Synthesis of Photoaffinity Labelling Analogues of the Peptide Hormone Bradykinin

Fourteen peptides, analogues of bradykinin and (des-Arg⁹)bradykinin, have been synthesized by the solid-phase method or have been obtained through chemical modification. In all these peptides an aromatic residue has been substituted by (4′-NO₂)Phe, (4′-NH₂)Phe, (4′-N₃)Phe or (4′-NH₂-3′, 5′-I₂)Phe. These peptides will be used as photoaffinity labelling or affinity labelling probes for peptide hormone receptors.     

Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I

Background: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyp hosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility. We sought to investigate the site of interaction of profilin with phosphoinositides.Results: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P₂). The P-1-tethered D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of Ptdlns(4,5)P₂ but not with Ins(1,4,5)P₃. The acyl-modified Ptdlns(4,5)P₂ analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P₂ phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [³H]BZDC-Ins(1,4,5)P₃-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix.Conclusions: The Ptdlns(4,5)P₂-binding site of profilin I includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of Ptdlns(4,5)P₂ and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.     

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 of [Sar1, Val5, (4′-Azido-3′,5′-ditritio)Phe8] Angiotensin II,a Photoaffinity Label for the Isolation of Angiotensin II Receptors Communication 1

The synthesis of [Sar¹, Val⁵, (4′-azido-3′,5′-ditritio)Phe⁸] angiotensin II from a iodinated precursor peptide is described. The principal problems of this synthesis and their resolution are discussed: (i) The β-induced autophotolysis of the highly tritiated (73 Ci/mmol) and photosensitive label, and (ii) the absorption problems encountered during the purification of microgramm quantities. Such photoaffinity labels are being used for specific labeling and isolation of peptide hormone receptors.     

Synthesis of [D-alanine, 4'-azido-3',5'-ditritio-L-phenylalanine, norvaline4[alpha-melanotropin as a 'photoaffinity probe' for hormone-receptor interactions (author's transl)]

Synthesis of [D -alanine¹, 4′-azido-3′, 5′-ditritio-L -phenylalanine², norvaline⁴]α-melanotropin as a ‘photoaffinity probe’ for hormone-receptor interactions. The synthesis of an α-MSH derivative containing 4′-azido-3′,5′-ditritio-L -phenylalanine is described: Ac · D -Ala-Pap(³H₂)-Ser-Nva-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val · NH₂. This hormone analogue is being used for specific photoaffinity labelling of receptor molecules. The synthesis was performed in a way to minimize the number of radioactive steps and to introduce the radio-active and the photoaffinity label exclusively into position 2. The dipeptide N(α)-acetyl-D -alanyl- (4′-amino-3′,5′-diiodo)-L -phenylalanine was tritriated and transformed into the azido compound, N(α)-acetyl-D -alanyl-(4′-azido-3′,5′-ditritio)-L -phenylalanine which was then condensed with H · Ser-Nva-Glu(OtBu)-His-Phe-Arg-Trp-Gly-Lys(BOC)-Pro-Val · NH₂ to the tridecapeptide. The α-MSH analog displayed a specific activity of 11 Ci/mmol, and a biological activity of about 4 · 10⁹ U/mmol (10% of α-MSH).     

Isotope-coded, fluorous photoaffinity labeling reagents

A pair of isotope-coded, fluorous photoaffinity labeling reagents has been developed and coupled with a peptide. The modified peptides form adducts with methanol upon light illumination, which show characteristic isotope labeling patterns in mass spectra and can be separated from other peptides through fluorous silica.     

A method combining SPITC and 18O labeling for simultaneous protein identification and relative quantification

The relative quantification and identification of proteins by matrix‐assisted laser desorption ionization time‐of‐flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N‐terminal derivatization with 4‐sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while ¹⁸O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4‐sulphophenyl isothiocyanate derivatization with ¹⁸O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R² value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis of protein phosphorylation combining capillary electrophoresis with ATP analog labeling technique

Protein phosphorylation is one of the most basic mechanisms for regulating and controlling protein biological activity and function, and it is also a very important posttranslational modification process. Protein phosphorylation participates in and regulates many life activities such as signal transduction, gene expression, cell cycle, and so on. In this paper, we propose a method for the determination of the protein phosphorylation combining capillary electrophoresis (CE) with ATP analog labeling technique. We synthesized two new ATP analogs (ATP-NB and ATP-TATD-NB) functionalized by norbornene. Using Abl kinase as a model, we established a method for the determination of the kinase activity in solution and lysate by CE with laser-induced fluorescence detection (CE-LIF). This method was used to evaluate the efficiencies of kinase inhibitors. The IC₅₀ values obtained are basically consistent with the reports. By D–A reaction (inverse electron demand Diels–Alder reaction) to label TZ-BODIPY fluorescence, we also realized the phosphorylation fluorescence detection of substrate peptide. Then, we used fluorescence confocal microscopy imaging technology to study the phosphorylation of proteins in vivo by the D–A reaction of ATP-NB and TZ-BODIPY. Our preliminary results documented that the combination of CE-LIF with analog ATP-NB labeling technique is an effective strategy for the determination of the protein phosphorylation and the kinase activity and for screening of kinase inhibitors. The D–A reaction of ATP-NB and TZ-BODIPY also laid the foundation for the subsequent in situ study of protein phosphorylation.     

Design and Synthesis of Photoaffinity Probe Candidates for the GABA-gated Chloride Channel

In order to characterize binding sites of insecticidal compounds on GABA gated chloride channel,new photoaf-finity probe candidates based on 5e-t-butyl-2e-[4-(substituted-propynyl)phenyl]-1,3-dithiane for the noncompetitiveblocker(NCB)site of the γ-aminobutyric acid(GABA)-gated chloride channel were designed and synthesized,andtheir potency as an inhibitor on NCB was measured by 4'-ethynyl-4-n-[2,3-~3H_2]-propylbicycloorthobenzoate(~3HEBOB)assay.The synthesized compounds showed high inhibition activities with half maximum inhibition concen-trations(IC_(50))of lower than 35 nmol/L and were very stable in binding conditions as well photoreacted quickly at300 nm light.These new compounds are expected to be good photoaffinity labeling probes if radioisotope iodine isincorporated.     

Integration of High Accuracy N-Terminus Identification in Peptide Sequencing and Comparative Protein Analysis Via Isothiocyanate-Based Isotope Labeling Reagent with ESI Ion-trap TOF MS

A multifunctional isothiocyanate-based isotope labeling reagent, [d ₀]-/[d ₆]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement, and facilitating peptide sequencing. More significantly, DMPITC-based labeling strategy possessed the capacity of higher reliable N-terminus identification owning to the high-yield b₁ ion combined with the isotope validation of 6 Da. Meanwhile, it also showed potential in differentiating isomeric residues of leucine and isoleucine at N-terminus on the basis of the relative abundance ratios between the fragment ions of their respective b₁ ions. The strategy not only allows accurate interpretation for peptide but also ensures rapid and sensitive comparative analysis for protein by direct MS analysis. Using trypsin-digested bovine serum albumin (BSA), both peptide N-terminus identification and quantitative analysis were accomplished with high accuracy, efficiency, and reproducibility. The application of DMPITC-based labeling strategy is expected to serve as a promising tool for proteome research.     

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。     

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.     

Tag‐Convertible Photocrosslinker with Click‐On/Off N‐Acylsulfonamide Linkage for Protein Identification

The N‐acylsulfonamide group, known as a safety‐catch linker, has been applied to photoaffinity labeling (PAL) using a cinnamate‐type photocrosslinker to improve the efficiency of PAL‐based target identification. A bioorthogonal sulfo‐click reaction was used to stably link a photocrosslinker unit with N‐acylsulfonamide linkage to produce a photoactivatable probe without any protection. In addition, the crosslinked protein was selectively isolated with a small cinnamate tag via linkage disruption upon N‐alkylation. Furthermore, the tag moiety was photochemically converted to a stable coumarin derivative by losing a water molecule, which is a useful property in MS‐based identification.     

On Penicillin‐Binding Protein 1b Affinity‐Labeling Reagents

The synthesis of some 3‐aryl‐3‐(trifluoromethyl)3H‐diazirine and benzophenone‐based photoaffinity labels is reported. The photolabile group is bound to a scaffold that also accommodates functional groups to which an indicator unit (biotin) and the bioactive ligand can be attached orthogonally. To three of the labels, moenomycin was conjugated with the aim to provide tools for the identification of the moenomycin binding site within the transglycosylase domain of the enzyme PBP 1b. Some preliminary photoaffinity‐labeling experiments were carried out.     

EFFICIENCY OF PHOTOAFFINITY LABELING DNA HOMOPOLYMERS AND COPOLYMERS WITH ETHIDIUM MONOAZIDE *

Abstract— Photoaffinity labeling of synthetic DN As with ethidium monoazide was studied to determine if the efficiency of adduct formation was related to DNA sequence. Equilibrium drug binding to DNA homopolymers and copolymers was quanitified by phase partition techniques. The amount of drug bound to a deoxypolymer at equilibrium was then compared to the fraction of ethidium analog covalently-linked following photoactivation at the same drug/DNA input ratio. There were significant sequence-related differences in the ability of the photoaffinity probe to label DNA covalently. The efficiency of covalent-adduct formation decreased in the order poly(dG-dC)^. poly(dG-dC)> poly-(dG). poly(dC)poly(dA-dT). poly(dA-dT)poly(dA). poly(dT). Ethidium monoazide was about 2-fold more efficient in labeling deoxyhomopolymers and deoxycopolymers composed of G-C pairs than the A-T base counterparts. In low ionic buffers (0.015 M Na⁺), the efficiency of photoactivation decreased with increasing ethidium monoazide concentrations. However. the base sequence effect was observed over a 40-fold range of drug concentrations. Therefore, the amount of ethidium monoazide bound to a DNA site after irradiation does not appear to represent the true affinity of the drug for that site.     

Heterogeneity of peptide adducts with carbonylated lipid peroxidation products

Highly reactive lipid peroxidation‐derived carbonyls (oxoLPP) modify protein nucleophiles via Michael addition or Schiff base formation. Once formed, Michael adducts can be further stabilized via cyclic hemiacetals with or without loss of water. Depending on the mechanism of their formation, peptide–oxoLPP can carry aldehyde or keto groups and thus be a part of the total protein carbonylation level. If a carbonyl function is lost during consecutive reactions, the oxoLPP–peptide adducts will not be detected using the common carbonyl labeling protocols. Because of the differences in adduct stabilities, it is possible to address the heterogeneity of peptide/protein–oxoLPP adducts by careful evaluation of tandem mass spectra of modified peptides. Here, we used hydrophilic interaction liquid chromatography–tandem mass spectrometry analysis of lysine, cysteine and histidine containing model peptides co‐incubated with oxidized 1‐palmitoyl‐2‐linoleoyl‐sn‐glycerophosphatidylcholine to characterize the collision‐induced dissociation behavior of peptide–carbonyl adducts. Numerous modifications were detected based on the analysis of tandem mass spectra, including Schiff bases on lysine (two), Michael adducts on lysine (six), cysteine (eleven) and histidine (two), as well as 4‐hydroxy‐2‐aldehydes derived dehydrated cyclic hemiacetals on cysteine (five) and histidine (one). Additionally, cysteine and histidine side chains were modified by lipid‐bound aldehydes as Michael adducts and dehydrated hemiacetals. The tandem mass spectra revealed collision‐induced dissociation characteristics specific for each class of oxoLPP–peptide adducts. Copyright © 2015 John Wiley & Sons, Ltd.     

应用光亲和分子进行PEG的光照修饰及蛋白质的光照偶联

合成了一种光活性标记分子-对叠氮苯甲酸,将其偶联到具有双羟基的碳酸酯与乳酸的共聚物P (LA-co-DHP)上,获得了具有光反应活性的可生物降解共聚物P(LA-co-DAP),在光照条件下,可以将蛋白质方便快捷地共价偶联到P(LA-co-DHP)聚合物纤维上.在溶液中进行PEG与对叠氮苯甲酸的光照反应,通过核磁共振光谱...     

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.     

Mass spectra of Sceletium alkaloids

The mass spectra of several naturally occuring Sceletium alkaloids, as well as several synthetic variants, have been investigated. Techniques used include high resolution mass measurements, analysis of metastable transitions by the technique of ion defocusing and specific deuterium labeling in conjunction with low resolution mass spectrometry. Characteristic major fragmentation pathways have been determined for four basic structure types studied: the cis-3a-aryl-octahydroindole ring system, exemplified by mesembrine; its 2,3-pyrido analog, Sceletium A₄; the seco series of pyridine bases, represented by tortuosamine; and the seco analog of the mesembrine-type represented by tortuosamine; and the seco analog of the mesembrine-type represented by dehydrojoubertiamine.     

Photoaffinity labeling of bovine rhodopsin

Photoaffinity labeled (3-diazoacetoxy)-9-cis-retinal (1) and (9-methylenediazoacetoxy)-9-cis-retinal (20) were synthesized and bound to absorption maxima at 465 and 460 nm respectively. Binding studies established that synthetic retinals 1 and 2 bind to the natural binding site and that the integrity of the diazoacetoxy photoaffinity label is preserved in the process. Incorporation of 3-(O¹⁴COCHN₂)-labeled 9-cis retinal could be conveniently carried out in high yield using apomembrane solubilized in CHAPS as detergent to afford the pigment analog in a pure form. Photolysis of the diazoacetoxy group within the binding site led to 15–20%, crosslinking of rhodopsin as estimated by using radiocarbon containing labeled retinal 1 thus showing that this synthetic retinal is suitable for photoaffinity labeling of the active site in rhodopsin. Subsequent experiments to establish the site(s) of crosslinking by sequencing studies will then contribute to our knowledge of the structure of rhodopsin.