This work was supported by a Grant-in-Aid for Scientific Research No. 22750122 (S.H.), No. 26288099 (T.Y.), and No. 23350050 (Y.T.) of the Japan Society for the Promotion of Science. S.H. is grateful for The Kurata Memorial Hitachi Science and Technology Foundation. The research reported in this publication was supported by the King Abdullah University of Science and Technology (S.H.).

<ol>
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Chem. 6 (22), 4109-4115 (2015).</li><li>Habuchi, S., Fujiwara, S., Yamamoto, T., Vacha, M., Tezuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Study+on+Polymer+Diffusion+in+a+Melt+State:+Effect+of+Chain+Topology.">Single-Molecule Study on Polymer Diffusion in a Melt State: Effect of Chain Topology.</a> Anal. Chem. 85 (15), 7369-7376 (2013).</li><li>Habuchi, S., Satoh, N., Yamamoto, T., Tezuka, Y., Vacha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimode+Diffusion+of+Ring+Polymer+Molecules+Revealed+by+a+Single-Molecule+Study.">Multimode Diffusion of Ring Polymer Molecules Revealed by a Single-Molecule Study.</a> Angew. Chem. Int. Ed. 49 (8), 1418-1421 (2010).</li><li>Habuchi, S., Tezuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topological Polymer Chemistry: Progress of Cyclic Polymers in Syntheses, Properties and Functions. , 265-290 (2013).</li><li>Fernandez, P., Bayona, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+off-line+gel-remeation+chromatography+normal-phase+liquid-chromatography+fro+the+determination+of+polycyclic+aromatic-compounds+in+environmental-samples+and+standard+reference+materials+(air+particulate+matter+and+marine+sediment).">Use of off-line gel-remeation chromatography normal-phase liquid-chromatography fro the determination of polycyclic aromatic-compounds in environmental-samples and standard reference materials (air particulate matter and marine sediment).</a> J. 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The authors have nothing to disclose.

4-武装と8の字型ポリマーは、合成のための重要なステップであるESA-CFプロトコル（ 図1）を介して調製した。12,24単官能性および二官能性直鎖ポリ（THF）類をN -phenylpiperidinium末端基としました前述の手順に従って合成。11イオン交換は、カルボン酸の過剰量を含有する水溶液にトリフレート対アニオンを有するポリマー前駆体のアセトン溶液の再沈殿により行いました。 4アーム星型ポリマーのためのイオン交換生成物の共有結合性変換を4時間還流することにより、トルエン（4.9グラム/ L）で実施しました。変換が不十分である場合、反応時間を延長します。共有結合した生成物を、水中で、アセトン/ n-ヘキサンおよび再沈殿を用いてシリカゲルカラムクロマトグラフィーにより得ました。 4アーム重合体の1 H NMRは、 図2aに示されています。ティS ESA-CFの手順では、環状ポリマーの効果的な合成を可能にします。しかしながら、このプロトコルは、環状オニウム末端基を有するポリマーに限定されます。 8状ポリマー生成のために、反応を4時間還流希釈（0.3グラム/ L）トルエン中で行い、不溶性画分の大部分の形成をもたらし、おそらく分子間に製品を反応させました。可溶性部分は、n個のヘキサン中に再沈殿させ、アセトン/ n-ヘキサンを用いてシリカゲルカラムクロマトグラフィーに付し、水で再沈殿させました。得られた粗生成物を、サイズ排除ビーズをカラムクロマトグラフィーに付し、8状ポリマー生成物の単離を可能にするためにGPCにリサイクルしました。 8状ポリマーの1 H NMRは、 図2bに示されています。このESA-CFプロトコルは、さらに複雑なトポロジカルポリマーに適用することができます。 高品質の蛍光画像であるessenti分子の拡散運動の正確な分析のためにアル。 1）蛍光不純物が試料中に存在する場合、蛍光画像が著しく劣化し、2）組み込まれ、フルオロフォアの蛍光量子収率が低く、3）画像のフレームレートは、ポリマー分子の拡散の動きよりも遅いです。室温以下の温度（20℃）で、または37°C以上に設定すると、記録された蛍光画像の品質が低下する屈折率の不一致を引き起こします。フィルターキューブに取り付けられた狭帯域発光帯域通過フィルタを使用すると、いつか蛍光画像の品質を向上させることができます。画像化実験に用いたEM-CCDカメラの露光時間は、通常、ミリ秒に制限されているので、拡散の動きが速く、この時間スケールよりも、この方法によって捕捉することができません。 MSD分析における統計誤差の影響の評価はfoの重要なステップです異質拡散の特性をrは。統計誤差を注意深く異種の拡散を議論する前に、均一な拡散モデル22を用いて拡散係数の確率分布を計算することによって評価されるべきです。異種の拡散も慎重CDF分析によって評価されるべきです。 CDFは、均質な拡散モデル（ すなわち 、単一指数関数減衰曲線）から明らかな偏差を表示すると、これは、複数の拡散成分の存在を示唆しています。異種の拡散の定量的な特徴づけは、結合されたMSD、CDFを必要とし、確率分布を分析します。14,15 拡散運動を含むポリマーのダイナミクスは、例えば、NMR、7光散乱、8および粘度測定のような従来の方法ではアンサンブル平均値として記載されている。9実際、SINGLによって明らかに異種の拡散運動を電子分子イメージング16は、アンサンブル平均法で検出することはしばしば非常に困難です。ポリマーの固有の不均一な性質を考慮すると、このプロトコルで報告25-27方法は、トポロジカルポリマーの特性に制限されないが、絡み合った条件下でのポリマーのすべての種類に適用可能である。28また、このプロトコルで報告アプローチが見つかりますこのようなメソ多孔性材料を介して分子拡散などの複雑なシステム、不均質拡散の解析に広く応用。29

Cyclic polymers are unique in that they do not have chain ends. They often exhibit unusual behaviors which is distinct from their linear counterpart, including increased thermal stability of polymer micelles by a linear-to-cyclic conversion,1,2 and spatial organization of DNA in bacterial cells by a loop formation.3 Topological interactions between the cyclic chains are believed to be the critical factor for such unusual behaviors.4,5 Therefore, characterizing the motion and relaxation of cyclic polymers under entangled conditions has been an important research topic in polymer science for decades.6
Cyclic polymer dynamics has been investigated using both synthetic and naturally occurring molecules by means of ensemble averaged experimental methods such as nuclear magnetic resonance (NMR), light scattering, and viscosity measurements.7-9 However, these studies often suffer from impurity molecules in the samples.10 Furthermore, spatiotemporal heterogeneities of the motion of individual molecules caused by inherent structural heterogeneity of entangled polymers are often hidden behind the ensemble averaging in these studies. In order to characterize molecular level dynamics of cyclic polymers, a synthesis method that provides high purity cyclic polymers and an experimental and analysis methods that allow for quantitative characterization of molecular motion at the single molecule level have to be developed. Here, we will show a method to synthesize high-purity cyclic and dicyclic poly(THF)s that incorporate a fluorophore unit using an electrostatic self-assembly and covalent fixation (ESA-CF) process11-13 and a method to analyze the motion of the individual fluorophore-incorporated polymer chains using a combination of mean-squared displacement (MSD) and cumulative distribution function (CDF) analyses.
A proper data processing has been shown to be essential for the accurate characterization of the diffusive motion. With an adequate MSD and CDF analyses, a multiple-mode diffusion of the cyclic and dicyclic polymers in the melt and semi-dilute solution of the linear polymer chains has been revealed,14-16 suggesting the significant effects of the topological states of the polymers on the diffusive motion of the chains under entangled conditions.17 While the experimental and analytical approaches to characterize the motion of the cyclic polymers are described in this protocol, the same method can be used to quantitatively characterize the diffusive motion in many other heterogeneous systems. The approach would be especially suitable when multiple diffusion components existing in the samples are to be analyzed.

<table border="0" cellpadding="0" cellspacing="0" width="846">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Materials</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">THF</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Wakosil C-300</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Acetone</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Toluene</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">n-Hexane</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">CHCl3</td>
			<td style="width:240px;">Kanto Chemical</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bio-Beads S-X1</td>
			<td style="width:240px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Methyl triflate</td>
			<td style="width:240px;">Nacalai Tesque</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Triflic anhydride</td>
			<td style="width:240px;">Nacalai Tesque</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Potassium Hydroxide</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ethanol</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Poly(tetrahydrofuran)</td>
			<td style="width:240px;">Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Chloroform</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Immersion oil</td>
			<td style="width:240px;">Cargille</td>
			<td style="width:119px;">Type 37 / Type A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"></td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"></td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Equipment</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">2-Neck 100-mL round-bottom flask</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Flask</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Beaker</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Funnel</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Filter paper</td>
			<td style="width:240px;">Whatman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Reflux condenser</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Syringe</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Water bath</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Magnetic stirrer</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Rotary evaporator</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Microscope cover slips (24 x 24 mm, No. 1)</td>
			<td style="width:240px;">Matsunami Glass</td>
			<td style="width:119px;">CO22241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Staining jar</td>
			<td style="width:240px;">AS ONE Corporation</td>
			<td style="width:119px;">1-7934-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ultrasonic cleaner</td>
			<td style="width:240px;">VWR International&#160;</td>
			<td style="width:119px;">142-0047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Inverted microscope</td>
			<td style="width:240px;">Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ar-Kr ion laser</td>
			<td style="width:240px;">Coherent</td>
			<td style="width:119px;">Innova 70C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Berek compensator</td>
			<td style="width:240px;">Newport</td>
			<td align="right" style="width:119px;">5540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Excitation filter</td>
			<td style="width:240px;">Semrock</td>
			<td style="width:119px;">LL01-488-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dichloric mirror</td>
			<td style="width:240px;">Omega optical</td>
			<td style="width:119px;">500DRLP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Emission filter</td>
			<td style="width:240px;">Semrock</td>
			<td style="width:119px;">BLP01-488R-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Lens and mirror</td>
			<td style="width:240px;">Thorlabs</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">EM-CCD camera</td>
			<td style="width:240px;">Andor Technology</td>
			<td style="width:119px;">iXon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Objective lens (x100, N.A. = 1.3)</td>
			<td style="width:240px;">Olympus</td>
			<td style="width:119px;">UPLFLN 100XOP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Objective heater</td>
			<td style="width:240px;">Bioptechs</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Preparative GPC</td>
			<td style="width:240px;">Japan Analytical Industry</td>
			<td style="width:119px;">LC-908</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of cyclic polymers and characterization of their diffusive motion in the melt state at the single molecule level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

ペリレンテトラカルボン酸ジイミド内蔵4アーム星8状二環ポリ（THF）の静電自己集合と共有結合固定（ESA-CF）工程（ 図1、 図2）を用いて合成しました。タイムラプス単一分子蛍光画像は、4アーム（ 図3a）と8の字（ 図3b）ポリマーを測定しました。タイムラプス蛍光画像（ 図3）によるチェーンに高度に蛍光ペリレンテトラカルボン酸ジイミド、フルオロフォア23の取り込みに空間的に分離された、明るくシャープなスポットを示しています。拡散係数の頻度ヒストグラムは、タイムラプス画像の平均二乗変位（MSD）分析により、4-武装（ 図4a）のために計算され、8の字型（ 図4b）ポリマーました。 MSDプロットとのCDFの計算は、MATLABで記述されたルーチンを使用して行われています。フィッティング実験から得られたCDFのような起源プロのデータ処理ソフトウェアを使用して行われます。 MSD分析により決定拡散係数の周波数ヒストグラムは、拡散の解析及び異質の統計誤差の両方から得られた広い分布（ 図4）を表示します 。頻度ヒストグラムは、透明で均一な拡散モデルからのずれポリマー分子の不均一な拡散を示す、（ 図4の緑色の線）。14累積分布関数（CDFの）4-武装（ 図5a）を計算したと示して8字型（ 図5b）ポリマーおよび単一のガウス（ 図5a）とダブルガウス（ 図5b）モデルでフィッティング。拡散係数の統計的確率分布は、4アーム（ 図4a）について計算し、8字型（ 図4b）POL<html>単一ガウス、またはダブルガウスモデルによってymers。 （ 図5a）のシングルとダブル（ 図5b）ガウスモデルはよく実験的に得られたのCDFにフィット。これらの結果は、8状ポリマーは、2つの別個の拡散モードを表示するのに対し、4アームポリマーの拡散は、拡散係数の広い分布によって記述されていることを示しています。 <img alt="図1" src="/files/ftp_upload/54503/54503fig1.jpg" /> ペリレンジイミド内蔵ポリ図1.合成経路（THF）■。（a） は、4本腕星型ポリマーおよび（b）は 8の字型二環ポリマーの合成経路は<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54503/54503fig1large.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <html> 54503fig2.jpg &quot;/&gt; 合成されたポリマーの図2.キャラクタリゼーション。（a） は、4本腕星型ポリマーおよび（b）は 8の字型二環ポリマーのNMRスペクトル。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54503/54503fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54503/54503fig3.jpg" /> 図3（映画）。ペリレンジイミド内蔵ポリの1分子蛍光イメージング（THF）■。タイムラプス蛍光（a）は、4本腕星型ポリマーの画像および非標識線状ポリの溶融物中の（b）は 8の字型二環ポリマー（THF ）。スケールバー=5μmです。映画見るにはこちらをクリックしてください<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54503/Figure_3a_submit.mov" target="_blank">（A）を</a>し、3 / Figure_3b_submit.mov &quot;ターゲット=&quot; _ブランク」&gt;（B）。 <img alt="図4" src="/files/ftp_upload/54503/54503fig4.jpg" /> ペリレンジイミド-組み込まれたポリ（THF）の図4. MSD分析は、溶融物中に拡散するよ。拡散個々の（a）は、4本腕星型ポリマーのために決定される係数及び溶融物中の（b）は 8の字型二環ポリマーの頻度ヒストグラム線状ポリ（THF）非標識の。実線は、3つの異なる拡散モデルに基づいて拡散係数の理論的に計算された確率分布を示す図です。均質な拡散モデル（緑色の線、4.3.1を参照）、単一ガウスモデル（朱書き、4.3.2を参照）、およびダブルガウスモデル（青線は、4.3.3を参照してください）。14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54503/54503fig4large.jpg" target="_blank">本の拡大版をご覧になるにはこちらをクリックしてください。図。</a> ペリレンジイミド-組み込まれたポリ（THF）の図5. CDF分析は、溶融物中に拡散するsを実験的に得られた累積分布関数（iΔt= 7.5から75ミリ秒）。（a）は、4本腕スターポリマーのための1-Pの形で非標識線状ポリ（THF）のメルトおよび（b）は 8の字型二環ポリマー。破線は4.2.3.3で4.2.3.2および（b）式中の（a）の式でフィッティングを示す。14は、 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54503/54503fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level at JoVE.com

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

単一分子レベルでの環状ポリマーの拡散運動の合成と特性評価のためのプロトコルが提示されています。

単一分子レベルでの環状ポリマーの合成と溶融状態におけるそれらの拡散運動のキャラクタリゼーション

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

synthesis-cyclic-polymers-characterization-their-diffusive-motion

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.8K ビュー.  King Abdullah University of Science and Technology (KAUST). 単一分子レベルでの環状ポリマーの拡散運動の合成と特性評価のためのプロトコルが提示されています。 

ビデオ: Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

グラム陰性菌からの単離とリピドAの化学特性評価

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

化学

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

ペプチド第三級アミドライブラリのスプリット·アンド·プールの合成とキャラクタリゼーション

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is 1H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

ビスピリジン系リガンドの合成と二核抗癌剤として白金彼らの複合体形成のためのアミドカップリング反応

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

ルテニウムのシリーズの合成、キャラクタリゼーションと反応性<em&gt; N</em&gt; -triphos<sup&gt; Phで</sup&gt;錯体

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

合成、キャラクタリゼーション、およびハイブリッドのAu / CdSのとAu / ZnSのコア/シェルナノ粒子の機能化

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

の合成のためのプロトコル<em&gt;オルソ</em&gt; -trifluoromethoxylatedアニリン誘導体

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

超分子コロイドの合成とキャラクタリゼーション

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

タンパク質のバイオコンジュゲートの合成<em&gt;経由で</em&gt;システイン - マレイミドケミストリー

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

単一分子力分光法を用いて無機材料とアミノ酸とペプチドの相互作用への洞察

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

サイクリックボルタンメトリー、紫外-可視-近赤外、および EPR 分光電気化学を使用して有機性化合物を分析するには

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...