This study was supported by the US Department of Energy (DE-FG02-09ER09-08 and DE-PS02-09ER09-18), the Jonsson Comprehensive Cancer Center at UCLA, and the Industry-University Cooperative Research Program (UC Discovery Grant, bio07-10665). We thank Dr. Nagichettiar Satyamurthy and staffs at the UCLA Biomedical Cyclotron Facility for providing the F-18 radioisotope and many insightful discussions. We thank Drs. Michael Collins, Greg Leblanc, Joseph Lambert, and Keller Barnhardt from CEM for their technical advice and support. We thank Dirk Williams, Darin Williams, Drs. Joseph Hong Dun Lin, and Michael van Dam for designing and machining parts to modify the CEM microwave reactor and for SPE purification modules.

1. Initial preparations
<ol>
<li>A V-vial (5-mL) RV1 (with stirring bar) is used as the main reaction vessel for performing microwave synthesis. It is connected to a PEEK adaptor with seven inlet/outlet ports connects and placed inside the microwave cavity (see Figure 2). RV2 is connected to SPE cartridge (I) to collect the crude [18F]SFB. RV3 is connected to SPE cartridge (II) for collecting the final [18F]SFB solution. It can be placed in a warm water bath (40&deg;C) to concentrate the corresponding solution before reconstituting in PBS buffer, especially for the downstream radiolabeling of biomolecules.</li>
<li>Setup for collecting crude [18F]SFB: Fill up MeCN/H2O [6 mL; 1:4 (v:v)] solution, 5% aqueous AcOH (8 mL), MeCN (2 mL) for reservoir A,B and C, respectively. Then activate a SPE cartridge (I) (polystyrene, Merck LiCholut EN) with ethanol (10 mL), followed by 5% aqueous AcOH (10 mL) washing.</li>
<li>Setup for collecting purified [18F]SFB: Prepare reservoir D and E filled up with 10 ml of H2O and 3 ml of diethyl ether respectively. The second SPE cartridge (II) (polystyrene, Merck LiCholut EN) is activated by the same procedure mentioned above.</li>
<li>Start the HPLC (elution buffer: MeCN/ H2O, 1:1 (v/v) containing 0.2% TFA; flow rate: 3 ml/min) for pre-conditioning the HPLC column [a reverse-phase semi-prep column (Luna, 5 &mu;m C18(2) 100 &Aring;, 250 x 10 mm), Phenomenex, Torrance, CA, USA].</li>
</ol>
2. Preparation of dried [i.e. non-carrier-added, (n.c.a)] [18F]fluoride
<ol>
<li>[18F]fluoride solution in [18O]H2O (100 &mu;L) was added to a mixture of Kryptofix 222 (20 mg), 1M aqueous K2CO3 (26 &mu;L) and MeCN (0.8 mL) in an Eppendorf tube. The entire solution is then mixed well before transferring to the RV1 via inlet line 1. The [18F]fluoride solution can be also passed through an anionic exchange cartridge (e.g. QMA-light Sep-Pak from Waters) to trap the fluoride-18 and then eluted out with a mixture of K2CO3 and Kryptofix in MeCN.</li>
<li>Execute the Drying sequence (20W, 3 min) under the Microwave control program to remove residual water in RV1 [under vacuum]. After the cooling down as system temperature is below 50&deg;C, additional MeCN (1.0 mL) was introduced into the reactor and the sequence is repeated once.</li>
</ol>
3. Synthesis of ethyl 4-[18F]fluorobenzoate
<ol>
<li>To a DMSO solution (0.4 mL) containing ethyl 4-(N,N,N-trimethylammonium)benzoate triflate (1.5 mg) was added into RV1 via inlet line 2.</li>
<li>Execute the Labeling sequence (50W, 1 min) under the Microwave control program with stirring, vessel cooling and all valves closed to afford ethyl 4-[18F]fluorobenzoate ([18F]2).</li>
</ol>
4. Synthesis of potassium 4-[18F]fluorobenzoate
<ol>
<li>To a DMSO solution (0.5 mL) containing KOtBu (13 mg) was added into RV1 via inlet line 3.</li>
<li>Execute Deprotect program (40 W, 1 min) under the Microwave control program with stirring, vessel cooling and all valves closed to afford the 4-[18F]fluorobenzoate salt ([18F]3).</li>
</ol>
5. Synthesis of crude [18F]SFB
<ol>
<li>To an acetonitrile solution (2.5 mL) containing TSTU (30 mg) was added to RV1 via inlet line 6. TSTU is moisture- and light-sensitive. It should be aliquoted into small vial and stored at 4&deg;C in a closed container covered by aluminum foil.</li>
<li>Execute Coupling sequence (30W, 2 min) under the Microwave control program with stirring, vessel cooling and all valves closed to afford the crude [18F]SFB.</li>
</ol>
6. The Preparation of SPE-purified [18F]SFB
<ol>
<li>5% aqueous AcOH (1.0 mL) was added to RV1 via inlet line 7 to neutralize the reaction mixture. The solution was then transferred into vial B containing 8 ml of 5% aqueous AcOH (Figure 2).</li>
<li>Pass the diluted reaction mixture through SPE cartridge (I) to trap crude [18F]SFB using nitrogen (10 psi).</li>
<li>WASH SPE cartridge (I) with a mixture of MeCN and H2O [10 mL, 1:4 (v:v)] from reservoir A.</li>
<li>[18F]SFB was eluted out into RV2 using MeCN (2 mL) from reservoir C.</li>
</ol>
7. Purification of Crude [18F]SFB with Radio-HPLC
<ol>
<li>Dilute either crude [18F]SFB or SPE-purified [18F]SFB with H2O (2 mL) in RV2 and transfer the mixture into the HPLC loop (5 mL). The the solution was injected into radio-HPLC [MeCN/ H2O, 1:1 (v/v) containing 0.2% TFA; flow rate: 3 mL/min]. </li>
<li>Collect the fraction containing purified [18F]SFB (retention time: 8-10 minutes) into the vial D (pre-filled with 10 ml of H2O) (Figure 2). Critical step: If performed correctly, the fraction volume collected here should be 4-5 ml.</li>
<li>Pass the diluted reaction mixture through SPE cartridge (II) to trap purified [18F]SFB using nitrogen (10 psi). Dry the cartridge with a stream of nitrogen for 2-3 minutes.</li>
<li>[18F]SFB was eluted out into RV3 using diethylether (3 mL) from reservoir E..</li>
<li>Evaporate the solvent in RV3 to dryness by a gentle stream of nitrogen gas (10 psi) using a water bath (40&deg; C). The final dried [18F]SFB can be reconstituted into PBS buffer for the downstream application.</li>
</ol>
8. Representative Results:
We developed a simplified, rapid, one-pot method for synthesizing [18F]SFB using a deprotection strategy under anhydrous conditions and microwave heating during each radiochemical/chemical transformation. Figure 1 presents the details of our radiosynthesis. The identity of final product was confirmed by comparison of HPLC retention time with a non-radioactive SFB reference. The purified [18F]SFB was also analyzed through radio-TLC and -HPLC to determine its radiochemical and chemical purity. The RCY of [18F]SFB was 35 &plusmn; 5% within 60 min after HPLC purification (n &gt; 30), with high radiochemical purity (&gt;99%) and good chemical purity (see the UV trace in the HPLC profile, Figure 3). The specific activity was ca. 67-330 GBq/&mu;mol (1.8-9.0 Ci/&mu;mol), depending on the starting radioactivity.
<img src="/files/ftp_upload/2755/2755fig1.jpg" alt="Figure 1" /> 
Figure 1. Microwave-assisted one-pot radiosynthesis of [18F]SFB. First, the radiofluorination of ethyl 4-(N,N,N-trimethylammonium)benzoate triflate (1) was performed under microwave heating (50 W, 1 min) in the presence of [K&sup;2.2.2][18F]F- complex in dimethylsulfoxide (DMSO) to afford ethyl 4-[18F]fluorobenzoate ([18F]2). Without purification, a DMSO solution of potassium tert-butoxide (tBuOK) was added and the reaction vessel was microwave irradiated (40 W, 1 min) to complete the anhydrous deprotection. The final conversion of [18F]3 into [18F]SFB was achieved using O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) activation. TSTU in acetonitrile was added to the reaction mixture containing the 4-[18F]fluorobenzoate ([18F]3) salt; this last synthetic step yielded crude [18F]SFB after heating (30 W, 2 min).
<img src="/files/ftp_upload/2755/2755fig2.jpg" alt="Figure 2" /> 
Figure 2. The schematic diagram of setup for microwave-assisted one-pot [18F]SFB synthesis.
<img src="/files/ftp_upload/2755/2755fig3.jpg" alt="Figure 3" /> 
Figure 3. Radio-HPL chromatograms of final [18F]SFB. Top: UV signal at 254 nm; bottom: radioactive signal; inset: UV signal at 254 nm (x 33.3).
<img src="/files/ftp_upload/2755/2755table1.jpg" alt="Table 1" /> 
Table 1. Summary of [18F]SFB radiosyntheses reported in the literature using alkyl 4-(trimethylammonium)benzoate triflate as precursors.

This method has been submitted for US patent application.

This simplified three-step, one-pot radiosynthesis of the 18F-acylation reagent [18F]SFB is developed based on non-aqueous chemistry. This process has excellent reproducibility and could be used reliably for the production of [18F]SFB in automated radiochemistry modules, owing to two key modifications described as followings: 1. We employ a deprotection/saponification step in anhydrous KOtBu/DMSO system to replace the common aqueous basic or acidic solution. Our non-aqueous deprotection ...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>acetic acid in aqueous solution (5%, v/v)</td>
 <td>Fisher </td>
 <td>A38-500 </td>
 <td>Prepared in our lab</td>
 </tr>
 <tr>
 <td>Acetonitrile</td>
 <td>Sigma-Aldrich</td>
 <td>75-05-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Diethyl ether </td>
 <td>Sigma-Aldrich</td>
 <td>14775</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl Sulfoxide (DMSO)</td>
 <td>Sigma-Aldrich</td>
 <td>472301</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethyl 4-(N,N,N-trimethylammonium)benzoate triflate </td>
 <td>Prepared in lab</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222)</td>
 <td>Sigma-Aldrich</td>
 <td>29,111-0</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) </td>
 <td>Sigma-Aldrich</td>
 <td>105832-38-0</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium carbonate in aqueous solution (1M)</td>
 <td>Sigma-Aldrich</td>
 <td>209619</td>
 <td>Prepared in our lab</td>
 </tr>
 <tr>
 <td>Potassium tert-butoxide</td>
 <td>Sigma-Aldrich</td>
 <td>156671</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.15-18 Therefore, significant effort has devoted to the synthesis and exploration of 18F-labeled biomolecules. Although there are elegant examples of the direct 18F-labeling of peptides,19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.23,24
N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB),25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the 18F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [18F]SFB, the resulting [18F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.1 Most [18F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [18F]SFB syntheses have been reported,29-32, 41-42 they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [18F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.
Herein, we report a concise preparation of [18F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.38 Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.28,43 The novelty of our improved [18F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [18F]SFB.

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

A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

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

Research

JoVE Journal

Biology

12.9K Aufrufe.  University of California at Los Angeles. Eine einfache, one-pot Synthese von N-Succinimidyl-4-[ 18 F] fluorbenzoat ([ 18 F] SFB) wurde auf Basis einer nicht-wässrigen, Drei-Stufen-radiochemische Verfahren entwickelt. Durch Mikrowellen-Erhitzung, kann das gesamte Verfahren in weniger als 30 min oder 60 min mit weiteren Reinigung durch präparative HPLC abgeschlossen sein. Der Zerfall korrigierten radiochemischen Ausbeuten (RCYs) waren 35-5% (n> 30).

Serie: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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