The cell membrane is an incredibly sophisticated environment, the complex composition of which regulates the variety of chemical and physical processes that occur inside and outside the cell. A wide range of reactive functional groups, including amines, thiols, and carbonyls, offer great opportunities for the conjugation of exogenous molecules. Introducing functionalities such as small molecules, proteins, peptides, polymers, and nanoparticles in such a diverse environment provides an excellent platform to manipulate cell behavior. Recent developments in polymer conjugation to the cell membrane have allowed modification of cell-cell, and cell-extracellular matrix (ECM) interactions, as well as targeted delivery of therapeutic agents. Common methods used to achieve polymer cell surface engineering have employed covalent strategies, via conjugation to amines or thiols, physical approaches, including electrostatic interactions and insertion through the phospholipid structure, or metabolic labeling.
Fig.1 Schematic illustration of the cell membrane highlighting the diversity of functional groups (native and non-native) expressed on cell surfaces. (Thomsen & Klok, 2021)
Protein Functional Groups
Primary Amines (–NH2): This group is found on the side chain of lysine residues, referred to as the epsilon-amine, and at the N-terminus of every polypeptide chain, referred to as the alpha-amine. Primary amines are normally exposed on the outside of proteins due to their physiologically generated positive charge. This makes them easily accessible for conjugation without denaturing the protein structure.
Carboxyls (–COOH): The carboxyl group may be found in the side chains of aspartic and glutamic acids, as well as in the C-terminus of every polypeptide chain. Carboxyl groups are typically found on the surface of the protein structure, just as primary amines.
Sulfhydryls (–SH): Cysteine has sulfhydryl groups on its side chain. Often, disulfide linkages join cysteines via their side chains in the secondary or tertiary structure of proteins. Most kinds of reactive groups cannot be used to crosslink these bonds until they are reduced to sulfhydryls.
Carbonyls (–CHO): By oxidizing the polysaccharide post-translational modifications (glycosylation) with sodium meta-periodate, glycoproteins can produce ketone or aldehyde groups.
Here are some types of crosslinkers CD BioGlyco used for this purpose, and we also provide some custom crosslinkers for clients according to their requirements. Chemical crosslinkers used to attach glycans to cell surface proteins are typically bifunctional reagents that have two reactive groups. One group reacts with specific functional groups on the glycan, while the other reacts with functional groups on the cell surface protein. The choice of crosslinker depends on the specific functional groups present in the glycan and the protein.
Fig.2 Some common types of crosslinkers. (CD BioGlyco)
These crosslinkers have an N-hydroxysuccinimide (NHS) ester group that interacts with the glycan and protein's main amines. N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and N-succinimidyl-4-(maleimidomethyl) cyclohexane-1-carboxylate (SMCC) are two notable instances.
Hydrazide crosslinkers create stable hydrazone linkages by reacting with aldehyde or ketone groups in the glycan and periodate-oxidized sugar residues on the protein. Adipic acid dihydrazide (ADH) is a good example.
Utilized when the glycan contains thiol (sulfhydryl) groups, such as in cysteine residues or thiolated glycans. Maleimide groups specifically react with thiols, forming thioether bonds.
Examples like 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) activate carboxylic acid groups in the glycan or protein to create amide bonds with primary amines. They are frequently used alongside NHS ester crosslinkers.
These are photoactivatable crosslinkers containing a diazirine group, activated using UV light. Diazirine crosslinkers enable precise control of cross-linking in terms of time and location.
Some crosslinkers are engineered to specifically target glycan structures, often containing lectin or antibody domains that recognize particular glycans.
The choice of crosslinker should be based on the specific properties of the glycan and the cell surface protein. CD BioGlyco has a well-equipment lab and a professional R&D team with adventure chemical knowledge, which helps us have the ability to generate the most suitable chemical conjugation solution and provide professional answers for our clients. Additionally, it's essential to consider the desired stability of the crosslinked complex and the potential impact of the crosslinker on the biological activity of the protein or glycan. Careful optimization and validation of the crosslinking conditions are necessary to achieve the desired results.
The custom cell surface chemical conjugation glycoengineering service from CD BioGlyco is an excellent choice for clients, besides, CD BioGlyco also provides other Chemical Conjugation-based Cell Surface Glycoengineering Services such as Click Chemistry-based, Fluorophore-based, and Biotin-based chemical conjugation glycoengineering services. Our Technology Developed for Cell Surface Glycoengineering is a powerful support for these custom services. Please do not hesitate to contact us If you are interested in our custom services and would like to communicate with us for details.
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