Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Chemical aftertreatment – e.g. – acylation – methylation – etc.
Reexamination Certificate
2001-03-22
2004-10-05
Russel, Jeffrey E. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Chemical aftertreatment, e.g., acylation, methylation, etc.
C530S391100, C530S408000, C530S409000, C530S810000, C536S023100, C548S542000, C564S017000, C564S018000, C564S034000, C564S037000
Reexamination Certificate
active
06800728
ABSTRACT:
FIELD OF THE INVENTION
The present disclosure may be applied in general to the field of chemistry, more particularly in the area of crosslinking reagents.
BACKGROUND OF THE INVENTION
Methods to crosslink biomolecules such as proteins, oligonucleotides and carbohydrates to each other, to radioactive and non-radioactive metal chelates, to drugs and to surfaces have allowed development of both in vitro and in vivo diagnostic assays as well as in vivo therapies. A wide variety of methods have been developed and reviewed (Greg T. Hermanson, Bioconjugate Techniques, Academic Press).
There are a limited number of crosslinking couples, i.e., maleimide/thiol and bromoacetamide/thiol, that are routinely used to prepare conjugates for diagnostic and therapeutic uses. These reagents have limitations in that at high protein concentrations (i.e., >5 mg/mL) protein/protein crosslinking may occur. Also, the maleimido-modified moieties have a limited half-life due to hydrolysis at neutral and basic pH. Incorporation of thiol moieties on biomolecules requires both a coupling and a subsequent activation step. The resultant thiol-modified proteins can readily oxidize to form disulfide polymerized proteins. Also macromolecules containing disulfide bonds, i.e., antibodies, are readily cleaved following activation of the thiol moiety by a reductant. Also, quantitation of the maleimido moiety is somewhat difficult and there is no means to quantify directly the level of conjugation. Therefore, it is advantageous to have a crosslinking couple that does not have these limitations.
Consequently there is a need for crosslinking couples that: bind more efficiently to surfaces; may be controlled to achieve desired crosslinking; do not lead to homobifunctional crosslinking following modification of aggregated proteins; are stable to biological conditions of varying pH and temperature; are stable in solution or when lyophilized; are one step modifications unlike those reagents currently used in the art, e.g., SATA, SPDP type reagents; can be indirectly quantified by a spectrophotometric assay; and can be used to quantify the level of conjugation by spectrophotometric means utilizing the bond formed following conjugation.
Therefore, it is an object herein to provide reagents and methods for crosslinking biomolecules to other biomolecules, polymers, metals or drugs that meet the above needs and have improved properties over known crosslinking reagents and methods.
SUMMARY OF THE INVENTION
Reagents and methods for crosslinking biomolecules to other biomolecules, polymers, metals or drugs are provided. The reagents are heterobifunctional compounds possessing, as one of the functionalities a hydrazino group, a carbonyl group, or an oxyamino group, all as defined herein. The reagents are used in the methods provided herein to afford improved crosslinking for both in vitro and in vivo diagnostic assays as well as in vivo therapies.
Provided herein are bifunctional compounds containing amine or thiol reactive moieties and a hydrazino or oxyamino moiety that may be utilized to modify small molecules, macromolecules, biomolecules and solid surfaces. A number of hydrazino moieties may be utilized including aliphatic and aromatic hydrazine derivatives, including, but not limited to, hydrazines, hydrazides, semicarbazides, carbazides, thiosemicarbazides, thiocarbazides, hydrazine carboxylates and carbonic acid hydrazines (see, e.g., FIG.
1
).
In one embodiment, the reagents for use in the methods provided herein have the formula:
B—R—Y
or a derivative thereof, where B is an amino or thiol reactive moiety; Y is a hydrazino group, as defined herein, an oxyamino group or a carbonyl group; and R is a divalent group having any combination of the following groups, which are combined in any order: arylene, heteroarylene, cycloalkylene, C(R
10
)
2
, —C(R
10
)═C(R
10
)—, >C═C(R
12
)(R
13
), >C(R
12
)(R
13
), —C≡C—, O, S(G)
a
, P(J)
b
(R
10
), P(J)
b
(LR
10
), N(R
10
), >N
+
(R
12
)(R
13
) and C(L); where a is 0, 1 or 2; b is 0, 1, 2 or 3; G is O or NR
10
; J is S or O; and L is S, O or NR
10
; each R
10
is a monovalent group independently selected from hydrogen and M
1
—R
14
; each M
1
is a divalent group independently having any combination of the following groups, which groups are combined in any order: a direct link, arylene, heteroarylene, cycloalkylene, C(R
15
)
2
, —C(R
15
)═C(R
15
)—, >C═C(R
12
)(R
13
), >C(R
12
)(R
13
), —C≡C—, O, S(G
1
)
a
, P(J)
b
(R
15
), P(J)
b
(LR
15
), N(R
15
), N(COR
15
), >N
+
(R
12
)(R
13
) and C(L); where a is 0, 1 or 2; b is 0, 1, 2 or 3; G
1
is O or NR
15
; J is S or O; and L is S, O or NR
15
; R
14
and R
15
are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR
16
R
17
R
18
, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR
19
R
20
; R
19
and R
20
are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl; R
12
and R
13
are selected from (i) or (ii) as follows: (i) R
12
and R
13
are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl; or (ii) R
12
and R
13
together form alkylene, alkenylene or cycloalkylene; R
16
, R
17
and R
18
are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR
19
R
20
; and
R
11
, R
12
, R
13
, R
14
, R
15
, R
16
, R
17
, R
18
, R
19
and R
20
can be substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)
h
R
30
, NR
30
R
31
, COOR
30
, COR
30
, CONR
30
R
31
, OC(O)NR
30
R
31
, N(R
30
)C(O)R
31
, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido; h is 0, 1 or 2; and R
30
and R
31
are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.
Thus, the reagents provided herein are aliphatic and aromatic crosslinking compounds that possess (i) a thiol or amine reactive group; and (ii) a hydrazino, oxyamino or carbonyl group. Thiol reactive groups are moieties that react directly with sulfhydryl groups forming stable thioether bonds. These thiol reactive groups include, but are not limted to, maleimido, &agr;-bromoacetamido and pyridyldisulfides. Amino reactive moieties are those that react directly with amine moieties forming amide bonds. These amino reactive groups include, but are not limited to, N-hydroxysuccinimidyl, p-nitrophenyl, pentafluorophenyl and N-hydroxybenzotriazolyl esters.
Hydrazino groups, as defined herein, include, but are not limited to, hydrazines, hydrazides, semicarbazides, carbazides, thiosemicarbazides, thiocarbazides, hydrazine carboxylates and carbonic acid hydrazines (see, e.g., FIG.
1
). Oxyamino groups have the formula R—O—NH
2
.
In certain embodiments herein, R is an al
Heller Ehrman White & McAuliffe LLP
Russel Jeffrey E.
Solulink Biosciences, Inc.
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