Metal chelating compositions

Details Metal chelating compositions Metal chelating compositions

2000-04-24

2003-09-23

Warden, Jill (Department: 1743)

Compositions

Miscellaneous

C436S166000, C436S086000, C252S390000, C562S512000, C562S553000, C562S571000

Reexamination Certificate

active

06623655

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention is generally directed to metal chelating compositions and to methods for making and using the same for protein purification, detection or binding and, in particular, to nitrilotriacetic acid derivatives that have improved binding specificity and stability and to methods for making and using these nitrilotriacetic acid derivatives for protein purification, protein detection or protein binding.
Metal chelate affinity chromatography has been used as a technique for the purification of proteins for many years. Early resins used in this process were simple chelators such as iminodiacetic acid (IDA) coupled to agarose supports (Porath et al.
Nature,
258:598-599, 1975) and charged with various metals such as Cu
2+
, Zn
2+
and Ni
2+
. These resins were found to selectively capture proteins and peptides from natural sources (Porath and Olin,
Biochemistry,
22:1621, 1983; Lonnerdal and Keen,
J. Appl. Biochem.,
4:203, 1983; Sulkowski,
Protein Purification: Micro to Macro,
pages 149-162, Edited by R. Burgess, Published by Liss New York, N.Y., 1987). With the advent of molecular biological techniques, metal chelate chromatography assumed a more important role in the purification of proteins with the use of a 6-histidine tag. See, for example, Dobeli et al., U.S. Pat. No. 5,284,933. The poly histidine tag bound very strongly to the immobilized nickel and could be used for the identification and purification of these recombinant molecules. The tridentate chelator IDA was quite selective for these tagged proteins but the nickel was found to leach slowly from the resin reducing the capacity and causing interference with some downstream uses of the proteins.
More recently, a tetradentate chelator known as nitrilotriacetic acid resin was developed for use with metals having six coordination sites. This resin has become the preferred resin for the purification of poly histidine containing proteins since it has very little metal leaching and good selectivity. However, considerable amount of effort is required to obtain this selectivity. For example, the addition of various amounts of imidazole is necessary to determine whether the resin will bind the protein selectively and the capacity of the resin for the protein must be optimized to achieve the desired results (Janknecht et all,
Proc. Natl. Acad. Sci.,
88:8972-8976, 1991, Schmitt et all.,
Molecular Biology Reports,
88:223-230, 1993).
In U.S. Pat. No. 4,877,830, Dobeli et al. describe nitrilotriacetic acid resins suitable for protein purification represented by the general formula:
[carrier matrix]-spacer-NH—(CH
2
)
x
—CH(COOH)—N(CH
2
COO—)
2
Ni
2+
wherein x is 2, 3 or 4, the carrier matrix is one used in affinity or gel chromatography such as cross-linked dextrans, agarose or polyacrylamides, and the spacer is preferably —O—CH
2
—CH(OH)—CH
2
— or —O—CO—. Dobeli et al., U.S. Pat. No. 4,877,830 at col. 2, lines 23-37. These resins are prepared by reacting an N-terminal protected compound of the formula:
R—HN—(CH
2
)
x
—CH(NH
2
)—COOH
wherein R is an amino protecting group and x is 2,3 or 4, with bromoacetic acid in an alkaline medium and subsequently cleaving off the protecting group and reacting this product with an activated resin. See, e.g., Hochuli et al.,
Journal of Chromatography,
411(1987) 177-184.
In U.S. Pat. No. 5,625,075, Srinivasan et al. describe a metal radionuclide chelating compound having multiple sulfur and nitrogen atoms. These chelating compounds incorporate two nitrogen atoms and three sulfur atoms, two nitrogen atoms and four sulfur atoms, or three nitrogen atoms and three sulfur atoms.
While these compounds provide improved specificity relative to some resins containing nitrilotriacetic acid derivatives, a need remains for chelating compounds having greater binding specificity for polyhistidine containing proteins.
SUMMARY OF THE INVENTION
Among the objects of the present invention is the provision of metal chelating compositions and to metal chelates which are relatively stable and provide superior binding specificity for protein or polypeptide purification, protein or polypeptide detection or protein or polypeptide binding, and the provision of processes for the preparation and use of such compositions.
Briefly, therefore, the present invention is directed to a metal chelating composition having the formula:
wherein
Q is a carrier;
S
1
is a spacer;
L is —A—T—CH(X)— or —C(═O)—;
A is an ether, thioether, selenoether, or amide linkage;
T is a bond or substituted or unsubstituted alkyl or alkenyl;
X is —(CH
2
)
k
CH
3
, —(CH
2
)
k
COOH, —(CH
2
)
k
SO
3
H, —(CH
2
)
k
PO
3
H
2
, —(CH
2
)
k
N(J)
2
, or —(CH
2
)
k
P(J)
2
;
k is an integer from 0 to 2;
J is hydrocarbyl or substituted hydrocarbyl;
Y is —COOH, —H, —SO
3
H, —PO
3
H
2
, —N(J)
2
, or —P(J)
2
;
Z is —COOH, —H, —SO
3
H, —PO
3
H
2
, —N(J)
2
, or —P(J)
2
; and
i is an integer from 0 to 4.
The present invention is further directed to a metal chelate comprising a metal and the metal chelating composition of the present invention.
The present invention is further directed to a process for the purification or detection of a polypeptide or other composition having an affinity for a metal chelate. The process comprising contacting the composition with a metal chelate, the metal chelate comprising a metal and the metal chelating composition of the present invention.
The present invention is further directed to a process for the preparation of a mono- or dicarboxylated amine. The process comprises combining an amine and an oxoacid in the presence of a reducing agent. The amine has the formula R
2
R
3
NH wherein R
2
is hydrocarbyl or substituted hydrocarbyl and R
3
is hydrogen, hydrocarbyl or substituted hydrocarbyl.
Other objects and features will be in part apparent and in part pointed out hereinafter.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The linkage between the chelator and the resin was found by us to be an important parameter for the selectivity of the resin for polyhistidine tagged proteins. Conventional nitrilotriacetic acid resin has a positively charged amine linkage that acts as a binding site for any negatively charged molecule which may interfere with the binding of the protein to the coordination sites offered by the immobilized metal. Oxygen, sulfur, selenium and amides have some affinity for metals which may provide enhanced chelation properties, binding the metal more firmly than traditional tetradentate chelators having positive amine linkages. In addition, the use of a non-charged atoms between the nitrilo nitrogen and the carrier appears to reduce non-specific binding of proteins.
The metal chelating compositions of the present invention are capable of forming relatively stable chelates with metal ions and, advantageously, the presence of ether (—O—), thioether (—S—), selenoether (—Se—) or amide ((—NR
1
(C═O)—) or (—(C═O)NR
1
—) wherein R
1
is hydrogen or hydrocarbyl) linkages within the chelating composition contributes to the specificity of the resulting chelate when it is used for the separation or purification of molecules such as proteins, phosphoproteins, peptides, phosphopeptides, DNA, RNA, oligonucleotides, drugs, and synthetic and natural products that have an affinity for metal chelates such as clustered histidines or poly histidines.
In general, the chelating compositions of the present invention correspond to composition (1) shown in the structure below:
wherein
Q is a carrier;
S
1
is a spacer;
L is —A—T—CH(X)— or —C(═O)—;
A is an ether, thioether, selenoether, or amide linkage;
T is a bond or substituted or unsubstituted alkyl or alkenyl;
X is —(CH
2
)
k
CH
3
, —(CH
2
)
k
COOH, —(CH
2
)
k
SO
3
H, —(CH
2
)
k
PO
3
H
2
, —(CH
2
)
k
N(J)
2
, or —(CH
2
)
k
P(J)
2
, preferably —(CH
2
)
k
COOH or —(CH
2
)
k
SO
3
H;
k is an integer from 0 to 2;
J is hydrocarbyl or substituted hydrocarbyl;
Y is —COOH, —H, —SO
3
H, —PO
3
H
2
, —N(J)
2
, or —P(J)
2
, preferably, —COOH;
Z is —COOH, —H, —SO
3
H, —PO
3
H
2
, —N(J)
2
, or —P(J)
2
,

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