Monitoring the cellular surface display of recombinant proteins by cysteine labeling and flow cytometry

A general method is described that allows one to follow the surface display of recombinant proteins in Escherichia coli without having to use specific antibodies or enzymatic reactions. The method is based on cysteine-specific labeling through Michael addition to the double bond of maleimide and its derivatives, and takes advantage of the fact that naturally occurring surface proteins in E. coli contain no accessible cysteine residues. The method is easy to perform and could be simply applied to different analytic procedures including Western blot, spectral photometry, and flow cytometry. By using this new labeling method, single cells bearing a distinct protein at the surface could be selected by fluorescence-activated cell sorting. The data were obtained by using autodisplay, an efficient surface display system established for E. coli, but the method presented here represents rather a general solution for analyzing the surface display of recombinant proteins, independent of the cellular system used.     

Reactivity of intein thioesters: appending a functional group to a protein

The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein-mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small-molecule thioester revealed that the alpha-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an alpha-hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3-dipolar azide-alkyne cycloaddition. Thus, the mechanism of intein-mediated protein splicing provides the means to install a useful functional group at a specific site-the C terminus-of virtually any protein.     

Native Chemical Ligation Combined with Desulfurization and Deselenization: A General Strategy for Chemical Protein Synthesis

Of the many approaches proposed to generalize the native chemical ligation approach for protein synthesis, the simple procedure of global desulfurization of peptide thiols has become the most widely adopted. In this review, the development of the native ligation–desulfurization strategy is described, focusing on the conversion of Cys to Ala following ligation at N-terminal Cys residues. Subsequent variations on this theme have broadened the scope to other natural amino acids including Phe, Leu, Val, and Lys, and even non-native peptide linkages such as isopeptide bonds on lysine side chains. Using insights from both selenocysteine–peptide side reactions and radical initiated desulfurization procedures, a new method for the selective deselenization of peptides containing both selenocysteine and cysteine residues has been developed. Together, these approaches represent a robust and flexible methodology for the synthesis of complex polypeptides without the use of protecting groups.     

Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells

An ideal technology for direct imaging of post-translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein-activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full-length form. We also prepared a nonphosphorylated, caged full-length Smad2 analogue labeled with an orthogonal fluorophore, and simultaneously imaged the phosphorylated and nonphosphorylated forms of the protein in the same cell. This strategy should enable the dissection of the cellular consequences of post-translational modifications (PTMs) by direct comparison of the behavior of the modified and unmodified forms of the protein following uncaging.     

Phosphine-Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small-molecule triggers that turn on proteins through a Staudinger reduction/self-immolation cascade with substantially improved kinetics and yields. We achieved this through site-specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post-translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction-based activation, paving the way for its application in other proteins and organisms.     

Harnessing Split-Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Biochemical studies of integral membrane proteins are often hampered by low purification yields and technical limitations such as aggregation causing in vitro manipulations to be challenging. The ability of controlling proteins in live cells bypasses these limitations while broadening the scope of accessible questions owing to the proteins being in their native environment. Here we take advantage of the intein biorthogonality to mammalian systems, site specificity, fast kinetics, and auto-processing nature as an attractive option for modifying surface proteins. Using EGFR as a model, we demonstrate that the split-intein pair Ava^N/Npu^C can be used to efficiently and specifically modify target membrane proteins with a synthetic adduct for downstream live cell application.     

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.     

Electrochemical oxidation of dopamine in the presence of sulfhydryl compounds: Application to the square-wave voltammetric detection of penicillamine and cysteine

Electro-oxidation of dopamine at a glassy carbon electrode was investigated in the presence of some biologically important thiols (R-SH), e.g. cysteine and penicillamine. Results of cyclic voltammetric studies together with the spectrophotometric foundations via Ellman's test during the controlled-potential coulometry show a nucleophilic addition/reduction of thiol to the electrochemically generated dopaminoquinone by a 1 + 4 Michael addition. The resulting ring substituted substrate (as RS-form) is more easily oxidized leading to an increase in the anodic current of dopamine, which is proportional to the concentration of thiol. The square-wave voltammetry (SWV) were applied as a very sensitive voltammetric detection method for the detection of cysteine and penicillamine. A linear response from 0.10 to 2.5 μM with a detection limit of 0.08 μM is resulted by the voltammetric determinations for penicillamine. The effect of other biological thiols, such as N-acetyl cysteine, glutathione, methionine, captopril and mercaptoglycine were also assessed and found to present no appreciable change in the voltammetric response of dopamine. The proposed method can be considered as a relatively selective method for the voltammetric detection of penicillamine and cysteine. The practical utility of the method was investigated in the determination of thiol species in pharmaceutical preparations.     

Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells

Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.     

Promiscuous enzyme‐catalyzed Michael addition: synthesis of warfarin and derivatives

BACKGROUND: Biocatalytic promiscuity has attracted much attention from chemists and biochemists in recent years. Warfarin, one of the most effective anticoagulants, has been introduced for clinical use as a racemate for more than half a century. Although some different chemical strategies towards the synthesis of optically active warfarin have been reported, biocatalytic preparation of warfarin remains unexploited. RESULTS: Lipase from porcine pancreas (PPL) was used as a biocatalyst to catalyze the Michael addition of 4‐hydroxycoumarin to α,β‐unsaturated enones in organic medium in the presence of water to synthesize warfarin and derivatives. The products were obtained in moderate to high yields (up to 95%) with none or low enantioselectivities (up to 28% ee). The influence of reaction conditions including solvents, temperature and molar ratio of substrates was systematically investigated. CONCLUSION: Among the many reported lipase‐catalyzed Michael additions, only a few showed enantioselectivity. Therefore, this Michael addition activity of, for example, PPL is a valuable case of enantioselective lipase catalytic promiscuity. In addition, it was the first time warfarin and derivatives were prepared using a biocatalyst. Copyright © 2012 Society of Chemical Industry     

Cotranslational incorporation of a structurally diverse series of proline analogues in an Escherichia coli expression system

A set of Escherichia coli expression strains have been defined that are competent for the incorporation of a structurally diverse series of proline analogues under culture conditions that are compatible with high levels of analogue substitution within a proline-rich protein substrate. These bacterial strains have been employed to assay the efficacy of incorporation of noncanonical amino acids into a recombinant-protein test substrate and to create variant polypeptides in which native protein sequences have been globally substituted with imino acid analogues in response to proline codons. We envision that these methods may be used to interrogate the effect of imino acid substitution on protein structure and function and may be particularly informative in the context of structural comparison of a series of modified proteins with respect to the stereoelectronic differences between the incorporated proline analogues.     

Synthesis and characterization of in situ cross-linked hydrogel based on self-assembly of thiol-modified chitosan with PEG diacrylate using Michael type addition

A novel injectable in situ cross-linked hydrogel has been designed via Michael type addition between thiol-modified chitosan (CS-NAC) and PEG diacrylate (PEGDA). Hydrogel was rapidly formed in situ under physiological conditions. The gelation time depended on the content of free thiols in CS-NAC, temperature, and concentration of CS-NAC and PEGDA. Thermogravimetric analysis showed the thermal stabilities of hydrogels. SEM observation results confirmed a porous 3D structure. Rheological studies showed that the cross-linking density and elasticity of hydrogel had a correlation to the content of CS-NAC and PEGDA. Swelling studies revealed that these hydrogels had a high initial swelling and were degradable under physiological conditions. And swelling was highly temperature-dependent and was directly related to the amount of cross-linking. Biological activities of the hydrogels were evaluated by in vitro cell compatibility on HDFs and A549 cells and the results indicated that the hydrogel was biocompatible.     

新型聚天冬氨酸酯聚脲的合成、结构与性能研究

通过二步法:(1) 4,4'-二氨基环己基甲烷(PACM)或3,3'-二甲基-4,4'-二氨基环己基甲烷(Laromin C260)与马来酸二乙酯的Michael加成反应,(2) 反应(1)产物与E-51环氧树脂的加成反应,合成了新型聚天冬氨酸酯(PAEs).与现有Michael加成法相比,二步法能明显缩短合成反应时间,更适合工业化生产.进一步通过PAEs与4,4'-二环已基甲烷二异氰酸酯(H12MDI)/聚四亚甲基二醇(PTMG)预聚物反应,常温合成了2种PAEs聚脲.采用FT-IR、1H-NMR和GPC等表征了PAEs,并采用FT-IR和DMA考察了PAEs聚脲的结构形态、反应活性和力学性质.研究结果表明,聚脲硬段氨基氢键化程度高,氢键键长为3.04 ?;脲羰基的氢键化程度分别为74.4%和73.2%,提高固化温度,有助于脲羰基氢键的形成.PAEs聚脲呈现微相分离的形态,是低活性、高强度的弹性体涂层材料.     

Multicomponent Reactions of Phosphines,Enynedioates and Cinnamaldimines Give γ‐Lactams with a 1,3,5‐Hexatriene Moiety for Facile 6π Electrocyclization: Access to Oxindoles,Isatins and Isoxazolinones

Multicomponent reactions of phosphines, enynedioates and cinnamaldimines generated 3‐phosphorus ylide γ‐lactams having a 1,3,5‐hexatriene moiety with low activation energy barrier for 6π electrocyclization, through initial formation of 1,3‐dipoles from the α(δ′)‐Michael addition of phosphines to enynedioates. The reactive 1,3‐dipoles underwent addition to cinnamaldimines, lactamization, 6π electrocyclization and oxidation to give 3‐phosphorus ylide oxindoles as platform molecules toward isatins and isoxazolinones. The key step, 6π electrocyclization, was further examined by a kinetic and a computational study.

     

N-硝基苯基脯氨酰胺小分子催化剂的合成及其在环己酮与硝基烯烃不对称Michael加成中的应用

以苄氧羰基保护的L-脯氨酸和硝基苯胺类化合物为起始原料,通过酰胺化反应和脱苄氧羰基反应,合成了两个结构新颖的N-硝基苯基脯氨酰胺类催化剂,其结构经~~1HNMR、~(13)CNMR和HR-ESI-MS确证。该类催化剂用于环己酮与硝基烯烃的不对称Michael加成反应,其中化合物(S)-N-(2-甲氧基-4-硝基苯基)脯氨酰胺催化所得产物具有优异的收率(82%)、较高的非对映体比例(94∶6 dr)和一定的对映选择性(21%e.e.)。     

Primary Amine‐Thioureas with Improved Catalytic Properties for “Difficult” Michael Reactions: Efficient Organocatalytic Syntheses of (S)‐Baclofen, (R)‐Baclofen and (S)‐Phenibut

Among the class of primary amine‐thioureas based on tert‐butyl esters of α‐amino acids, the most efficient organocatalyst for “difficult” Michael reactions was identified. The derivative based on (S)‐di‐tert‐butyl aspartate and (1R,2R)‐diphenylethylenediamine provided the products of the reaction between aryl methyl ketones and nitroolefins in excellent yields and enantioselectivities. In addition, this new catalyst can be used at low catalyst loading (5 mol%). The utility of this methodology was highlighted by the efficient synthesis of (S)‐baclofen, (R)‐baclofen and (S)‐phenibut.     

Facile,Efficient and Diastereoselective Construction of Indane‐Fused Pyrrolidin‐2‐ones via a Potassium Hydroxide‐Catalyzed Domino Reaction

A facile and efficient potassium hydroxide (KOH)‐catalyzed tandem reaction involving sequential Michael addition of electron‐deficient alkenes with malonic esters, ring opening of activated aziridines and lactamization has been described. A highly functionalized indane‐fused tricyclic scaffold containing three rings, three adjacent stereocenters and a quaternary center was constructed by the formation of two new C–C bonds and one new C–N bond in a diastereoselective manner.

     

Asymmetric Domino Nitro‐Michael/Horner–Wadsworth–Emmons Reaction for Disubstituted Cyclohexenecarboxylate Annulation: Efficient Synthesis of Dipeptidyl Peptidase IV Inhibitor ABT‐341 and Influenza Neuraminidase Inhibitor

An asymmetric domino nitro‐Michael/Horner–Wadsworth–Emmons (HWE) reaction involving α,β‐unsaturated aldehydes and nitro phosphonates has been developed, which gave 4,5‐disubstituted cyclohexenecarboxylates with high stereoselectivities (dr up to >20:1, ee 83–92%) in good yields (44–76%). Furthermore, using this methodology as a key step, a short and practical synthesis of pharmaceutically useful compounds (such as the dipeptidyl peptidase IV inhibitor ABT‐341 and an influenza neuraminidase inhibitor) has also been accomplished.