Room-Temperature Grafting from Synthesis of Protein–Polydisulfide Conjugates via Aggregation-Induced Polymerization

Abstract

The reversible modification of proteins with lipoic acid (LPA)-derived polydisulfides (PDS) is an important approach toward the transient regulation and on-demand recovery of protein functions. The in situ growth of PDS from the cysteine (Cys) residue of a protein, however, has been challenging due to the nearequilibrium thermodynamics of the ring-opening polymerization of LPA. Here, we report the protein-mediated, aggregation-induced polymerization (AIP) of amphiphilic LPA-derived monomers at room temperature, which can be performed at a concentration as low as ∼2% of the equilibrium monomer concentration normally needed. The aggregation of monomers increases the effective monomer concentration in aqueous solutions to the degree that the polymerizations behave similarly to those in bulk. The PDS conjugation enhances the thermostability, protease resistance, and tolerance to freeze−thaw treatments of the target proteins. Moreover, the PDS conjugation allows rapid and convenient purification of Cys-bearing proteins by taking advantage of the liquid− liquid phase separation of the protein−PDS conjugates and the full recovery of native proteins under mild reducing conditions. This AIP effect may shed light on facilitating other polymerizations with a similar near-equilibrium character. The PDS conjugation can open up new avenues to protein delivery, dynamic and reversible protein engineering, enzyme preservation, and recycling.

Tetrel‐Centered Exchange Cascades to Decouple Inhibition and Induction of Thiol‐Mediated Uptake: Introducing Cell‐Penetrating Thiolactones, Focus on Reversible Michael Acceptor Dimers

“…In the less potent thiolactone series, the application of ring tension in β thiolactones [67 -69] could decelerate ring closure to access covalent cascades along disulfide arrays ('disul-fide walking', Figure 3,b) and thus shift from inhibition ('thiol/ate walking', Figure 3,d) to induction of thiolmediated uptake. Respecting the emerging importance of thiol-mediated uptake as a general network to control cellular entry [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][22][23][24] and the unique properties found for tetrel-centered CAXs, continuing studies on tetrel-centered CAXs along these lines are considered worthwhile and have been initiated.…”

“…It stands for 1 ) the emergence of cell‐penetrating activity in a substrate appended with a dynamic covalent cascade exchanger (CAX), and 2 ) the inhibition of this cell penetration with thiol‐reactive agents, often also by the same CAX. Since the introduction of cell‐penetrating poly(disulfide)s to thiol‐mediated uptake in 2014, [2] various CAXs have been explored to deliver a wide variety of substrates to the cytosol, [3–17] including challenging objects like quantum dots, [17] genes [7–11] or proteins, [6,16] from antibodies [6] to genome editing machinery, also in vivo [7] . Although weak and unreliable, [18] the uptake inhibition with Ellman ’s reagent has traditionally been accepted as experimental evidence for the occurrence of thiol‐mediated uptake [19–21] .…”

The Dawn of a New Era: Targeting the “Undruggables” with Antibody-Based Therapeutics

“…The cryopolymerization of 1,2-dithiolanes to prepare reversible protein-polydisulfide conjugates by grafting- from synthesis may shed new lights on the preparation of antibody-CPD conjugates. Very recently, room-temperature grafting-from synthesis was also achieved by the same research group, in which amphiphilic lipoic acid-derived monomers were used to facilitate the polymerization at a more biologically relevant temperature …”

“…Very recently, room-temperature grafting-from synthesis was also achieved by the same research group, in which amphiphilic lipoic acid-derived monomers were used to facilitate the polymerization at a more biologically relevant temperature. 639 In contrast to the conjugation approaches, monomers of CPDs have received comparably less attention since the original CPDs made from cationic guanidium-containing monomers, M1 or M2, showed good intracellular delivery efficiency, 630 and M1/M2 has thus been widely used (Figure 34a). The glycosylation of M2 (GlyM) was introduced by Matile et al to increase the solubility of CPDs as well as to achieve multifunctional cellular uptake (Figure 34b).…”

Visible Light Photocatalytic Hydrogen Evolution Paired with Excellent Intramolecular Coupling of Dithiols into Sulfurous Heterocyclic Compounds over CdS/P25/Ni2P

“…Heterocyclic compounds have attracted considerable interest in the field of biology, medicine, and chemistry owing to their great significance for the preparation of new materials and pharmaceuticals. − Among them, sulfurous heterocyclic compounds containing disulfide bonds (S–S) are pivotal structural motifs in numerous proteins and medical intermediates. , For example, lipoic acid has attracted extensive attention as an important antioxidant and insulin-mimetic supplement, which has been used to remedy multiple sclerosis, schizophrenia, and diabetes . A variety of methods have been explored for the direct synthesis of sulfurous heterocyclic compounds at present; however, most of them rely on lots of energy supplements and can release highly toxic substances in the presence of various additives under harsh reaction conditions. − Therefore, it is indispensable to develop a mild and nontoxic method for the synthesis of sulfurous heterocyclic compounds.…”