This work was supported by the Lehigh University Start-up Package. We thank Dr. Walter Fast for providing the GST-PAD4 plasmid for protein expression.

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The authors have nothing to disclose.

Herein, a fluorescence based assay was successfully developed that monitors the activity of PAD4. The assay has proven to be incredibly robust in a number of high-throughput conditions and is also compatible with whole cell lysates37.
The affinity between the histone proteins and the DNA strands surrounding it loosens due the neutralization of the positive charge on the arginine side chain upon citrullination. It was envisioned that the neutralization of the arginine side-chain coul...

يتم تعديل عدد كبير من البروتينات الثدييات بشكل كبير بفعل الإنزيمات التالية الحيوي من البروتينات التي الريبوسوم. ويمكن لهذه التعديلات بعد متعدية (PTMs) زيادة كبيرة في التنوع الوظيفي للبروتيوم عن طريق تغيير حجم، وتهمة، وهيكل، والدولة oligomerization (من بين الميزات الأخرى) البروتينات 1-3. ونتيجة لذلك، فإن التغيير في بنية البروتين يمكن أن يؤدي إلى عواقب فيزيولوجية، مثل تدهور البروتين، التمايز الخلوي، مما يشير، في تعديل التعبير الجيني، وتفاعلات البروتين البروتين. في حين أن هذه التعديلات هي السائدة في نسبة كبيرة من جميع البروتينات البشرية، ومحطة تنتهي في البروتينات هيستون الخضوع لعدد كبير بشكل غير عادي من التعديلات التساهمية 4. البروتينات هيستون هي عائلة من البروتينات الهيكلية التي تسهل التكثيف من الحمض النووي الجيني. وتجرى التعديلات التساهمية من ذيول هيستون غير منظم بها وينظمها دوري الدرجة الاولى الايطاليS من الانزيمات التي يمكن أن تحفز تعديل التساهمية المخلفات (الكتاب)، عكس نفس التعديلات (المحايات)، والتمييز بين التغييرات التي مطبوع على الذيول هيستون (القراء) 5-7. في الواقع، فإن معظم PTMs معروفة يمكن ملاحظتها ضمن هذه الشريحة قصيرة من هيستون بما مثيلة، الفسفرة، أستلة، sumoylation، ubiquitination، وcitrullination 8. Citrullination ينطوي على تحويل ببتيديل أرجينين إلى غير المشفرة ببتيديل ر RNA-سيترولين (الشكل 1A). البروتينات المسؤولة عن هذه السلسلة تحييد الجانب، هم أعضاء في PAD (ص eptide على rginine د eiminase) عائلة بروتين، وكلها تعتمد على الانزيمات الكالسيوم. 9،10. حتى الآن، وقد وصفت خمسة أفراد من عائلة PAD (PAD1، PAD2، PAD3، PAD4، وPAD6). يبدو أن كل عضو من أعضاء هذه الأسرة لاستهداف البروتينات الخلوية متميزة وأيضا يعرض أحاديكيو ملامح توزيع الأنسجة. PAD4 هو العضو الوحيد من هذه العائلة البروتين المعروف أن تكون مترجمة داخل النواة عبر تسلسل توطين النووية 11. تبعا لذلك، فقد تبين أن deiminate عددا من الأهداف النووية، بما في ذلك السلاسل الجانبية أرجينين على ذيول N-الطرفية من الهستونات H2A أرجينين بقايا 3 (H2R3)، H3 (H3R2، H3R17، وH3R26) وH4 (H4R3) 12، 13. في حين أن كل من isozymes PAD لها وظائف فسيولوجية معينة وحاسمة، تلقت PAD4 إيلاءها مزيدا من الاهتمام نظرا لدوره في عدد من العمليات الإنسانية في كل الخلايا المريضة والسليمة. في الآونة الأخيرة، وقد تبين PAD4 أن تكون عضوا في شبكة النسخي تعدد القدرات 14. وعرضت كل من مستويات التعبير PAD4 والنشاط لتكون مرتفعة خلال إعادة برمجة وللدولة الارض الدول المحفزة في الفئران. عن طريق التحكم في تنظيم جينات الخلايا الجذعية، قد PAD4 الاحتفاظ دورا محوريا في كفاءة إعادة برمجة الخلايا. PAD4 كما تم المتورطين فيتشكيل الفخاخ خارج الخلية المتعادلة، التي تقوم عليها ملزمة من مسببات الأمراض تمكين إزالة النظام الخاص بهم. وhypercitrullination من البروتينات هيستون من قبل PAD4 يدفع decondensation من لونين، والتي هي بمثابة المادة الأساسية للتغليف من البكتيريا المسببة للأمراض من فخ خارج الخلية، وبالتالي درء العدوى البكتيرية 15،16. بالإضافة إلى ذلك، تم العثور PAD4 للعب دور نشط في عدد من الأمراض التي تصيب الإنسان. وقد تبين سابقا أن التعبير المنحرف من PAD4 يرتبط مع بداية وشدة التهاب المفاصل الروماتويدي، مرض الزهايمر، مرض باركنسون، والتصلب المتعدد 17. في الواقع، فإن وجود الأجسام المضادة بروتين مكافحة citrullinated هو واحد من المؤشرات الحيوية التشخيصية والنذير الأكثر موثوقية ونهائية من التهاب المفاصل الروماتويدي 18. وبالمثل، فقد لوحظ مؤخرا نشاط PAD4 dysregulated في عدد من السرطانات البشرية بما في ذلك المبيض والثدي والرئة، وسرطان المريء د 19-22. وقد تبين الصلة بين PAD4 والسرطان إلى أن توسطت من خلال الجين الورمي ELK1 أو عبر الورم البروتين p53 البروتين القامع 22،23، واقترح العمل السابق الذي PAD4 يمكن أن تكون مضادة للسرطان الهدف العلاجي رواية 17،24،25. كدليل على دراسة المبدأ، تم الكشف عن نضوب PAD4 عبر shRNA في القولون والمستقيم خط الخلايا السرطانية HCT116 أن تكون كافية لإحداث موت الخلايا المبرمج ودورة الخلية اعتقال 26. أدى ضعت مؤخرا لا رجعة فيه PAD4 المانع إلى انخفاض سبعين في المئة في كتلة الورم في الفئران (27). بشكل ملحوظ جدا، ويبدو PAD4 تثبيط لتكون بمثابة العلاج المستهدفة التي أسفرت عن مقتل الانتقائي للخلايا السرطانية في حين أن يجنب الخلايا untransformed. استخدام جزيئات صغيرة لإيقاف وظيفة PAD4 قد يثبت أنه استراتيجية جديدة وقوية لاستهداف الخلايا السرطانية أو لزيادة مبحث المعالجة الكيميائية السرطانية الموجودة 28. للأسف، صوبعد أن اكتشف خيمة PAD4 عكسها المانع. وقد تم تطوير عدد من مثبطات التساهمية باستخدام الكلور / مقبض imidine الفلوري الذي يحاكي الركيزة أرجينين 27،29،30 وأثبتت أنها أدوات عملية في فهم دور PAD4 في كل خلايا الدولة السليمة والمريضة. ومع ذلك، هذه الجزيئات تمنع كل من منصات نشطة مع قوة مماثلة. وبالتالي، فإن الحاجة لفحص سطحي التي تفيد عن نشاط PAD4 أمر بالغ الأهمية. حتى الآن، وقد وصفت PAD4 المقايسات التي تربط الإفراج عن الأمونيا من رد الفعل إلى قراءات اللونية 31، الاستفادة من التناظرية chloroamidine الركيزة fluorescently المسمى لمضان الاستقطاب فحص 32، تعتمد على رد فعل حمض بمساعدة بين الجليوآسال وسيترولين 33، و زوجان النشاط PAD4 إلى مضان dequenching خطوة 34. من هذه، وقد أثبتت هذه الاستراتيجية haloacetamidine معدل التساهمية فقط لتكون متوافقة مع scre عالية الإنتاجيةمنصات ملمسه 32،35،36. نحن تصف فحص سطحي مضان القائم الذي يقيس موثوق نشاط PAD4. الفحص، الذي يعرض نسبة قوية إشارة إلى الضجيج، وسرعة تحليلها، وقياس متانة، لديه القدرة على اكتشاف قوية وانتقائية حقا PAD4 المانع.

<table border="0" cellpadding="0" cellspacing="0" width="716">
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			<td height="46" style="height:46px;width:192px;">Name of the Material/Equipment</td>
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			<td height="66" style="height:66px;width:192px;">LB Broth, Miller (LURIA-BERTANI)</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:135px;">J106-2KG</td>
			<td style="width:267px;"><a href="http://www.amresco-inc.com/">http://www.amresco-inc.com</a></td>
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			<td height="63" style="height:63px;width:192px;">Ampicillin Trihydrate (Off-white Powder), Fisher BioReagents</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td>BP902-25&#160;</td>
			<td style="width:267px;"><a href="http://www.fishersci.com/">http://www.fishersci.com</a></td>
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			<td height="68" style="height:68px;width:192px;">Isopropylthiagalactonse (IPTG)</td>
			<td style="width:123px;">Life Technogolies</td>
			<td>15529-019</td>
			<td style="width:267px;"><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com</a></td>
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			<td height="62" style="height:62px;width:192px;">All Buffer Salts&#160;</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Can purchase from Fisher Scieintifc; VWR; Acros; Sigma-Aldrich; etc.</td>
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			<td height="51" style="height:51px;width:192px;">Protino Glutathione (GSH) agarose&#160;</td>
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			<td style="width:135px;">745500.10</td>
			<td style="width:267px;">Store at 4 &#176;C; http://www.mn-net.com/</td>
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			<td height="71" style="height:71px;width:192px;">Z-&#8203;Arg-&#8203;Arg-&#8203;7-&#8203;amido-&#8203;4-&#8203;methylcoumarin hydrochloride (Zcoum)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>C5429</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
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			<td height="58" style="height:58px;width:192px;">Chloroamidine&#160;</td>
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			<td style="width:135px;">10599</td>
			<td style="width:267px;">Store at -20 &#176;C; https://www.caymanchem.com/app/template/Home.vm</td>
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			<td height="66" style="height:66px;width:192px;">Tris(2-&#8203;carboxyethyl)&#8203;phosphine hydrochloride (TCEP HCl)</td>
			<td style="width:123px;">Thermo Scientifc</td>
			<td style="width:135px;">20490</td>
			<td style="width:267px;"><a href="http://www.piercenet.com/">http://www.piercenet.com</a></td>
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			<td height="50" style="height:50px;width:192px;">Triton X-100</td>
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			<td style="width:135px;">X100-100ML</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
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		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Corning/Nunclon MicroWell plates (96 and 384)</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Purchase from Corning; Sigma-Aldrich</td>
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		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Tecan Infinite 200 / Tecan i-control microplate reader software</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.tecan.com/">www.tecan.com</a></td>
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			<td height="65" style="height:65px;width:192px;">Europium Ex. 340/40 Em. 475/15 filter</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.perkinelmer.com/">http://www.perkinelmer.com</a></td>
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</table>

fluorescence-based monitoring of pad4 activity via a pro-fluorescence substrate analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

في البداية، وقد تبين أن ZRcoum يمكن أن تقدم تقريرا عن نشاط PAD4 في شكل لوحة 96-جيدا. حضنت الآبار مع ZRcoum الركيزة وحضور / غياب PAD4. بعد فترة حضانة المرض من 45 دقيقة عند 37 درجة مئوية، وقد تم قياس مضان مع 340/40 - 475/15 نانومتر مرشح (الشكل 2A). كما هو متوقع، ظلت مس?...

Watch this Scientific Journal Video about Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog at JoVE.com

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

رصد القائم على مضان آخر PAD4 عبر برو-مضان الركيزة النظير

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

fluorescence-based-monitoring-pad4-activity-via-pro-fluorescence

Research

JoVE Journal

Chemistry

9.8K Views.  Lehigh University. PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Video: Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

تحديد الطائرات الملزمة للجليد من البروتينات المضادة للتجمد من قبل تقارب طائرة الجليد القائم على الفلورسينس

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

إعداد السيليكا Aerogel كتل عن طريق استخراج الطريقة السريعة فوق الحرجة

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

رصد في الوقت الحقيقي من ردود الفعل تنفيذها باستخدام معالجة التدفق المستمر: إعداد 3-Acetylcoumarin كمثال

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

قياس واحد من HDAC1 والنشاط HDAC6 في الحلة الخلايا باستخدام أوهبلك--مرض التصلب العصبي المتعدد

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

تصنيع واختبار أيروجيلس الحفاز أعدت عن طريق استخراج فوق الحرجة السريع

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

تركيب أسلاك الاتحاد الأفريقي الركازة زمنياً عبر إليه نمو نشط السطح

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

A Two-Step Protocol for Umpolung Functionalization of Ketones Via Enolonium Species

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. Recently, we have developed a two-step strategy for ketone enolate umpolung that has enabled the development of methods for chlorination, azidation, and amination using azoles. In addition, we have developed C-C bond&#8211;forming arylation and allylation reactions. At the heart of these methods is the preparation of the intermediate and highly reactive enolonium species prior to addition of a reactive nucleophile. This strategy is thus reminiscent of the preparation and use of metal enolates in classical synthetic chemistry. This strategy allows the use of nucleophiles that would otherwise be incompatible with the strongly oxidizing hypervalent iodine reagents. In this paper we present a detailed protocol for chlorination, azidation, N-heteroarylation, arylation, and allylation. The products include motifs prevalent in medicinally active products. This article will greatly assist others in using these methods.

A two step one-pot protocol for the umpolung of ketone enolates to enolonium species and addition of a nucleophile to the &#945;-position is described. Nucleophiles include chloride, azide, azoles, allyl-silanes, and aromatic compounds.

بروتوكول خطوتين الروغان أومبولونج الكيتونات عن طريق الأنواع انولونيوم

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

توليف استرات عبر أكثر اخضرارا الأسترة ستيجليتش في الاسيتو الانيتريل

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR القائمة على تحليل النشاط لتحديد تثبيط مركب, قيم IC50, نشاط Artifactual, ونشاط الخلية الكاملة من النوكليوسيد الريبوهيدرولاسيس

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.