This work was supported financially by the Netherlands Organization for Scientific Research (NWO-CW), the European Research Council (ERC; advanced grant no. 694345 to B.L.F.), and the Ministry of Education, Culture and Science (Gravitation program no. 024.001.035).

NOTE: Organic synthesis is the major core of this project. Figure 1 shows the key steps and how to obtain the target molecule.
1. Preparation of 1b
NOTE: Solvents were purchased in analytical grade.
<ol>
	<li>Synthesis of ketone (3) 
	NOTE: At first, a five-member ring ketone 2 with an iodo group is functionalized because it is more reactive. This reaction is carried out under argon atmosphere.
	<ol>
		<li>In a sealed tube (100 mL) containing 2 (640 mg, 2.3 mmol), CuI (219 mg, 1.1 mmol), and NaI (3.44 g, 23 mmol), add 1,4-dioxane (50 mL) and N,N&#39;-dimethyl ethylenediamine (202 mg, 2.3 mmol).</li>
		<li>Set the reaction temperature to 140 &#176;C and stir for 24 h.</li>
		<li>Cool the mixture to room temperature (RT), remove the solvent in vacuo, and purify the remaining material by flash chromatography: SiO2 (40 g); eluent: pentane:EtOAc = 10:1 (total = 550 mL). The product should be obtained as a yellow, sticky oil (642 mg, 91%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 5 
	NOTE: Use the Barton-Kellogg reaction to form the central double bond. This reaction is carried out under argon.
	<ol>
		<li>In a 50 mL round-bottom flask, add Lawesson&#39;s reagent (415 mg, 1.1 mmol), ketone 3 (219 mg, 0.68 mmol), and toluene (10 mL).</li>
		<li>Heat the mixture at reflux for 2 h and subsequently evaporate the solvent.</li>
		<li>Purify the residue by flash column: SiO2 (24 g), eluent: pentane:ethyl acetate = 30:1 (total = 155 mL) to obtain the corresponding thioketone, which is subsequently redissolved in 20 mL of THF to yield a blue solution.</li>
		<li>Add a THF solution (20 mL) of diazo compound 4&#160;(476 mg, 1.37 mmol) to the blue solution, and stir the newly mixed solution at 50 &#176;C overnight. 
		NOTE: This is the most crucial step of the entire synthesis. The diazo compound and thioketone solution must be fresh and should be made prior to the reaction.</li>
		<li>Evaporate the solvent and purify the residue by chromatography: SiO2 (24 g), eluent: pentane:CH2Cl2 = 10:1 (total = 220 mL) to yield motor 5 (250 mg, 50%) as a red solid.</li>
	</ol>
	</li>
	<li>Synthesis of motor 6 
	NOTE: When the central double bond is formed, replace the iodo group by an acetylene moiety.
	<ol>
		<li>To a 20 mL Schlenk tube, add 3 (165 mg, 0.26 mmol), Pd(PPh3)2Cl2 (2.5 mole %), CuI (5 mole %). Then, add THF (10 mL) and (i-Pr)2NH (2 mL) that should be bubbled with argon for 10 min before.</li>
		<li>Stir the mixture at RT for 10 min. Then, add triisopropylsilyl acetylene (42 mg, 0.27 mmol).</li>
		<li>Stir the mixture for 15 h, then pour it into an aqueous saturated NH4Cl solution (25 mL).</li>
		<li>Extract the mixture with CH2Cl2 (3 x 20 mL). Further wash the combined organic layers with saturated brine and dry (Na2SO4).</li>
		<li>Remove the solvent and purify the residue by flash chromatography: SiO2 (12 g), eluent: pentane:CH2Cl2 = 10:1 (220 mL) to yield 8 as a brown oil (171 mg, 99%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 8 
	NOTE: This reaction is carried out under argon.
	<ol>
		<li>Stir a mixture of 6 (161 mg, 0.24 mmol), pinacol ester 7 (240 mg, 0.71 mmol), K3PO4 (300 mg, 1.44 mmol), and Pd(PPh3)4 (98 mg, 0.096 mmol) in 1,4dioxane (20 mL) at 90 &#176;C in a 50 mL Schlenk tube for 16 h.</li>
		<li>Cool the mixture to RT, dilute it with ethyl acetate (30 mL), and perform the filtration with a glass filter.</li>
		<li>Remove the solvent. Purify the residue by flash column chromatography: SiO2 (12 g), eluent: pentane: CH2Cl2= 1:6 (total = 122 mL) to yield ester 8 as a brown oil (156 mg, 56%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 9
	<ol>
		<li>Add TBAF (0.1 mL) to a solution of 8 (120 mg, 0.13 mmol) in THF (10 mL) in a 20 mL Schlenk tube at 0 &#176;C.</li>
		<li>Stir the mixture at 0 &#176;C for 1 h, then pour it into an aqueous saturated NH4Cl solution (20 mL).</li>
		<li>After extraction with CHCl3 (3 x 10 mL), wash the combined organic layers with saturated brine and dry (Na2SO4).</li>
		<li>Remove the solvent and purify the residue by flash chromatography: SiO2 (12 g), eluent: pentane:ethyl acetate = 1:3 (124 mL) to yield 9 as a dark red oil (116 mg, 95%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 12 
	NOTE: This reaction is carried out under argon.
	<ol>
		<li>To a 20 mL Schlenk tube, add motor 9 (75 mg, 0.10 mmol), PBI 11 (68 mg, 0.10 mmol) Pd(PPh3)2Cl2 (2.5 mol%), CuI (5 mol%). Then, add THF (10 mL) and (i-Pr)2NH (2 mL) that should be bubbled with argon for 10 min before.</li>
		<li>Stir the mixture overnight and pour it into an aqueous saturated NH4Cl solution.</li>
		<li>After extraction with CHCl3 (3 x 20 mL), wash the combined organic layers with saturated brine and dry (Na2SO4).</li>
		<li>Remove the solvent and purify the residue by flash chromatography: SiO2 (12 g), eluent: CHCl3 (100 mL) to yield motor 12 as a dark red solid (66 mg, 57%).</li>
	</ol>
	</li>
	<li>Synthesis of motor 1b 
	NOTE: When the ester compound is obtained, hydrolyze it to make the final target molecule.
	<ol>
		<li>Dissolve the ester 12 (90 mg, 0.038 mmol) in THF (5 mL), MeOH (5 mL), and NaOH(aq.) (1 M, 5 mL) in a 50 mL flask and heat the mixture to 75 &#176;C for 16 h.</li>
		<li>Cool the mixture to RT and add 5 mL of water. Remove the THF and MeOH by rotary evaporation.</li>
		<li>Titrate the mixture with HCl(aq.) (1 M) until reaching a pH of 1 to form a brown precipitate. Filtrate the mixture and wash the brown solid with cold water (10 mL) and dry in vacuo. This brown solid is the target molecule 1b (55 mg, 85%).</li>
	</ol>
	</li>
</ol>
2. Preparation of motor functionalized monolayer MS-1b
<ol>
	<li>Clean quartz slides by immersing them in a piranha solution (3:7 ratio of 30% H2O2 in H2SO4) at 90 &#176;C for 1 h. Rinse copiously with doubly distilled water 3x, then rinse with MeOH. Dry the slides under a stream of N2 before surface modification.</li>
	<li>Silanize the piranha-cleaned quartz slides by immersing in a 1 mM solution of 3-aminopropyl(diethoxy)methylsilane in freshly distilled toluene at RT for 12 h. Rinse copiously with toluene and MeOH.</li>
	<li>Sonicate the quartz first in toluene, then in MeOH, and dry them under a stream of argon.</li>
	<li>Immerse the amine-coated slides in DMF solution of 1b (10-4 M) at RT for 12 h.</li>
	<li>Wash the slides with DMF, water, and MeOH, then dry them under a stream of argon. After drying, the slides are ready for use.</li>
</ol>

<ol>
	<li>Berg, J. M., Tynoczko, J. L., Styer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry 5th ed. , (2006).</li><li>Erbas-Cakmak, S., Leigh, D. A., McTernan, C. T., Nussbaumer, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Molecular+Machines.">Artificial Molecular Machines.</a> Chemical Reviews. 115, 10081-10206 (2015).</li><li>Feringa, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Art+of+Building+Small:+From+Molecular+Switches+to+Motors.">The Art of Building Small: From Molecular Switches to Motors.</a> Angewandte Chemie: International Edition. 56, 11059-11078 (2017).</li><li>Stoddart, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Machines.">Molecular Machines.</a> Accounts of Chemical Research. 34, 410-411 (2001).</li><li>Kinbara, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Intelligent+Molecular+Machines:+Directed+Motions+of+Biological+and+Artificial+Molecules+and+Assemblies.">Toward Intelligent Molecular Machines: Directed Motions of Biological and Artificial Molecules and Assemblies.</a> Chemical Reviews. 105, 1377-1400 (2005).</li><li>Kay, E. R., Leigh, D. A., Zerbetto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+Molecular+Motors+and+Mechanical+Machines.">Synthetic Molecular Motors and Mechanical Machines.</a> Angewandte Chemie: International Edition. 46, 72-191 (2007).</li><li>Kottas, G. S., Clarke, I. L., Horinek, D., Michl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Molecular+Rotors.">Artificial Molecular Rotors.</a> Chemical Reviews. 105, 1281-1376 (2005).</li><li>Watson, M. A., Cockroft, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Man-made+Molecular+Machines:+Membrane+Bound.">Man-made Molecular Machines: Membrane Bound.</a> Chemical Society Reviews. 45, 6118-6129 (2016).</li><li>Kassem, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Molecular+Motors.">Artificial Molecular Motors.</a> Chemical Society Reviews. 46, 2592-2621 (2017).</li><li>Sauvage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Machines and Motors. , (2001).</li><li>van Delden, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unidirectional+molecular+motor+on+a+gold+surface.">Unidirectional molecular motor on a gold surface.</a> Nature. 437, 1337-1340 (2005).</li><li>Noji, H., Yasuda, R., Yoshida, M., Kinosita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+the+rotation+of+F1-ATPase.">Direct observation of the rotation of F1-ATPase.</a> Nature. 386, 299-302 (1997).</li><li>Hutchison, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface-bound+molecule+that+undergoes+optically+biased+Brownian+rotation.">A surface-bound molecule that undergoes optically biased Brownian rotation.</a> Nature Nanotechnology. 9, 131-136 (2014).</li><li>Krajnik, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defocused+Imaging+of+UV-Driven+Surface-Bound+Molecular+Motors.">Defocused Imaging of UV-Driven Surface-Bound Molecular Motors.</a> Journal of the American Chemical Society. 139, 7156-7159 (2017).</li></ol>

The authors have nothing to disclose.

This project involves a significant amount of synthetic work; therefore, the most critical step is organic synthesis towards the final molecule. Among the total synthesis, the Barton-Kellogg reaction is the key step, since it is the reaction in which the central double bond of the molecular motor is formed. Currently, several methods have been used to form these types of structures. Here, diazo-thioketone coupling is used, and the upper and lower halves have been prepared as the diazo and thioketone compounds, respectively. Thioketone and diazo compounds are usually not stable under air; therefore, the reaction requires fast operating under a strictly inert atmosphere. 
Existing methods to confine molecular motors on the surfaces are mostly based on bipodal systems. However, the isomerization processes of previously designed bipodal motors were obstructed due to intermolecular interactions. In addition, some of the bipodal examples requires further activation prior to attachment. The current method accomplishes this in a tetrapodal manner, which provides robust attachment of the motor on surfaces with sufficient isolated space. 
A limitation of this method is the choice of fluorescent tag. Only dyes with specific wavelengths are allowed, as the rotation of motor is triggered by the 365 nm wavelength and thus should not be overlapped. In addition, the synthetic route employed in the described protocol towards the target molecule requires several steps in which harsh conditions are needed for completion of the reaction. In the future, a more facile synthetic design is probably needed if a more advanced molecule for single molecular imaging is required. 
In conclusion, the design and synthesis of a highly functionalized light-driven molecular motor is described for the first time. Some details of the synthetic effort are discussed, as well. Furthermore, methods to graft the motor onto a quartz slide surface are demonstrated, and the sample can be further tested for visualization of single molecular motion14.

In living organisms, there are abundant molecular motors functioning to sustain daily life. They are able to perform various tasks such as fuel production, transport, mobility, etc.1. Drawing from the inspiration of these fascinating examples in Nature, scientists have developed a series of artificial molecular motors over the last several decades to convert different types of energy into controlled motion at the molecular level2,3,4,5,6,7,8,9,10. The Nobel Prize in Chemistry in 2016 was awarded to three pioneers in this field. Ben Feringa, one of the laureates, has developed the light-driven molecular motor that is able to undergo continuous unidirectional rotary motion.
However, at a molecular level, Brownian motion, also known as the random motion due to molecular collisions and vibrations, is usually the major obstacle for further application of these molecular motors. Brownian motion can disrupt any directed motion. Confining the molecular motors on surfaces may be one of the options to overcome this problem. By doing so, the relative rotation of one part of the molecule with respect to the other is converted to the absolute rotation of the rotor relative to the surface11. In addition, use of the single molecular imaging technique may help visualize the motion. Therefore, the results obtained by this work will help to gain more insight into the synthetic molecular motor.
The pioneering work of Yoshida and Kinosita (Figure 2a)12,13 has served as the inspiration for the design in the current work, shown in Figure 2b. The lower half of a light-driven molecular motor is attached to a surface to serve as the stator. The rotor part is functionalized with a rigid arm and fluorescent label. When applying two different irradiation wavelengths to the system, one will trigger the rotation of the motor, while the other will excite the fluorescent tag. In principle, the rotary motion of the rotor part triggers the rotation of the fluorescent group. Therefore, the rotation of the fluorescent tag can be followed by defocused wide-field fluorescence microscopy. This method provides, for the first time, a method to convert the relative rotation of a molecular motor into absolute rotation, and therefore a way to visualize the rotation of a synthetic motor.
This article provides details on the design, total synthesis, and solution isomerization studies of a molecular motor that is used for single molecular imaging. The molecular structure is shown in Figure 3. In addition, the method to attach molecular motors on quartz surface is described.

<table border="0" cellpadding="0" cellspacing="0" style="width:827px;" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">NMR spectrometer</td>
			<td style="width:73px;">Varian</td>
			<td style="width:228px;">AMX400</td>
			<td style="width:172px;">for proton nmr study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Reagent for organic reactions</td>
			<td style="width:73px;">Sigma</td>
			<td style="width:228px;">analytical grade</td>
			<td>reagent for organic reactions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silica gel&#160;</td>
			<td style="width:73px;">Merck</td>
			<td>230-400 mesh ASTM</td>
			<td>Flash chromatography&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Solvent</td>
			<td style="width:73px;">Acros</td>
			<td style="width:228px;">spectrophotometric grade&#160;</td>
			<td>Flash chromatography&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV lamp</td>
			<td style="width:73px;">ENB</td>
			<td style="width:228px;">280C</td>
			<td>for UV-vis irradation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">UV-vis absorption spectrophotometer</td>
			<td>JASCO</td>
			<td>V-630&#160;</td>
			<td>UV-vis measurment</td>
		</tr>
	</tbody>
</table>

light-driven molecular motors on surfaces for single molecular imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

Irradiation of the molecular motor is performed with UV light (&#955;max = 365 nm). Upon irradiation, a photo-induced E-Z isomerization around the central double bond occurs. During this process, the molecule is transformed from a stable to an unstable isomer. A thermally activated helix inversion step then follows to release the strain of the whole molecule. This results in the original stable state. 1H NMR spectroscopy is then employed to evaluate the rotary process (Figure 4a). A solution of the sample is prepared in an NMR tube, then a lamp of UV-light (&#955;max = 365 nm) is placed next to the tube. After 2 h of irradiation, distinct changes can be found in the 1H NMR spectrum. These changes indicate the generation of a new isomer that is considered to be unstable-1b (Figure 4b). It is seen in the 1H NMR spectroscopy that Ha shifts from 2.9 ppm (doublet) to 3.3 ppm (double doublet). The signal at 1.4 ppm can be assigned as the absorption of the methyl group, and it downfield shifts from 1.4 ppm to 1.6 ppm. When the sample is kept overnight at room temperature in the dark, the original spectrum can be recovered (Figure 4a). It indicates the process of the thermal helix inversion that converts unstable-1b to stable-1b.
In order to study the rotary motion of motor 1b on surfaces, the surface-attached motor assemblies MS-1b (MS = motor on surfaces) are prepared. The quartz slides are first functionalized with amine. After this step, the quartz is immersed in a DMF solution (10-4 M) of 1b at RT overnight. The resultant quartz is rinsed with DMF, water, and MeOH. The prepared quartz slides are then submitted for UV/vis studies. A UV/vis absorption spectrum of MS-1b (solid line) is shown in Figure 5b. As seen in the spectrum, the major absorption band and absorption profile are similar to that observed in solution (Figure 5a). It also shows the characteristic absorptions for motor (420 nm) and PBI (456 nm, 490 nm, 524 nm). These peaks suggest the successful attachment of motor 1b to the amine-coated surfaces. In addition, the quartz slide is irradiated for 15 min, and spectral changes are observed similar to that of the solution, indicating generation of the unstable MS-1b.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58750/58750fig1.jpg" /> 
Figure 1: Synthetic scheme towards the preparation of target molecule 1b. The scheme shows the reagents, solvents, and reaction conditions that are used in each step.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58750/58750fig2.jpg" /> 
Figure 2: (a) Schematic illustration of the structure of F0F1-ATPase grafted on a surface for visualization of unidirectional rotation (reproduced with permission12). (b) Conceptual design of a synthetic surface-bound light-driven molecular motor for single molecule imaging.&#160;
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58750/58750fig3.jpg" /> 
Figure 3: Structure of a surface-bound molecular motor 1b, bearing a rigid long arm between the motor core and PBI label.&#160;
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58750/58750fig4.jpg" /> 
Figure 4: Aliphatic region of the 1H-NMR spectra of motor 1b (CD2Cl2, -20 &#176;C, c = 10-3 M) (a) stable-1b, before irradiation (365 nm). (b) Photo stationary state mixture after irradiation. <a href="https://www.jove.com/files/ftp_upload/58750/58750fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58750/58750fig5v2.jpg" /> 
Figure 5: UV/vis absorption spectra. UV/vis absorption spectra of (a) motor 1b (CH2Cl2, 0 &#176;C), stable isomer (solid line) and unstable isomer (dashed line) at PSS. (b) MS-1b (quartz, 4 &#176;C) before (solid line) and after (dashed line) irradiation.&#160;

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Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

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11.3K Views.  University of Groningen. The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

Video: Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

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Direct Imaging of Laser-driven Ultrafast Molecular Rotation

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb explosion imaging setup in which a hitherto-impractical camera angle is realized. In our imaging technique, diatomic molecules are irradiated with a circularly polarized strong laser pulse. The ejected atomic ions are accelerated perpendicularly to the laser propagation. The ions lying in the laser polarization plane are selected through the use of a mechanical slit and imaged with a high-throughput, 2-dimensional detector installed parallel to the polarization plane. Because a circularly polarized (isotropic) Coulomb exploding pulse is used, the observed angular distribution of the ejected ions directly corresponds to the squared rotational wave function at the time of the pulse irradiation. To create a real-time movie of molecular rotation, the present imaging technique is combined with a femtosecond pump-probe optical setup in which the pump pulses create unidirectionally rotating molecular ensembles. Due to the high image throughput of our detection system, the pump-probe experimental condition can be easily optimized by monitoring a real-time snapshot. As a result, the quality of the observed movie is sufficiently high for visualizing the detailed wave nature of motion. We also note that the present technique can be implemented in existing standard ion imaging setups, offering a new camera angle or viewpoint for the molecular systems without the need for extensive modification.

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The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

Deposition of Porous Sorbents on Fabric Supports

A microwave deposition technique for silanes, previously described for production of oleophobic fabrics, is adapted to provide a fabric support material that can be subsequently treated by dip coating. Dip coating with a sol preparation provides a supported porous layer on the fabric. In this case, the porous layer is a porphyrin functionalized sorbent system based on a powdered material that has been demonstrated previously for the capture and conversion of phosgene. A representative coating is applied to cotton fabric at a loading level of 10 mg/g. This coating has minimal impact on water vapor transport through the fabric (93% of the support fabric rate) while significantly reducing transport of 2-chloroethyl ethyl sulfide (CEES) through the material (7% of support fabric rate). The described approaches are suitable for use with other fabrics providing amine and hydroxyl groups for modification and can be used in combination with other sol preparations to produce varying functionality.

This report details a microwave-initiated approach for deposition of porphyrin functionalized porous organosilicate sorbents on a cotton fabric and demonstrates reduction in 2-chloroethyl ethyl sulfide (CEES) transport through the fabric resulting from this treatment.

A microwave deposition technique for silanes, previously described for production of oleophobic fabrics, is adapted to provide a fabric support material ...

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Automation of a Positron-emission Tomography PET Radiotracer Synthesis Protocol for Clinical Production

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases. Here, we demonstrate light-triggered nuclear translocation of guests using caged molecular glue (CagedGlue-R) tags, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Guests tagged with CagedGlue-R are taken up into living cells via endocytosis and remain in endosomes. However, upon photoirradiation, CagedGlue-R is converted into uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which facilitates the endosomal escape and subsequent nuclear translocation of the guests. This method is promising for site-selective nuclear-targeting drug delivery, since the tagged guests can migrate into the cytoplasm followed by the cell nucleus only when photoirradiated. CagedGlue-R tags can deliver macromolecular guests such as quantum dots (QDs) as well as small-molecule guests. CagedGlue-R tags can be uncaged with not only UV light but also two-photon near-infrared (NIR) light, which can deeply penetrate into tissue.

This protocol describes light-triggered nuclear translocation of guests in living cells using caged molecular glue tags. This method is promising for site-selective nuclear-targeting drug delivery.

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is ...

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Excitonic Hamiltonians for Calculating Optical Absorption Spectra and Optoelectronic Properties of Molecular Aggregates and Solids

Rational design of disordered molecular aggregates and solids for optoelectronic applications relies on our ability to predict the properties of such materials using theoretical and computational methods. However, large molecular systems where disorder is too significant to be considered in the perturbative limit cannot be described using either first principles quantum chemistry or band theory. Multiscale modeling is a promising approach to understanding and optimizing the optoelectronic properties of such systems. It uses first-principles quantum chemical methods to calculate the properties of individual molecules, then constructs model Hamiltonians of molecular aggregates or bulk materials based on these calculations. In this paper, we present a protocol for constructing a tight-binding Hamiltonian that represents the excited states of a molecular material in the basis of Frenckel excitons: electron-hole pairs that are localized on individual molecules that make up the material. The Hamiltonian parametrization proposed here accounts for excitonic couplings between molecules, as well as for electrostatic polarization of the electron density on a molecule by the charge distribution on surrounding molecules. Such model Hamiltonians can be used to calculate optical absorption spectra and other optoelectronic properties of molecular aggregates and solids.

Here, we present a protocol for parametrizing a tight-binding excitonic Hamiltonian for calculating optical absorption spectra and optoelectronic properties of molecular materials from first-principles quantum chemical calculations.

Rational design of disordered molecular aggregates and solids for optoelectronic applications relies on our ability to predict the properties of such ...