Name: synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants
Uploaded: 2017-03-25T13:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants at JoVE.com

This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.

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All authors declare no competing financial interests in this manuscript.

في الجزء الأول من هذا البروتوكول، وصفنا توليفة معدلة من 1،2-azaborines على أساس أساليب ذكرت سابقا 12 و 13. وقد استخدم Triallylborane 22 كبديل للطرق باستخدام allyltriphenyl القصدير أو البوتاسيوم allyltrifluoroborate لإعداد N -allyl- N -TBS- ب -allyl ن?...

وضعت البورون النيتروجين التي تحتوي على حلقات غير متجانسة (أي 1،2-azaborines) في الآونة الأخيرة اهتماما كبيرا كما isosteres من أرينية. هذا isosterism يمكن أن يؤدي إلى تنويع الأشكال الهيكلية القائمة لتوسيع مساحة الكيميائية 2، 3، 4. Azaborines لها فائدة محتملة لتطبيقها في البحوث الطبية الحيوية 5، 6، 7، 8، خصوصا في مجال الكيمياء الطبية التي الكيميائيين إجراء توليف مكتبات هيكليا والجزيئات ذات الصلة وظيفيا. إلى حد كبير، ومع ذلك، في حين أن هناك العديد من طرق اصطناعية متطورة إلى الجزيئات التي تحتوي على دآرين المتاحة، تم الإبلاغ سوى عدد محدود من الطرق لتركيب azaborines 9، 10، </sتصل&gt; 11، 12، 13. ويرجع ذلك أساسا إلى عدد محدود من الخيارات لمصدر البورون والجوي وطبيعة رطوبة الحساسة من جزيء في مرحلة مبكرة من تسلسل الاصطناعية. في الجزء الأول من هذه المقالة، فإننا سوف تصف توليفة نطاق متعدد غرام من N -TBS- ب -Cl-1،2-azaborine (3) باستخدام تقنيات خالية من الهواء القياسية. يخدم هذا المركب كمادة وسيطة تنوعا التي يمكن زيادة functionalized لهيكليا أكثر تعقيدا الجزيئات 14 و 15. بدءا من 3، وسيتم وصف التوليف وتنقية N -H- ب -ethyl-1،2-azaborine (5) لاستخدامها في دراسات ملزمة البروتين. نظرا لتقلب 5، عزلتها فعالة تتطلب مراقبة دقيقة من درجة حرارة التفاعل، والوقت، وشعبة نظماستنتاج الظروف. في الجزء الثاني، وبروتوكولات للتعبير البروتين وعزل المسوخ T4 الليزوزيم (L99A وL99A / M102Q) 17، 18، 19، وستقدم 20، تليها بلورة البروتين وإعداد المجمعات الكريستال البروتين يجند. وقد تم اختيار المسوخ T4 الليزوزيم L99A وL99A / M102Q كما نظم نموذج البيولوجية لدراسة قدرة الرابطة الهيدروجينية من NH تحتوي على جزيئات azaborine 17. استخدام معيار بروتوكول البيولوجيا الجزيئية، ويتم التعبير عن هذا البروتين في سلالة كولاي RR1 والتي يسببها مع الآيزوبروبيل-β-D-1-thiogalactopyranoside (IPTG). ويتم تنقية البروتين خارج باستخدام التبادل الأيوني العمود اللوني. يتم تنفيذ البروتين تبلور مع الحل عالية التركيز النقي البروتين (&gt; 95٪ نقاء من قبل الكهربائي للهلام) باستخدام شنقاإسقاط طريقة نشر بخار. ونظرا لحساسية بروابط هذه الدراسة إلى الأكسجين، يتم إعداد المجمعات البروتين يجند في ظل ظروف خالية من الهواء.

<table><tbody><tr><td>Tetrahydrofuran (THF), inhibitor-free, for HPLC, &amp;ge;99.9%</td><td>Sigma Aldrich</td><td>34865</td><td></td></tr><tr><td>Diethyl ether (Et&lt;sub&gt;2&lt;/sub&gt;O), for HPLC, &amp;ge;99.9%, inhibitor-free</td><td>Sigma Aldrich</td><td>309966</td><td></td></tr><tr><td>Methylene chloride&amp;nbsp; (CH&lt;sub&gt;2&lt;/sub&gt;Cl&lt;sub&gt;2&lt;/sub&gt;), (Stabilized/Certified ACS)</td><td>Fisher</td><td>D37-20</td><td></td></tr><tr><td>Toluene</td><td>Fisher</td><td>T290-4</td><td></td></tr><tr><td>Pentane, HPLC</td><td>Fisher</td><td>P399-4</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A21-4</td><td></td></tr><tr><td>Calcium hydride (CaH&lt;sub&gt;2&lt;/sub&gt;), reagent grade, 95%</td><td>Sigma Aldrich</td><td>208027</td><td>Pyrophoric</td></tr><tr><td>Palladium on activated carbon (Pd/C), 10 wt% Pd</td><td>Strem</td><td>46-1900</td><td></td></tr><tr><td>1.0 M Boron trichloride solution in hexane</td><td>Sigma Aldrich</td><td>211249</td><td>Highly toxic/Pyrophoric</td></tr><tr><td>Triethylamine, &amp;ge;99.5%</td><td>Sigma Aldrich</td><td>471283</td><td></td></tr><tr><td>Grubbs 1&lt;sup&gt;st&lt;/sup&gt; generation catalyst&amp;nbsp;</td><td>materia</td><td>C823</td><td></td></tr><tr><td>Acetamide</td><td>Sigma Aldrich</td><td>A0500</td><td></td></tr><tr><td>n-Butanol, anhydrous, 99.8%</td><td>Sigma Aldrich</td><td>281549</td><td></td></tr><tr><td>Ethyllithium solution, 0.5&amp;nbsp;M in benzene/cyclohexane</td><td>Sigma Aldrich</td><td>561452</td><td>Highly toxic/ Pyrophoric</td></tr><tr><td>HCl solution, 2.0 M in Et&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>455180</td><td></td></tr><tr><td>2-Methylbutane, anhydrous, &amp;ge;99%</td><td>Sigma Aldrich</td><td>277258</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;, (Migula) Castellani and Chalmers (ATCC&amp;reg; 31343&amp;trade;)</td><td>ATCC</td><td>31343</td><td></td></tr><tr><td>T4 lysozyme WT* (L99A)</td><td>Addgene</td><td>18476</td><td></td></tr><tr><td>T4 lysozyme mutant (S38D L99A M102Q N144D)</td><td>Addgene</td><td>18477</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A0166</td><td></td></tr><tr><td>isopropyl-&amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Invitrogen</td><td>AM9464</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous</td><td>Fisher</td><td>BP329</td><td></td></tr><tr><td>Sodium Phosphate dibasic anhydrous</td><td>Fisher</td><td>BP332</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>S642212&amp;nbsp;</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Fisher</td><td>BP118</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M4880</td><td>Corrosive</td></tr><tr><td>Thermo scientific pierce DNaseI</td><td>Fisher</td><td>PI-90083</td><td></td></tr><tr><td>GE Healthcare&amp;nbsp;Sepharose Fast Flow Cation Exchange Media</td><td>Fisher</td><td>45-002-931</td><td></td></tr><tr><td>Tris-base</td><td>Fisher</td><td>BP152-500&amp;nbsp;</td><td></td></tr><tr><td>Sodium azide</td><td>TCI</td><td>S0489</td><td>Highly toxic</td></tr><tr><td>2-Mercaptoethanol</td><td>Fisher</td><td>ICN806443&amp;nbsp;</td><td></td></tr><tr><td>Sartorius&amp;nbsp;Vivaspin 20 Centrifugal Concentrators</td><td>Fisher</td><td>14-558-501</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5379</td><td></td></tr><tr><td>2-Hydroxyethyl disulfide</td><td>Sigma Aldrich</td><td>380474</td><td></td></tr><tr><td>N-paratone&amp;nbsp;</td><td>Hampton Research</td><td>HR2-643</td><td></td></tr><tr><td>4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO&amp;nbsp;</td><td>Fisher</td><td>08-667E</td><td></td></tr><tr><td>&amp;nbsp;CryoLoop</td><td>Hampton Research</td><td></td><td>cryogenic tubing shaped into a loop</td></tr><tr><td>CryoTong</td><td>Thermo Fisher</td><td></td><td>cryogenic tong</td></tr><tr><td>Coot</td><td></td><td></td><td>Electron density images are generated from the software</td></tr></tbody></table>

synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

ويظهر الطريق الاصطناعية التخطيطي ل1،2-azaborines في الشكل 1. ينطبق هذا البروتوكول لتخليق خمسة مختلفة البورون النيتروجين التي تحتوي على جزيئات. يمثل الرقم 2 11 ب NMR الأطياف قياسها أثناء الخطوة 1.3 لمراقبة تشكيل المنتج المطلوب (3).</s...

Watch this Scientific Journal Video about Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants at JoVE.com

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

توليف 1،2-Azaborines وإعداد مجمعات البروتين مع T4 الليزوزيم المسوخ

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

synthesis-12-azaborines-preparation-their-protein-complexes-with-t4

Universidad de Cartagena UDEC

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Biochemistry

7.5K Views.  Boston College. A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

Video: Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

السريع خطوة واحدة الأنزيمية توليف وجميع المائيه تنقية طرهالوز النظائر

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

التوليف وتنقية Iodoaziridines إشراك الكمي التحديد الأمثل للقرطاسية المرحلة اللوني لل

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Chemistry

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

Chemoselective تعديل السطوح الفيروسية عن طريق الكيمياء نقرة Bioorthogonal

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Immunology and Infection

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

إعداد أيونات أزيريدينيوم تربين مستقرة وعلى فتح الحلقة لتوليف أزاهيتيروسيكليس

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1 بوساطة التوليف الأنزيمي وتجميد البروتين المبلمر للمطاطي للقوة أحادية الجزيء

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

استخدام تحسين الفلورية المستحث بالبروتين الذي تم حله زمنيا لتحديد التركيبات المحلية المستقرة α-Synuclein Monomer في كل مرة

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very difficult and laborious to make aziridines in optically pure forms on a large scale to apply asymmetric synthesis of aza compounds. Fortunately, we successfully achieved both enantiomers (2R)- and (2S)-aziridine-2-carboxylates with the electron-donating &#945;-methylbenzyl group at the ring nitrogen as non-activated aziridines. These starting aziridines have two distinct functional groups-highly reactive three-membered ring and versatile carboxylate. They are applicable in ring-opening or ring-transformation with aziridine and in functional group transformation to others from carboxylate. Both of these enantiomers were utilized in the preparation of biologically important amino acyclic and/or aza-heterocyclic compounds in an asymmetric manner. Specifically, this report describes the first expedient asymmetric synthesis of both enantiomers of 5, 6-dihydrouracil-type marine natural products biemamide B and D as potential TGF-&#946; inhibitors. This synthesis consisted of regio- and the stereoselective ring-opening reaction of aziridine-2-carboxylate and subsequent formation of 4-aminoteterahydropyrimidine-2,4-dione. One more example in this protocol dealt a highly stereoselective Mukaiyama reaction of aziridine-2-carboxylate and silyl enol ether, following intramolecular aziridine ring-opening to provide easy and facile access to (-)-epiallo-isomuscarine.

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

In this study, we prepare both enantiomers of aziridine-2-carboxylate, which are used in the asymmetric synthesis of alkaloids, including biemamide B and D, and (-)-epiallo-isomuscarine.

تحضير الأزيريدين غير المنشط Enantiopure وتخليق البيماميد B و D و epiallo-isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very ...

Preparation of Contiguous Bisaziridines for Regioselective Ring-Opening Reactions

Aziridines, a class of reactive organic molecules containing a three-membered ring, are important synthons for the synthesis of a large variety of functionalized nitrogen-containing target compounds through the regiocontrolled ring-opening of C-substituted aziridines. Despite the tremendous progress in aziridine synthesis over the past decade, accessing contiguous bisaziridines efficiently remains difficult. Therefore, we were interested in synthesizing contiguous bisaziridines bearing an electronically diverse set of N-substituents beyond the single aziridine backbone for regioselective ring-opening reactions with diverse nucleophiles. In this study, chiral contiguous bisaziridines were prepared by organocatalytic asymmetric aziridination of chiral (E)-3-((S)-1-((R)-1-phenylethyl)aziridin-2-yl)acrylaldehyde with N-Ts-O-tosyl or N-Boc-O-tosyl hydroxylamine as the nitrogen source in the presence of (2S)-[diphenyl(trimethylsilyloxy)methyl]pyrrolidine as a chiral organocatalyst. Also demonstrated here are representative examples of regioselective ring-opening reactions of contiguous bisaziridines with a variety of nucleophiles such as sulfur, nitrogen, carbon, and oxygen, and the application of contiguous bisaziridines to the synthesis of multi-substituted chiral pyrrolidines by Pd-catalyzed hydrogenation.

Contiguous bisaziridines containing non-activated and activated aziridines were synthesized by asymmetric organocatalytic aziridinations and then subjected to chemoselective ring-opening reactions under acidic or basic conditions. The non-activated aziridine ring opens with less reactive nucleophiles under acidic conditions, whereas the activated aziridine ring opens with more reactive nucleophiles under basic conditions.

تحضير البيسازيريدين المتجاورين لتفاعلات فتح الحلقة الانتقائية

Aziridines, a class of reactive organic molecules containing a three-membered ring, are important synthons for the synthesis of a large variety of ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

تصور المقاومة البكتيرية باستخدام تحقيقات المضادات الحيوية الفلورية

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

كشف اللونية من البكتيريا عن طريق محك الاختبار

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...