Chemistry of Bioconjugates

1

COVALENT AND NONCOVALENT BIOCONJUGATION STRATEGIES

RAJESH SUNASEE1 AND RAVIN NARAIN2

1Department of Chemistry, State University of New York, Plattsburgh, NY, USA

2Department of Chemical and Materials Engineering, Alberta Glycomics Centre, University of Alberta, Edmonton, AB, Canada

1.1 INTRODUCTION

Bioconjugation—the process of covalently or noncovalently linking a biomolecule to other biomolecules or small molecules to create new molecules—is a growing field of research that encompasses a wide range of science between chemistry and molecular biology. The tremendous achievement of modern synthetic organic chemistry has led to a variety of bioconjugation techniques [1] available for application in research laboratories, medical clinics, and industrial facilities. While bioconjugation involves the fusion of two biomolecules, for example protein–protein, polymer–protein, carbohydrate–protein conjugates, it also involves the attachment of synthetic labels (isotope labels, fluorescent dyes, affinity tags, biotin) to biological entities such as carbohydrates, proteins, peptides, synthetic polymers, enzymes, glycans, antibodies, nucleic acids, and oligonucleotides (ONTs). The product of a bioconjugation reaction is usually termed as a “bioconjugate” and synthetic macromolecules produced by bioconjugation approaches are commonly referred to as biohybrids, polymer bioconjugates, or molecular chimeras. Modification of biomolecules is an important technique for modulating the function of biomolecules and understanding their roles in complex biological systems [1a]. However, selective biomolecule modification remains challenging and the ease of generating the desired bioconjugate rapidly under physiological conditions is vital for many applications, such as disease diagnosis, biochemical assays, ligand discovery, and molecular sensing. As applications of bioconjugates continue to grow, an expanded toolkit of chemical methods will be required to add new functionality to specific locations with high yield and chemoselectivity.

The aim of this chapter is to provide a comprehensive review of the different types of bioconjugation methods (covalent and noncovalent approaches) available for the modification of biomolecules (proteins, peptides, carbohydrates, polymers, DNA, etc.). Traditional bioconjugation methods will first be elaborated upon, followed by some modern bioconjugation techniques, particularly the emerging role of bioorthogonal chemistry, where the translation of knowledge of chemical reactions to reactions in living systems can be achieved. While the synthetic aspects of the bioconjugates will be the main focus, a brief description of their applications will also be presented.

1.2 COVALENT BIOCONJUGATION STRATEGIES

The covalent bond is the most common form of linkage between atoms in organic chemistry and biochemistry. The reaction of one functional group with another leads to the formation of a covalent bond via the sharing of electrons between atoms (Figure 1.1).

FIGURE 1.1 Schematic representation of covalent bioconjugation strategy.

Covalent bioconjugation strategies are generally categorized as random (modification at multiple sites) or site-specific (modification at a single site) bioconjugation. Traditional covalent bioconjugation strategies preclude control over the regiochemistry of reactions, thereby leading to heterogeneous reaction products and eventually, loss of the biological function of the target biomolecule [1(d)]. However, new methods of bioconjugation that are highly site specific and cause minimal change to the active form of the biomolecule have been developed. For instance, bioorthogonal reactions have recently emerged as essential tools for chemical biologists [1(e)]. The following sections survey the covalent modifications of several reactive functional groups (carboxylic acids, aldehydes, ketones, amines, thiols, and alcohols), which are generally present or can be introduced onto macromolecules (proteins, peptides, carbohydrates, nucleic acids, ONTs, etc.).

1.2.1 Carboxyl Modifications

Carboxyl groups are commonly found on the C-terminal ends of proteins and on glutamate (Glu) and aspartate (Asp) amino acid side chains. Carboxylic acids are strong organic acids and the fastest reaction with a nucleophile is removal of the acidic hydrogen to form the carboxylate anion. The resulting anion is resistant to addition reaction with a second nucleophile, and thus makes conjugation through carboxylate group via nucleophilic addition a difficult process. Usually, harsher conditions, acid catalysis, or special reagents are required to promote carboxylic acid-mediated reactions. However, some carboxylate-reactive chemical reactions have been achieved with diazoalkanes and diazoacetyl derivatives (diazoacetate esters and diazoacetamides) and common activating agents such as carbonyldiimidazole (CDI) and carbodiimides to derivatize carboxylic acids. These reactions generate stable covalent linkages namely esters and amides.

1.2.1.1 Diazoalkanes and Derivatives

Diazoalkanes, in particular, diazomethane [2] is a powerful reagent for esterification of carboxylic acids. They react instantaneously with carboxylic acids without the addition of catalysts and may be useful for direct carboxylic acid modification of proteins and synthetic polymers. The reaction mechanism involves nucleophilic attack of the resulting carboxylate anion onto the diazonium ion, followed by an alkylation step to furnish a covalent ester linkage. The driving force of the reaction is the formation of nitrogen, which is a superb leaving group (Scheme 1.1).

SCHEME 1.1 Mechanism of diazomethane esterification reaction.

FIGURE 1.2 Fluorescent diazomethane derivatives as labeling reagents.

Diazomethane, though easily made, is quite toxic, highly explosive, and requires special glassware for reactions. A less explosive and commercially available reagent, trimethylsilyldiazomethane [3], is commonly employed; however, toxicity is still a major concern. In the past, fluorescent diazomethane derivatives have gained much attention for the derivatization of biologically important molecules, especially the nonchromophoric fatty acids [4], bile acids, and prostaglandins. 9-anthryldiazomethane (ADAM) [5,6] and 1-pyrenyldiazomethane (PDAM) [7,8] are diazomethane derivatives of the fluorescent dyes anthracene and pyrene, respectively, that have commonly been used as fluorescent labeling reagents for liquid chromatographic determination of carboxylic acids. ADAM and PDAM react readily with carboxylic acids at room temperature in both protic and aprotic solvents. ADAM was found to be unstable and decomposed easily upon storage, while PDAM has a much better chemical stability (a 0.1% (w/v) of PDAM in ethyl acetate solution is stable for 1 week at ≤−20°C) [9]. Furthermore, the detection limit for PDAM conjugates (about 20–30 fmol) is reported to be five times better than reported for detection of ADAM conjugates. Fatty acids derivatized with these reagents have been used to measure phospholipase A2 activity [10].

Protocol for reaction of PDAM with fatty acids [9]:

1.2.1.2 Activating Agents

The direct conversion of a carboxylic acid to an amide with amines is a very difficult process as an acid–base reaction to form a carboxylate ammonium salt occurs first before any nucleophilic substitution reaction happens. As such, amide formation from carboxylic acid is much easier if the acid is first activated (Scheme 1.2) prior to nucleophilic attack by the amine. This strategy converts the poor carboxy −OH leaving group into a better one. Ester linkages can also be formed using this strategy in the presence of alcohols.

SCHEME 1.2 General strategies for the conjugation of carboxylic acid with amines or alcohols via an activating agent.

The explosion in the field of peptide chemistry has led to the development of many activating agents that greatly enhance amide formation, but only the most commonly used ones, such as CDI and carbodiimides, will be discussed here (Table 1.1). N, N′-Carbonyldiimidazole (CDI) [11] is a white crystalline solid that is useful for activating carboxylic acids to form amide, ester, and thioester linkages. During the reaction, a reactive intermediate, N-acylimidazole is formed with liberation of carbon dioxide and imidazole as innocuous side products. The N-acylimidazole can then react with amines or alcohols to form stable covalent amide or ester linkages, respectively. CDI is not commonly used in routine peptide synthesis, but nevertheless is quite useful for coupling peptide fragments to form large peptides and small proteins [12]. One unique application of CDI is the synthesis of urea dipeptides [13]. Dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) are commonly used in organic synthesis for the preparation amides, esters, and acid anhydrides from carboxylic acids. These reagents can also transform primary amides to nitriles, which is a...