Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation.

Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biological activity. This perspective focuses on chemical and enzymatic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chemical modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymatic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges associated with using chemical and enzymatic approaches are highlighted and areas requiring further development are discussed.

Photochemical Alkene Hydrophosphination with Bis(trichlorosilyl)phosphine.

Bis(trichlorosilyl)phosphine (HP(SiCl3)2, 1) was prepared from [TBA][P(SiCl3)2] ([TBA]2, TBA = tetra-n-butylammonium) and triflic acid in 36% yield. Phosphine 1 is an efficient reagent for hydrophosphination of unactivated terminal olefins under UV irradiation (15-60 min) and gives rise to bis(trichlorosilyl)alkylphosphines (RP(SiCl3)2, R = (CH2)5CH3, 88%; (CH2)7CH3, 98%; (CH2)2C(CH3)3, 76%; CH2Cy, 93%; (CH2)2Cy, 95%; CH2CH(CH3)(CH2)2CH3, 82%; (CH2)3O(CH2)3CH3, 95%; (CH2)3Cl, 83%; (CH2)2SiMe3, 92%; (CH2)5C(H)CH2, 44%) in excellent yields. The products require no further purification beyond filtration and removal of volatile material under reduced pressure. The P-Si bonds of prototypical products RP(SiCl3)2 (R = -(CH2)5CH3, -(CH2)7CH3) are readily functionalized to give further phosphorus-containing products: H3C(CH2)7PCl2 (56%), [H3C(CH2)5P(CH2Ph)3]Br (84%), H3C(CH2)7PH2 (61%), H3C(CH2)5P(O)(H)(OH) (81%), and H3C(CH2)5P(O)(OH)2 (55%). Experimental mechanistic investigations, accompanied by quantum chemical calculations, point toward a radical-chain mechanism. Phosphine 1 enables the fast, high-yielding, and atom-efficient preparation of compounds that contain phosphorus-carbon bonds in procedures that bypass white phosphorus (P4), a toxic and high-energy intermediate of the phosphorus industry.

π-Clamp-Mediated Homo- and Heterodimerization of Single-Domain Antibodies via Site-Specific Homobifunctional Conjugation.

Post-translational protein-protein conjugation produces bioconjugates that are unavailable via genetic fusion approaches. A method for preparing protein-protein conjugates using π-clamp-mediated cysteine arylation with pentafluorophenyl sulfonamide functional groups is described. Two computationally designed antibodies targeting the SARS-CoV-2 receptor binding domain were produced (KD = 146, 581 nM) with a π-clamp sequence near the C-terminus and dimerized using this method to provide a 10-60-fold increase in binding (KD = 8-15 nM). When two solvent-exposed cysteine residues were present on the second protein domain, the π-clamp cysteine residue was selectively modified over an Asp-Cys-Glu cysteine residue, allowing for subsequent small-molecule conjugation. With this strategy, we build molecule-protein-protein conjugates with complete chemical control over the sites of modification.

Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents.

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody-drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the ß-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.

Bacterial Phosphate Granules Contain Cyclic Polyphosphates: Evidence from 31P Solid-State NMR.

Polyphosphates (polyPs) are ubiquitous polymers in living organisms from bacteria to mammals. They serve a wide variety of biological functions, ranging from energy storage to stress response. In the last two decades, polyPs have been primarily viewed as linear polymers with varying chain lengths. However, recent biochemical data show that small metaphosphates, cyclic oligomers of [PO3](-), can bind to the enzymes ribonuclease A and NAD kinase, raising the question of whether metaphosphates can occur naturally as products of biological activity. Before the 1980s, metaphosphates had been reported in polyPs extracted from various organisms, but these results are considered artifactual due to the extraction and purification protocols. Here, we employ nondestructive 31P solid-state NMR spectroscopy to investigate the chemical structure of polyphosphates in whole cells as well as insoluble fractions of the bacterium Xanthobacter autotrophicus. Isotropic and anisotropic 31P chemical shifts of hydrated whole cells indicate the coexistence of linear and cyclic phosphates. Under our cell growth conditions and the concentrated conditions of the solid-state NMR samples, we found substantial amounts of cyclic phosphates in X. autotrophicus, suggesting that in fresh cells metaphosphate concentrations can be significant. The cellular metaphosphates are identified by comparison with the 31P chemical shift anisotropy of synthetic metaphosphates of known structures. In X. autotrophicus, the metaphosphates have a chemical shift anisotropy that is consistent with an average size of 3-8 phosphate units. These metaphosphates are enriched in insoluble and electron-dense granules. Exogenous hexametaphosphate added to X. autotrophicus cell extracts is metabolized to trimetaphosphates, supporting the presence and biological role of metaphosphates in cells. The definitive evidence for the presence of metaphosphates, reported here in whole bacterial cells for the first time, opens the path for future investigations of the biological function of metaphosphates in many organisms.

Organoiron- and Fluoride-Catalyzed Phosphinidene Transfer to Styrenic Olefins in a Stereoselective Synthesis of Unprotected Phosphiranes.

Catalytic phosphiranation has been achieved, allowing preparation of trans-1-R-2-phenylphosphiranes (R = t-Bu: 1-t-Bu; i-Pr: 1-i-Pr) from the corresponding dibenzo-7-(R)-7-phospha-norbornadiene (RPA, A = C14H10, anthracene) and styrene in 73% and 57% isolated yields, respectively. The cocatalyst system requires tetramethylammonium fluoride (TMAF) and [Fp(THF)][BF4] (Fp = Fe(η5-C5H5)(CO)2). In the case of the t-Bu derivative, the reaction mechanism was probed using stoichiometric reaction studies, a Hammett analysis, and a deuterium labeling experiment. Together, these suggest the intermediacy of iron-phosphido FpP(F)(t-Bu) (2), generated independently from the stoichiometric reaction of [Fp(t-BuPA)][BF4] with TMAF. Two other plausible reaction intermediates, [Fp(t-BuPA)][BF4] and [Fp(1-t-Bu)][BF4], were prepared independently and structurally characterized.

Orthophosphate and Sulfate Utilization for C-E (E = P, S) Bond Formation via Trichlorosilyl Phosphide and Sulfide Anions.

Reduction of phosphoric acid (H3PO4) or tetra- n-butylammonium bisulfate ([TBA][HSO4]) with trichlorosilane leads to the formation of the bis(trichlorosilyl)phosphide ([P(SiCl3)2]-, 1) and trichlorosilylsulfide ([Cl3SiS]-, 2) anions, respectively. Balanced equations for the formation of the TBA salts of anions 1 and 2 were formulated based on the identification of hexachlorodisiloxane and hydrogen gas as byproducts arising from these reductive processes: i) [H2PO4]- + 10HSiCl3 → 1 + 4O(SiCl3)2 + 6H2 for P and ii) [HSO4]- + 9HSiCl3 → 2 + 4O(SiCl3)2 + 5H2 for S. Hydrogen gas was identified by its subsequent use to hydrogenate an alkene ((-)-terpinen-4-ol) using Crabtree's catalyst ([(COD)Ir(py)(PCy3)][PF6], COD = 1,5-cyclooctadiene, py = pyridine, Cy = cyclohexyl). Phosphide 1 was generated in situ by the reaction of phosphoric acid and trichlorosilane and used to convert an alkyl chloride (1-chlorooctane) to the corresponding primary phosphine, which was isolated in 41% yield. Anion 1 was also prepared from [TBA][H2PO4] and isolated in 62% yield on a gram scale. Treatment of [TBA]1 with an excess of benzyl chloride leads to the formation of tetrabenzylphosphonium chloride, which was isolated in 61% yield. Sulfide 2 was used as a thionation reagent, converting benzophenone to thiobenzophenone in 62% yield. It also converted benzyl bromide to benzyl mercaptan in 55% yield. The TBA salt of trimetaphosphate ([TBA]3[P3O9]·2H2O), also a precursor to anion 1, was found to react with either trichlorosilane or silicon(IV) chloride to provide bis(trimetaphosphate)silicate, [TBA]2[Si(P3O9)2], characterized by NMR spectroscopy, X-ray crystallography, and elemental analysis. Trichlorosilane reduction of [TBA]2[Si(P3O9)2] also provided anion 1. The electronic structures of 1 and 2 were investigated using a suite of theoretical methods; the computational studies suggest that the trichlorosilyl ligand is a good π-acceptor and forms σ-bonds with a high degree of s character.

Ambient-Temperature Synthesis of 2-Phosphathioethynolate, PCS-, and the Ligand Properties of ECX- (E = N, P; X = O, S).

A synthesis of the 2-phosphathioethynolate anion, PCS-, under ambient conditions is reported. The coordination chemistry of PCO-, PCS- and their nitrogen-containing congeners is also explored. Photolysis of a solution of W(CO)6 in the presence of PCO- [or a simple ligand displacement reaction using W(CO)5(MeCN)] affords [W(CO)5(PCO)]- (1). The cyanate and thiocyanate analogues, [W(CO)5(NCO)]- (2) and [W(CO)5(NCS)]- (3), are also synthesised using a similar methodology, allowing for an in-depth study of the bonding properties of this family of related ligands. Our studies reveal that, in the coordination sphere of tungsten(0), the PCO- anion preferentially binds through the phosphorus atom in a strongly bent fashion, while NCO- and NCS- coordinate linearly through the nitrogen atom. Reactions between PCS- and W(CO)5(MeCN) similarly afford [W(CO)5(PCS)]-; however, due to the ambidentate nature of the anion, a mixture of both the phosphorus- and sulfur-bonded complexes (4a and 4b, respectively) is obtained. It was possible to establish that, as with PCO-, the PCS- ion also coordinates to the metal centre in a bent fashion.

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond.

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged AlphaFold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond.

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged Alphafold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

Sustainable Production of Reduced Phosphorus Compounds: Mechanochemical Hydride Phosphorylation Using Condensed Phosphates as a Route to Phosphite.

In pursuit of a more sustainable production of phosphorous acid (H3PO3), a versatile chemical with phosphorus in the +3 oxidation state, we herein report that condensed phosphates can be employed to phosphorylate hydride reagents under solvent-free mechanochemical conditions to furnish phosphite (HPO3 2-). Using potassium hydride as the hydride source, sodium trimetaphosphate (Na3P3O9), triphosphate (Na5P3O10), pyrophosphate (Na4P2O7), fluorophosphate (Na2PO3F), and polyphosphate ("(NaPO3) n ") engendered phosphite in optimized yields of 44, 58, 44, 84, and 55% based on total P content, respectively. Formation of overreduced products including hypophosphite (H2PO2 -) was identified as a competing process, and mechanistic investigations revealed that hydride attack on in-situ-generated phosphorylated phosphite species is a potent pathway for overreduction. The phosphite generated from our method was easily isolated in the form of barium phosphite, a useful intermediate for production of phosphorous acid. This method circumvents the need to pass through white phosphorus (P4) as a high-energy intermediate and mitigates involvement of environmentally hazardous chemicals. A bioproduced polyphosphate was found to be a viable starting material for the production of phosphite. These results demonstrate the possibility of accessing reduced phosphorus compounds in a more sustainable manner and, more importantly, a means to close the modern phosphorus cycle.

Synthesis of phosphiranes via organoiron-catalyzed phosphinidene transfer to electron-deficient olefins.

Herein is reported the structural characterization and scalable preparation of the elusive iron-phosphido complex FpP( t Bu)(F) (2-F, Fp = (Fe(η5-C5H5)(CO)2)) and its precursor FpP( t Bu)(Cl) (2-Cl) in 51% and 71% yields, respectively. These phosphide complexes are proposed to be relevant to an organoiron catalytic cycle for phosphinidene transfer to electron-deficient alkenes. Examination of their properties led to the discovery of a more efficient catalytic system involving the simple, commercially available organoiron catalyst Fp2. This improved catalysis also enabled the preparation of new phosphiranes with high yields ( t BuPCH2CHR; R = CO2Me, 41%; R = CN, 83%; R = 4-biphenyl, 73%; R = SO2Ph, 71%; R = POPh2, 70%; R = 4-pyridyl, 82%; R = 2-pyridyl, 67%; R = PPh3 +, 64%) and good diastereoselectivity, demonstrating the feasibility of the phosphinidene group-transfer strategy in synthetic chemistry. Experimental and theoretical studies suggest that the original catalysis involves 2-X as the nucleophile, while for the new Fp2-catalyzed reaction they implicate a diiron-phosphido complex Fp2(P t Bu), 4, as the nucleophile which attacks the electron-deficient olefin in the key first P-C bond-forming step. In both systems, the initial nucleophilic attack may be accompanied by favorable five-membered ring formation involving a carbonyl ligand, a (reversible) pathway competitive with formation of the three-membered ring found in the phosphirane product. A novel radical mechanism is suggested for the new Fp2-catalyzed system.

Let's Make White Phosphorus Obsolete.

Industrial and laboratory methods for incorporating phosphorus atoms into molecules within the framework of Green Chemistry are in their infancy. Current practice requires large inputs of energy, involves toxic intermediates, and generates substantial waste. Furthermore, a negligible fraction of phosphorus-containing waste is recycled which in turn contributes to negative environmental impacts, such as eutrophication. Methods that begin to address some of these drawbacks are reviewed, and some key opportunities to be realized by pursuing organophosphorus chemistry under the principles of Green Chemistry are highlighted. Methods used by nature, or in the chemistry of other elements such as silicon, are discussed as model processes for the future of phosphorus in chemical synthesis.

Synthesis of acyl(chloro)phosphines enabled by phosphinidene transfer.

Acyl(chloro)phosphines RC(O)P(Cl)(t-Bu) have been prepared by formal insertion of tert-butyl phosphinidene (t-Bu-P) from t-BuPA (A = C14H10 or anthracene) into the C-Cl bond of acyl chlorides. We show that the under-explored acyl(chloro)phosphine functional group provides an efficient method to prepare bis(acyl)phosphines, which are important precursors to compounds used industrially as radical polymerization initiators. Experimental and computational investigations into the mechanism of formation of acyl(chloro)phosphines by our synthetic method reveal a pathway in which chloride attacks a phosphonium intermediate and leads to the reductive loss of anthracene from the phosphorus center in a P(v) to P(iii) process. The synthetic applicability of the acyl(chloro)phosphine functional group has been demonstrated by reduction to an acylphosphide anion, which can in turn be treated with an acyl chloride to furnish dissymmetric bis(acyl)phosphines.

Phosphoric acid as a precursor to chemicals traditionally synthesized from white phosphorus.

White phosphorus, generated in the legacy thermal process for phosphate rock upgrading, has long been the key industrial intermediate for the synthesis of phosphorus-containing chemicals, including herbicides, flame-retardants, catalyst ligands, battery electrolytes, pharmaceuticals, and detergents. In contrast, phosphate fertilizers are made on a much larger scale from phosphoric acid, obtained by treating phosphate rock with sulfuric acid. Dehydration of phosphoric acid using sodium chloride gives trimetaphosphate, and here we report that trichlorosilane, primarily used for the production of high-purity silicon, reduces trimetaphosphate to the previously unknown bis(trichlorosilyl)phosphide anion. This anion offers an entry point to value-added organophosphorus chemicals such as primary and secondary alkyl phosphines, and thus to organophosphinates, and can also be used to prepare phosphine gas and the hexafluorophosphate anion, all previously available only downstream from white phosphorus.