Title: N-aryl-carbamic acid ester-derived and valeric acid ester-derived cross-linkers and conjugates, and methods for their synthesis and use
Abstract: The present invention describes carbamic acid ester-derived and valeric acid ester-derived polyfunctional cross-linker molecules, and methods for their synthesis and use. The inclusion of polymeric moieties such as poly(alkylene oxide) in the cross-linkers of the present invention can provide advantageous solubility properties in aqueous environments. Such cross-linkers may be used to form conjugates for use in a variety of assay formats.
Patent Number: 6,967,107 Issued on 11/22/2005 to Buechler, et al.
| Inventors:
|
Buechler; Kenneth F. (San Diego, CA);
Banaszczyk; Mariusz G. (San Marcos, CA);
Noar; Joseph Barry (Solana Beach, CA)
|
| Assignee:
|
Biosite, Inc. (San Diego, CA)
|
| Appl. No.:
|
080211 |
| Filed:
|
March 14, 2005 |
| Current U.S. Class: |
436/533; 436/532; 436/543; 525/54.1; 525/532; 548/534; 548/545; 548/563 |
| Intern'l Class: |
G01N 033/54.7; G01N 033/53.2; C07D 207/22; C07D 207/30; C07L 089/00 |
| Field of Search: |
436/533,532,543
525/532,541
548/534,545,563
|
References Cited [Referenced By]
U.S. Patent Documents
| 5643575 | Jul., 1997 | Martinez et al.
| |
| 5672662 | Sep., 1997 | Harris et al.
| |
| 5705153 | Jan., 1998 | Shorr et al.
| |
| 5730990 | Mar., 1998 | Greenwald et al.
| |
| 5763189 | Jun., 1998 | Buechler et al.
| |
| 5902588 | May., 1999 | Greenwald et al.
| |
| 5932462 | Aug., 1999 | Harris et al.
| |
| 6238931 | May., 2001 | Buechler et al.
| |
| 6251687 | Jun., 2001 | Buechler et al.
| |
| Foreign Patent Documents |
| WO 95/0877/2 | Mar., 1995 | WO.
| |
Other References
Gibson et al., "Nonpeptidic α84β3 Integrin
Antagonist Libraries: On-Bead Screening and Mass Spectrometric Identification without
Tagging," Agnew. Chem. Int. Ed. 40: 165-169, 2001.
Gottschling et al., "Cellular Solid-Phase Binding Assay and Mass Spectrometry
for Screening of α4β7 Integrin Antagonists," Bioorg. Med. Chem. Lett.
11:2997-3000, 2001.
Leon et al., "Evaluation of Resins for On-Bead Screening: A Study of Papain and
Chymotrypsin Specificity Using Pega-Bound Combinatorial Peptide Libraries," Bioorg.
Med. Chem. Lett. 8:2997-3002, 1998.
Orain and Bradley, "Solid Phase synthesis of tyrpanothione reductase inhibitors—towards
single bead screening," Tetrahedron Lett. 42:515-518, 2001.
Papanikos et al., "α-Ketocarbonyl Peptides: A General Approach to Reactive
Resin-Bound Intermediates in the Synthesis of Peptide Isosteres for Protease Inhibitor
Screening on Solid Support," J. Am. Chem. Soc. 123:2176-2181, 2001.
Smith and Bradley, "comparison of Resin and Solution Screening Methodologies
in Combinatorial Chemistry and the Identification of a 100 nM Inhibitor of Trypanothione
Reductases," J. Comb. Med. 1:326-332, 1999.
Topchieva et al., "Synthesis and Physiochemical Properties of Protein Conjugates
with Water-Soluble Poly(alkylene oxides)," Bioconjung. Chem. 6:380-8, 1995.
Ward et al., "Binding activities of a repertoire of single immunoglobulin variable
domins secreted from Escherichia coli," Nature 341:544-546, 1989.
Wilson, "Simplified conjugation chemistry for coupling peptides to F(ab') fragments:
autologous red cell agglutination assay for HIV-1 antibodies," J. Immunol. Methods
175:267-273; 1994.
Yarmush, "coupling of antibody-binding fragments to solid-phase supports: site-directed
,binding of F(ab)2 fragments," J. Biochem. Biophys. Methods 25:85-97, 1992.
|
Primary Examiner: Le; Long V.
Assistant Examiner: Haq; Shafiqul
Attorney, Agent or Firm: Foley & Lardner LLP
Parent Case Text
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a Division of U.S. application Ser. No. 10/778,919, filed
Feb. 12, 2004 now U.S. Pat. No. 6,887,952, incorporated herein by reference in
its entirety.
Claims
1. A method of conjugating a first species selected from the group consisting
of a protein, a polypeptide, an antibody, a nucleic acid, a small molecule, an
aptamer, and a carbohydrate, to a second species selected from the group consisting
of a detectable label, a solid phase, a protein, a polypeptide, an antibody, a
nucleic acid, a small molecule, an aptamer, and a carbohydrate, the method comprising:
contacting said first and second species with a crosslinker of the formula:
![embedded image]()
wherein X is (alkylene oxide)
n;
n is between about 40 to about 450; and
Y and Z are independently arylene or heteroarylene units having 5 or 6 ring atoms,
optionally substituted with from 1 to 4 substituents independently selected from
the group consisting of C
1-6 alkyl straight or branched chain, halogen,
trihalomethyl, C
1-6 alkoxy, —NO
2, —NH
2,
—OH, —COOR′, where R′ is H or lower alkyl, —CH
2OH,
—CONH
2, and a linkage to a poly(alkylene oxide) moiety, wherein
Z is optionally present;
under conditions selected to conjugate said first species to said second species,
wherein one of the first or second species becomes covalently bound to said crosslinker
through a moiety reactive with an N-hydroxysuccinimide functional group, and the
other of the first or second species becomes covalently bound to said crosslinker
through a moiety reactive with a maleimide functional group wherein the conjugating
step comprises (i) covalent binding of the crosslinker to the first or second species
through a moiety reactive with a first functional group that is an N-hydroxysuccinimide
or a maleimide functional group, to form a covalently bound crosslinker intermediate;
(ii) removal of unreacted crosslinker from the covalently bound crosslinker intermediate;
and (iii) covalent binding of the crosslinker to the other of the first or second
species through a moiety reactive with a second functional group that is the other
of the N-hydroxysuccinimide or maleimide functional group.
2. A method according to
![embedded image]()
wherein X is (alkylene oxide)
n;
n is between about 40 to about 450; and
each R is independently selected from the group consisting of C
1-6
alkyl straight or branched chain, halogen, trihalomethyl, C
1-6 alkoxy,
NO
2, NH
2, OH, —COOR′, where R′ is H or
lower alkyl, CH
2OH, CONH
2, and a linkage to a polyalkylene
oxide moiety.
3. A method according to claim 1, wherein the (alkylene oxide)
n group
is a copolymer of two or more units selected from the group consisting of methylene
oxide, ethylene oxide, propylene oxide, isopropylene oxide, and butylene oxide.
4. A method according to claim 1, wherein the (alkylene oxide)n group is a homopolymer
of units selected from the group consisting of methylene oxide, ethylene oxide,
propylene oxide, isopropylene oxide, and butylene oxide.
5. A method according to
![embedded image]()
wherein n is between about 40 to about 450.
6. A method according to claim 5, wherein n is between 60 and 100.
7. A method according to claim 5, wherein n is between 70 and 90.
8. A method according to claim 5, wherein n averages about 77.
9. A method according to claim 1, wherein the conjugating step comprises (i)
covalent binding of the crosslinker to the first or second species through a moiety
reactive with an N-hydroxysuccinimide functional group to form a covalently bound
crosslinker intermediate; (ii) removal of unreacted crosslinker from the covalently
bound crosslinker intermediate; and (iii) covalent binding of the crosslinker to
the other of the first or second species through a moiety reactive with a maleimide
functional group.
10. A method according to claim 1, wherein the first or second species is an antibody.
11. A method according to claim 1, wherein the second species is a latex particle
comprising a moiety reactive with a N-hydroxysuccinimide functional group or reactive
with a maleimide functional group.
12. A method according to claim 1, wherein the second species is a solid phase
modified to provide linkage sites for the crosslinker.
13. A method according to claim 12, wherein the second species is a latex particle,
and the linkage sites are provided by an intermediary protein.
14. A method according to claim 1, wherein the first species is an antibody,
and the second species is a solid phase comprising linkage sites for the crosslinker.
15. A method according to claim 14, wherein the second species is a latex particle
comprising a moiety reactive with a N-hydroxysuccinimide functional group or reactive
with a maleimide functional group.
16. A method according to claim 15, wherein the latex particle is covalently
bound to the crosslinker through a moiety reactive with an N-hydroxysuccinimide
functional group provided on an intermediary protein, and wherein the antibody
is covalently bound through a moiety reactive with a maleimide functional group
provided by cystenylation of the antibody.
Description
FIELD OF THE INVENTION
The present invention relates to novel carbamic acid and valeric acid esters
useful as cross-linkers, to conjugates comprising such cross-linkers, and to methods
for their synthesis and use.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention is merely provided
to aid the reader in understanding the invention and is not admitted to describe
or constitute prior art to the present invention.
Chemical cross-linkers are valuable tools for scientists and are discussed
in numerous books and catalogues. See, e.g., Wong,
Chemistry of Protein Conjugation
and Cross-
linking, CRC Press, Boca Raton, Fla., 1991. These reagents
may be used in a variety of ways, such as to assist in the determination of near-neighbor
relationships in proteins, molecular associations in cell membranes, three-dimensional
structures of proteins, enzyme-substrate orientation, solid-phase immobilization,
and hapten-carrier protein conjugation. They are also useful for preparing antibody-detectable
label conjugates, immunotoxins and other labeled protein and nucleic acid reagents.
Cross-linking agents often employ functional groups that couple to amino acid side
chains of peptides. These reagents may be classified on the basis of the following:
- 1. Functional groups and chemical specificity;
- 2. length and composition of the cross-bridge;
- 3. whether the cross-linking groups are similar (homobifunctional) or
different (heterobifunctional);
- 4. whether the groups react chemically or photochemically;
- 5. whether the reagent is cleavable; and
- 6. whether the reagent can be radiolabeled or tagged with another label.
Reactive groups that can be targeted using a cross-linker include primary
amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids. In addition,
many reactive groups can be coupled nonselectively using a cross-linker such as
photoreactive phenyl azides.
Cross-linking reagents contain at least two reactive groups, and are
divided generally into homofunctional cross-linkers (containing identical reactive
groups) and heterofunctional cross-linkers (containing non-identical reactive groups).
While for convenience the following discussion refers to homobifunctional and heterobifunctional
cross-linkers (where "bifunctional" refers to the presence of two functional groups),
cross-linking reagents having more than two functional groups are well known to
the artisan and are within the scope of the invention described herein.
Homobifunctional cross-linkers that couple through amines, sulfhydryls
or react non-specifically are available from many commercial sources. Maleimides,
alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive
groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls
to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce
mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also
very useful for protein-protein cross-links. These cross-linkers can penetrate
cell membranes and cross-link proteins within the membrane to study membrane composition,
structure and protein-protein and protein-lipid interactions. Imidoesters are also
useful for oligomer formation. For example, cross-linking proteins to form oligomers
may reveal if a bivalent, dimeric or trimeric form of the protein is responsible
for activity.
A nonselective homobifunctional cross-linker is useful for conjugating functional
groups, such as hydroxyls for which specific cross-linkers are not available. An
example of a nonselective homobifunctional cross-linker is BASED (Product #21564
Pierce Co.). This cross-linker has a long spacer arm and 2 aromatic rings which
makes it very hydrophobic with a limited solubility in aqueous systems. This cross-linker
also has a large diffusion capacity and may be useful for permeation of biological
membranes before conjugation initiates.
Heterobifunctional cross-linkers possess two or more different
reactive groups that allow for sequential conjugations with specific groups of
proteins, minimizing undesirable polymerization or self-conjugation. Heterobifunctional
reagents are also used when modification of amines is problematic. Amines may sometimes
be found at the active sites of macromolecules, and the modification of these may
lead to the loss of activity. Other moieties such as sulfhydryls, carboxyls, phenols
and carbohydrates may be more appropriate targets. A two-step strategy allows for
the coupling of a protein that can tolerate the modification of its amines to a
protein with other accessible groups. A variety of heterobifunctional cross-linkers,
each combining different attributes for successful conjugation are commercially
available. Cross-linkers that are amine-reactive at one end and sulfhydryl-reactive
at the other end are quite common.
If using heterobifunctional reagents, the most labile group is typically reacted
first to ensure effective cross-linking and avoid unwanted polymerization. A selection
of heterobifunctional reagents that contain at least one photoaffinity group are
also commercially available. This selection includes iodinatable and cleavable
reagents that react nonspecifically at the azido group and with amines, sulfhydryls,
carbohydrates and carbonyls.
Many factors must be considered to determine optimum cross-linker-to-target
molar ratios. Depending on the application, the degree of conjugation is an important
factor. For example, when preparing immunogen conjugates, a high degree of conjugation
is normally desired to increase the immunogenicity of the antigen. However, when
conjugating to an antibody or an enzyme, a low-to-moderate degree of conjugation
may be optimal to ensure that the biological activity of the protein is retained.
It is also important to consider the number of reactive groups on the surface of
the protein. If there are numerous target groups, a lower cross-linker-to-protein
ratio can be used. For a limited number of potential targets, a higher cross-linker-to-protein
ratio may be required. This translates into more cross-linker per gram for a small
molecular weight protein.
Conformational changes of proteins associated with a particular interaction
may also be analyzed by performing cross-linking studies before and after the interaction.
A comparison is made by using different arm-length cross-linkers and analyzing
the success of conjugation. The use of cross-linkers with different reactive groups
and/or spacer arms may be desirable when the conformation of the protein changes
such that hindered amino acids become available for cross-linking.
Cross-linkers are available with varying lengths of spacer arms or
bridges connecting the reactive ends. The most apparent attribute of the bridge
is its ability to deal with steric considerations of the moieties to be linked.
Because steric effects dictate the distance between potential reaction sites for
cross-linking, different lengths of bridges may be considered for the interaction.
Shorter spacer arms are often used in intramolecular cross-linking studies, while
intermolecular cross-linking is favored with a cross-linker containing a longer
spacer arm.
The inclusion of polymer portions (e.g., polyethylene glycol ("PEG") homopolymers,
polypropylene glycol homopolymers, other alkyl-polyethylene oxides, bis-polyethylene
oxides and co-polymers or block co-polymers of poly(alkylene oxides)) in cross-linkers
can, under certain circumstances be advantageous. See, e.g., U.S. Pat. Nos. 5,643,575,
5,672,662, 5,705,153, 5,730,990, 5,902,588, and 5,932,462; and Topchieva et al.,
Bioconjug. Chem. 6: 380-8, 1995). For example, U.S. Pat. No. 5,672,662 discloses
bifunctional cross-linkers comprising a PEG polymer portion and a single ester
linkage. Such molecules are said to provide a half-life of about 10 to 25 minutes
in water.
Each reference cited in the preceding section is hereby incorporated by reference
in its entirety, including all tables, figures, and claims.
BRIEF SUMMARY OF THE INVENTION
It is an object of the invention to provide N-aryl-carbamic acid ester derived
cross-linkers, and methods of their synthesis and use. In a first aspect, the invention
relates to cross-linkers having the general formula:
where A is a first functional moiety directly or indirectly covalently linked
to one terminus of a polymeric moiety C through an N-aryl (or heteroaryl) carbamic
acid ester B, and a second functional moiety D directly or indirectly covalently
linked to a second terminus of the polymeric moiety C. The first functional moiety
A may be linked to the aryl (or heteroaryl) portion of the N-aryl carbamic acid
ester via the ortho, meta, or para position relative to the polymeric moiety C.
In addition, the aryl (or heteroaryl) portion of the N-aryl carbamic acid ester
may be further substituted by one or more additional moieties.
Such cross-linkers, which may be homobifunctional or heterobifunctional, advantageously
provide simplified synthetic routes and the ability to monitor synthesis due to
the light absorptive properties of the aryl portion of the N-aryl carbamic acid
ester. In addition, the polymeric moiety can impart sufficient water solubility
to the cross-linkers to compensate for the relatively hydrophobic character of
the N-aryl carbamic acid ester portion of the molecule resulting from the presence
of the aryl group.
In preferred embodiments, the linker connecting second functional moiety D to
the polymeric moiety is a second aryl (or heteroaryl)-containing ester linkage.
In these preferred embodiments, second functional moiety D may be linked to this
aryl (or heteroaryl) portion of the linker in the ortho, meta, or para position
relative to the polymeric moiety C. In addition, the aryl (or heteroaryl) portion
of the linker may be substituted by one or more additional moieties.
As is well understood in the art, the functional moieties selected depend upon
the group being targeted for attachment of the crosslinker. Preferred functional
moieties include primary amine-reactive moieties, sulfhydryl-reactive moieties,
nonselective moieties, photoreactive moieties, carboxyl-reactive moieties, arginine-reactive
moieties, and carbonyl-reactive moieties. Specific examples of these moieties are
described hereinafter.
The polymeric moieties that may find use in the present invention include polyalkylene
oxides, including homopolymers and copolymers comprising methylene oxide, ethylene
oxide, propylene oxide, isopropylene oxide, and butylene oxide. Additional examples
of polymeric moieties are described below. Particularly preferred are polyethylene
oxides (i.e., (—CH
2CH
2O—) n), which are often
referred to in the art as PEGs due to their derivation from polyethylene glycol.
Preferably, n=from about 40 to about 450 (i.e., the polymeric moieties comprise
from about 40 to about 450 monomer units). Preferred cross-linkers have between
about 50 and about 150 monomer units, more preferably between about 60 and about
100 monomer units, still more preferably between about 70 and about 90 monomer
units, and most preferably about 77 monomer units. The term "about" in this context
refers to +/-10% of a given measurement.
The skilled artisan will understand that the commercially available polyalkylene
glycol molecules used in the synthesis of the cross-linkers of the present invention
are often not pure, and instead are provided as a pool of molecules differing in
the number of monomeric units, but having a specified "average molecular weight"
as that term is defined hereinafter. Thus, the present invention also relates to
compositions comprising a plurality of cross-linkers described herein that may
differ in molecular weight (e.g., due to differences in the number of monomeric
units in the molymeric moiety), but which comprise polymeric moieties having an
average number of monomer units of between about 40 to about 450. The term "about"
in this context refers to +/-10% of a given measurement. Preferred compositions
comprise cross-linkers having an average number of monomer units of between about
60 and about 100, more preferably between about 70 and about 90, and most preferably
about 77.
In certain preferred embodiments, the N-aryl-carbamic acid ester derived cross-linkers
of the present invention have the following formula:
![embedded image]()
wherein X is (alkylene oxide)n;
n is between about 40 to about 450; and
Y and Z are independently arylene or heteroarylene units having 5 or 6 ring
atoms, optionally substituted with from 1 to 4 substituents independently selected
from the group consisting of C1-6 alkyl straight or branched chain,
halogen, trihalomethyl, C1-6 alkoxy, —NO2, —NH2,
—OH, —COOR′, where R′ is H or lower alkyl, —CH2OH,
—CONH2, and a linkage to a poly(alkylene oxide) moiety, wherein
Z is optionally present.
More preferred N-aryl-carbamic acid ester derived cross-linkers of the present
invention have the following formula:
![embedded image]()
wherein X is (alkylene oxide)n;
n is between about 40 to about 450; and each R is independently selected
from the group consisting of C1-6 alkyl straight or branched chain,
halogen, trihalomethyl, C1-6 alkoxy, —NO2, —NH2,
—OH, —COOR′, where R′ is H or lower alkyl, —CH2OH,
—CONH2, and a linkage to a poly(alkylene oxide) moiety.
Particularly preferred N-aryl-carbamic acid ester derived cross-linkers
of the present invention have the following formula:
![embedded image]()
wherein n is between about 40 to about 450.
As described herein, the N-aryl-carbamic acid ester derived cross-linkers of
the
present invention find use in forming a covalent linkage between two species, such
as between a first and second protein, polypeptide, nucleic acid, small molecule,
aptamer, carbohydrate, or peptidomimetic; between a protein, polypeptide, nucleic
acid, small molecule, aptamer, carbohydrate, peptidomimetic, etc., and a detectable
label; between a hapten and a protein or polypeptide acting as an antigenic carrier;
and/or between a protein, polypeptide, nucleic acid, small molecule, aptamer, carbohydrate,
peptidomimetic, etc., and a solid phase.
In another aspect, then, the present invention provides conjugates formed by
the
foregoing cross-linker molecules and compositions. Such conjugates have the following
general formula:
where E is a first protein, polypeptide, nucleic acid, small molecule, aptamer,
carbohydrate, peptidomimetic, detectable label, or solid phase directly or indirectly
covalently linked to one terminus of a polymeric moiety C through an N-aryl (or
heteroaryl) carbamic acid ester B, and a second protein, polypeptide, nucleic acid,
small molecule, aptamer, carbohydrate, peptidomimetic, detectable label, or solid
phase F directly or indirectly covalently linked to a second terminus of the polymeric
moiety C. As discussed above, the linkage between E and the aryl (or heteroaryl)
portion of the N-aryl carbamic acid ester may be made via the ortho, meta, or para
position relative to the polymeric moiety C. In addition, the aryl (or heteroaryl)
portion of the N-aryl carbamic acid ester may be further substituted by one or
more additional moieties.
In certain preferred embodiments, the conjugates of the present invention have
the following formula:
![embedded image]()
wherein X is (alkylene oxide)n;
n is between about 40 to about 450;
Y and Z are independently arylene or heteroarylene units having 5 or 6 ring
atoms, optionally substituted with from 1 to 4 substituents independently selected
from the group consisting of C1-6 alkyl straight or branched chain,
halogen, trihalomethyl, C1-6 alkoxy, —NO2, —NH2,
—OH, —COOR′, where R′ is H or lower alkyl, —CH2OH,
—CONH2, and a linkage to a poly(alkylene oxide) moiety, wherein
Z is optionally present; and
R1 and R2 are independently covalent linkages to a
protein, polypeptide, signal development element, or solid phase.
In more preferred embodiments, the conjugates of the present invention have the
following formula:
![embedded image]()
wherein X is (alkylene oxide)n;
n is between about 40 to about 450; and
each R is independently selected from the group consisting of C1-6
alkyl straight or branched chain, halogen, trihalomethyl, C1-6 alkoxy,
—NO2, —NH2, —OH, —COOR′, where
R′ is H or lower alkyl, —CH2OH, —CONH2,
and a linkage to a poly(alkylene oxide) moiety; and
R1 and R2 are independently covalent linkages to a
protein, polypeptide, signal development element, or solid phase.
In certain particularly preferred embodiments, the conjugates of the present
invention
have the following formula:
![embedded image]()
wherein n is between about 40 to about 450; and
R1 and R2 are independently covalent linkages to a
protein, polypeptide, signal development element, or solid phase.
Preferably, one of R
1 or R
2 is selected from the
group consisting of a protein, a polypeptide, an antibody, an antibody fragment,
a single-chain variable region fragment, a small molecule, a nucleic acid, an oligosaccharide,
a polysaccharide, a cyclic polypeptide, a peptidomimetic, and an aptamer; and the
other of R
1 or R
2 is selected from the group consisting of
a detectable label and a solid phase. Such conjugates are particularly useful in
receptor binding assays. Thus, in another aspect, the present invention relates
to receptor binding assays in which the conjugates of the present invention are
used. Preferred assay methods include immunoassay, (e.g., competitive assays, non-competitive
assays, sandwich assays, homogenous assays, etc.) and nucleic acid hybridization.
Examples of such assays are described hereinafter.
It is another object of the present invention to provide valeric acid ester derived
cross-linkers, and methods of their synthesis and use. In various aspects, the
invention relates to cross-linkers having the general formula:
where G is a first functional moiety directly or indirectly covalently linked
to one terminus of a polymeric moiety H, and a second functional moiety J covalently
linked to a second terminus of the polymeric moiety H through a valeric acid moiety I.
As discussed above, the functional moieties selected depend upon the group being
targeted for attachment of the crosslinker, and the resulting crosslinker may be
homobifunctional or heterobifunctional. Preferred functional moieties include those
described for the foregoing N-aryl-carbamic acid ester derived cross-linkers. Likewise,
as discussed above, the polymeric moieties that may find use in the present invention
include polyalkylene oxides, including homopolymers and copolymers comprising methylene
oxide, ethylene oxide, propylene oxide, isopropylene oxide, and butylene oxide.
Particularly preferred are polyethylene oxides. Preferably, the polymeric moieties
comprise from about 40 to about 450 monomer units. Preferred cross-linkers have
between about 60 and about 90 monomer units, more preferably between about 70 and
about 80 monomer units, and most preferably about 77 monomer units.
In certain preferred embodiments, the valeric acid ester derived crosslinkers
of the present invention have the following formula:
![embedded image]()
wherein X is (alkylene oxide)n;
n is between about 40 to about 450; and
Y is C1-10 alkylene straight or branched chain comprising from
0-4 backbone (i.e., non-substituent) heteroatoms, optionally substituted with from
1 to 4 substituents independently selected from the group consisting of C1-6
alkyl straight or branched chain, halogen, trihalomethyl, C1-6 alkoxy,
—NO2, —NH2, ═O, —OH, —CH2OH,
—C(O)NH2, and a linkage to a polyalkylene oxide moiety.
More preferred valeric acid ester derived cross-linkers of the present invention
have the following formula:
![embedded image]()
wherein X is (alkylene oxide)n, and n is between about 40 to
about 450.
Particularly preferred valeric acid ester derived cross-linkers of the
present invention have the following formula:
![embedded image]()
wherein n is between about 40 to about 450.
As in the case of the N-aryl-carbamic acid ester derived cross-linkers, the valeric
acid ester derived cross-linkers of the present invention find use in forming a
covalent linkage between two species, such as between a first and second protein,
polypeptide, nucleic acid, small molecule, aptamer, carbohydrate, or peptidomimetic;
between a protein, polypeptide, nucleic acid, small molecule, aptamer, carbohydrate,
peptidomimetic, etc., and a detectable label; between a hapten and a protein or
polypeptide acting as an antigenic carrier; and/or between a protein, polypeptide,
nucleic acid, small molecule, aptamer, carbohydrate, peptidomimetic, etc., and
a solid phase.
In another aspect, then, the present invention relates to conjugates formed by
the foregoing cross-linker molecules and compositions. Such conjugates have the
following general formula:
where K is a first protein, polypeptide, nucleic acid, small molecule, aptamer,
carbohydrate, peptidomimetic, detectable label, or solid phase directly or indirectly
covalently linked to one terminus of a polymeric moiety H, and L is a second protein,
polypeptide, nucleic acid, small molecule, aptamer, carbohydrate, peptidomimetic,
detectable label, or solid phase covalently linked to a second terminus of the
polymeric moiety H through a valeric acid moiety I.
In certain preferred embodiments, the conjugates of the present invention have
the following formula:
![embedded image]()
wherein X is (alkylene oxide)n;
n is between about 40 to about 450;
Y is C1-10 alkylene straight or branched chain comprising from
0-4 backbone heteroatoms, optionally substituted with from 1 to 4 substituents
independently selected from the group consisting of C1-6 alkyl straight
or branched chain, halogen, trihalomethyl, C1-6 alkoxy, —NO2,
—NH2, ═O, —OH, —CH2OH, —C(O)NH2,
and a linkage to a polyalkylene oxide moiety; and
R1 and R2 are independently covalent linkages to a
protein, polypeptide, signal development element, or solid phase.
In more preferred embodiments, the conjugates of the present invention have the
following formula:
![embedded image]()
wherein X is (alkylene oxide)n;
n is between about 40 to about 450; and
R1 and R2 are independently covalent linkages to a
protein, polypeptide, signal development element, or solid phase.
In particularly preferred embodiments, the conjugates of the present invention
have the following formula:
![embedded image]()
wherein n is between about 40 to about 450; and
R1 and R2 are independently covalent linkages to a
protein, polypeptide, signal development element, or solid phase.
Preferably, one of R
1 or R
2 is selected from the
group consisting of a protein, a polypeptide, an antibody, an antibody fragment,
a single-chain variable region fragment, a small molecule, a nucleic acid, an oligosaccharide,
a polysaccharide, a cyclic polypeptide, a peptidomimetic, and an aptamer; and the
other of R
1 or R
2 is selected from the group consisting of
a detectable label and a solid phase. Such conjugates are particularly useful in
receptor binding assays. Thus, in another aspect, the present invention relates
to receptor binding assays in which the conjugates of the present invention are
used. Preferred assay methods include immunoassay, (e.g., competitive assays, non-competitive
assays, sandwich assays, homogenous assays, etc.) and nucleic acid hybridization.
Examples of such assays are described hereinafter.
It is another object of the present invention to provide assays and devices for
performing assays. Such assays and devices comprise a conjugate as described herein
comprising an antibody or binding fragment thereof covalently linked to a detectable
label; and/or a conjugate as described herein comprising an antibody or binding
fragment thereof covalently linked to a solid phase. The devices of the present
invention preferably contain a plurality of diagnostic zones, each of which is
related to a particular analyte of interest. Such devices may be referred to as
"arrays" or "microarrays." Following reaction of a sample with the devices, a signal
may be generated from the diagnostic zone(s), which may then be correlated to the
presence or amount of the analyte(s) of interest. Numerous suitable devices are
known to those of skill in the art, and exemplary devices are described hereinafter.
It is another object of the present invention to provide methods for producing
the cross-linker molecules and compositions of the present invention, and methods
for producing conjugates using the cross-linker molecules and compositions described herein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to N-aryl carbamic acid ester derived crosslinkers
and methods and for their production and use.
N-aryl Carbamic Acid Esters
N-aryl carbamic acid esters (see, e.g., U.S. Pat. No. 3,915,984) have the
general formula:
![embedded image]()
While Ar is most preferably a monocyclic carbocyclic aromatic ring having 5
or 6 ring atoms (and is most preferably phenyl), the aryl or heteroaryl Ar group
(formed into an arylene or heteroarylene in the crosslinkers described herein by
elaboration from a ring atom) generally may contain up to ten ring atoms, although
the skilled artisan will recognize that Ar groups with more than ten ring atoms
are within the scope of the invention. Ar can be a monocyclic or fused bicyclic
aryl, alkaryl, heteroaryl or heteroarylalkyl group. The ring systems encompassed
by Ar can contain up to four heteroatoms, independently selected from the group
consisting of N, S, and O. When Ar is a heteroaryl ring or ring system, it preferably
contains one or two heteroatoms.
Monocyclic Ar groups include, but are not limited to: phenyl, thiazoyl,
furyl, pyranyl, 2H-pyrrolyl, thienyl, pyrroyl, imidazoyl, pyrazoyl, pyridyl, pyrazinyl,
pyrimidinyl, and pyridazinyl moieties. Fused bicyclic Ar groups include, but are
not limited to: benzothiazole, benzimidazole, 3H-indolyl, indolyl, indazoyl, purinyl,
quinolizinyl, isoquinolyl, quinolyl, phthalizinyl, naphthyridinyl, quinazolinyl,
cinnolinyl, isothiazolyl, quinoxalinyl indolizinyl, isoindolyl, benzothienyl, benzofuranyl,
isobenzofuranyl, and chromenyl moieties.
As used herein, the term "arylene" refers to a divalent all-carbon monocyclic
or fused ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms)
Ar groups in which one or more of the rings has a completely conjugated pi-electron
system. A "heteroarylene" group refers to a divalent monocyclic or fused ring group
having in the ring(s) one or more atoms selected from the group consisting of nitrogen,
oxygen and sulfur; in addition, at least one of the rings has a completely conjugated
pi-electron system.
The term "heteroatom" as used herein refers to non-carbon, non-hydrogen atoms
such as N, O, and S.
The Ar group may also be optionally substituted by replacement of one or more
hydrogen atoms by another chemical moiety. Preferred substituents include C
1-6
alkyl straight or branched (e.g. isopropyl) chain, halogen, trihalomethyl, alkoxy,
NO
2, NH
2, OH, —COOR′, where R′ is H or
lower alkyl, CH
2OH, and CONH
2. Substitution of the Ar group
also provides an attractive point for a linkage to which one or more additional
polymeric moieties (e.g., polyalkylene oxide moieties) may be attached to the cross-linker.
As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon
including
straight chain and branched chain groups. Preferably, the alkyl group has 1 to
20 carbon atoms. More preferably, it is a medium alkyl (having 1 to 10 carbon atoms).
Most preferably, it is a lower alkyl (having 1 to 4 carbon atoms). The alkyl group
may be substituted or unsubstituted. An "alkoxy" group refers to both an —O-alkyl
and an —O-cycloalkyl group; preferably an alkoxy group refers to a lower
alkoxy, and most preferably methoxy or ethoxy.
Valeric Acid Esters
Valeric acid esters have the general formula:
![embedded image]()
Functional Moieties
Designing a cross-linker involves selection of the functional moieties
to be employed. The choice of functional moieties is entirely dependent upon the
target sites available on the species to be crosslinked. Some species (e.g., proteins)
may present a number of available sites for targeting (e.g., lysine ε-amino
groups, cysteine sulfhydryl groups, glutamic acid carboxyl groups, etc.), and selection
of a particular functional moiety may be made empirically in order to best preserve
a biological property of interest (e.g., binding affinity of an antibody, catalytic
activity of an enzyme, etc.)
1. Coupling Through Amine Groups
Imidoester and N-hydroxysuccinimidyl ("NHS") esters are typically employed
as amine-specific functional moieties. NHS esters yield stable products upon reaction
with primary or secondary amines. Coupling is efficient at physiological pH, and
NHS-ester cross-linkers are more stable in solution than their imidate counterparts.
Homobifunctional NHS-ester conjugations are commonly used to cross-link amine-containing
proteins in either one-step or two-step reactions. Primary amines are the principle
targets for NHS-esters. Accessible α-amine groups present on the N-termini
of proteins react with NHS-esters to form amides. However, because α-amines
on a protein are not always available, the reaction with side chains of amino acids
become important. While five amino acids have nitrogen in their side chains, only
the ε-amino group of lysine reacts significantly with NHS-esters. A covalent
amide bond is formed when the NHS-ester cross-linking agent reacts with primary
amines, releasing N-hydroxysuccinimide.
2. Coupling Through Sulfhydryl Groups
Maleimides, alkyl and aryl halides, α-haloacyls, and pyridyl disulfides
are typically employed as sulfhydryl-specific functional moieties. The maleimide
group is specific for sulfhydryl groups when the pH of the reaction mixture is
kept between pH 6.5 and 7.5. At pH 7, the reaction of the maleimides with sulfhydryls
is 1000-fold faster than with amines. Maleimides do not react with tyrosines, histidines
or methionines. When free sulfhydryls are not present in sufficient quantities,
they can often be generated by reduction of available disulfide bonds.
3. Coupling Through Carboxyl Groups
Carbodiimides couple carboxyls to primary amines or hydrazides, resulting
in formation of amide or hydrazone bonds. Carbodiimides are unlike other conjugation
reactions in that no cross-bridge is formed between the carbodiimide and the molecules
being coupled; rather, a peptide bond is formed between an available carboxyl group
and an available amine group. Carboxy termini of proteins can be targeted, as well
as glutamic and aspartic acid side chains. In the presence of excess cross-linker,
polymerization may occur because proteins contain both carboxyls and amines. No
cross-bridge is formed, and the amide bond is the same as a peptide bond, so reversal
of the cross-linking is impossible without destruction of the protein.
4. Nonselective Labeling
A photoaffinity reagent is a compound that is chemically inert but becomes reactive
when exposed to ultraviolet or visible light. Arylazides are photoaffinity reagents
that are photolyzed at wavelengths between 250-460 nm, forming a reactive aryl
nitrene. The aryl nitrene reacts nonselectively to form a covalent bond. Reducing
agents must be used with caution because they can reduce the azido group.
5. Arginine Specific Cross-linkers
Glyoxals are useful compounds for targeting the guanidinyl portion of arginine
residues. Glyoxals will target arginines at mildly alkaline pH. There is some cross-reactivity
(the greatest at higher pH) with lysines.
6. Carbonyl Specific Cross-Linkers
Carbonyls (aldehydes and ketones) react with amines and hydrazides at pH
5-7. The reaction with hydrazides is faster than with amines, making this useful
for site-specific cross-linking. Carbonyls do not readily exist in proteins; however,
mild oxidation of sugar moieties using sodium metaperiodate will convert vicinal
hydroxyls to aldehydes or ketones.
Polymeric Moieties
The polymer substances included in the cross-linkers are preferably poly(alkylene
oxides). As used herein, the term "alkylene oxide" refers to the structure, —X—O—,
where X is an alkylene moiety covalently linked to oxygen O; thus poly(alkylene
oxide) refers to the structure —(X—O—)
m)—. It
is preferred that the poly(alkylene oxide) polymer be a nonbranched homopolymer
(i.e., a polymer of the structure —((CH
2)
n—O—)
m)—
in which n does not vary) such as poly(ethylene oxide) derived from ethylene glycol.
Alternative polymers such as other polyalkylene oxide homopolymers (e.g., methylene
oxide, propylene oxide, isopropylene oxide, and butylene oxide polymers) and co-polymers
or block co-polymers of poly(alkylene oxides) may also be used. In those aspects
of the invention where PEG-based polymers are used, it is preferred that they have
average molecular weights of from about 1,000 to about 25,000. Average molecular
weights of about 2,000 to 5,000 are preferred and average molecular weights of
from about 3,000 to about 3,500 are especially preferred. Molar equivalent amounts
of the other alkylene oxides may be determined readily by those of ordinary skill
in the art to arrive at preferred average molecular weights for other homopolymers
and copolymers.
Average molecular weights of the present invention are measured using the
"number-average" method. In a mixture of polymer molecules with different molecular
weights in which the number of molecules having a particular molecular weight,
M
i, is given by N
i, the "number-average" probability of a
given mass being present is
##EQU1##
and the number-average molecular weight is given by the formula
##EQU2##
The number average is the simple arithmetic mean, representing the total weight
of the molecules present divided by the total number of molecules. The number-average
molecular weight of a polymer may be measured by vapor pressure osmometry using
methods and apparatuses well known to those of skill in the art.
Alternative polymeric substances which may be used in place of poly(alkylene
oxides) include materials such as dextran, polyvinyl pyrrolidones, polysaccharides,
starches, polyvinyl alcohols, polyacryl amides or other similar polymers. Those
of ordinary skill in the art will realize that the foregoing is merely illustrative
and not intended to restrict the type of non-antigenic polymeric substances suitable
for use herein.
The polymers are preferably activated in order to affect the desired linkages.
By "activation," it is understood by those of ordinary skill in the art that the
polymer is functionalized to directly or indirectly attach to a desired functional
moiety as described herein.
Indirect Linkages to Functional Moieties
As described herein, a first functional moiety is directly or indirectly covalently
linked to the aryl (or heteroaryl) portion of the N-aryl carbamic acid ester at
one terminus of a polymeric moiety, and a second functional moiety is directly
or indirectly covalently linked to another terminus of the polymeric moiety of
the bifunctional cross-linkers described herein. The following exemplary molecule
provides an example of each type of linkage:
![embedded image]()
The term "directly covalently linked" indicates that no additional linkage chemistry
is present between the functional moiety and the group on the N-aryl carbamic acid
ester, or polymeric moiety terminus, to which the functional moiety is bound. In
the exemplary molecule, a maleimide group is directly covalently linked at the
para position to the phenyl portion of the N-phenyl carbamic acid ester. The term
"indirectly covalently linked" indicates that some additional linkage chemistry
is present. In the exemplary molecule, an N-hydroxy succinimide ester is linked
via a phenylethyl group to a terminus of the polymeric moiety. Thus, the exemplary
molecule comprises two "activated" ester linkages flanking the poly(ethylene oxide)
core. Indirect linkages of the present invention may be from about 1 to 30 atoms,
usually 1 to 15 atoms, where the atoms include C, N, O, S, P, etc., and will generally
have from about 1 to 12 carbon atoms and from about 0 to 8, and usually 0 to 6,
heteroatoms. The number of atoms referred to above are exclusive of hydrogen. In
preferred embodiments, the indirect linkage chemistry is used to incorporate one
or more aryl or heteroaryl ring structures into the molecule (in addition to that
provided in the N-aryl carbamic acid ester).
Applications for Use of Cross-Linkers
1. Cell Surface Cross-Linking
To ensure cell-surface specific cross-linking for identification of surface receptors
or their ligands, it is preferred to use membrane-impermeable cross-linkers. In
the past, researchers used water-insoluble cross-linkers and carefully controlled
the amount of cross-linker and the cross-linking duration. This prevented penetration
of the membrane by the cross-linker and subsequent reaction with membrane proteins.
Many references cite the use of membrane-permeable cross-linkers for cell surface cross-linking.
2. Subunit Cross-Linking and Protein Structural Studies
Cross-linkers can be used to study the structure and composition of
proteins in biological samples. Some proteins are difficult to study because they
exist in different conformations under varying pH or salt conditions. One way to
avoid conformational changes is to cross-link the subunits together. Amine-, carboxyl-
or sulfhydryl-reactive reagents are employed for identification of particular amino
acids or for the determination of the number, location and size of subunits in
a protein. Short-to-medium spacer arm cross-linkers are typically selected when
intramolecular cross-linking is performed. If the spacer arm is too long, intermolecular
cross-linking can occur.
3. Intermolecular Cross-Linking for the Study of Protein Interactions and Associations
Cross-linkers are widely used for identification of near-neighbor protein
relationships, ligand-receptor identification and interactions, and enzyme substrate
orientations. The cross-linkers chosen for these applications are usually longer
than those used for subunit cross-linking. Homobifunctional, amine-reactive NHS-esters
or imidates and heterobifunctional, amine-reactive, photoactivatable phenyl azides
are the most commonly-used cross-linkers for these procedures. Occasionally, a
sulfhydryl- and amine-reactive cross-linker may be employed if one of the two proteins
or molecules is know to contain sulthydryls. Cleavable or noncleavable cross-linkers
are typically used. Because the distances between two molecules are not always
known, the optimum length of the spacer arm of the cross-linker may be determined
by the use of a panel of similar cross-linkers with different lengths. NHS-ester,
phenylazides are very useful for this type of cross-linking because they usually
result in some successful, if not efficient, cross-linking.
Cross-linkers can be used to determine whether a particular protein
is located on the surface or the integral part of the membrane. These studies are
possible because water-soluble cross-linkers are membrane-impermeable, while water-insoluble
cross-linkers are membrane permeable.
4. Cell Membrane Structural Studies
Cell membrane structural studies require reagents of varying hydrophobicity
to determine the location and the environment within a cell's lipid bilayer. Fluorescent
tags are used to locate proteins, lipids or other molecules inside and outside
the membrane. Various cross-linkers with differing spacer arm lengths can be used
to cross-link proteins to associated molecules within the membrane to determine
the distance between molecules. Successful cross-linking with shorter cross-linkers
is a strong indication that two molecules are interacting in some manner. Failure
to obtain cross-linking with a panel of shorter cross-linkers, while obtaining
conjugation with the use of longer reagents, generally indicates that the molecules
are located in the same part of the membrane but are not interacting. Homobifunctional
NHS-esters, imidates or heterobifunctional NHS-esters, photoactivatable, phenyl
azides are commonly used for these procedures.
5. Immunotoxins
Specific antibodies can be covalently linked to toxic molecules and then
used to target antigens on cells. Often these antibodies are specific for tumor
associated antigens. Immunotoxins are brought into the cell by surface antigens
and, once internalized, they proceed to kill the cell by ribosome inactivation
or other means. The type of cross-linker used to make an immunotoxin can affect
its ability to locate and kill the appropriate cells. For immunotoxins to be effective,
the conjugate must be stable in vivo. In addition, once the immunotoxin reaches
its target, it is important that the antibody be separable from the toxin to allow
the toxin to kill the cell. Thiol-cleavable, disulfide-containing conjugates have
been shown to be more cytotoxic to tumor cells than noncleavable conjugates of
ricin A immunotoxins. Cells are able to break the disulfide bond in the cross-linker,
allowing the release of the toxin within the targeted cell.
6. Carrier Protein-Hapten/Peptide/Polypeptide Conjugates for Use as Immunogens
Numerous companies offer commercially available products in this area of
immunological research. There are many cross-linkers used for the production of
these conjugates, and the best choice is dependent on the reactive groups present
on the hapten and the ability of the hapten carrier conjugate to function successfully
as an immunogen after its injection. Carbodiimides are good choices for producing
peptide carrier conjugates because both proteins and peptides usually contain several
carboxyls and primary amines.
Other heterobifunctional cross-linkers can also be used to make immunogen conjugates.
Often peptides are synthesized with terminal cysteines to allow for their attachment
to supports or to carrier proteins through a part of the molecule that is not important
for activity or recognition. Sulfhydryl-reactive, heterobifunctional cross-linkers
can be coupled to carrier proteins through their other functional group and then
can be linked to peptides through terminal cysteines. This method can be very efficient
and yield an immunogen that is capable of eliciting a good response upon injection.
7. Solid-Phase Immobilization
Proteins, peptides and other molecules can be immobilized on solid-phase
matrices for use as affinity supports or for sample analysis. The term "solid phase"
as used herein refers to a wide variety of materials including solids, semi-solids,
gels, films, membranes, meshes, felts, composites, particles, papers and the like
typically used by those of skill in the art to sequester molecules. The solid phase
can be nonporous or porous. Suitable solid phases include those developed and/or
used as solid phases in solid phase binding assays. See, e.g., chapter 9 of
Immunoassay,
E. P. Dianiandis and T. K. Christopoulos eds., Academic Press: New York, 1996,
hereby incorporated by reference. Examples of suitable solid phases inc
