KGは、RAウェルチ財団補助金P-1760、（KG）は数学と科学教育のTCUアンドリュース研究所、（KG）はTCU研究と創造活動グラントと（JHSへ）TCU SERCグラントによってサポートされていました。

ビオチンFcのシステインの1の合成（1） <ol><li>樹脂に結合した1を製造する固相法。 <ol><li>フリットシリンジにビオチンロードされた樹脂（250mgを、0.145ミリモル）を配置し、ジメチルホルムアミド（5ml）に策定し、20分間ラボシェーカー上で注射器を振盪することにより樹脂を膨潤させる。ソリューションを追放し、1より多くの時間を腫れジメチルホルムアミドを繰り返します。 </li><li>振とうの10〜15分、続いて注射器に、ジメチルホルムアミド中20％ピペリジン4-6を加えてFmoc保護基を除去する。ピペリジンの別の4〜6ミリリットルで脱保護処理を繰り返します。 3×ホルムアミド、3倍のジメチルホルムアミド順序で樹脂を洗浄：メタノール（1：1）、3回メタノール：ジクロロメタン（1：1）、3回ジクロロメタン、約5 mlずつ。加熱時のブルーの存在によって成功した脱保護を確認するためにビーズの小さなサンプリング（〜10）にニンヒドリンテスト（+）を行います。 </li><li> 1&#39;-のFmoc-アミノフェロセン-1-カルボン酸を含む溶液を混合する（203.3ミリグラム、0.4350ミリモル）、1-ヒドロキシベンゾトリアゾール水和物（58.8ミリグラム、0.413ミリモル）、ジイソプロピルカルボジイミド（0.0673ミリリットル、0.435ミリモル）、ジイソプロピルエチルアミン（0.0757ミリリットル、0.435ミリモル）、および4：ジクロロメタンの1：1混合物とジメチルホルムアミド。フリットシリンジにこれを描画し、軽く6時間ラボシェーカーで振る。その後、注射器からソリューションを追放し、前述のように洗う。 </li><li>カップリングを確認するために、上記のように - （）ニンヒドリンテストを実行します。ニンヒドリン試験は、まだ部分を含む鉄の結合から誘導されるビーズのオレンジ色にもかかわらず、結合を確認するのに有用であり得る。 </li><li>その後、ジメチルホルムアミド中20％ピペリジンを添加することによりFmoc基を除去し、上記のように洗浄した。ニンヒドリン試験（+）は、Fmoc除去を確認するために使用されるべきである。 </li><li>のFmoc-Cysの（Trt）と-OH（254.8ミリグラム、0.4350ミリモル）、ジイソプロピルカルボジイミド1-ヒドロキシベンゾトリアゾール水和物（58.8 mgの、0.4125ミリモル）、（0.0673ミリリットル、0.4350からなる溶液を準備ミリモル）、ジイソプロピルエチルアミン（0.0757ミリリットル、0.4350ミリモル）、および4：ジクロロメタンとジメチルホルムアミドの1：1混合物。このシステインカップリングカクテルのフリットシリンジを加え、穏やかに6時間振る。以前に記載されたプロトコルを使用して洗ってください。 </li><li> 20％ピペリジンと洗浄とのFmoc成分を除去し、（ -​​ ）ニンヒドリン試験を用いてカップリングすることを確認します。ニンヒドリン試験（+）を使用して、アミン遊離ターミナルを確認してください。 </li></ol></li><li>樹脂からの1の切断。 <ol><li> TFAの溶液（9.45ミリリットル）、水（0.25 ml）を、1,2-エタンジチオール（0.25 ml）を確認し、トリイソプロピルシラン（0.1mL）を、シリンジに追加し、4時間穏やかに振とうする。 </li><li>エッペンドルフチュー​​ブ中で、得られた赤茶色の溶液を収集し、空気流を用いてゆっくりとTFAを蒸発させる。 </li><li>穏やかな撹拌を形成するであろう、1を沈殿さエッペンドルフチューブに冷ジエチルエーテル（〜15ml）に加える。遠心分離を介して生成物（1 gで5分）を単離する。 Tジエチルエーテル洗浄液（~60 mlの総）及び遠心分離機の鶏反復サイクルは、赤/褐色の固体として1を得た。 </li></ol></li></ol> 1の2。評価と解析<ol><li>アイデンティティは1 H（16スキャン）と重水素化メタノール（300μL）およびESI-MS分析における13 C NMR（512スキャン）を使用して、 図2に示す接続性や組成と一致していることを確認してください。 以下の結果を期待する： 1 H NMRスペクトル（CD 3 OD）δ/ ppmで：1.407から1.684（メートル、6H）、2.245（トン、2H）、2.665から3.150（メートル、12H）、4.015（トン、1H）、4.104の（d、2H ）、4.274（qは、1H）、4.426の（d、2H）、4.479（qは、1H）、4.595（tは、1 H）及び13 C NMRスペクトル（CD 3 OD）δ/ ppmで：24.644（CH 2）、25.472 （CH 2）、28.051（CH 2）、28.300（CH 2）、35.474（CH 2）、38.698（CH 2）、39.241（CH 2）、39.717（CH 2）、55.340 / 55.538（Cpのリング） 60.286（CH）、61.964（CH）、62.521 / 62.821（CPリング）、66.038 / 66.170（CPリング）、69.153 / 69.328（CPリング）、71.468 / 71.593（CPリング）、76.466（CH）、171.770 （C = O）、175.361（C = O）。 ESI-MS（M / z）は：実測：639.00 [1 +のNa] +、理論値：639.1 [1 +のNa] +とHR-MS（M / Z）：実測：617.2049 [1 + H] +、理論値： 617.1622 [1 + H] +。 </li><li>孤立した1の組成を確認するために、HPLC、元素分析を行います。 C8を使用して実施するHPLCクロマトグラムを0.5ml /分の流速で100％MeOHを用いて逆相カラム。注：HPLC保持時間は以下の通りであった：3.198から4.674分。 </li></ol>金表面上の1の3の固定化<ol><li> 2〜0.25の正方形にポリマー担保金のスライドをカット。 </li><li> 1（〜1ミリモル）のDI水溶液で50mlのビーカーを埋める。 </li><li>ビーカーに金のスライドを追加し、時計皿でカバーしています。すべてフローガラススライドを撹拌せずに室温でO / Nインキュベートした。 </li><li>溶液からの金のスライドを削除し、空気中のそれが乾燥許す。 </li><li> 1を固定化した観察する走査型電子顕微鏡（または同等品）を用いた走査型電子顕微鏡像を得る。 </li></ol>

The authors have nothing to disclose.

非対称のフェロセン誘導体の合成は、溶液中に挑戦されている。例えば、溶液中の1を製造する試みは、所望の生成物（20％未満）の低い収率をもたらした。同様に、1&#39;-アミノフェロセンカルボン酸（ サンセリフのFmoc）および樹脂結合ビオチンを利用する反応はBaristic らによって報告重合生成物と一致して不溶性生成物をもたらした。そして最小限の製品。47<...

バイオセンサは、選択的分析のためのプラットフォームとして、生体分子認識技術を採用し、その特異性、速度、および低コストのために利用される小型の装置である。生体分子の検出のための電気化学的バイオセンサは、そのシンプルさ、コスト効率、高感度のために、この分野の最前線にある。これらのセンサの1,3一般的な解剖学は、対象とする生物学的マーカーに特異的な認識分子を搭載した電極である。認識分子によりバイオマーカーの結合は、簡単な測定によって検出され得る電位または電流の局所的な変化をもたらす。認識部分とデートするには、酵素、4-8抗体、9-12細胞全体、13-16受容体、17-20ペプチド21-23とDNA 24の範囲とすることができる、大部分が大きく、生物学的分子に焦点を当ててきた。25-28リサーチこの分野での取り組みは、主にWHE免疫センサーに集中している免疫再（フェロセンなど）、レドックス活性コアを固定化し、目的の抗体を検出するために使用される。これらの研究は、抗原/抗体の使用に起因する合併症に起因する不良による精度と時間消費への臨床応用から除外されている。1,3栽培注意が生物医学の小分子の検出（未満1キロ/モル）に焦点を当てている、食品や国家安全保障に加えて、環境の関心。29バイオセンサーデバイスの最もよく知られている例は、ポケットサイズの電流測定メーターに接続されたスクリーン印刷された酵素電極を有するセルフテストグルコースモニター、である。これらのシステムは、典型的には、グルコース酸化反応で発生した電荷の総量は、ある期間にわたって測定された電量分析法を利用する。市場性のあるデバイスは、携帯用堅牢でなければならない一般の集団のために容易に利用するためにハンドヘルド。 フェロセン等のレドックスタグがnecessaですほとんどのバイオマーカーは、本質的に電気化学的に活性でないように、溶液中のバイオマーカーまたは小分子の電気化学的検出を提供するために、RY。30-38フェロセンは、電気化学的バイオセンサーへの統合のための優れた選択肢となり電気化学のゴールドスタンダードであり、有機金属分子である。フェロセンベースの酸化還元活性種はすでにサイズが小さいため、優れた安定性、便利な合成アクセス、容易な化学修飾、相対的な親油性、及び酸化還元チューニングのしやすさにかなりの注目を集めている。3,30-42小分子を有するフェロセンコアをベース金属イオンや小分子の検出器として広く使用され、このような生体分子のようなより大きな化学種を標的32-38,43システムは、電気化学的表面上に埋め込 ​​まれたフェロセン誘導体大きな抗体または免疫グロブリンの結合を利用している。1,3,39 、それぞれの場合には44、潜在的な、現在のintensi鉄III / II鉄の酸化還元対のティは、このように、分析物分子の存在を示す新たな分光ハンドルを生成する、分子の結合の際に変化した。この変更は、シクロペンタジエニル環のπシステムと鉄のd軌道との間で発生した大規模なオーバーラップから生じる。パイ-システムが変更された場合、 すなわち、誘導体化するか、または、その後軌道相互作用が意志、順番に、変更を反応させた。これは、鉄芯に影響を与えるとFe III /鉄II対の電位の変化として観察することができる。40,45,46これらのプロパティは、電気化学的イムノアッセイまたはバイオセンサーで定量化剤として使用するためのそのようなシステムは魅力的である。 バイオセンサーの能力のために特定のフェロセンを含むシステムを製造するためには、標的分子に特異的なバイオレセプター、ワンCp環を変更して、電気化学的読み出しまたはELECの分子テザーのような他のCp環を利用することが最適であるtrode（ 図1）。これらの非対称のフェロセン誘導体の合成は、副反応および分子間架橋の際に形成された二量体およびポリマー種の形成によって攻撃される。47が、アミド結合を生成するカップリング化学は、このような生物学的成分を含むフェロセンの単純な誘導体を提供するための最も直接的なルートであるペプチドおよびその代謝物など。したがって、第一のペプチド合成メリフィールドによって、1950年代に開発された固相技術は、フェロセンを含有する有機金属化合物に適用することができる。有する直交置換1&#39;-のFmoc-アミノフェロセン-1-カルボン酸分子、受容体部分（ビオチン）、電気化学的な読み出し（フェロセン）と固定リンカー成分（システイン）を含むことができるフェロセンシステムの使用を介して構築され、本明細書に詳述されて。このバイオコンジュゲートの合成は、金表面上に固定化するための証拠としてだけでなく、議論されている。この作品のrepresenビオチン、フェロセン及び金表面上に固定化するためのアミノ酸から構成されるシステムの最初のプレゼンテーションのts。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Biotin Novatag Resin</td>
			<td>NovaBiochem</td>
			<td>8550510001</td>
			<td></td>
		</tr>
		<tr>
			<td>TORVIQ 10ML LUER LOCK FRITTED SYRINGE</td>
			<td>Fisher</td>
			<td>NC9299151</td>
			<td></td>
		</tr>
		<tr>
			<td>piperdine</td>
			<td>Acros</td>
			<td>P/3520/PB05</td>
			<td></td>
		</tr>
		<tr>
			<td>ninhydrin test</td>
			<td>Sigma-Aldrich</td>
			<td>60017-1ea</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#8217;-Fmoc-amino-ferrocene-1-carboxylic acid</td>
			<td>Omm Scientific</td>
			<td>Special Order</td>
			<td></td>
		</tr>
		<tr>
			<td>1-&#8203;Hydroxybenzotriazole hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>157260-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Diisopropylcarbodiimide</td>
			<td>Sigma-Aldrich</td>
			<td>D125407-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-&#8203;Diisopropylethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>496219-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Cys(Trt)-OH</td>
			<td>Novabiochem</td>
			<td>8520080025</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T5408</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-ethanedithiol</td>
			<td>Sigma-Aldrich</td>
			<td>2930</td>
			<td></td>
		</tr>
		<tr>
			<td>triisopropyl silane</td>
			<td>Sigma-Aldrich</td>
			<td>233781</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes (20 mL)</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology

早期発見は、ほとんどの疾患の治療の成功の鍵であり、多くの型の癌の診断および治療のために特に不可欠である。利用される最も一般的な技術は、磁気共鳴イメージング（MRI）、陽電子放射断層撮影（PET）、及びコンピュータ地形（CT）のようなモダリティを撮像し、疾患の物理的構造を把握するために最適であるが、一度だけ実行されるすべての4つまですることができている造影剤と全体的なコストを使用しているため6週間。同様にこのことを念頭に置いて、そのような臨床医のオフィスで、疾患の段階及び/又は治療の有効性を評価し、適時にそうバイオセンサ、などの「ポイントオブケア」の開発技術は、治療プロトコルに革命をもたらすであろう。1生物学的に関連する分子2の検出のためのフェロセンベースのバイオセンサーを探求する手段は、方法は、本明細書に記載フェロセン-ビオチンバイオコンジュゲートを生成するために開発された。このレポートは、金表面に固定化することができるビオチンフェロセン - システインシステムに焦点を当てます。

1の樹脂結合形態は、 図2に示されている。フェロセン成分の共有結合により、鉄吸収とは異なり連続的な洗浄で永続と複合体を含む固定化された鉄の指標である樹脂ビーズのオレンジ色合いを生じさせる樹脂ビーズのPEG成分。 1の樹脂を含まない形態では、樹脂ビーズの色が同じである。樹脂ビーズから化合物を除去した後、方法から得られた純度およ?...

Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

固相樹脂系の方法論を経由して、非対称フェロセン派生バイオコンジュゲートシステムのための合成方法論

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

synthetic-methodology-for-asymmetric-ferrocene-derived-bio-conjugate

Research

JoVE Journal

Chemistry

9.6K ビュー.  Texas Christian University. The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

ビデオ: Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO2 system was studied at 25 and 40&#160;&#176;C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO2 pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.
The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

COの相平衡測定のための高圧サファイアセル<sub&gt; 2</sub&gt; /有機/水システム

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

化学

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

最適化された合成表面を使用した肝細胞表現型の安定化

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

保存科学における粉末X線回折：遺産アートオブジェクトに対する腐食製品のルーチンの結晶構造の決意に向けて

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

動的光散乱顕微鏡による濁りソリューションにおける粒度分布の測定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

臨床応用のためのヒト尿中の3-ニトロチロシンの小型化固相抽出およびLC-MS / MS検出の統合

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

フッ素含有医薬品の量を測定するアプリケーションと非破壊19F 時間ドメインの NMR 手法論

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to investigate the lipid raft hypothesis. Numerous studies, however, neglect the impact of physiological solution conditions. On that account, the current work presents the effect of high-salinity buffer and trans-membrane solution asymmetry on liquid-liquid phase separation in charged GUVs grown from dioleylphosphatidylglycerol, egg sphingomyelin, and cholesterol. The effects were studied under isothermal and varying temperature conditions.
We describe equipment and experimental strategies applicable for monitoring the stability of coexisting liquid domains in charged vesicles under symmetric and asymmetric high-salinity solution conditions. This includes an approach to prepare charged multicomponent GUVs in high-salinity buffer at high temperatures. The protocol entails the option to perform a partial exchange of the external solution by a simple dilution step while minimizing the vesicle dilution. An alternative approach is presented utilizing a microfluidic device that allows for a complete external solution exchange. The solution effects on phase separation were also studied under varying temperatures. To this end, we present the basic design and utility of an in-house built temperature control chamber. Furthermore, we reflect on the assessment of the GUV phase state, pitfalls associated with it and how to circumvent them.

Experiments on phase separated giant unilamellar vesicles (GUVs) frequently neglect physiological solution conditions. This work presents approaches to study the effect of high-salinity buffer on liquid-liquid phase separation in charged multicomponent GUVs as a function of trans-membrane solution asymmetry and temperature.

蛍光顕微鏡による監視対称および非対称のソリューションの条件下での荷電ベシクルの相挙動

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2&#039;-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

2 &#39;末端を含むRNAのオリゴマーの固相合成のためのプロトコール<em&gt; O</em&gt;チオフェンメチル修飾と環状二色性によるキャラクタリゼーション

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

マトリクスによる合成高分子のキャラクタリゼーション支援レーザー脱離イオン化時間 (MALDI Tof) 質量分析装置

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

[4.4] スピロ エナンチオマーの固相合成

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...