U.S. patent application number 12/054871 was filed with the patent office on 2008-07-24 for labeling reactant.
This patent application is currently assigned to WALLAC OY. Invention is credited to Jari HOVINEN, Veli-Matti Mukkala, Jari Peuralahti.
Application Number | 20080176338 12/054871 |
Document ID | / |
Family ID | 35510731 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080176338 |
Kind Code |
A1 |
HOVINEN; Jari ; et
al. |
July 24, 2008 |
LABELING REACTANT
Abstract
This invention concerns novel labeling reactants suitable for
labeling of a biospecific binding reactant using solid-phase
synthesis. The novel reactants are derivatives of
diethylenetriaminepentaacetic acid (DTPA), wherein a suitable group
is linked to the DTPA molecule, thus allowing site specific
introduction of the ligand of said derivatives to bioactive
molecules on solid phase in an oligopeptide synthesizer.
Inventors: |
HOVINEN; Jari; (Raisio,
FI) ; Peuralahti; Jari; (Turku, FI) ; Mukkala;
Veli-Matti; (Kaarina, FI) |
Correspondence
Address: |
BUCHANAN, INGERSOLL & ROONEY PC
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
WALLAC OY
Turku
FI
|
Family ID: |
35510731 |
Appl. No.: |
12/054871 |
Filed: |
March 25, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11607045 |
Dec 1, 2006 |
7381420 |
|
|
12054871 |
|
|
|
|
60748195 |
Dec 8, 2005 |
|
|
|
Current U.S.
Class: |
436/501 ;
560/33 |
Current CPC
Class: |
C07C 2603/18 20170501;
C07C 229/24 20130101; A61K 49/085 20130101; G01N 33/532 20130101;
G01N 33/58 20130101; C07K 1/13 20130101; C07C 271/22 20130101; A61K
49/14 20130101 |
Class at
Publication: |
436/501 ;
560/33 |
International
Class: |
C07C 271/06 20060101
C07C271/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 2005 |
FI |
20055653 |
Claims
1. A labeling reactant of formula (I) ##STR00007## wherein: -A- is
a linker formed from one to ten moieties, each moiety being
selected from the group consisting of phenylene, alkyl containing
1-12 carbon atoms, ethynediyl (--C.ident.C--), ethylenediyl
(--C.dbd.C--), ether (--O--), thioether (--S--), amide (--CO--NH--
and --NH--CO-- and --CO--NR'' and --NR''-CO--), carbonyl (--CO--),
ester (--COO-- and --OOC--), disulfide (--SS--), diaza
(--N.dbd.N--) and tertiary amine (--NR''--), wherein R'' represents
an alkyl containing less than 5 carbon atoms; R is independently
--COOR' or --CONHR', wherein R' is an alkyl of 1 to 4 carbon atoms,
phenyl or benzyl, and wherein said phenyl or benzyl is substituted
or unsubstituted; Z is a transient protecting group; and X is a
carboxylic acid, or an organic or inorganic salt, active ester or
acid halide thereof.
2. The labeling reactant according to claim 1, wherein X is an
active ester of carboxylic acid and said active ester of carboxylic
acid is selected from the group consisting of an
N-hydroxysuccinimido, p-nitrophenol and pentafluorophenol
ester.
3. The labeling reactant according to claim 1, wherein X is an acid
halide and said acid halide is selected from the group consisting
of chloride and fluoride.
4. The labeling reactant according claim 1, wherein Z is selected
from the group consisting of fluorenylmethoxycarbonyl (Fmoc),
nitrobenzenesulfonyl (Ns), tert-butoxycarbonyl (Boc) and
1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc).
5. The labeling reactant according to claim 1, wherein the labeling
reactant is penta-tert-butyl 2-{4'-{2-[4-carboxy-4-fluorenyl
methyloxycarbonylamino)]butyrylamido}benzyl}diethylenetriamine-pentakis(a-
cetate).
6. A biospecific binding reactant labeled with the labeling
reactant according to claim 1, wherein the biospecific labelling
reactant is selected from the group consisting of an oligopeptide,
protein, oligosaccaride, polysaccaride, phospholipid, PNA, LNA,
antibody, hapten, drug, receptor binding ligand and lectine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
11/607,045, filed on Dec. 1, 2006, which claims priority to U.S.
Provisional Application No. 60/748,195 filed on Dec. 8, 2005, the
disclosure of which are incorporated herein in their entirety by
reference. This application also claims priority under 35 U.S.C.
.sctn. 119 to Finnish Patent Application No. 20055653, filed on
Dec. 8, 2005, in the Finnish Intellectual Property Office, the
disclosure of which is incorporated herein in its entirety by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to novel derivatives of
diethylenetriaminepentaacetic acid which allow site specific
introduction of the ligand of said derivatives to bioactive
molecules on solid phase.
BACKGROUND OF THE INVENTION
[0003] The publications and other materials used herein to
illuminate the background of the invention, and in particular,
cases to provide additional details respecting the practice, are
incorporated by reference.
[0004] Because of its excellent metal chelating properties
diethylenetriaminepentaacetic acid (DTPA) is one of the most widely
used organic ligands in magnetic resonance imaging (MRI) and
positron emission tomography (PET) [Aime, S., Botta, M., Fasano, M.
and Terrano, E. 1998, Chem. Soc. Rev., 27, 19, Caravan, P.,
Ellison, J. J., McMurry, T. J. and Lauffer, R. B., 1999, Chem.
Rev., 99, 2293, Woods, M., Kovacs, Z. and Sherry, A. D., 2002, J.
Supramol. Chem., 2, 1]. Indeed, the first FDA approved contrast
agent in clinical use is the Gd.sup.3+ DTPA chelate [Runge, V. M.,
2000, J. Magn. Res. Imaging, 12, 205.]. The corresponding .sup.111
in and .sup.68Ga chelates, in turn, are suitable for PET
applications [Anderson, C. J. and Welch, M. J., 1999, Chem. Rev.
99, 2219], while Eu.sup.3+, Tb.sup.3+, Sm.sup.3+ and
Dy.sup.3+chelates can be used in applications based on
disassociation enhanced lanthanide fluorescence immunoassay
(DELFIA) [PCT WO 03/076939A1]. .sup.99mTc DTPA in turn, is suitable
for single positron emission computed tomography (SPECT)
[Lorberboym, M., Lampl, Y. and Sadeh, M., 2003, J. Nucl. Med. 44,
1898, Galuska, L., Leovey, A., Szucs-Farkas, Z., Garai, I., Szabo,
J., Varga, J. and Nagy, E. V., 2002, Nucl. Med. Commun. 23, 1211].
Bioactive molecules labeled with .sup.111In or .sup.117mSn DTPA may
find applications as target-specific radiopharmaceuticals [Volkert,
W. A. and Hoffman, T. J., 1999, Chem. Rev. 99, 2269].
[0005] In several applications, covalent conjugation of DTPA to
bioactive molecules is required. Often, isothiocyanato,
N-hydroxysuccinimide or maleimide derivatives of the chelate are
used in the labeling the target molecules such as oligonucleotides
and oligopeptides. Several bifunctional DTPA derivatives are
currently commercially available. Because in all of these cases the
labeling reaction is performed in the presence of an excess of an
activated label, laborious purification procedures cannot be
prevented. Especially, when attachment of several label molecules
is needed, purification and characterization of the desired
biomolecule conjugate may be extremely difficult.
[0006] The purification problems can be avoided by performing the
labeling reaction on solid phase. Hence, most of the impurities can
be removed by washings when the biomolecule conjugate is still
anchored to the solid support, and after release to the solution,
only one chromatographic purification is needed. Several such
blocks have been published. They include organic dyes [Loshe, J.,
Nielsen, P. E., Harrit, N. and Dahl, O., 1997, Bioconjugate Chem.
8, 503, McCafferty, D. G., Bishop, B. M., Wall, C. G., Hughes, S.
G., Mecklenberg, S. L., Meyer, T. J., and Erickson, D. W., 1995,
Tetrahedron, 51, 1093, WO 96/03409, Cuppoletti, A., Cho, Y., Park,
J.-C., Strassler, G. and Kool, E. T. 2005, Bioconjugate Chem. 16,
528, Bethelot, T., Lain, G., Latxague, L. and Deleris, G., 2004, J.
Fluorescence, 14, 671], derivatives of EDTA [Sluka, J. P., Griffin,
J. H., Mack, D. P. and Dervan, P. B. 1990, J. Am. Chem. Soc, 112,
6369, Arya, R. and Gariepy, J. 1991, Bioconjugate Chem., 2, 323,
Cuenoud, B. and Schepartz, A. 1991, Tetrahedron, 47, 2535, Rana, T.
M., Ban, M. and Hearst, J. E., 1992, Tetrahedron Lett, 33, 4521,
Song, A. I. and Rana, T. A., 1997, Bioconjugate Chem., 8, 249,
Davies, J. C., Al-Jamri, L., 2002, J. peptide Sci., 8, 663, U.S.
Pat. No. 5,637,759], DOTA [Bhorade, R., Weissleder, R., Nakakoshi,
T., Moore, A. and Tung, C.-H., 2000, Bioconjugate Chem., 11, 301.,
Gallazzi, F., Wang, Y., Jia, F., Shenoy, N., Landon, L. A.,
Hannink, M., Lever, S. Z. and Lewis, M. R. 2003, Bioconjugate
Chem., 14, 1083.] and luminescent and non-luminescent lanthanide
chelates [U.S. Pat. No. 6,080,839; 6,949,696; Peuralahti, J.,
Hakala, H., Mukkala, V.-M., Hurskainen, P., Mulari, O. and Hovinen,
J. 2002 Bioconjugate Chem. 13, 876.].
[0007] Although DTPA molecule is known for decades, and although
reagents for solid phase oligonucleotide derivatization with DTPA
has been demonstrated [U.S. Pat. No. 6,949,639], no reactants which
allow its direct solid phase conjugation to oligopeptides have been
synthesized. The solid phase methods published involve synthesis of
oligopeptides, where one .epsilon.-amino group of lysine is
selectively deprotected while the oligomer is still anchored to the
resin [Handl, H. L., Vagner, J., Yamamura, H. I., Hruby, V. J. and
Gilles, R. J. 2005, Anal. Biochem. 343, 299, Nagy, I. B., Vagra, I.
and Hudecz, F, 2000, Anal. Biochem., 287, 17] Then, an activated
DTPA molecule (as an anhydride or an HOBt ester) is coupled to the
primary amino function, the oligopeptide is deprotected and
converted to the appropriate DTPA chelate. However, this
methodology has some drawbacks. First, practically only one DTPA
molecule can be introduced. This may be problematic in applications
were high detection sensitivity is required. Second, since one of
the iminoacetic acid groups is used for conjugation, the resulting
chelate is less stable than the parent DTPA molecule [Paul-Roth, C.
and Raymond, K. N. 1995, Inorg. Chem. 34, 1408, Li, W. P., Ma, D.
S., Higginbotham, C., Hoffman, T., Ketring, A. R., Cutler, C. S.
and Jurisson, S. S. 2001, Nucl. Med. Biol. 28, 145.]. This may be a
serious problem in vivo applications especially in MRI due to the
high toxicity of free Gd(III) ion.
[0008] A schematic preparation of stable DTPA derivatives
applicable to solid phase peptide incorporation have been proposed
[U.S. Pat. No. 5,637,759], but the method of their preparation is
challenging due to the carboxyl protecting strategy. There,
selective deprotection of a single and specic carboxylic acid group
out of six of similar reactivities is required. This problem can be
avoided by changing the protecting group strategy, but the
synthetic route will be considerably longer [WO 03/011115].
SUMMARY OF THE INVENTION
[0009] The main object of the present invention is to provide DTPA
derivatives which allow solid phase introduction of the chelate to
bioactive molecules using a standard oligopeptide synthesizer. The
bioconjugates thus obtained are highly suitable for magnetic
resonance imaging (MRI), positron emission tomography (PET), single
positron emission computed tomography (SPECT) and dissossiation
enhanced lanthanide fluorescence immunoassay (DELFIA) as well as
target-specific radiopharmaceuticals. The major advantage of the
present invention are:
[0010] (i) synthesis of the building block is simple and thus these
molecules can be synthesized in large scale;
[0011] (ii) the blocks can be introduced to the biomolecule
structure with standard oligopeptide synthesizer in high efficiency
using normal procedures;
[0012] (iii) since the metal is introduced after the chain assembly
is completed, the molecule synthesized can be used in various
applications simply by changing the metal;
[0013] (iv) since none of the DTPA carboxylic acid residues are
used for conjugation the stability of the chelate does not
change;
[0014] (v) the chelate formation is considerably faster than in the
case of DOTA (the other most commonly used chelator). This is
advantageous while working with short-living radioisotopes.
[0015] (vi) the labeling reactant can be used in the labeling of a
large variety of bioactive molecules such as oligopeptides,
steroids and drugs.
[0016] In some applications it is advantageous that the chelate is
neutral. Then, two of the acetate groups can be substituted with
amides. Naturally, the stability of these chelates is lower than
that of the corresponding acetates.
[0017] Thus, the present invention concerns a labeling reactant of
formula (I) or (Ia) suitable for labeling of a biospecific binding
reactant
##STR00001##
[0018] wherein,
[0019] -A- is a linker, which is formed from one to ten moieties,
each moiety being selected from the group consisting of phenylene,
alkyl containing 1-12 carbon atoms, ethynediyl (--C.ident.C--),
ethylenediyl (--C.dbd.C--); ether (--O--), thioether (--S--), amide
(--CO--NH-- and --CO--NR''), carbonyl (--CO--), ester (--COO-- and
--OOOC--), disulfide (--SS--), diaza (--N.dbd.N--) or a tertiary
amine (--NR''--), where R'' represents an alkyl containing less
than 5 carbon atoms,
[0020] each R is independently --COOR' or --CONHR', where R' is an
alkyl of 1 to 4 carbon atoms, phenyl or benzyl, which phenyl or
benzyl is substituted or unsubstituted;
[0021] Z is a transient protecting group;
[0022] X is a carboxylic acid, its organic or inorganic salt or
active ester or acid halide.
[0023] The present invention concerns a method for the preparation
of labeling reactants of formula (I) and (Ia) as defined in claim
9.
DETAILED DESCRIPTION OF THE INVENTION
[0024] In case R' as defined above is a substituted phenyl or
substituted benzyl, the most preferable substituents include
chloride.
[0025] Where X is an active ester of a carboxylic acid, said ester
is preferably an N-hydroxysuccinimido, p-nitrophenol or
pentafluorophenol ester.
[0026] Where X is an acid halide of a carboxylic acid, said halide
is preferably chloride or fluoride.
[0027] The transient protecting group Z is preferably
fluorenylmethoxycarbonyl (Fmoc); nitrobenzenesulfonyl (Ns);
tert-butoxycarbonyl (Boc) or 1,1-dioxobenzo[b]thio-phen-2-yl
methyloxycarbonyl (Bsmoc).
[0028] The biospecific binding reactant to be labeled is, for
example, an oligopeptide, protein, oligosaccaride, polysaccaride,
phospholipid, PNA, LNA, antibody, hapten, drug, receptor binding
ligand or lectine. Most preferably, the biospecific binding
reactant is an oligopeptide.
[0029] The present invention concerns also a method for the
preparation of labeling reactants of formula (I) and (Ia)
##STR00002##
[0030] comprising
[0031] reaction of the molecule of structure formula (II) or
(IIa)
##STR00003##
[0032] with a compound of the formula (III),
##STR00004##
[0033] wherein Z.sup.2 is a transient protecting group, most
preferably allyl or benzyl, A.sup.2 is a linker formed from one to
ten moieties, each moiety being selected from the group consisting
of phenylene, alkyl containing 1-12 carbon atoms, ethynediyl
(--C.ident.C--), ethylenediyl (--C.dbd.C--); ether (--O--),
thioether (--S--), amide (--CO--NH-- and --NH--CO-- and --CO--NR''
and --NR''--CO--), carbonyl (--CO--), ester (--COO-- and --OOO--C),
disulfide (--SS--), diaza (--N.dbd.N--) and tertiary amine
(--NR''--), where R'' represents an alkyl containing less than 5
carbon atoms, X.sup.2 carboxylic acid, acid chloride, acid bromide,
acid fluoride or an active ester, most preferably pentafluorophenyl
or N-hydroxysuccininyl ester, optionally in the presence of an
activator to give compound (IV) or (IVa)
##STR00005##
[0034] which is deprotected to give compound of formula I or Ia in
which X is COOH, after which X optionally is converted to the
corresponding organic or inorganic salt or active ester or acid
halide.
[0035] The invention will be illuminated by the following
non-restrictive Experimental Section.
EXPERIMENTAL SECTION
[0036] The invention is further elucidated by the following
examples. The structures and synthetic routes employed in the
experimental part are depicted in Scheme 1. Experimental details
are given in examples 1 and 2. Coupling of the oligopeptide
building block to oligopeptide structure on solid phase,
deprotection and convertion to the corresponding gadolinium(III)
chelate is given in Example 3.
Procedures
[0037] Adsorption column chromatography was performed on columns
packed with silica gel 60 (Merck). Reagents for oligopeptide
synthesis were purchased from Nova Biochem. Sodium sulfinate resin
(200-400 mesh, 1% DVB, 1.3 mmol g.sup.-1) was purchased from
Tianjin Nankai Hecheng Science & technology Company Limited
(China). The oligopeptides were assembled on an Applied Biosystems
433A instrument, using recommended protocols. HPLC purifications
were performed using a Shimazu LC 10 AT instrument equipped with a
diode array detector, a fraction collector and a reversed phase
column (LICHROCART.RTM. 125-3 PUROSPHER.RTM. RP-18e 5 .mu.m).
Mobile phase: (Buffer A): 0.02 M triethylammonium acetate (pH 7.0);
(Buffer B): A in 50% (v/v) acetonitrile. Gradient: from 0 to 1 min
95% A, from 1 to 21 min from 95% A to 100% B. Flow rate was 0.6 mL
min.sup.-1. All dry solvents were from Merck and they were used as
received. NMR spectra were recorded on a Bruker 250 spectrometer
operating at 250.13 MHz for .sup.1H. The signal of TMS was used as
an internal reference. ESI-TOF mass spectra and IR spectra were
recorded on Applied Biosystems MARINER.TM. and PerkinElmer
SPECTRUM.TM. ONE instruments, respectively.
Examples
Example 1
The synthesis of penta-tert-butyl
2-{4'-{2-[4-allyloxycarbonyl-4-(fluorenylmethyloxycarbonylamino)]butyryla-
mido}benzyl}-diethylenetriamine-pentakis(acetate), 2
[0038] Fmoc-Glu-OAII (1.31 g, 3.21 mmol), HATU (1.22 g, 1.32 mmol)
and DIPEA (0.57 mL, 3.21 mmol) were dissolved in dry DMF (5 mL),
and the mixture was stirred for 15 min at RT. Compound 1, disclosed
in Corson, D. T., Meares, C. F., 2000, Bioconjugate Chem., 11, 292
(2.50 g, 3.21 mmol; predissolved in 2 mL of dry DMF) was added and
the mixture was stirred for an additional 2 h. The mixture was
diluted with dichloromethane (50 mL), washed twice with 10% citric
acid and dried over Na.sub.2SO.sub.4. Purification on silica gel
(eluent CH.sub.2Cl.sub.2/MeOH 9:1, v/v) gave 2.84 g (82%) of
compound 2. .sup.1H NMR (CDCl.sub.3): .delta. 8.31 (2H, br s); 7.75
(2H, d, J 7.3); 7.61 (4H, m); 7.39 (2H, t, J 7.3); 7.30 (2H, m);
7.02 (2H, d, J 8.6); 5.96 (1H, m); 5.89 (1H, m); 5.26 (1H, m); 4.65
(2H, d, J 5.4); 4.41 (2H, m); 4.22 (1H, t, J 6.7); 3.40-2.38 (24H);
1.48 (36H, s); 1.42 (9H, s). ESI-TOF-MS for
C.sub.64H.sub.92N.sub.5O.sub.15 (M+H).sup.+: calcd, 1170.66; found,
1170.59.
Example 2
The synthesis of penta-tert-butyl
2-{4'-{2-[4-carboxy-4-(fluorenylmethyloxycarbonylamino)]butyrylamido}benz-
yl}diethylenetriaminepentakis(acetate), 3
[0039] Compound 2 (1.00 g, 0.90 mmol) was dissolved in dry THF (20
mL) and deaerated with argon. Pd(Ph.sub.3P).sub.4 (63 mg) and
sodium sulfinate resin (1.0 g) were added, and the mixture was
stirred for 2 h at RT. The resin was filtered off, washed with THF
and the filtrate was concentrated. The residue was dissolved in
dichloromethane, washed with 10% citric acid, dried over 4 .ANG.
molecular sieves. Concentration in vacuo yielded compound 3.
ESI-TOF-MS for C.sub.61H.sub.88N.sub.5O.sub.15 (M+H).sup.+: calcd,
1130.63; found, 1130.65.
Example 3
The Synthesis of Oligopeptide Conjugates
[0040] A model sequence (RKEMSIKVAVS) was synthesized in 10 .mu.mol
scale using Fmoc chemistry and recommended protocols (coupling time
30 min for natural amino acid analogues, and 2 h for 3). One or
five blocks 3 was coupled to its carboxy terminus. When the chain
assembly was completed, the resin was treated with the mixture of
crystalline phenol (75 mg), ethanedithiol (25 mL), thioanisole (50
.mu.L), water (50 .mu.L) and trifluoroacetic acid (1 mL) for 4 h.
The resin was removed by filtration, and the solution was
concentrated in vacuo. The crude oligopeptide was precipitated with
diethyl ether. The precipitate was redissolved in water and treated
with gadolinium(III) citrate (5 equiv per ligand). Purification was
performed on HPLC.
[0041] It will be appreciated that the methods of the present
invention can be incorporated in the form of a variety of
embodiments, only a few of which are disclosed herein. It will be
apparent for the expert skilled in the field that other embodiments
exist and do not depart from the spirit of the invention. Thus, the
described embodiments are illustrative and should not be construed
as restrictive.
##STR00006##
* * * * *