Bu çalışma ABD Enerji Bakanlığı (DE-FG02-09ER09-08 ve DE-PS02-09ER09-18), UCLA Jonsson Kapsamlı Kanser Merkezi, Üniversite Sanayi İşbirliği Araştırma Programı (UC Discovery Grant bio07 tarafından desteklenmiştir. -10.665). Biz F-18 radyoizotop ve pek çok anlayışlı tartışmalar sağlamak için UCLA Biyomedikal Siklotron Tesisleri&#39;nde Dr Nagichettiar Satyamurthy ve kurmayları teşekkür ederiz. Biz Dr teşekkür ederim. Michael Collins, Greg Leblanc Joseph Lambert ve teknik danışmanlık ve destek için CEM Keller Barnhardt. Biz Dirk Williams, Darin Williams, Dr teşekkür ederim. Joseph Hong Dun Lin, tasarım ve işleme parçaları için Michael van Dam CEM mikrodalga reaktör değiştirmek ve SPE arıtma modülleri için.

1. İlk hazırlıkları <ol><li> V-flakon (5 ml) RV1 (bar karıştırarak), mikrodalga sentezini gerçekleştirmek için ana reaksiyon kabı olarak kullanılır. (Bkz. Şekil 2) bağlayan yedi giriş / çıkış portları ile bir PEEK adaptöre bağlı ve mikrodalga boşluğunun içine konur. RV2 ham toplamak için SPE kartuşu (I) bağlı [18 F] SFB. Rv3 final [18 F] SFB çözüm toplamak için SPE kartuşu (II) bağlıdır. Özellikle Biyomoleküllerin downstream Radyoaktif, PBS yeniden oluşturmak önce ilgili çözüm konsantre bir sıcak su banyosu (40 ° C) yerleştirilebilir. </li><li> Çözümü,% 5 sulu AcOH (8 ml) A rezervuar MeCN (2 ml), doldurun MeCN / H 2 O [01:04 (v v) 6 mL:] SFB ham [18] toplama için kurulum sırasıyla B ve C. Sonra,% 5 sulu AcOH (10 ml) yıkama takip etanol (10 ml) ile SPE kartuş (I) (polistiren, Merck LiCholut TR), etkinleştirin. </li><li> Saflaştırılmış toplamak için Kurulum [18 F] SFB: hazırlayın rezervuar D ve E, H 2 O 10 ml ve 3 ml dietil eter sırasıyla ile doldurulur . Yukarıda belirtilen aynı usulle ikinci SPE kartuşu (II) (polistiren, Merck LiCholut TR) devreye girer. </li><li> Başlangıç ​​ön şartlandırma için HPLC sütun [ters fazlı yarı hazırlık sütun:; HPLC (3 ml / dak akış hızı MeCN / H 2 O, 1:1 (v / v)% 0.2 TFA içeren elüsyon tampon) (Luna, 5 mikron C18 (2) 100 Å, 250 x 10 mm), Phenomenex, Torrance, CA, USA]. </li></ol> 2. Kurutulmuş [yani taşıyıcı eklendi (NCA)] [18 F] florür hazırlanması <ol><li> [18 F] [18 O] H 2 O (100 mcL) florür çözüm Kryptofix 222 (20 mg), 1M sulu K 2 CO 3 (26 mcL) ve MeCN (0.8 ml) karışımı bir Eppendorf eklendi tüp. Tüm çözüm sonra giriş hattı 1 ile RV1 aktarmadan önce iyice karıştırılır. [18 F] florür çözüm de, flor-18 yakalamak için anyonik değişim kartuşu (Waters örneğin QMA ışık Sep-Pak) üzerinden geçti ve sonra MeCN K 2 CO 3 ve Kryptofix karışımı ile yıkandı, olabilir . </li><li> [Vakum altında] RV1 kalan su çıkarmak için Kuruma dizisi (20W, 3 dk) Mikrodalga kontrol programı altında çalıştırın. , Sistem sıcaklığı 50 olarak soğuma sonra ° C, ek MeCN (1.0 mL) reaktör içine tanıtıldı ve sıra bir kez tekrarlanır. </li></ol> 3. Sentezi etil 4 - [18 F] fluorobenzoate <ol><li> Etil 4 içeren bir DMSO solüsyonu (0.4 ml) - (N, N, N-trimethylammonium) benzoat triflate (1.5 mg) giriş hattı 2 üzerinden RV1 içine eklendi. </li><li> Mikrodalga kontrol programı kapsamında karıştırma, gemi soğutma ve tüm vanaları ile Etiketleme dizisi (50W, 1 dak) Yürütme etil 4 göze kapalı - [18 F] fluorobenzoate ([18] 2). </li></ol> 4. Sentez potasyum 4 - [18 F] fluorobenzoate <ol><li> KOtBu (13 mg) içeren bir DMSO solüsyonu (0.5 mL) giriş hattı 3 üzerinden RV1 içine eklendi. </li><li> Mikrodalga kontrol programı kapsamında karıştırma, gemi soğutma ve tüm vanaları ile korumanızı programı (40 W, 1 dak.) Yürütme 4 göze kapalı - [18 F] fluorobenzoate tuz ([18 F] 3). </li></ol> 5. Ham [18 F] SFB Sentezi <ol><li> TSTU (30 mg) içeren asetonitril solüsyonu (2.5 mL) RV1 giriş hattını 6 ile eklendi. TSTU, nem ve ışığa duyarlı. Bu, küçük bir şişenin içine aliquoted ve 4 ° C, alüminyum folyo ile kaplı kapalı bir kapta muhafaza edilmelidir. </li><li> Karıştırma, gemi soğutma ve tüm vanaları ile Mikrodalga kontrol programı kapsamında Kaplin dizisi (30W, 2 dak) Yürütme [18 F] SFB ham göze kapalı. </li></ol> 6. SPE-saflaştırılmış [18 F] SFB Hazırlık <ol><li> Reaksiyon karışımı nötralize etmek için giriş hattını 7 ile% 5 sulu AcOH (1.0 mL) RV1 için eklendi. Bu çözüm daha sonra 8 ml% 5 sulu AcOH (Şekil 2) içeren flakon B aktarılır. </li><li> Tuzak SPE kartuşu (I) aracılığıyla Pass seyreltilmiş reaksiyon karışımı ham [18 F] SFB kullanarak azot (10 psi). </li><li> Rezervuar A.: MeCN ve H 2 O [10 ml, 1:4 (v v)] karışımı ile YIKAMA SPE kartuşu (I) </li><li> [18 F] SFB rezervuar C. MeCN (2 mL) kullanarak RV2 içine madde elde edilmiştir </li></ol> 7. Ham Arıtma [18 F] SFB Radyo-HPLC ile <ol><li> Ya ham sulandırınız [18 F] SFB veya SPE-saflaştırılmış [18 F] RV2 H 2 O (2 ml) ile SFB ve HPLC döngü (5 ml) karışımı aktarın. Çözüm radyo-HPLC enjekte edildi [MeCN / H 2 O,% 0.2 TFA içeren 1:1 (v / v); debisi: 3 ml / dk]. </li><li> Fraksiyonu içeren saflaştırılmış [18 F] SFB (retansiyonu tim toplayıne: 8-10 dakika) D (10 ml H 2 O ile önceden doldurulmuş) (Şekil 2) flakon içine. Kritik adım: Doğru şekilde gerçekleştirilmemesi durumunda, burada toplanan kesir hacmi 4-5 ml olmalıdır. </li><li> Tuzak SPE kartuşu (II) aracılığıyla Pass seyreltilmiş reaksiyon karışımı saflaştırılmış [18 F] SFB kullanarak azot (10 psi). Kartuşu 2-3 dakika azot akışı ile kurulayın. </li><li> [18 F] SFB rezervuar E. dietileter (3 mL) kullanarak rv3 içine madde elde edilmiştir. </li><li> Bir su banyosu (40 ° C) azot gazı kullanarak yumuşak bir akışı (10 psi), kuruluk için rv3 çözücü buharlaşır. Son kurutulmuş [18 F] SFB alt uygulama için PBS içine sulandırılmış olabilir. </li></ol> 8. Temsilcisi Sonuçlar: Biz hızlı bir basitleştirilmiş, sentezlemek için bir pot yöntemi geliştirdi [18 F] SFB her bir kimyasal / radyokimyasal dönüşüm sırasında susuz koşulları ve mikrodalga ısıtma altında deprotection strateji kullanarak. Şekil 1 bizim radiosynthesis detayları sunar. Nihai ürün kimliği olmayan bir radyoaktif SFB referans HPLC tutma süresi karşılaştırma tarafından da teyit edildi. Saflaştırılmış [18 F] SFB radyokimyasal ve kimyasal saflık belirlemek için radyo-TLC ve HPLC ile analiz edildi. RCY [18 F] SFB radyokimyasal saflık yüksek (&gt;% 99) ve iyi kimyasal saflığı (HPLC profili, UV izleme bakınız, Şekil 3, HPLC arıtma (n&gt; 30) sonra 60 dakika içinde 35 ±% 5 .) Ca spesifik aktivite oldu. 67-330 GBq / mol (1,8-9,0 Ci / mmol), başlangıç ​​radyoaktivite bağlı. <img src="/files/ftp_upload/2755/2755fig1.jpg" alt="Şekil 1" /> Şekil 1. Mikrodalga destekli tek pota radiosynthesis [18] SFB. Radiofluorination etil 4 - (N, N, N-trimethylammonium) benzoat triflate (1) Öncelikle, mikrodalga ısıtma (50 W, 1 dak.) Varlığı altında yapıldı ⊃ 2.2.2 [K] [18 F] F - etil 4 göze dimethylsülfoksit (DMSO) karmaşık - [18 F] fluorobenzoate ([18] 2). Arıtma olmadan, potasyum tert-butoxide (t BuOK) bir DMSO solüsyonu eklenir ve reaksiyon kabı susuz deprotection tamamlamak için ışınlanmış mikrodalga (40 W, 1 dak.) Oldu. Son dönüşüm [18 F] içine 3 [18 F] SFB O-(N-succinimidyl) kullanılarak elde edildi - N, N, N, N-tetramethyluronium tetrafloroborat (TSTU) aktivasyonu. Asetonitril&#39;de TSTU 4 içeren reaksiyon karışımı eklendi - [18 F] fluorobenzoate ([18 F] 3) tuz, bu son sentetik adım vermiştir ham [18 F] SFB ısıttıktan sonra (30 W, 2 dak). <img src="/files/ftp_upload/2755/2755fig2.jpg" alt="Şekil 2" /> Şekil 2 [18 F] SFB sentezi tek pot mikrodalga destekli kurulum şematik diyagramı. <img src="/files/ftp_upload/2755/2755fig3.jpg" alt="Şekil 3" /> Şekil 3 final Radyo-HPL kromatogramlar [18] SFB. Üst: 254 nm UV sinyal, alt: Radyoaktif sinyal içerlek: 254 nm (x 33.3) UV sinyal. <img src="/files/ftp_upload/2755/2755table1.jpg" alt="Tablo 1" /> Tablo 1: Özet alkil 4 kullanarak literatürde bildirilmiştir [18 F] SFB radiosyntheses - (trimethylammonium) öncüleri olarak benzoat triflate.

Bu yöntem, ABD patent başvurusu için teslim edilmiştir.

Bu basitleştirilmiş üç adım, bir pot 18 F-asilleşme reaktif radiosynthesis [18 F] SFB susuz kimya dayalı geliştirilmiştir. Bu süreç mükemmel tekrarlanabilirlik ve üretimi için güvenilir olabilir [18 F] aşağıdaki gibi açıklanan iki anahtar değişiklikler nedeniyle otomatik Radyokimya modülleri, SFB: 1. Biz yaygın sulu bazik veya asidik bir çözüm yerine, susuz KOtBu / DMSO sistemi bir deprotection / sabunlaşma adım kullanmaktayız. Susuz deprotection strateji ar...

<table border="1"><tbody><tr><td> Reaktifin Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> asetik asit sulu çözeltisi (% 5, v / v) </td><td> Balıkçı </td><td> A38-500 </td><td> Laboratuarda hazırlanan </td></tr><tr><td> Asetonitril </td><td> Sigma-Aldrich </td><td> 75-05-8 </td><td></td></tr><tr><td> Dietil eter </td><td> Sigma-Aldrich </td><td> 14775 </td><td></td></tr><tr><td> Dimetil sülfoksit (DMSO) </td><td> Sigma-Aldrich </td><td> 472301 </td><td></td></tr><tr><td> Etil 4 - (N, N, N-trimethylammonium) benzoat triflate </td><td> Laboratuarda hazırlanan </td><td></td><td></td></tr><tr><td> 4,7,13,16,21,24-Hexaoxa 1 ,10-diazobisiklo [8.8.8] hexacosane (K222) </td><td> Sigma-Aldrich </td><td> 29,111-0 </td><td></td></tr><tr><td> O - (N-succinimidyl) - N, N, N &#39;, N&#39; tetramethyluronium tetrafloroborat (TSTU) </td><td> Sigma-Aldrich </td><td> 105832-38-0 </td><td></td></tr><tr><td> Potasyum karbonat (1M) sulu çözeltisi </td><td> Sigma-Aldrich </td><td> 209619 </td><td> Laboratuarda hazırlanan </td></tr><tr><td> Potasyum tert-butoxide </td><td> Sigma-Aldrich </td><td> 156671 </td><td></td></tr></tbody></table>

microwave-assisted one-pot synthesis of n-succinimidyl-4-[18f]fluorobenzoate ([18f]sfb)

Biyomoleküller, peptidler, proteinler 1-9, 10,11 ve antikorlar ve bunların mühendislik parçaları da dahil olmak üzere, 12-14, potansiyel terapötikler ve moleküler görüntüleme ajanları olarak önem kazanıyor. Özellikle, pozitron yayan radyoizotoplar (örneğin, Cu-64, Ga-68, F-18) ile etiketlenmiş, birçok fizyolojik ve patolojik süreçler hedeflenen görüntüleme için problar olarak kullanılmalıdır. 15-18 Bu nedenle, önemli çaba 18 F-etiketli biyomoleküllerin sentezi ve keşif için ayrılmıştır. Peptidlerin doğrudan 18 F-etiketleme zarif örnekler, 19-22 sert reaksiyon şartları olmasına rağmen (yani, organik çözücü, aşırı pH, yüksek sıcaklık) doğrudan radiofluorination ilişkili genellikle kırılgan protein örnekleri ile uyumsuz. Bugüne kadar, bu nedenle, biyomoleküllerin içine radyoaktif protez grupların birleşmesiyle tercih edilen yöntem olmaya devam etmektedir. 23,24 N Succinimidyl-4-[18 F] fluorobenzoate 18 F-etiketleme ([18 F] SFB), 25-37 biyomoleküllerin temel amino grupları ile reaksiyona girerek bir Bolton-Hunter tipi reaktif, çok yönlü bir protez grup , biyolojik varlıklar açısından, in vivo istikrar ve yüksek Radyoaktif verimi belirgin çok geniş bir yelpaze . F ile etiketleme sonra [18] SFB [18 F] fluorobenzoylated biyomoleküllerin in vivo görüntüleme çalışmaları için potansiyel PET tracer olarak araştırılmalıdır olabilir. 1. En [18 F] edebiyatlarında açıklanan SFB radiosyntheses iki, hatta üç reaktörler gerektirir ve iki katı faz ekstraksiyon (SPE) ya da yüksek performanslı sıvı kromatografi (HPLC) kullanarak birden fazla arındırmalardan. Bu tür uzun süreçler, rutin üretim ve biyomoleküllerin Radyoaktif yaygın uygulamalar engel. Rağmen, birkaç modül destekli [18 F] SFB sentezlere bildirilmiştir, 29-32, 41-42, onlar çok pahalı piyasada bulunan Radyokimya kutular (Tablo 1) kullanılarak karmaşık ve uzun prosedürler dayanmaktadır . Bu nedenle, radiosynthesis daha basitleştirilmesi [18 F] SFB düşük maliyetli kurulum kullanarak otomatik bir süreç adaptasyonu için çok yararlı olacaktır. Burada, biz kısa bir rapor hazırlanması [18 F] basitleştirilmiş bir tek pot mikrodalga yardımlı sentez (Şekil 1) dayalı SFB. Bizim yaklaşımımız adımları ya da herhangi bir sulu reaktifler arasındaki arıtma gerektirmez. Buna ek olarak, birkaç PET tracer sentezlere kullanılmakta olan mikrodalga ışınlama, 38-41 RCYs yüksek ve karşılık gelen termal reaksiyonlar daha iyi seçicilik verir ya da daha kısa tepki süreleri benzer verim sağlar. 38 En önemlisi, biyomoleküllerin etiketleme kaydettiğiniz zaman, sonraki bioconjugation veya PET adım için yönlendirilir olabilir bizim geliştirilmiş 28,43 yenilik [18 F] SFB sentezi iki yönlüdür: (1) susuz deprotection stratejisi arasında orta (ler) hiçbir arıtma gerektirir. her adımda ve (2) [18 F] SFB mikrodalga destekli radyokimyasal dönüşümler, hızlı, güvenilir üretimini sağlayacak .

Watch this Scientific Journal Video about Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB at JoVE.com

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

Bir uyduruk bir pot sentez N succinimidyl-4-[<sup&gt; 18</sup&gt; F] fluorobenzoate ([<sup&gt; 18</sup&gt; F] SFB) susuz, üç adım radyokimyasal süreci dayalı geliştirilmiştir. Mikrodalga ısıtma kullanarak, tüm işlemleri en az 30 dakika veya daha fazla arıtma preparatif HPLC ile 60 dakika içinde tamamlanabilir. Çürüme düzeltilmiş radyokimyasal verimleri (RCYs) 35-5 (% n&gt; 30).

Mikrodalga-destekli-pot Bir Sentez N Succinimidyl-4-[ 18 F] fluorobenzoate ([ 18 F] SFB)

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

microwave-assisted-one-pot-synthesis-n-succinimidyl-4

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Biology

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

12.9K Görüntüleme.  University of California at Los Angeles. Bir uyduruk bir pot sentez N succinimidyl-4-[ 18 F] fluorobenzoate ([ 18 F] SFB) susuz, üç adım radyokimyasal süreci dayalı geliştirilmiştir. Mikrodalga ısıtma kullanarak, tüm işlemleri en az 30 dakika veya daha fazla arıtma preparatif HPLC ile 60 dakika içinde tamamlanabilir. Çürüme düzeltilmiş radyokimyasal verimleri (RCYs) 35-5 (% n> 30).

Video: Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

Paralel Sentezi ve Otomatik Liquid Handling kullanma Ugi Reaksiyon optimizasyonu

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

Biyoloji

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Biyolojik Sistemleri Oksijen Görüntüleme Fosforlu Nanoprobes sentezi ve Kalibrasyon

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Bir sentezi<em&gt; In vivo</em&gt; MR-saptanabilir Apoptoz Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Solid Phase Synthesis of a Functionalized Bis-Peptide Using &quot;Safety Catch&quot; Methodology

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

Bir Fonksiyonlu Bis-Peptid Katı Faz Sentezi Metodolojisi &quot;Güvenlik&quot; Catch Kullanımı

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

In vitro sentezi insan hücrelerinde protein sentezlenmesinin uyarımından için mRNA&#39;nın Değiştirilmiş

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Işığa siyanin boyası kullanarak Dalgaboyu değiştiren DNA Hibridizasyon Sondalar Sentezi

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

Yüksek basınç dondurucu, mikrodalga destekli kontrast geliştirme ve en az reçine birim görüntüleme için katıştırma tarafından biyolojik örnek hazırlama

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Caenorhabditis elegans de novo Protein Sentez Oranlarının Değerlendirilmesi

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...