Site‐Selective Labeling of a Lysine Residue in Human Serum Albumin

Abstract

Conjugation to human serum albumin (HSA) has emerged as a powerful approach for extending the in vivo halflife of many small molecule and peptide/protein drugs. Current HSA conjugation strategies, however, can often yield heterogeneous mixtures with inadequate pharmacokinetics, low efficacies, and variable safety profiles. Here, we designed and synthesized analogues of TAK-242, a small molecule inhibitor of Toll-like receptor 4, that primarily reacted with a single lysine residue of HSA. These TAK-242-based cyclohexene compounds demonstrated robust reactivity, and Lys64 was identified as the primary conjugation site. A bivalent HSA conjugate was also prepared in a site-specific manner. Additionally, HSA-cyclohexene conjugates maintained higher levels of stability both in human plasma and in mice than the corresponding maleimide conjugates. This new conjugation strategy promises to broadly enhance the performance of HSA conjugates for numerous applications.

Recent Advances on the Development of Pharmacotherapeutic Agents on the Basis of Human Serum Albumin

“…As indicated in Fig. 11, the biotinylated HSA conjugate formed by their present strategy was more stable than that prepared by maleimide-thiol chemistry [64]. This new conjugation method may find great utility for the generation of drug-HSA therapeutics with well defined components and improved stability.…”

“…Moreover, there is no doubt that the well established albuminbased targeting strategy could facilitate to accumulate in malignant and inflammatory tissues due to the EPR effect, the specific affinity to the targeted disease sites remains a pressing and challenging concern. Incorporation of additional targeting ligands fused with HSA may compensate its shortcomings in improving the specific targeting [64]. However, the variety of active groups on HSA leads to failure of commonly used conjugating methods with siteselectivity, thus making it quite difficult to get a precisely defined product [10,147,149,157].…”

“…Therefore, reliable methods for site-specific labeling on HSA for the purpose of obtaining new homogeneous protein therapeutics are highly desirable [63]. Besides the conjuga- tion of lysine residues in HSA structure through N-hydroxysuccinimide ester based chemistry, some other reactions have also been found to react with HSA lysine residues in a highly specific manner, which could serve as new targets to achieve site-specific labeling of drugs with HSA protein carrier [64]. For example, Carlos and co-workers developed a series of labeling reagents based on the structure of TAK-242 (As shown in Fig.…”

Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells

“…Another way of achieving genetic encoding is the use of modifiable amino acids like cysteine or lysine that covalently react with labile functional groups in the fluorescent label. 11 , 12 While practical for some purified proteins, this approach lacks specificity in the cellular context, 13 and there is an effort to improve selectivity using unnatural amino acids with bio-orthogonal coupling chemistry. 14 A variety of methods use enzymes, either expressed as fusion proteins or preloaded with fluorescent substrates to catalyze covalent ligation of a dye onto an encoded tagging domain on a protein of interest (POI).…”

Chemoselective and photocleavable cysteine modification of peptides and proteins using isoxazoliniums

“…Among the 20 natural amino acids, lysine and cysteine are prominent targets for chemical modification due to their high nucleophilicity. However, the prevalence of lysine residues on protein surface results in a difficulty to control the level and regioselectivity of the modification, and only a few examples of site-selective lysine labeling have been reported 1,[7][8][9][10] . In comparison, the low abundance (1.7%) and possible incorporation by site-directed mutagenesis allow cysteine to serve as an ideal residue for labeling 11,12 .…”