those with a metal binding moietp
attached, either directly or via a spacer group, to the
pharmacophore via the side chain amine of a lysine or
other bis-amino acid residue. Specific linear chelating
derivatives of these general structures include, but are
not limited to:
<GHWSYK(MaGC)LRPG-NFI, (SEQ iD NO: 6)
<GHYSLK{MaGC)WKPG-NHS (SEQ ID NO: 7)
<GHWSYK(Ma-azaGC)LRPG-NH, (SEQ ID NO: 6)
<GHYSLK(PtscGC)WKPG-NHi (SEQ ID NO: 7)
<GHYSLK(PtscGDap)WKPG-NHS (SEQ ID NO: 7)
<GHWSYItd {MaGC) LRPG-NHi (SEQ ID NO: 13)
<GHYSLK(azaGGC)WKPG-NFh (SEQ ID NO: 7)
<GHWSYK ( iECG) LRPG-NH, ( S$Q ID rro: 6 )
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<GI~WSYKd (MtaGC,) LRPG-NH2 ( SEQ ID NO: 13 )
<GHYSLK(iECiD)WKPG-NHi (SEQ ID NO: 7)
<GHYSLK(DiGIyGDap)WRPG-NH, (SEQ ID NO: 7)
<GHYSLK(iDGDap)WKPG-NH, (SEQ ID NO: 7)
<GHWSYK(MtaGC)LRPG-NH: (SEQ ID NO: 6)
<GHWSK (MaGC) WeLRPG-NH, ( SEQ ID NO: 26)
<GHWSYIt~(MtaGDap)LRPG-NH= (SEQ ID NO: 13)
<GHWSYF~, (PtscGC) LRPG-NHi ( SEQ ID NO: 13 )
<GHWSYItd (E) LRPG-NHi (SEQ ID NO: 13 )
<GHWSYI~ (MtscGC ) LRPG-NH2 ( SEQ ID NO: 13 )
<GHWSYKd (Mta (hqss ) GDap) LRpG-~2 : (SEQ
ID NO: 13 )
AcNalaCpadWdSRIta (MaGC) LRPAd-NFi2 (SEQ ID NO: 8 )
NalaCpadWaSRIt~ ( PtscGC ) LRPA,d-NH2 ( SsQ NO: 8 )
ID
AcNaldCpadWaSRKd (MaFC ) LRPAd-NH2 ( SEQ ID NO: 8 )
AcNalaCpadWaSRKd ( azaGFC ) LRPAd-NH2 ( SEQ ID NO: 8
)
where:
<G is pyroglutamic acid,
Ma is mercaptoacetic acid
azaG is azaglycine or H2NNHCHiCOZH,
PtscG is 2-(4-phenyl-3-thiosemicarbazidyl)acetic
acid or PhNHCSNHNHCH2C0,H,
Dap is 2,3-diaminopropionic acid
iD is an aspartic acid coupled via the side chain
carboxyl group,
iE is a glutamic acid coupled via the side chain
acid group,
DiGly is HOOCCH=NHCFI,C00- ,
Mta(hqss) is S-(2,5-dihydroxyphenyl-S-
methyl)sulfoniumacetyl
C, is an Acm protected cysteine
Mta is the methylthioether of mercaptoacetic acid,
Nal is 2-naphthylalanine,
Cpa is 4-chlorophenylalanine,
in IC,,, the subscript d denotes that the D isomer was
used, and
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in K(MaGC), the parentheses denote that enclosed
amino acids are attached to the a amine of
lysine and the first amino acid attached is C
followed by G and ending in Ma.
Additionally, complexes of these peptides with non-
radioactive metals may be prepared. Such complexes
include:
<GHWSYK(MaGC)LRPG-NH, Re0 (SEQ ID NO: 6)
<GHYSLK(MaGC)WKPG-NHi Re0 (SEQ ID NO: 7)
<GHYSLI~ (MaGC) LRPG-NHS Re0 ( SEQ ID NO: 27 )
In each of the compounds described above, the
chelating moiety may be replaced by a chelator of the
general formula I, as described above.
(v) Linear a-MSH Receptor Targeting Agents
Naturally occurring a-MSH has the sequence:
Ac-SYSMEHFRWGKPV-NHz (SEQ ID NO: 28)
It had previously been shown that the cyclic peptide
NIeDHFaRWK-NHz (where Nle is norleucine and Fd indicates
D-Phe) has a high affinity for the a-MSH receptor and is
~°~ to be relatively stable in-vivo. See A1-Obeidi et
al. J. Amer. Chem. Soc. 111:3413 (1989); Haskell-Luevano
et al. J. Med. Chew. 39:432 (1996). The underlined
portion indicates those residues within the cyclized
portion of the peptide, and also the termini of the_
cyclic structure, i.e. the peptide is cyclized by an
amide bond from the side chains of aspartic acid and
lysine.
Linear chelating derivatives based upon the
structures of these known a-MSH receptor binding peptides
include those with a chelating derivative attached to the
N-terminus of the peptide, either directly or via a
spacer group, such as y-amino butyric acid (y-Abu).
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Specific linear chelating derivatives with this general
structure include, but are not limited to:
MaGC~yAbuSYSNIeDBFdRWK-NHt ( SEQ ID NO: 1 ) , and
MaGC~yAbuSYSNIeDHFdRnWK-NH2 ( SEQ iD No: 29 )
where Y-Abu is y-aminobutyric acid and Rn is a nitrated
arginine residue.
In each of the compounds described above, the
chelating moiety may be replaced by a chelator of the
general formula I, as described above.
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B. Design and Synthesis of Cyclic Peptides
Incorporating Chelating Amino Acid Derivatives
(i) In General
The process of preparing a cyclic metal-
s chelator/peptide complex is analogous to that described
above for linear peptides, except that at some point
during or subsequent to synthesis of the peptide chain
cyclization is carried out. The cyclization can be
between any two functional groups on the peptide such as
the peptide termini or amino acid side chains. The
cyclization can be achieved by disulfide or sulfide
formation, or preferably by lactam formation. Site-
selective cyclization requires selective deprotection of
two functional groups on the peptide. For lactam
formation this requires using an amino and a carboxyl
protecting group that can be deprotected in the presence
of other amino and carboxyl protecting groups. This task
is made more difficult when the peptide synthesis also
requires that selective deprotection be achieved between
these other protecting groups. Accordingly, such a
sophisticated protecting group strategy has heretofore
proved difficult to achieve in practice.
The methods of the present invention allow both
cyclization and coupling of the chelator moiety to the
peptide to be achieved at any point during peptide
synthesis. Use of appropriate protecting groups allows
synthesis of the peptide, assembly of the ligand and
cyclization of the peptide to be achieved in any order
that is desired. This approach is more efficient than
either solid-phase methods which cyclize the peptide off
the resin or methods that attach ligands in solution
following synthesis of the cyclic peptide.
The methods of the present invention may be used in
solution phase, but preferably are carried out using an
automated solid-phase peptide synthesizer. Using a
multi-well automated synthesizer allows a large number of
peptides, differing in the point of attachment of the
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chelator moiety or in the site of cyclization, to be
prepared simultaneously. These so-called "combinatorial
libraries," wherein the Iigand containing peptides are
deprotected while still attached to the solid support,
can be reacted with the appropriate metal to form
complexes and then screened in an appropriate bioactivity
assay to select the compound having the optimally desired
characteristics of receptor binding and stability.
Combinatorial synthesis can be carried out in "split"
syntheses or by "parallel" syntheses. In split
synthesis, synthetic peptide intermediates bound to beads
are subdivided into different groups for addition of the
next amino acid in each successive step. After each step
the beads are divided into different groups for the next
reaction. In parallel synthesis, different compounds are
synthesized in different reactions vessels, such as the
wells of a peptide synthesizer. Split synthesis provides
small quantities of large numbers of compounds, wherea$
parallel synthesis provides larger quantities of a
smaller number of compounds.
Combinatorial synthesis also requires that each
individual compound be labeled in some way in order that
it might be identified in the screening step. Various
means of labeling compounds for this purpose are known in
the art. Fox example, inert halogenated aromatic
compounds are used as labels that can be identified by
gas chromatography (Borman, Chem. Eng. News 74:29 (1996)).
In a preferred embodiment of the invention
cyclization is achieved by lactam formation. Most
preferably the amino function is protected using an aloc
group, and the carboxyl function is protected as an allyl
ester. - This allows simultaneous deprotection of the
amino and carboxy functions using Pd(PPh3), in the
presence of a nucleophile for the allyl group. The
nucleophile typically used is tri-n-butyl tin hydride
(°BujSnH) .
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Prior to the present invention it was known that
amines would react with allyl ions under Pd° catalysis.
See, for example, Roos et al., J. Org. Chem. 60:1733
(1995) and Heck, PALLADIUM REAGENTS IN ORGANIC SYNTHESES,
Academic Press, 1995, pp. 122-131. The present inventors
found that this caused a problem for simultaneous
deprotection of allyl-protected amino and carboxyl
functions because of the side reaction wherein the newly
deprotected amino function was alkylated with the allyl
group. It was found, however, that addition of
piperidine as an allyl scavenger during the Pd-catalyzed
aloc cleavage reaction inhibited this unwanted side-
reaction. This allowed deprotection of, for example,
aspartic acid and lysine side chains selectively and
simultaneously while greatly reducing formation of the
undesired NE-allyl lysine. Those skilled in the art will
recognize that other primary or secondary amines will
also be suitable allyl scavengers.
This method is useful and advantageous because it is
compatible with Fmoc based peptide synthesis. It allows
preparation of cyclic peptides on HMP and PAM resins
where both the Boc or Fmoc side chain protection can be
used in addition to Aloc side chain protection. This
technique furthermore allows synthesis of cyclic peptides
containing a metal-binding ligand attached to the side
chain of an amino acid at any point in a peptide chain.
Thus three orthogonal nitrogen protecting groups are
used: one for building the peptide chain, such as Fmoc or
Boc; a second for attaching a metal-binding ligand to a
side chain of a bisaminoacid such as 4-methyltrityl(mtt)
or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl
(Dde); and a third, such as Aloc, for achieving side-
chain to side-chain cyclization. Other protecting,
groups, such as Boc, Cbz, 'Bu and benzyl groups can be
used for protection of other side-chain amino and carboy
functions that are not deprotected until the peptide
synthesis is finished. Combinations of these protecting
groups allow the use of Rink, Wang, Merrifield, PAM and
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HMP-type resins for solid-phase peptide synthesis.
Cleavage of the peptides from the resin can be
accomplished with trifiuoroacetic acid (for Fmoc-based
syntheses) or HF or trimethylsilyl
trifluoromethanesulfonate (for Boc-based syntheses).
The cyclic peptides of the invention are from 4 to
100 residues long, and have up to 5 metal chelating
groups. The peptides can contain cyclized regions that
are between 2 and 60 amino acids in length, and can
l0 contain more than one cyclic portion.
Cyclization is preferably between amino and carboxy
amino acid side chains to form lactam bridges, but may
also be between a side chain amine and the carboxy
terminus to form a lactam bridge, between a side chain
acid and the N-terminal amine to form lactam, between
thiols to form disulfide bridges, between hydrazines and
esters to form hydrazides, between hydrazines and
aldehydes to from hydrazones, between a thiol and a
suitable leaving group to form a sulfide, or between a
hydroxyl group and another suitable leaving group to form
an ether. When more than one cyclic region is present in
the compound, the bridges in the cyclic regions may be of
the same or different types.
When a cyclic disulfide is to be formed, the peptide
of interest is preferably synthesized using Acm
protection on thiols and aloc protection on amino side
chains. The aloc is cleaved and the chelating ligand is
coupled to the peptide. The Acm group is then cleaved
and the disulfide cyclized using thallium
trifluoroacetate. Alternatively, the thiols can be
protected using S-trityl groups, and the cyclization can
be carried out in solution after cleavage from the resin.
Preparation of a cyclic sulfide may be achieved by,
for example, cyclization of a thiol onto an a-haloamido
function present on an amine side chain. Thus, for
example, the peptide may be synthesized with Fmoc
protection on the N-terminus and S-trityl protection on
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the thiol. An aloc-protected lysine side chain is then
deprotected and the chelator is coupled to the lysine as
described above. The Fmoc is cleaved and reacted with
chloroacetyl chloride or an equivalent reagent. If an
acid stable resin such as the photocleavable BromoWang
resin, Wang, J. Org. Chem. 41:3258 (1976), is used the
thiol protecting group is removed and the peptide is
cyclized on the resin.
Cyclic sulfides between side chain residues are
l0 prepared by using a suitable protecting group on the N
terminus of the peptide that allows selective
deprotection at two differentially protected amino side
chains. For example, the N-terminus can be protected as
an acetyl or Boc group, and the side chains can be
protected as Fmoc and aloc groups. Following peptide
synthesis, one amino acid side chain (for example, Fmoc)
is selectively deprotected and coupled with the chelate
moiety as described above . Another amino side chain ( for
example aloc or Dde) is then deprotected and coupled to
a sulfide electrophile such as haloacetyl or maleimide.
The protecting group on the sulfur of interest is cleaved
and cyclization is carried out on the resin using a base
catalyst. Alternatively, the peptide can be cleaved and
the cyclization can be carried out in solution.
As set forth above, a wide variety of cyclic peptides
may be prepared using the methods of the present
invention. Additional methods of preparing cyclic metal
chelating peptides using the methods of the claimed
invention will be apparent to the skilled artisan.
Specific applications using these methods are set forth
below to further exemplify the invention, but it will be
appreciated that these examples are merely illustrative
and are not meant to limit the scope of application of
the invention.
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(ii) Cyclic a-MSH Receptor Targeting Agents
Naturally occurring a-MSH has the sequence Ac-
SYSMEHFRWGKPV-NH2 (SEQ ID N0: 28). It had previously been
shown that the cyclic peptide NIeDHFdRWK-NH2 (SEQ ID N0: 1)
(where Nle is norleucine and Fd indicates D-Phe) has a high
affinity for the a-MSH receptor and is known to be
relatively stable in-vivo. See A1-Obeidi et al. J. Amer.
Chem. Soc. 111:3413 (1989) Haskell-Luevano et a1. J. Med.
Chem. 39:432 (1996). The underlined portion indicates those
residues within the cyclized portion of the peptide, and
also the termini of the cyclic structure, i . e. the peptide
is cyclized by an amide bond from the side chains of
aspartic acid and lysine. This cyclic structure is used as
a basis for constructing labeled peptides according to the
present invention.
Cyclic chelating derivatives based upon the structure
of the known a-MSH receptor binding ligand include those
with a chelating derivative attached to the N-terminus of
the peptide, either directly or via a spacer group, such
as ~-amino butyric acid (7-Abu). Specific chelating
derivatives of this general structure include, but are
not limited to:
MaGCYAbuNleDFiFdRWK-NH, ( SEQ ID NO: 1)
PtscGCNIeD RWK-NH, (SEQ ID NO: 30)
AcCGCNleDHF~RWK-NH= (SEQ ID NO: 31)
DTPA-NIeDHF~RWK-NHZ ( SEQ ID NO: 1)
where
Ma is mercaptoacetic acid,
Abu is y-aminobutyric acid,
PtscG is 2-(4-phenyl-3-thiosemicarbazidyl)acetic
acid, and
DTPA is diethylenetriaminepentaacetic acid.
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In each of the compounds described above, the chelating
moiety may be replaced by a chelator of the general formula
I, as described above.
(iii) Cyclic VIP Receptor Targeting Agents
Naturally occurring VIP has the sequence:
HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2 (SEQ ID N0: 2)
Native VIP is thought to form a helical structure in
solution. See Musso et al. Hiocheraistry 27:8174 (1988) .
The putative helix structure can be stabilized by
intramolecular cyclization via the side chains of
residues placed in spatial proximity by the helical
structure. Examples include:
Ac-HSDAVFTENYTKLRKQNIeAAKKYLNDLKKGGT-NH2 (SEQ ID N0:10)
Ac-HSDAVFTDNYTKLRKQNIeAVKKYLNSVLT-NH2 (SEQ ID NO: 32)
(where Nle is aorleucine). See O~Donnell et al. J.
Pharm. Exp. Ther. 270:1282; US Patent 4,822,890; Holin,
Eur Pat Appl 0 536 741 A2. The underlined portion
indicates the residues within the cyclized portion of, the
peptide, and also the termini of the cyclized portion,
i.e. the peptide is cyclized via the formation of an
amide bond between the side chains of the aspartic acid
and the lysine. These cyclic structures are used as a
basis for constructing labeled peptides according to the
present invention.
Cyclic chelating derivatives based on these
structures include, but are not limited to, those with a
metal binding moiety attached, either directly or via a
spacer group, to the pharmacophore via the side chain
amine of a lysine or other bis-amino acid residue.
Specific chelatiag derivatives of this general structure
include, but are not limited to:
Ac-HSDAVFTENYTKLRK(PtscGC)QNIeAAKKYLNDLKKGGT-NH2 (SEQ
ID N0: 10)
. ,
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(where PtscG - 2-(4-phenyl-3-thiosemicarba-
zidyl)acetic acid); and
Ac-HSDAVFTENYTKLRK(DTPA)QNIeAAKKYLNDLKKGGT-NH2 (SEQ ID
NO: 10) (where DTPA = diethylenetriaminepentaacetic acid)
In each of the compounds described above, the chelating
moiety may be replaced by a chelator of the general formula
I, as described above.
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The present invention, thus generally described, will
be understood more readily by reference to the following
examples, which are provided by way of illustration and
are not intended to be limiting of the present invention.
Examples
Example 1: Synthesis of N"Alloc-NE-Fmoc-L-Lysine
NE-Fmoc-L-Lysine (10.00 g, 27.1 mmol, 100 mol%,
Bachem Biosciences, Inc.) was suspended in dioxane (100
ml ) and Na,C03 ( 1M, 3 3 ml ) to form a milky suspension .
Allyl chloroformate (3.2 ml, 30.2 mmol, 111 mol%) was
added to dioxane (10 ml) and this solution was added
dropwise to the suspension of N'-Fmoc-L-Lysine over l0
min. Sodium carbonate, (1M, 20 ml) was added in two
portions and an additional quantity of allyl
chloroformate (0.3 ml) was added. The reaction was
stirred at room temperature for 16 hours. The volatile
solvents were removed under reduced pressure and the
residue was washed with diethyl ether (50 ml). The
residual liquid was then acidified with HC1 (1M) and
extracted with ethyl acetate (2x150 ml). The organic
layers were combined, washed with saturated NaCl (50 ml),
dried over Na,S04, evaporated under reduced pressure to
obtain a crude oily product (16g). The crude product was
dissolved in ether (100 ml) and a white solid formed and
was removed by filtration. The solvent from the filtrate
was removed under reduced pressure to afford a viscous
pale yellow oil (8.34 g, 68% yield) which eventually
formed a glassy solid.
Example 2: Synthesis of 2-(triphenylmethylmercapto)
acetyl hydrazide
2-(triphenylmethylmercapto) acetic acid (20.35 g,
60.9 mmol, 100 mol%) was dissolved in anhydrous THF (150
ml) and cooled in an ice water bath. t-Butylcarbazate
(8.61 g, 65.1 mmol, 107 mol%) was added to the reaction
solution followed by diisopropylcarbodiimide (10.0 ml,
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63.9 mmol, 105 mol%). The reaction was allowed to warm
slowly to room temperature and stirred for 28 hours . The
reaction mixture was filtered to remove the white
precipitate that had formed and the filtrate was
concentrated to a white foam by removal of the solvent
under reduced pressure. This material was dissolved in
chloroform (75 ml). Then acetic acid (75 ml) was added
followed by the addition of borontrifluoride etherate
(10.0 ml, 81 mmol, 134 mol%). The reaction was stirred
at room temperature for 6 hours and then quenched by
pouring the reaction mixture into water (200 ml)
containing sodium acetate (30 g). This mixture was
extracted with chloroform (2x100 ml) . The organic layers
were combined, washed with saturated NaCl solution
(150 ml) , dried over Na2S04 and filtered. The solvent was
removed under reduced pressure to obtain a pale gold oil
which solidified on standing. The solid was suspended in
1:1 diethylether/hexanes (20o ml) and collected by
filtration. The solid was washed with an additional
quantity of 1:1 diethylether/hexanes (100 ml) and dried
to afford the desired product (15.44 g, 73% yield) having
ESMS MHT calculated 349, observed 349.
Example 3: Synthesis of N~-[2-(triphenylmethylthio)
acetyl]azaglycine
Glyoxylic acid monohydrate (0.59 g, 6.41 mmol,
110 mol%) was dissolved in methanol (20 ml) and 2-
(triphenylmethylmercapto)acetyl hydrazide (2.03 g, 5.82
mmol, 100 mol%) was added. Dioxane (20 ml) was added to
the cloudy reaction mixture and the reaction was stirred
at room temperature for 18 hours. Sodium borohydride
(1.76 g) was added to the reaction mixture and after 30
minutes, another quantity of sodium borohydride (0.60 g)
was added. The reaction was stirred for 3 hours at room
temperature, then quenched by pouring the reaction
mixture into HC1 (1M, 60 ml). The mixture was extracted
with ethyl acetate (2x50 ml). The organic layers were
combined, washed with saturated NaCl solution (40 ml),
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dried over Na2S04, filtered, and concentrated under
reduced pressure on the rotary evaporator to afford a
solid (2.5 g) having ESMS MH+ calculated 407, found 407.
Example 4: Synthesis of Na-Boc-Ns- (2-
(triphenylmethylthio)acetyl] azaglycine
N~- [2- (triphenylmethylthio) acetyl] azaglycine (2 .39 g,
5.89 mmol, 100 mol%) was dissolved in dioxane (50 ml).
Di-t-butyl dicarbonate (BOC)20, (2.07 g, 9.48 mmol, 161
mol%) was added to the reaction solution followed by the
l0 addition of Na2C03 (1M, 15 ml). This mixture was stirred
at room temperature for 15 minutes, then additional
quantities of Na~C03 ( 1M, 10 ml ) and (BOC1 z0 ( 1. 41 g) were
added. The solution was stirred at room temperature for
18 hours then reacted with NaOH (6M, 3 ml) and (BOC),0
(1.4 g) for 1 hour. The crude reaction mixture was then
acidified to pH 3 with citric acid (1M) and extracted
with ethyl acetate (200 ml). The organic layer was
washed with saturated sodium chloride solution (60 ml),
dried over Na2S04, filtered and concentrated under reduced
pressure to obtain the crude product. The crude product
was dissolved in ether and diluted to obtain a 1:1
mixture with hexanes causing a white precipitate to form.
The white solid was collected by filtration to obtain the
desired product (1.48 g, 50% yield) having ESMS MH'
calculated 507, found 507.
Example 5: Synthesis of 2-(4-Phenyl-3-
thiosemicarbazidyl)acetic acid
4-Phenyl-3-thiosemicarbazide (6.02 g, 36 mmol,
100 mol%) was suspended in methanol (40 ml). Glyoxylic
acid monohydrate (3.32 g, 36.1 mmol, 100 mol%) was added
and the reaction was stirred at room temperature for 2
hours. Sodium borohydride (1.50 g) was added carefully,
and the reaction mixture bubbled very vigorously. The
reaction mixture was stirred at room temperature for 1
hour, then NaBH4 (0.66 g) was added, followed by the
addition of glacial acetic acid (6 ml). After 15
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minutes, NaBH4 (1.08 g) was added, and the reaction was
stirred at room temperature for 15 hours. An additional
quantity of NaHH4 (1.66 g) was then added and the
reaction was stirred at room temperature for 3 hours
before it was quenched with HCl (iM, 20o ml). The
mixture was then extracted with ethyl acetate (2x150 ml) .
The organic layers were combined, washed with saturated
NaCl solution (100 ml), dried over Na2S0" filtered, and
the solvent removed under reduced pressure to afford a
l0 yellow solid (9.03 g) having ESMS Negative ion mode M-H+
Calculated 224 Found 224.
Example 6: Synthesis of Ns-Boc-2-(4-Phenyl-3-
thiosemicarbazidyl)acetic acid
2-(4-Phenyl-3-thiosemicarbazidyl)acetic acid (8.93
g, 37.9 mmol, 100mo1%) and (BOC)20 (9.10 g) were
dissolved in dioxane (100 ml). Sodium carbonate (1M, 50
ml) and water (50 ml) were added and the mixture was
stirred at room temperature for 5 hours. Sodium
hydroxide (1M, 40 ml) and an additional quantity of
(BOC)20 (6.21 g) were added and the reaction was stirred
overnight at room temperature. The reaction was quenched
with citric acid (1M) and extracted with ethyl acetate
(2x100 ml). The organic layers were combined, washed
with saturated NaCl ( 5 0 ml ) , dried over Na2S04, and
filtered. The filtrate was concentrated under reduced
pressure to afford a gummy solid (19 g) . The crude solid
was suspended in ether and a white solid was collected by
filtration. The solid was washed with ether (200 ml) to
obtain the desired product (3.17 g) having ESMS MH'
calculated 326, found 326.
Example 7: Synthesis of N°'-(triphenylmethylsulfenyl)-N~-
(Boc)azaglycine
'-Butylcarbazate was condensed with glyoxylic acid
monohydrate in methanol. This crude hydrazone was then
reduced by catalytic hydrogenation over 10% Pd/C. This
product was then mixed with dioxane and base and a
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dioxane solution of triphenylmethanesulfenylchloride was
added dropwise . The desired N°'-
(triphenylmethylsulfenyl)-Na-(Boc)azaglycine (25 g) was
obtained on work-up.
Exa~aple 8s Solid Phase P.ptide Synthesis of Peptides
Using Alloc and finoc Protecting Groups
Solid phase peptide synthesis was carried out on a
0.050 mmol scale using an Advanced ChemTech mode? 348
peptide synthesizer modified to operate under nitrogen
pressure in the same manner as the model 396. The
allyloxycarbonyl (aloe) and 9-fluorenylmethyloxycarbonyl
(Fmoc) groups were employed for nitrogen protection and
diisopropylcarbodiimide (DIC)/ hydroxybenzotriazole
(IiOBT) were used to activate the carboxyl groups for
coupling. A variety of resins were used such as Sink,
Pal, and TentaGel'~S RAM for C-terminal amides and Tang,
2-chlorotrityl, or TentaGel S PHH for C-terminal acids.
To allow either introduction of the metal binding
chelate moiety and/or to allow cyclization via
selectively deprotected amino acid side chains a
differentially protected bis-amino acid was used fcr the
peptide synthesis. The differentially protected bis-
amino acid derivatives chosen were a-Aloc-Lys(e-Fnoc)OH
and a-Fmoc-hys(e-Aloc)OH. The a-Aloc-Lys(e-Fmoc)OH
derivative allowed the ligand pieces to be introduced on
the side chain using a routine Fmoc procedure.
The aloc groups were cleaved on the machine .a the
manual mode by washing the resin bound peptide with
dichloromethane (3 x 2 ml portions) and then mixing the
resin with a solution '(2 ml] containing
tetrakistriphenylphosphine palladium (0] (10 mg), and
acetic acid (0.1 ml). Tributyltinhydride (0.3 mli was
then added and the mixture was vortexed for one tour.
The reaction cell was then emptied, the resin was ~.:ashed
with dichloromethane (3 x.2 ml) and standard Fmoc
synthesis was then resumed. The peptides were c_eaved
* Trademark
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from the resin with a solution of trifluoroacetic acid
(TFA), anisole and ethane dithiol for 1 to 3 hours in the
ratio 23:3:1. The crude cleavage mixture was then poured
into ether to precipitate the crude peptide which was
then purified by reverse phase HPLC using a Waters Delta
Pak, Prep Pak C-18 cartridge system eluted with an
appropriate gradient of TFA ( 0 . 1 % ) in water and/or TFA
(0.1%) in acetonitrile (90%) and water (10%). The
fractions containing the desired purified peptides were
l0 collected and the volatile solvents were removed under
reduced pressure to obtain the aqueous solutions of the
peptides which were then lyophilized. Samples of the
lyophilized products were then sent for electrospray
(ESMS) or fast atom bombardment (FABMS) to confirm that
the observed mass of the products matched the calculated
mass of the desired peptide.
. r
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The table below shows some of the peptide sequences
synthesized by the methods described above.
Peptide HPLC2 MWb
<GHWSYGLRPG-NH2 (SEQ ID N0: 24) 6.1 1183
<GHYSLEWKPG-NH2 (SEQ ID N0: 33) 6.2 1227
HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2 6.7 3326
MaGCyAbuHSDAVFTDNYTRLRKQMAVKKY 7.3 3645
LNSILN-NH2 (SEQ ID NO: 2)
MaGC~yAbuVFTDNYTRLRKQMAVKKYLNSIL 7.5 3235
N-NH2 (SEQ ID N0: 4)
MaGCyAbuNleDHFdRWK-NH2c (SEQ ID N0: 1) 7~0 1302
<GHWSYK(MaGC)LRPG-NH2 (SEQ ID N0: 6) 6.3 1488
<GHYSLK(MaGC)WKPG-NH2 (SEQ ID N0: 7) 6.3 1460
<GHWSYK(Ma-azaGC)LRPG-NH2 (SEQ ID NO: 6) 6.1 1503
<GHYSLK(PtsGC)WKPG-NH2 (SEQ ID N0: 7) 6.9 1536
AcNal2CpadWdSRKd(MaGC)LRPAd-NH2 8.2 1668
{SEQ ID NO: 8)
<GHYSYLK(PtscGDap)WKPG-NH2 (SEQ ID N0: 11) 6.6 1519
<GHYSLK(azaGGC)WKPG-NH2 (SEQ ID N0: 7) 6.5 1474
NaldCpadWdSRKd(PtscGC)WKPG-NH2 8.1 1701
(SEQ ID N0: 12)
<GHWSYKd(MaGC)LRPG-NH2 (SEQ ID NO: 13) 6.3 1488
AcNaldCpadWdSRKd(AzaGFC)LRPAd-NH2
(SEQ ID N0: 8)
AcNaldCpadWdSRKd(MaFC)LRPAd-NH2
(SEQ ID N0: 8)
AcNaldCpadWdSRKd(PtscGC)LRPAd-NH2
(SEQ ID NO: 8)
<GHWSYK(iDGDap)LRPG-NH2 (SEQ ID NO: 6)
<GHWSYK(iECG)LRPG-NH2 (SEQ ID NO: 7)
c
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a HPLC Method [retention time in minutes] Solvent A is 0.1~
trifluoroacetic acid in water, Solvent B is
0.1$ trifluoroacetic acid in 90:10 aceto-
nitrile/water. Solvent flow rate is 3 ml/min
for 10 min, then 5 ml/min for 5 min. Gradient
is 0 to 100$ B over 10 min then 100~B for 5 min
b Electrospray mass spectrum values (MH+)
c Then underlined sequence is cyclized as the cyclic amide
connecting the side chain functional groups
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Abbreviations used is Table:
< G: pymglutamic acid
PtscG:2-(4-phenyl-3-thiosemicarbazidyl)acetic acid
or PhNHCSNHNHCH_CO,H
Ma: mercaptoacetic acid
azaG: azaglycine or H:NNHCH=COSH
Dap: 2,3-diaminopropionic acid
yAbu: y-aminobutyric acid
Nal: 2-ttaphchylalanine
Cpa: 4~chlorophenylalanine
K,: the subscript d denotes that the D isomer was used
K(MaGC): the parentheses denote that enclosed amino rids are attached to the s
amine of
lysine and the first amino acid attached is C followed by G and ending in Ma
iD: isoaspattic acid
iE: isoglutamic acid
Other peptides synthesized by these methods include:
Sequence MH+ HPLC RT
AcK(TscGC)FdCFW Ka TCT-OH (SEQ ID 14) 1436 7.7
N0:
AcK(TscGC)DF~CFW Kd TCT-OH (SEQ : 15) 1552 7.4
ID N0
TscGCDF~CFWdKTCT-OH (SEQ ID N0: 1381 7.7
34)
AcK(TscGC)FdCFWdKTCT-of (SEQ ID 14) 1422 7.6
N0:
AcK(MtscGC)FdCFWdKTCT-of (SEQ ID : 14) 1436 7.8
N0
AcK(TscGC)DF~CFW~KTCT-of (SEQ ID : 15 1537 7.4
N0
AcK(MaGG)FdCFWdKTCT-of (SEQ ID N0: 14) 1378 7.4
K(TscGC)DFdCFWdKTCT-NH2 (SEQ ID 15) 1508 7.1
N0:
K(TscGC)KKFdCFWdKTCT-of (SEQ ID 17) 1651 7.2
N0:
K(TscGC)KDFdCFWdKTCT-OH (SEQ ID 18) 1637 7.3
NO:
K(TscGC)DFacFwaKTCT-of (SEQ ID N0: 15) 1495 7.2
K(TscGC)DSFdCFWdKTCT-OH (SEQ ID 19) 1596 7.4
N0:
K(TscGC)DF~CFWdKTCT-OH (SEQ ID N0: 15) 1508 7.2
K(TscGC)DFdCFWdKTCD-NH2 (SEQ ID 20) 1521 7.1
NO:
K(TscGC)KDFdCFW~KTCT-NHNH2 (SEQ NO: 18) 1651 7.2
ID
AcK(TscGC)FaCFW~KTCT-NHNH2 (SEQ N0: 14) 1450 7.4
ID
K(AGC)FdCFWdKTCT-of (SEQ ID NO: 1379 6.8
14)
AcK(TscGC)DFdCFWdKTCT-of (SEQ ID 1537 7.4
N0: 15)
FdCFWdKTCTK(TscGC)-NH2 (SEQ ID N0: 21) 1393 6.8
,
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The underlined portion of the sequence is cyclic.
TscG is 3-thiosemicarbazonylglyoxyl, i.e. H~NCSNF~ICHCO-
MtscG is 4-methyl-3-thiosemicarbazonylglyoxyl,
i . a . cx,r~xcsr~xcxco-
Ma is mercaptoacetyl: HSCIi,CO-
Groups listed within parentheses are attached to the side
chain of the amino acid to the left of the parentheses
Example 9: Preparation of a cyclic MSH analogue containing
a chelating moiety
The method of synthesizing cyclic peptides was
demonstrated by preparing the cyclic a-melanocyte
stimulating hormone (aMSH) analogue MaGCY-AbuNIeDHFdRWK-NH2
(SEQ ID N0: 1) where the underlining indicates that the
peptide sequence is cyclized as a lactam through the
aspartic acid and lysine side chains. The residues to be
used for cyclization were side-chain protected as the aloe
group (for lysine) and as the allyl ester (for aspartate) .
The peptide was assembled using Fmoc chemistry as described
above, on a polystyrene-based Rink amide resin.
Allyl and aloe deprotection was first carried out
using Pd (PPhj)4, acetic acid, and Hu3SnH in the absence of
piperidine as an allyl scavenger. After cleavage of the
side chain protecting groups, the resin was washed and
the partially protected peptide was cyclized using the
method described by Felix et al., Int. J. Peptide Protein..
Res. 32:44 1 (1988). The peptide was then cleaved from
the resin and purified to isolate the N-allyl substituted
cyclic amide as the only clean peptide from the product
mixture.
The aloe cleavage reaction was then modified by the
addition of piperidine as an allyl scavenger.
When the aloe and allyl groups were cleaved using a
mixture containing 0.5 ml glacial acetic acid, 10 ml
d i c h 1 o r o m a t h a n a , 0 . 0 5 6 3 g
.
CA 02259950 2005-07-21
- 44 -
tetrakis(triphenylphosphine)palladium (0), plus 1.0 ml
piperidine as an allyl scavenger. Each well on the
peptide synthesizer contained 0.05 mmol of peptide on
Rink resin, and was treated with 0.3 ml/well tributyltin
hydride at room temperature for lhr with vortex mixing.
The resin was washed with: 2x2 ml/well dichloromethane,
2x1 ml/well methanol, 2x1 ml/well diisopropylethyl amine,
and 3x1 ml/well NMP after the cleavage of the side chain
protecting groups. The peptide side chains were then
coupled by the method of Felix supra ( 15 hr, using BOP
and DIEA). The peptide was~cleaved and purified 'as
described above to afford a pure peptide with the desired
ESMS MH+ of 1302. The N-allylated side product was
observed in only trace amounts.
Example 10s Radiolabaling with ~"'Tc
A Glucoscan* (DuPont) vial was reconstituted with 2.18
mCi of NaTc04 in 1 ml saline to form the 99mTc-gluceptate
complex. <GHWSYK(MaGC)LRPG amide (SEQ ID NO: 6) (IMP3) was
prepared as above. 99mTc-IMP3 was prepared by mixing 350 dal
(874 uCi) of 99mTc-gluceptate with 640 u1 of peptide in
saline. The initially formed precipitate disappeared upon
heating for 15 min at 75°C. An instant TLC (ITLC) strip
developed in H20:EtOH:NH40H mixture (5:2:1) showed 6.2~ of
the activity at the origin as colloids. HPLC showed 100 of
the activity bound to the peptide with a RT of 6.95 min,
whereas the unlabeled peptide eluted at 6.4 min under the
same HPLC conditions (reversed phase C-18 column,
gradient of o-100% B in 10 min at a flow rate of 3
ml/min, where A is 0.1% TFA in H,0 and B is 90% CH3CN,
0.l% TFA). Recovery from the HPLC column was 85% of the
injected activity.
IMP3 was formulated and lyophilized for ~"Tc labeling
in the amounts shown below:
* Trademark
CA 02259950 2005-07-21
- 44a -
IMP3 ( ~g ) ~ ( beg ) 9~/ Sn
1. 250 23 14
2. 100 23 14
3 . 250 15 14
where aDG is a-D-glucoheptonate. The lyophilized vials
were reconstituted with -900~CUCi. of NaTc04 in saline .
Cloudiness was observed in all the vials. The vials were
heated for 15 min at 75~C, but turbidity persisted. ITLC
analysis for colloids showed 14, 21 and 9~c colloids at
the origin for vials 1, 2, and 3, respectively.
In order to prevent the precipitation during '~'"Tc
labeling, a-D-glucoheptonate (aDG) and tartrate ratios to
Sn(II) were varied in the lyophilized vials. The
CA 02259950 1999-O1-11
WO 98/02192 PCT/US97/12084
-45-
following vials were formulated and lyophilized (250 ~,g
of IMP3 with 25 ~g Sn(II)) with tartrate and aDG ratios
as shown below. The vials were reconstituted with 500
~.Ci of NaTc04 in 1 ml saline . Observations are indicated
in the observation column. ITLC strips were developed
after 15 min at room temperature following heating at
75°C for 15 min.
tartrate/SnpH Observationcolloid, RTcolioid, 75 ~ C
1. 50 5.3 ppt
2. l00 5.3 ppt
3. 500 5.3 ppt clears l7 %2.4%
upon mixing,
aDG/Sn pH Observationcolloid, RTcolloid, 75 C
4. 25 5.3 ppt
5. 50 5.3 ppt
6. 100 5.3 turbid
7 . 500 5.3 slight turbidity25 % 3.5 %
8. 1000 5.3 clear 3.3 % 3.1 %o
The protocol above was repeated for vials 3, 7 and 8 and
colloids were determined to be 5.3, 3.8, and 4.6%,
respectively after heating 15 min at 75 C. A single
broad peak was observed on a reversed HPLC column at a RT
of 7 min .
Solubility of peptides that are poorly soluble in
saline alone is increased by the addition of a
solubilizing agent such as ethanol or 2-hydroxypropyl-~3
cyclodextrin.
CA 02259950 2005-07-21
c
- 46 -
Results from labeling other peptides with technetium-99
are shown in the table below:
Peptide HPLC HPLC
retention retention
timea timeb
MaGCyAbuHSDAVFTDNYTRLRKQMAVKKYLNSI 7.62 (99$) 7.65
LN-NH2 (SEQ ID N0: 2)
MaGCyAbuVFTDNYTRLRKQMAVKKYLNSILN- 7.8-9.7e 8.19c(99 $)
NH2 (SEQ ID N0: 4)
<GHWSYK(MaGC)LRPG.amide 6.59 (95$) 6,gOc (g2$)
(SEQ ID N0: 6)
<GHYSLK(MaGC)WKPG.amide NA 7.07 (100%)
(SEQ ID N0: 7)
<GHWSYK(Ma-azaGC)LRPG.amide 6.82 (100$) 7.02c (99%)
(SEQ ID N0: 6)
<GHYSLK(Ptsc-GC)WKPG amide 7.60 (100$) 7.67d
(SEQ ID N: 7) (100$)
AcNaldCpadWdSRKd(MaGC)LRPA-NH2 8.50 (27%)
(SEQ ID NO: 8) 9.00 (68$)
<GHWSYKd(MaGC)LRPG-NH2 6.83 (95%) 7,07c (g5$)
(SEQ ID N0: 13)
<GHYSYLK(PtscGDap)WKPG-NH2 7.08 (96%) 6_8e (g0$)
(SEQ ID NO: 11)
<GHYSLK(azaGGC)WKPG-NH2 6.60 (100$) 6.470 (99%)
(SEQ ID NO: 7)
NaldCpadWdSRKd(PtscGC)WKPG-NH2 8.43 (97%)
(SEQ ID NO: 12)
CA 02259950 2005-07-21
,
- 47 -
Abbreviations used in the table are the same as in
Example s supra. A change in HPLC retention time of the
complex formed by labeling at room temperature and that
formed by heating indicates a change in the binding of
the metal.
room temperature reaction 15 min [retention
time in minutes]
after heating in boiling water 15 min
significant change in peak shape and retention
after heating
no change ~n peak shape and retention time
difference a not significant
~ any peaks
In an alternative labeling method, dilute solutions
(30 ~g/mL) of the peptide were formulated into labeling
kits prior to addition of pertechnetate. The final
solution contained the peptide, 10~ hydroxypropyl-
~cyclodextrin (HPCD) , 200 mM glucoheptonate 21 mM acetate
buffer at pH 5 .3 , 2 . mg of ascorbic acid and l00 ~g of
stannous chloride in 1.5 mL total volume. In other
formulations, two equivalents of stannous ion relative to
the peptide were added, in a buffer containing 15
HPCD, 200 mM glucoheptonate, and 21 mM acetate buffer at
pH 5.6.
E~caatplo 11: xadiolabeliag of II~P-3 'ate
IMPS, (<GHWSYK(MaGC)LRPG amide) (SEQ ID N0: 6) was
synthesized as above. IMP 3 has a retention time of 6.4 min
on a reversed phase C-18 column using a gradient of 0-100
B in 10 min at a flow rate of 3 ml/min where A is 0.1~ TFA
in H20 and B is 90~ CH3CN, 0.1~ TFA.
IMF3 was formulated in 1 mg and 250 ~Cg amounts with
450 ~.g Sn(II) and a-D-glucoheptonate at a ratio of
1:17.5, and lyophilized. The lyophilized vials of IMP3_
(1 mg sad 250 ~Cg) were reconstituted with 617 and 578 ~cCi
CA 02259950 2005-07-21
- 47a -
of NaReO, in saliae. The vials were heated for 15 min at
75 C: HPLC analysis under the conditions described above
showed single peaks at RT of ~7 . 0 min for both vials . The
effluent was collected and counted on a y-counter. For
the 1 mg vial, the recovery of activity was 88~c whereas
the recovery was 77~c for the 250 ~g vial. Colloid
analyses on an ITLC strip developed in
H,O: Et~H :NH,OH ( 5 : 2 :1 ) showed 1. 4 and 1. 2 ~ of the act ivity
at the origin for 1 mg and 250 ~g vials, respectively.
'~°Re labeling at room temperature did not proceed as
well as at 75JC. At room temperature, only a few percent
of the activity (<5%) was incorporated into the peptide
and the rest of the activity eluted in the void volume
(1.2 min) .
e'acampla 12 s In vitro Receptor Binding Assays
The human breast adenocarcinoma cell lines MCF-7 , SK-
BR-3, and MDA-MH-231 were used for testing radiometal
labeled LHRH analogues. HT29 cells were used for testing
CA 02259950 1999-O1-11
WO 98/02192 PCT/US97112084
-48-
labeled VIP analogues. All cells lines were purchased
from the American Type Culture Collection, Rockwille, MD.
Cells were grown in DMEM supplemented with 5% fetal
bovine serum, 5% defined equine serum, penicillin (100
U/ml), streptomycin (100 ~g/ml), and L-glutamine (2 mM).
The cells were routinely passaged after detachment with
trypsin and 0.2% EDTA.
Specificity of the unlabeled LHRH analogue peptides
is determined by competitive cell binding assay. Target
l0 cells are washed with fresh medium, and adjusted to 5 x
105 cell/mi. 100.1 of the cell suspension (100 ~.1) is
added per well to a 96-well microtiter plate. The cells
are allowed to attach and are then treated with different
concentrations of the peptides in the presence of '25I-
LHRH (Amersham Life Science, Arlington Heights, IL, 2,000
Ci/mmol). Following a 2h incubation at room temperature
with shaking, the cells are washed twice and the
radioactivity associated with the cells is counted and
the concentration of the peptides that cause 50%
inhibition on the binding of the labeled LH-RH is
compared.
To determine receptor binding constants, serial
dilutions of radiolabeled LHRH are incubated with 5 x 105
cells in a 96-well plate. All assay are performed in
triplicates both with or without a high concentration of
unlabeled LHRH to allow determination of specifically
bound peptide. After a 2h incubation at room
temperature, the cells are washed and counted. The
equilibrium association constant, IC~, and the total
number of receptor sites per cell are determined by
Scatchard analysis. ,
For testing VIP analogues and their metal complexes
the protocol described Virgolini et al. (Cancer Res.
54:690 (1994)) is used. Briefly, '25I VIP is mixed with
increasing concentrations of test peptide in a solution
of binding buffer, following which each solution is added
to HT29 cells in a 48 well culture plate. Each
concentration is tested in triplicate. The cells are
CA 02259950 2005-07-21
,.
- 49 -
incubated at 4°C fvr 2h, followed by three washes with
ice-cold binding buffer. The cells are then lysed with
2M NaOH for 5 min and the liquid in the well is removed
with a cotton swab. The activity on the cotton swab is
counted using a gamma counter.
The invention has been disclosed broadly and
illustrated in reference to representative embodiments
described above. Those skilled in the art will recognize
that various modifications can be made to the present
invention without departing from the spirit and scope
thereof. Subject matter relating to radiometal-binding
peptides also is described in US patent No. 5,753,206,
issued May 19, 1998.
CA 02259950 1999-07-09
2259950.seq
' SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: IMMUNOMEDICS, INC.
(ii) TITLE OF INVENTION: RADIOMETAL-BINDING PEPTIDE ANALOGUES
(iii) NUMBER OF SEQUENCES: 34
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,259,950
(B) FILING DATE: 11-JUL-1997
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/021,662
(B) FILING DATE: 12-JUL-1996
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix ) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /product= "Nle"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Xaa Asp His Xaa Arg Trp Lys
1 5
Page 1
CA 02259950 1999-07-09
a~~~y5u.seq
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Lys Pro Arg Arg Pro Tyr Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Page 2
CA 02259950 1999-07-09
~~5~y5U.seq
Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys
1 5 10 15
Lys Tyr Leu Asn Ser Ile Leu Asn
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser
1 5 10 15
Ile Leu Asn
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDN~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is pyroglutamic acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Xaa His Trp Ser Tyr Lys Leu Arg Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
Page 3
CA 02259950 1999-07-09
. z259950.seq
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( i x ) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is pyroglutamic acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Xaa His Tyr Ser Leu Lys Trp Lys Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "D isomer of 2-naphthylalanine"
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) 'LOCATION: 2
(D) OTHER INF~RMATION: /note= "D isomer of 4-chlorophenylalanine"
( i x ) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Lys"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
Page 4
CA 02259950 1999-07-09
zZ59950.seq
- (B) LOCATION: 10
(D) OTHER INFORMATION: /note= "Xaa is D-Ala"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Xaa Xaa Xaa Ser Arg Xaa Leu Arg Pro Xaa
1 5 10
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDN~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /product= "Nle"
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Ser Tyr Ser Xaa Asp His Xaa Arg Trp Lys
1 5 10
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix ) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 17
(D) OTHER INFORMATION: /product= "Nle"
Page 5
CA 02259950 1999-07-09
~~5yy5U.seq
~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
His Ser Asp Ala Val Phe Thr Glu Asn Tyr Thr Lys Leu Arg Lys Gln
1 5 10 15
Xaa Ala Ala Lys Lys Tyr Leu Asn Asp Leu Lys Lys Gly Gly Thr
20 25 30
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is pyroglutamic acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Xaa His Tyr Ser Tyr Leu Lys Trp Lys Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "D isomer of 2-naphthylalanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "D isomer of 4-chlorophenylalanine"
Page 6
CA 02259950 1999-07-09
~~5yy5u.seq
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Lys"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Xaa Xaa Xaa Ser Arg Xaa Trp Lys Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is pyroglutamic acid"
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Lys"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Xaa His Trp Ser Tyx Xaa Leu Arg Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
Page 7
CA 02259950 1999-07-09
ZZ59950.seq
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Lys Xaa Cys Phe Xaa Lys Thr Cys Thr
1 5
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRTPTION: SEQ ID N0:15:
Lys Asp Xaa Cys Phe Xaa Lys Thr Cys Thr
1 5 10
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
Page 8
CA 02259950 1999-07-09
~~5yy5U.seq
(C) STRANDEDN~SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Asp Xaa Cys Phe Xaa Lys Thr Cys Thr
1 5
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Lys Lys Lys Xaa Cys Phe Xaa Lys Thr Cys Thr
1 5 10
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
Page 9
CA 02259950 1999-07-09
~~5yy5U.seq
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Lys Lys Asp Xaa Cys Phe Xaa Lys Thr Cys Thr
1 5 10
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRTPTION: SEQ ID N0:19:
Lys Asp Ser Xaa Cys Phe Xaa Lys Thr Cys Thr
1 5 10
(2) INFORMATION FOR SEQ ID N0:20:
Page 10
CA 02259950 1999-07-09
~~5~~5u.seq
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Lys Asp Xaa Cys Phe Xaa Lys Thr Cys Asp
1 5 10
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Xaa Cys Phe Xaa Lye Thr Cys Thr Lys
1 5
Page 1l
CA 02259950 1999-07-09
Z259950.seq
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Cys Gly Cys His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu
1 5 10 15
Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25 30
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Cys Gly Cys Val Phe ~hr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met
1 5 10 15
Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
Page 12
CA 02259950 1999-07-09
LG5yy5U.seq
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is pyroglutamic acid"
(xi) SEQUENCE DESCR$PTION: SEQ ID N0:24:
Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "D isomer of 2-naphthylalanine"
( i x ) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "D isomer of 4-chlorophenylalanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Arg"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Xaa Xaa Xaa Asp Glu Xaa Leu Lys Pro
1 5
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: ammo acid
Page 13
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Z25yy5U . SeCj
(C) STRANDEDNFSS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix ) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: l
(D) OTHER INFpRMATION: /note= "Xaa is pyroglutamic acid"
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Xaa His Trp Ser Lys Xaa Leu Arg Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is pyroglutamic acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Lys"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Xaa His Tyr Ser Leu Xaa Leu Arg Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
Page 14
CA 02259950 1999-07-09
GG5~y5U.seq
~ (A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDN$SS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val
1 5 10
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /product= "Nle"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "Xaa is a nitrated arginine residue
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Ser Tyr Ser Xaa Asp His Xaa Xaa Trp Lys
1 5 10
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
Page 15
CA 02259950 1999-07-09
zz5~y5~.seq
- (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /product= "Nle"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Gly Cys Xaa Asp His Xaa Arg Trp Lys
1 5
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARAOTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /product= "Nle"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Cys Gly Cys Xaa Asp His Xaa Arg Trp Lys
1 5 10
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
Page 16
CA 02259950 1999-07-09
~~5yy5U.seq
- (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 17
(D) OTHER INFORMATION: /product= "Nle"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Lys Leu Arg Lys Gln
1 5 10 15
Xaa Ala Val Lys Lys Tyr Leu Asn Ser Val Leu Thr
20 25
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( ix) FEATURE
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa is pyroglutamic acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Xaa His Tyr Ser Leu Glu Trp Lys Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
Page 17
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GG5yy5V . sect
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Xaa is D-Phe"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "Xaa is D-Trp"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Gly Cys Asp Xaa Cy~ Phe Xaa Lys Thr Cys Thr
1 5 10
Page 18
