Methods for preparing conjugates

Details Methods for preparing conjugates Methods for preparing conjugates

2001-10-17

2004-06-01

Ceperley, Mary E. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S188000, C436S525000, C436S527000, C436S529000, C436S530000, C436S531000, C436S532000, C436S829000, C530S391100, C530S402000, C530S409000

Reexamination Certificate

active

06743585

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the covalent attachment of one moiety to another moiety such as a ligand to a surface or to another ligand, a ligand to a receptor, a receptor to a surface, and the like. The invention also relates to reagents for conducting such covalent attachment and methods for using such reagents.
In the field of diagnostics and therapeutics it is often useful to attach species or to couple different species together so as to alter or improve their properties. For example, although haptens, short peptides and the like function poorly as immunogens, a vigorous immune response may be elicited by coupling these moieties to larger moieties such as a protein carrier. In general, haptens (antigens), biotin, drug derivatives or peptides may be covalently bonded to proteins or natural or synthetic polypeptides for use as immunogens or recognition tags, or to enzymes for use in homogeneous enzyme immunoassays or to other label moieties for use in specific binding assays.
Similarly, antibodies may be coupled to toxins for use as immunotoxins, or to enzymes for use in enzyme immunoassays. The attachment of cytotoxic drugs to tumor-localizing monoclonal antibodies is an approach to chemotherapy that is gaining acceptance. Many reagents for the preparation of such conjugates have been disclosed and studied extensively. The so-called cross-linking reagents that have been developed are typically designed to have specific reactivity with functional groups contained in each reactant. Both homo- and heterobifunctional reagents are known with the latter being most widely used.
Active agents that are preferably functional organic material such as proteins have been immobilized in the past on a wide variety of solid supports for various known applications including analysis, separation, synthesis and detection of biological and other materials. Often hydrophilic polymers have been used to immobilize proteins because it is less difficult to attach proteins to polymers than to inorganic materials. However, there is an increasing need to immobilize functional organic material such as proteins on inorganic material such as silica, glass, silicon, metals and the like. In solid phase technology the reagent or reagents used in the procedure are usually immobilized by being coated or bonded either covalently or by adsorption to the solid phase material.
Biologically active polypeptides or proteins that are attached to an insoluble carrier material, such as polymeric particles, have been used in a variety of ways. For example, the diagnosis of pathological or other conditions in human beings and animals is often carried out using immunological principles for the detection of an immunologically reactive species, for example, antibodies or an antigen, in the body fluids of the person or animal. Other proteins and amine-containing compounds, such as enzymes, avidin, biotin or polysaccharides, have been covalently linked to various carrier materials for use in affinity chromatography, enzymatic reactions, specific binding reactions and immunoassays.
In the field of bioscience, arrays of oligonucleotide probes, fabricated or deposited on a surface, are used to identify DNA sequences in cell matter. The arrays may be used for conducting cell study, for diagnosing disease, identifying gene expression, monitoring drug response, determination of viral load, identifying genetic polymorphisms, and the like. Significant morbidity and mortality are associated with infectious diseases and genetically inherited disorders. More rapid and accurate diagnostic methods are required for better monitoring and treatment of these conditions. Molecular methods using DNA probes, nucleic acid hybridization and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
A variety of methods have been reported for the covalent attachment of ligands to a surface or for the attachment of small molecule tags on to biomolecules or for the attachment of biomolecules to each other. Typically, these reactions are performed by the reaction of an active functional group on one molecule with an activated functional group on another. As an example, an amine containing fluorophor can be attached to a carboxylic acid containing surface by forming an activated ester of the carboxylic acid, such as an N-hydroxysuccinimide derivative. The amine readily reacts with this activated ester to form a stable amide bond. This reaction is useful under conditions whereby the reaction with the desired amine is significantly faster than with other nucleophiles in the system.
Methods previously described include the activation of surfaces with cyanogen bromide, N-hydroxysuccinimide esters, carbonyl diimidazole, carbodiimides, azlactones, cyanuric chlorides, organic sulfonyl chlorides, divinyl sulphone, nitrophenyl esters, iodoacetyl, maleimide, epoxy, hydrazide, reductive amination, diazonium salts and Mannich condensations. Ligands that react with the activated surface include amines, alcohols, carboxylic acids, thiols, carbonyls, and compounds containing active hydrogens.
The attachment of two generally polyfunctional molecules at defined reaction loci presents a considerable challenge. The bond that is formed must be stable under the desired conditions of use. Another factor is that the system must not produce side reactions that will destroy the ability of the ligand to react with an activated functionality. When the reaction is performed in aqueous or protic solvent systems, the desired reaction can compete with solvent molecules for product formation. The resulting side products thus significantly lower the yield of the desired reaction. Nearly all of the commonly used methods of surface attachment and bioconjugation are susceptible to these solvolysis problems. For example, in an aqueous solvent system, when coupling an amine to a surface activated with an N-hydroxysuccinimide ester, the hydrolysis reaction of the activated ester can complete with the desired amine coupling. The activated functionality can react with the solvent, usually water, instead of with the desired ligand. In order for the coupling reaction to be successful, the reaction rate of the ligand with the activated functionality must be fast relative to the deactivation of the functional group, or a large enough excess of activated functional group must be present.
2. Description of the Related Art
U.S. Pat. No. 4,623,629 (Kerachensteiner) discloses a solid phase immunoassay support and method of use thereof.
U.S. Pat. No. 5,766,963 (Baldwin, et al.) discusses a combination hydroxypropylamine library.
Conjugated polypeptides and methods for their preparation and use are discussed in PCT application WO 90/05749 (Schultz).
Stereochemistry of nucleophilic displacements by amines on activated vinyl halides is discussed by Truce, et al.,
J. Org. Chem
. (1970) 35(7):2113-2117.
Jones, et al., describe the substitution at an olefinic carbon, the reactions of the ethyl &bgr;-chlorocrotonates with nucleophiles (
J. Chem. Soc
. (1960) 2349-2366).
&bgr;-Chlorovinyl ketones are discussed by Pohland, et al., in
Chem. Rev
. (1966) 66:161-197.
The synthesis and reactions of &bgr;-chloroacrylonitrile are disclosed by Scotti, et al.,
J. Org. Chem
. (1964) 29:1800-1808.
Landini, et al.,
J. Chem. Soc. B
(1969) 243-247, describe the nucleophilic substitution and elimination in 2-halogenovinyl ketones.
Farina, et al.,
Synthesis
(1977) 642, describes the synthesis of a &bgr;-bromovinyl ester.
Yogo et al.
J. Chem. Soc.
, (1984), 2097, describes the synthesis of a &bgr;-bromovinyl imide.
Rao, et al.,
Synth. Comm
. (1989), 19 describe the synthesis of a &bgr;-chlorovinyl amide.
Bryant, et al.,
J. Heterocyclic Chem
. (1995), 32: 1473-1476 describes the reaction of an alcohol with a &bgr;-chlorovinyl amide.
Ichikawa, et al. Synlett (1966), 243, describe the reaction of a primary amine with a &bgr;-fluorovinyl ketone.
Jalander,
Acta Chem. Scand
. (1995), 49: 894-898, descr

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