One-step synthesis of biotinyl photoprobes from unprotected carbohydrates

A simple and versatile approach for the preparation of carbohydrate photoprobes has been developed. By a single-step reaction at 37 degrees C, a biotinylated carbene-generating unit was introduced to the reducing end of unprotected carbohydrates. Micromole quantities of N-acetyllactosamine, Lewis X trisaccharide, and sialyl Lewis X tetrasaccharide were easily converted to their biotinylated photoreactive analogues, which enabled the nonradioisotopic chemiluminescent detection of the photolabeled products. Thus, a sequence of lectin photoaffinity labeling, from the probe synthesis to the detection of labeled protein, was readily accomplished within one week. Our strategy may be applicable to any aldehyde-bearing ligand.     

Photochemical fishing approaches for identifying target proteins and elucidating the structure of a ligand-binding region using carbene-generating photoreactive probes.

Photoaffinity labeling enables the direct probing of a target protein through a covalent bond between a ligand and its binding protein, and even a complex formed by weak interactions can be isolated by the method. The photochemical fishing approach accelerates the throughput, isolating crosslinked complexes and analyzing the structure of the ligand binding site within the protein. We used carbene-generating phenyldiazirine for this approach because practical examinations had shown that the phenyldiazirine functioned as the powerful barb on the hook. Improving the synthetic pathways of the photoprobes and using chemoselective-integrated photoreactive units makes possible the easy and rapid preparation of carbene-generating photoreactive probes including the derivatives in peptides, proteins, DNAs, and carbohydrates. This review also shows several recent impacts of photoaffinity labeling, including the in vivo preparation of photoreactive proteins in living cells.     

Structure-Based Design of a Macrocyclic PROTAC

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co-crystal structure of macroPROTAC-1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12-fold loss of binary binding affinity for Brd4, macroPROTAC-1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.     

Structure‐Based Design of a Macrocyclic PROTAC

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co‐crystal structure of macroPROTAC‐1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12‐fold loss of binary binding affinity for Brd4, macroPROTAC‐1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.     

Design, synthesis, and biological evaluation of potent discodermolide fluorescent and photoaffinity molecular probes

[structure: see text] The design, synthesis, and biological evaluation of a series of (+)-discodermolide molecular probes possessing photoaffinity and fluorescent appendages has been achieved. Stereoselective olefin cross-metathesis comprised a key tactic for construction of two of the molecular probes. Three photoaffinity probes were radiolabeled with tritium.     

Simple and versatile method for tagging phenyldiazirine photophores

The first effective method for the introduction of a versatile substituent on 3-phenyl-3-trifluoromethyldiazirine has been developed. The simple preparation of a useful aldehyde intermediate allows easy access to various elaborated photoaffinity ligands, including a l-phenylalanine analog bearing a diazirine ring (TmdPhe). The asymmetric synthesis of TmdPhe was easily accomplished in gram quantities. Site-directed incorporation of this compound into the structure of a calmodulin-binding peptide using automated peptide synthesis afforded a photoreactive peptide that was successfully used for the specific labeling of calmodulin.     

Synthesis and characterization of photoaffinity probe of acetogenin, a strong inhibitor of mitochondrial complex I

Acetogenins are valuable inhibitor probes to get an insight into the structural and functional properties of mitochondrial NADH-ubiquinone oxidoreductase (complex I). We synthesized a photoreactive acetogenin mimic ([¹²⁵I]DANA) which retained a strong inhibitory activity. The preliminary photoaffinity labeling with bovine heart submitochondrial particles revealed that [¹²⁵I]DANA binds to the ND1 subunit among 45 different subunits with high specificity.     

携异戊烯基光亲和化学探针的合成和应用

应用“一锅法”耦连反应为关键步骤合成了3个同时含有异戊烯链和叠氮基团的光亲和探针分子。在光照条件下这些化合物和酿酒酵母总蛋白反应后,经过点击反应与含有生物素的报告分子连接,再进行亲和素印迹分析,初步表明它们可作为钓取与异戊烯链相互作用蛋白的化学探针。     

SYNTHESIS AND EVALUATION OF 2-DIAZO-3,3,3-TRIFLUOROPROPANOYL DERIVATIVES OF COLCHICINE AND PODOPHYLLOTOXIN AS PHOTOAFFINITY LABELS: REACTIVITY, PHOTOCHEMISTRY, AND TUBULIN BINDING

Derivatives of the tubulin polymerization inhibitors colchicine and podophyllotoxin bearing the photoreactive 2-diazo-3,3,3-trifluoropropanoyl (DTFP) group were synthesized for evaluation as potential photoaffinity labels of the tubulin binding site. All labels were assayed for their ability to inhibit tubulin polymerization, and N-DTFP-deacetylthiocolchicine was shown to competitively inhibit tubulin-colchicine binding with a Ki of 4-5 microM. The tubulin off-rate of this analog was similar to that of podophyllotoxin, rather than to the relatively irreversibly bound colchicine. Photochemical solvent insertion reactions of the labels were investigated. Radioactive samples of the two most active labels were prepared and used in initial protein-labeling experiments, during which the fractional occupancy of tubulin and extent of covalent incorporation were determined. A rearrangement of DTFP amides was encountered which is relevant to the utility of this moiety for use in synthesis of photoaffinity labels.     

Photolabile and paramagnetic reagents for the investigation of transmembrane signaling events.

To investigate the dynamics of membrane processes that may be integral components of specific transmembrane signaling events we have synthesized several novel paramagnetic probes and their photoreactive counterparts. The structure of these probes was designed to (1) restrict "flipping" across the membrane bilayer; (2) contain paramagnetic or photoreactive moieties that could be placed at specific depths within the bilayer; (3) provide information about membrane structure as well as dynamics of protein movement; and (4) in the case of the photoreactive probes, be of high specific radioactivity. The molecules described in this paper consist of amino acid, dipeptide, or carbohydrate groups attached to arylazide- or nitroxide-bearing fatty acids. The synthesis and initial characterization of these membrane probes is described.     

Convenient synthesis of photoaffinity probes and evaluation of their labeling abilities

Convenient synthesis of a variety of photoaffinity probes was accomplished by utilizing our Ns strategy and novel resin. The synthetic probes were evaluated via the labeling ability with the preseniline 1 C-terminal fragments, which was identified as a therapeutic target for Alzheimer's disease.     

Cross-linking chemistry and biology: development of multifunctional photoaffinity probes

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.     

Dehydrogenation of the NH−NH Bond Triggered by Potassium tert-Butoxide in Liquid Ammonia

A novel strategy for the dehydrogenation of the NH−NH bond is disclosed using potassium tert-butoxide (tBuOK) in liquid ammonia (NH₃) under air at room temperature. Its synthetic value is well demonstrated by the highly efficient synthesis of aromatic azo compounds (up to 100 % yield, 3 min), heterocyclic azo compounds, and dehydrazination of phenylhydrazine. The broad application of this strategy and its benefit to chemical biology is proved by a novel, convenient, one-pot synthesis of aliphatic diazirines, which are important photoreactive agents for photoaffinity labeling.     

Dehydrogenation of the NH−NH Bond Triggered by Potassium tert‐Butoxide in Liquid Ammonia

A novel strategy for the dehydrogenation of the NH−NH bond is disclosed using potassium tert ‐butoxide (t BuOK) in liquid ammonia (NH₃) under air at room temperature. Its synthetic value is well demonstrated by the highly efficient synthesis of aromatic azo compounds (up to 100 % yield, 3 min), heterocyclic azo compounds, and dehydrazination of phenylhydrazine. The broad application of this strategy and its benefit to chemical biology is proved by a novel, convenient, one‐pot synthesis of aliphatic diazirines, which are important photoreactive agents for photoaffinity labeling.     

A direct-to-biology high-throughput chemistry approach to reactive fragment screening

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines ‘direct-to-biology’ high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn²⁺ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design–make–test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

A photoreactive fragment screening platform employing direct-to-biology high-throughput chemistry (D2B-HTC) for the rapid iterative synthesis and screening of libraries of photoaffinity bits.     

Efficient solid-phase synthesis of trifunctional probes and their application to the detection of carbohydrate-binding proteins

[structure: see text] An efficient solid-phase synthesis of trifunctional probes containing a photoreactive group, a reporter tag, and a carbohydrate ligand was developed. Labeling studies with these probes demonstrate that specific lectins can be labeled with high sensitivity and selectivity. This technique serves as a powerful tool for the rapid detection and profiling of lectins.     

Photo-Brook rearrangement of acyl silanes as a strategy for photoaffinity probe design

Photoaffinity labeling (PAL) is a powerful tool for the identification of non-covalent small molecule–protein interactions that are critical to drug discovery and medicinal chemistry, but this approach is limited to only a small subset of robust photocrosslinkers. The identification of new photoreactive motifs capable of covalent target capture is therefore highly desirable. Herein, we report the design, synthesis, and evaluation of a new class of PAL warheads based on the UV-triggered 1,2-photo-Brook rearrangement of acyl silanes, which hitherto have not been explored for PAL workflows. Irradiation of a series of probes in cell lysate revealed an iPr-substituted acyl silane with superior photolabeling and minimal thermal background labeling compared to other substituted acyl silanes. Further, small molecule (+)-JQ1- and rapamycin-derived iPr acyl silanes were shown to selectively label recombinant BRD4-BD1 and FKBP12, respectively, with minimal background. Together, these data highlight the untapped potential of acyl silanes as a novel, tunable scaffold for photoaffinity labeling.

Irradiation initiated 1,2-photo Brook rearrangement of acyl silanes generated α-siloxycarbene intermediates that were used for photoaffinity labeling. Optimization of the acyl silane group produced a probe capable of capturing small molecule–protein interactions.     

A Series of Potent CREBBP Bromodomain Ligands Reveals an Induced‐Fit Pocket Stabilized by a Cation–π Interaction

The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein‐bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced‐fit pocket forming a cation–π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells.     

结构蛋白质组学研究进展

蛋白质结构与其生物学功能直接相关，蛋白质功能的调控也主要依赖于其构象和相互作用的动态调节。对蛋白质结构和功能的研究一直是生命科学领域的研究热点，也是当前蛋白质组学研究的重要发展方向。该综述重点讨论了近年来基于质谱的结构蛋白质组学主要分析方法的原理、进展和应用，主要包括非变性质谱法、限制性蛋白质酶切法、化学交联法、氢氘交换法、共价化学标记法、热稳定性分析法等；最后对结构蛋白质组学的发展进行了总结与展望。     

Novel bifunctional probe for radioisotope-free photoaffinity labeling: compact structure comprised of photospecific ligand ligation and detectable tag anchoring units

A novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.