Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) derivatives for chemoselective attachment to unprotected polyfunctionalized compounds

A convenient synthesis of novel bifunctional poly(amino carboxylate) chelating agents allowing chemoselective attachment to highly functionalized biomolecules is described. Based on the well known chelator 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA), we synthesized novel bifunctional chelating agents bearing additional functional groups by alkylating 1,4,7,10-tetraazacyclododecane (cyclen) with one equivalent of para-functionalized alkyl 2-bromophenyl-acetate and three equivalents of tert-butyl 2-bromoacetate. The resulting compounds, which contain an additional carbonyl or alkyne functionality, allow site specific labeling of appropriately functionalized unprotected biomolecules in a rapid manner via click reactions. This was demonstrated by the attachment of our new DOTA derivatives to the somatostatin analogue Tyr3-octreotate by chemoselective oxime ligation and CuI-catalyzed azide-alkyne cycloaddition. Initial biodistribution studies in mice with the radiometalated compound demonstrated the applicability of the described DOTA conjugation.     

Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.     

A bioorthogonal quadricyclane ligation

New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M(-1) s(-1), on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide-alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules.     

Reliable and efficient procedures for the conjugation of biomolecules through Huisgen azide-alkyne cycloadditions

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has been established as a powerful coupling technology for the conjugation of proteins, nucleic acids, and polysaccharides. Nevertheless, several shortcomings related to the presence of Cu, mainly oxidative degradation by reactive oxygen species and sample contamination by Cu, have been pointed out. This Minireview discusses key aspects found in the development of the efficient and benign functionalization of biomacromolecules through CuAAC, as well as the Cu-free strain-promoted azide-alkyne cycloaddition (SPAAC).     

Biocompatible copper(I) catalysts for in vivo imaging of glycans

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the standard method for bioorthogonal conjugation. However, current Cu(I) catalyst formulations are toxic, hindering their use in living systems. Here we report that BTTES, a tris(triazolylmethyl)amine-based ligand for Cu(I), promotes the cycloaddition reaction rapidly in living systems without apparent toxicity. This catalyst allows, for the first time, noninvasive imaging of fucosylated glycans during zebrafish early embryogenesis. We microinjected embryos with alkyne-bearing GDP-fucose at the one-cell stage and detected the metabolically incorporated unnatural sugars using the biocompatible click chemistry. Labeled glycans could be imaged in the enveloping layer of zebrafish embryos between blastula and early larval stages. This new method paves the way for rapid, noninvasive imaging of biomolecules in living organisms.     

Se-Adenosyl-l-selenomethionine Cofactor Analogue as a Reporter of Protein Methylation

Posttranslational methylation by S-adenosyl-l-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM's utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.     

Conferring Phosphorogenic Properties on Iridium(III)‐Based Bioorthogonal Probes through Modification with a Nitrone Unit

The use of bioorthogonal probes that display fluorogenic or phosphorogenic properties is advantageous to the labeling and imaging of biomolecules in live cells and organisms. Herein we present the design of three iridium(III) complexes containing a nitrone moiety as novel phosphorogenic bioorthogonal probes. These probes were non‐emissive owing to isomerization of the C=N group but showed significant emission enhancement upon cycloaddition reaction with strained cyclooctynes. Interestingly, the connection of the nitrone ligand to the cationic iridium(III) center led to accelerated reaction kinetics. These nitrone complexes were also identified as phosphorogenic bioorthogonal labels and imaging reagents for cyclooctyne‐modified proteins. These findings contribute to the development of phosphorogenic bioorthogonal probes and imaging reagents.     

Evidence for coupling between nitrile groups using DNA templates: a promising new method for monitoring structures with infrared spectroscopy

Infrared spectroscopy is a common method for monitoring biomolecular structures but suffers from spectral congestion. Non-natural vibrational probes provide a way to regain structural specificity because they provide a unique vibrational signature and can be incorporated into proteins or other biomolecules at specific locations. A popular probe is the nitrile group because its frequency is sensitive to the electrostatics of its environment. In this work, we show that pairs of nitrile groups can be used to directly probe distances and angles in dual labeled molecules. By labeling model DNA oligomers with pairs of nitrile tags, we demonstrate that the vibrational coupling between two nitrile groups is strong enough that Fourier transform infrared (FTIR) spectra can be used to probe relative nitrile distances >4.5 A. Our approach is similar in spirit to monitoring structures with fluorescence resonance energy transfer (FRET) using a pair of fluorescent labels or a pair of spin labels in electron spin resonance spectroscopy. The small sizes of nitrile groups make especially valuable probes of sterically confined regions like the inner cores of large biomolecules where other spectroscopic probes do not fit.     

Poly(Acrylic Acid-b-Styrene) Amphiphilic Multiblock Copolymers as Building Blocks for the Assembly of Discrete Nanoparticles

In order to expand the utility of current polymeric micellar systems, we have developed amphiphilic multiblock copolymers containing alternating blocks of poly(acrylic acid) and poly(styrene). Heterotelechelic poly(tert-butyl acrylate-b-styrene) diblock copolymers containing an α-alkyne and an ω-azide were synthesized by atom transfer radical polymerization (ATRP), allowing control over the molecular weight while maintaining narrow polydispersity indices. The multiblock copolymers were constructed by copper-catalyzed azide-alkyne cycloaddition of azide-alkyne end functional diblock copolymers which were then characterized by (1)H NMR, FT-IR and SEC. The tert-butyl moieties of the poly(tert-butyl acrylate-b-styrene) multiblock copolymers were easily removed to form the poly(acrylic acid-b-styrene) multiblock copolymer ((PAA-PS)(9)), which contained up to 9 diblock repeats. The amphiphilic multiblock (PAA-PS)(9) (M(n) = 73.3 kg/mol) was self-assembled by dissolution into tetrahydrofuran and extensive dialysis against deionized water for 4 days. The critical micelle concentration (CMC) for (PAA-PS)(9) was determined by fluorescence spectroscopy using pyrene as a fluorescent probe and was found to be very low at 2 × 10(-4) mg/mL. The (PAA-PS)(9) multiblock was also analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The hydrodynamic diameter of the particles was found to be 11 nm. Discrete spherical particles were observed by TEM with an average particle diameter of 14 nm. The poly(acrylic acid) periphery of the spherical particles should allow for future conjugation of biomolecules.     

New applications of multiply charged ionic probes as cleavable cross-linker and polymerization reagent

We have designed new cleavable cross-linkers for biomolecules containing the 2,6-bis(oxazolinyl)pyridine (pybox) lanthanum complex. These species can effectively donate multiple charges to afford cross-linker ions for target biomolecules. The cross-linkers are cleaved easily by adding water. Moreover, we were able to detect chain structures of target molecules, including biomolecules and carbon clusters, such as fullerene C₆₀, by the addition of some MS ionic probes. Cold-spray ionization mass spectrometry demonstrated the applications of multiply charged ionic probes as cleavable cross-linkers and polymerization reagents.     

Fast RNA conjugations on solid phase by strain-promoted cycloadditions

Strain promoted cycloaddition is presented as a tool for RNA conjugation on the solid phase; RNA-cyclooctyne conjugates are prepared by cycloaddition to both azide (strain-promoted azide-alkyne cycloaddition, SPAAC) and nitrile oxide dipoles (strain-promoted nitrile oxide-alkyne cycloaddition, SPNOAC). The conjugation is compatible with 2'-OMe blocks and with 2'-O-TBDMS protection on the ribose moieties of the sugar. Nitrile oxide dipoles are found to be more reactive click partners than azides. The conjugation proceeds within 10 min in aqueous solvents, at room temperature without any metal catalyst and tolerates dipoles of varying steric bulk and electronic demands, including pyrenyl, coumarin and dabcyl derivatives.     

基于3种非天然糖代谢标记的分泌蛋白质组分析性能对比

分泌蛋白质是调控细胞间信号转导的重要生物大分子。由于分泌蛋白的丰度相比于胞内蛋白以及培养基添加剂更低,因此分泌蛋白的高通量鉴定是目前蛋白质组学界研究的热点和难点。目前,基于生物质谱的分泌蛋白质组学分析一般均需要从无血清的条件培养基中获得分泌蛋白质,再对其进行富集和分析。该流程操作步骤繁琐,易造成分泌蛋白质的损失和降解。本工作采用基于生物正交化学生物学技术实现对分泌蛋白质的高选择性标记和高效富集。通过结合点击化学技术,综合评估了分泌蛋白质分析中用于代谢标记的不同糖类似物。采用3种最常用的商品化糖类似物,N-叠氮乙酰甘露糖胺(ManNAz)、N-叠氮乙酰半乳糖胺(GalNAz)和N-叠氮乙酰葡萄糖胺(GlcNAz)分别对HeLa细胞进行代谢标记,之后通过炔基生物素探针对条件培养基中的分泌蛋白进行富集,结合质谱分析来对比3种糖类似物对分泌蛋白的标记效率。最后通过无标定量蛋白质组学分析,系统评估了3种糖类似物用于分泌蛋白质组分析的性能。结果表明,基于ManNAz的分泌蛋白标记方法鉴定到了282个分泌蛋白、224个细胞质膜蛋白以及846个N-糖基化位点;对分泌蛋白的富集效率分别较GalNAz和GlcNAz提高了130%和67.2%;对细胞质膜蛋白的富集效率较GalNAz和GlcNAz分别提高了273.3%和148.7%,体现出了明显的优势。本研究的实验结果为分泌蛋白高选择性富集和系统分析提供了有益的对比分析和新技术策略。     

Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.     

Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics

The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na?S?O?-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.     

Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Dual‐labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.     

Development of molecular imaging tools to investigate protein functions by chemical probe design

Molecular imaging technologies, which enable the visualization of the behaviors or functions of biomolecules in living systems, have received considerable attention from life scientists. Novel imaging technologies that overcome the limitations of current imaging techniques are desired. In this review, two independent technologies that were recently developed by the authors are described. The first technology is for smart (19)F magnetic resonance imaging (MRI) probes that were developed for in vivo applications. These probes were developed by exploiting paramagnetic relaxation enhancement in order to detect hydrolase activity. With respect to cellular applications, gene expression in cells was visualized using one of the (19)F MRI probes. It was confirmed that this probe design principle is effective for various hydrolases, and broad applications are expected. The second technology is for practical protein labeling. This labeling method is based on a mutant β-lactamase and its specific labeling probes. Since the probe is fluorescence resonance energy transfer (FRET)-based, this labeling method achieves both specific and fluorogenic labeling of target proteins. In addition, derivatization of the probe enabled the labeling of intracellular proteins and the modification of various functional molecules.     

Reactivity of functional groups on the protein surface: development of epoxide probes for protein labeling

We present the development of new affinity probes for protein labeling based on an epoxide reactive group. Systematic screening revealed that an epoxide functionality possesses the special combination of stability and reactivity which renders it stable toward proteins in solution but reactive on the protein surface outside the active site (proximity-induced reactivity). Highly efficient and selective labeling of purified HCA II (human carbonic anhydrase II) was achieved. For instance, 2 equiv of epoxide probe 9 was sufficient for nearly quantitative labeling of HCA II (>90% yield, 20 h reaction time). MS analysis of the labeled protein revealed that 1 equiv of the probe was attached and that labeling occurred at a single residue (His 64) outside the active site. Importantly, epoxide probe 9 selectively labeled HCA II both in simple protein mixtures and in cellular extracts. In addition to the chemical insight and its relevance to many epoxide-containing natural products, this study generated a promising lead in the development of new affinity probes for protein labeling.     

A simple and effective strategy for labeling cysteine sulfenic acid in proteins by utilization of β-ketoesters as cleavable probes

β-ketoesters are robust probes for labeling sulfenic acid (-SOH) proteins allowing quantitative cleavage of the tag for improved analysis of the labeled peptides by MS.     

Developing photoactive affinity probes for proteomic profiling: hydroxamate-based probes for metalloproteases

The denaturing aspect of current activity-based protein profiling strategies limits the classes of chemical probes to those which irreversibly and covalently modify their targeting enzymes. Herein, we present a complimentary, affinity-based labeling approach to profile enzymes which do not possess covalently bound substrate intermediates. Using a variety of enzymes belonging to the class of metalloproteases, the feasibility of the approach was successfully demonstrated in several proof-of-concept experiments. The design template of affinity-based probes targeting metalloproteases consists of a peptidyl hydroxamate zinc-binding group (ZBG), a fluorescent reporter tag, and a photolabile diazirine group. Photolysis of the photolabile unit in the probe effectively generates a covalent, irreversible linkage between the probe and the target enzyme, rendering the enzyme distinguishable from unlabeled proteins upon separation on a SDS-PAGE gel. A variety of labeling studies were carried out to confirm that the affinity-based approach selectively labeled metalloproteases in the presence of a large excess of other proteins and that the success of the labeling reaction depends intimately upon the catalytic activity of the enzyme. Addition of competitive inhibitors proportionally diminished the extent of enzyme labeling, making the approach useful for potential in situ screening of metalloprotease inhibitors. Using different probes with varying P(1) amino acids, we were able to generate unique "fingerprint" profiles of enzymes which may be used to determine their substrate specificities. Finally, by testing against a panel of yeast metalloproteases, we demonstrated that the affinity-based approach may be used for the large-scale profiling of metalloproteases in future proteomic experiments.     

Profiling enzyme activities in vivo using click chemistry methods

Methods for profiling the activity of enzymes in vivo are needed to understand the role that these proteins and their endogenous regulators play in physiological and pathological processes. Recently, we introduced a tag-free strategy for activity-based protein profiling (ABPP) that utilizes the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry") to analyze the functional state of enzymes in living cells and organisms. Here, we report a detailed characterization of the reaction parameters that affect click chemistry-based ABPP and identify conditions that maximize the speed, sensitivity, and bioorthogonality of this approach. Using these optimized conditions, we compare the enzyme activity profiles of living and homogenized breast cancer cells, resulting in the identification of several enzymes that are labeled by activity-based probes in situ but not in vitro.