This research is supported by the National Key Research and Development Program of China (2017YFA0206801, 2018YFA0208600), Natural Science Foundation of Jiangsu Province,&#160;and National Natural Science Foundation of China (91645116). L.X is the Zhong-Wu Specially Appointed Professor of the Jiangsu University of Technology. The authors acknowledge the suggestions from Dr. Hao Hu and Dr. Mingjun Yang.

1. Building model
<ol>
	<li>Build porphycene structure: Open GaussView software by double-click the mouse. Then click Element Fragment button in the menu of the GaussView to choose the needed elements. Construct porphycene. Then click File button to save as the pdb file.</li>
	<li>Solvate the model: Solvate porphycene in a cubic TIP3P21 water box with an edge length of 38 &#197; by issuing the command in the linux operating system: genbox_d -cp prp-vac.pdb -cs spc216.gro -o solv.pdb -maxsol 1484 -box 3.8.</li>
	<li>Build deuterium porphycene: Issue the following command to generate topology file: cns &#60; ppi_solv.inp. Then open prp-wat.psf with vi command and change the mass of H1 from 1.00800 to 2.01600 to replace one intramolecular hydrogen atom of porphycene with deuterium to build single deuterium substituted porphycene.</li>
	<li>Set normal MD simulation parameters: Input method scctb, integral 0.5 fs, and cutoff 12 in the MD input file by opening it with vi command. 
	NOTE: Adopt a cutoff distance of 12 &#197; for calculating both vdW and electrostatic interactions. Simulate the porphycene molecule with DFTB/MIO method22. Set the integration time step as 0.5 fs for MD simulations. Maintain the temperature of the simulation system at 300 K with Langevin thermostat. Then perform the simulations with QM4D software following the steps below.</li>
</ol>
2. Pre-sits
<ol>
	<li>Set up temperature parameters: Input templow 260, temphigh 1100 and ntemp 160 in the input file. 
	NOTE: The temperature range from 260K to 1100K was spread out by QM4D software to 160 temperature points during MD simulations. The template input files are included in Supplementary Files.</li>
	<li>Initiate the pre-sits: Set runtype 100 and step 120,000 in the input file. Then issue the following command: $PATH/qm4d $INPUTFILE &#62; $OUTPUTFILE. 
	NOTE: The total step is 120,000 but can be adjusted depending on the specific need. The suggested parameters of MD simulations are saved in the $INPUTFILE. The same command is also used in the following opt-sits and production simulation steps, with the input file modified accordingly.</li>
	<li>Calculating the decomposed energies
	<ol>
		<li>Extract energy changes: During the pre-sits stage, monitor the energy of each term to calculate the mean values, as shown in Figure 1. Use grep Linux command to extract the energy as follows: 
		grep &#8216;SITS-ener0&#8217; $INPUTFILE | awk &#8216;{a+=$3;b+=$4;c+=$5}END{print a/NR,b/NR,c/NR}.</li>
		<li>Modify average energies in the MD input file: Calculate the average energies based on the output of the command line above, and modify the line vshift0 -30801.95; vshift1 -26.88; vshift2 -13888.28 in the input file with the newly generated averages. 
		NOTE: Numbers -30801.85, -26.88 and -13888.28 are the average energies in the current model system. Please modify the values based on the specific systems.</li>
	</ol>
	</li>
</ol>
3. Opt-sits
<ol>
	<li>Initiate opt-sits: Set runtype 0 in the input file. Then initiate the QM4D program by typing the command as shown in step 2.2 to start the optimization step.</li>
	<li>Monitoring the energy changes and nk values.
	<ol>
		<li>Plot the energy propagation with &#8220;grace&#8221; program and make sure the energy fluctuation can cover the lowest and the highest ends of the temperature range.</li>
		<li>After optimization, save the final nk values of the opt-sits step into a new file, which is named as nk.dat in this protocol.</li>
	</ol>
	</li>
</ol>
4. Running production simulations
<ol>
	<li>Prepare MD input file: Set runtype 1 in the new input file to start the production simulation step. Specify the file name of stored nk file as nkfile nk.dat in the input file. The number of time steps was set as 6,400,000 in the present systems.</li>
	<li>Initiate production MD simulation: Issue the following command to start MD simulations: $PATH/qm4d $INPUTFILE &#62; $OUTPUTFILE. 
	NOTE: Make sure the nk values can be read in by the QM4D software. The simulation time is system dependent so change the simulation step based on specific demands. Select a proper number of time steps to ensure enough simulation time for your own system. This step is likely time consuming, so save restart files to avoid restarting the production from the very beginning once being disrupted.</li>
</ol>
5 Data analysis
<ol>
	<li>Monitoring the distance changes
	<ol>
		<li>Monitor the bond forming and breaking process during the production phase, use the grep command to check the distance changes of H1-N1 and H1-N2 along the simulation time. The same operation can be conducted for H2-N3 and H2-N4. Then plot the distance propagation using the accumulated distance value during the production simulations.</li>
	</ol>
	</li>
	<li>Extracting reaction coordinates
	<ol>
		<li>Extract the reaction coordinates and the energy terms from the production output file generated by QM4D by grep command: 
		grep &#8216;dist 1&#8217; $OUTPUTFILE | awk &#8216;{print $5}&#8217; &#62; distance1; 
		grep &#8216;ener0&#8217; $OUTPUTFILE &#62; ener0.</li>
		<li>Organize data in four columns: q1, q2, U0 and U&#8217; (U0 and U&#8217; are the output normal energy and weighted energy), and write them into the data file at each time frame.</li>
	</ol>
	</li>
	<li>Calculating Free energy
	<ol>
		<li>Calculate the free energy by issuing the following command: 
		sits-pmf 300 $INPUTFILE PMF2 [hist_minx hist_maxx num_binsx] [hist_miny hist_maxy num_binsy] &#62; $OUTPUTFILE. 
		NOTE: sits-pmf is the histogram-based analysis method. [hist_minx hist_maxx num_binsx] defines the range and number of bins for the first reaction coordinate. The second reaction coordinates can be set by [hist_miny hist_maxy num_binsy].</li>
		<li>To project the free energy on the two-dimensional landscape, type the following command: 
		sits-pmf 300 h1-2d.dat PMF2 -0.6 0.6 24 2.45 4.25 36 &#62; sits-pmf.out. 
		NOTE: Use a total of 24 bins and 36 bins to cover the distance changes in two selected reaction coordinates, q1 and q2, respectively. Save the 2D PMF data for each hydrogen/deuterium to the sits-pmf.out file.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Waluk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground-+and+excited-state+tautomerism+in+porphycenes.">Ground- and excited-state tautomerism in porphycenes.</a> Accounts of Chemical Research. 39 (12), 945-952 (2006).</li><li>Waluk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopy+and+tautomerization+studies+of+porphycenes.">Spectroscopy and tautomerization studies of porphycenes.</a> Chemical Reviews. 117 (4), 2447-2480 (2017).</li><li>Kozlowski, P. M., Zgierski, M. Z., Baker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inner-hydrogen+migration+and+ground-state+structure+of+porphycene.">The inner-hydrogen migration and ground-state structure of porphycene.</a> The Journal of Chemical Physics. 109 (14), 5905-5913 (1998).</li><li>Yoshikawa, T., Sugawara, S., Takayanagi, T., Shiga, M., Tachikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+study+on+the+mechanism+of+double+proton+transfer+in+porphycene+by+path-integral+molecular+dynamics+simulations.">Theoretical study on the mechanism of double proton transfer in porphycene by path-integral molecular dynamics simulations.</a> Chemical Physics Letters. 496 (1-3), 14-19 (2010).</li><li>Smedarchina, Z., Shibl, M. F., K&#252;hn, O., Fern&#225;ndez-Ramos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tautomerization+dynamics+of+porphycene+and+its+isotopomers+-+concerted+versus+stepwise+mechanisms.">The tautomerization dynamics of porphycene and its isotopomers - concerted versus stepwise mechanisms.</a> Chemical Physics Letters. 436 (4-6), 314-321 (2007).</li><li>Wolfsberg, M., Hook, W. A., Paneth, P., Rebelo, L. P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope+effects.">Isotope effects.</a> The Chemical, Geological, and Bio Sciences. , (2009).</li><li>Pietrzak, M., Shibl, M. F., Br&#246;ring, M., K&#252;hn, O., Limbach, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1H/2H+NMR+studies+of+geometric+H/D+isotope+effects+on+the+coupled+hydrogen+bonds+in+porphycene+derivatives.">1H/2H NMR studies of geometric H/D isotope effects on the coupled hydrogen bonds in porphycene derivatives.</a> Journal of the American Chemical Society. 129 (2), 296-304 (2007).</li><li>Yoshikawa, T., Sugawara, S., Takayanagi, T., Shiga, M., Tachikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+tautomerization+in+porphycene+and+its+isotopomers:+Path-integral+molecular+dynamics+simulations.">Quantum tautomerization in porphycene and its isotopomers: Path-integral molecular dynamics simulations.</a> Chemical Physics. 394 (1), 46-51 (2012).</li><li>Ladenthin, J. N., 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=Force-induced+tautomerization+in+a+single+molecule.">Force-induced tautomerization in a single molecule.</a> Nature Chemistry. 8 (10), 935-940 (2016).</li><li>Dellago, C., Bolhuis, P. G., Csajka, F. S., Chandler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+path+sampling+and+the+calculation+of+rate+constants.">Transition path sampling and the calculation of rate constants.</a> The Journal of Chemical Physics. 108 (5), 1964-1977 (1998).</li><li>Bolhuis, P. G., Chandler, D., Dellago, C., Geissler, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+path+sampling:+Throwing+ropes+over+rough+mountain+passes,+in+the+dark.">Transition path sampling: Throwing ropes over rough mountain passes, in the dark.</a> Annual Review of Physical Chemistry. 53 (1), 291-318 (2002).</li><li>Torrie, G. M., Valleau, 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=Nonphysical+sampling+distributions+in+monte+carlo+free-energy+estimation:+Umbrella+sampling.">Nonphysical sampling distributions in monte carlo free-energy estimation: Umbrella sampling.</a> Journal of Computational Physics. 23 (2), 187-199 (1977).</li><li>K&#228;stner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Umbrella+sampling.">Umbrella sampling.</a> Wiley Interdisciplinary Reviews: Computational Molecular Science. 1 (6), 932-942 (2011).</li><li>Gao, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrate-over-temperature+approach+for+enhanced+sampling.">An integrate-over-temperature approach for enhanced sampling.</a> Journal of Chemical Physics. 128 (6), 064105 (2008).</li><li>Yang, L., Gao, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+selective+integrated+tempering+method.">A selective integrated tempering method.</a> Journal of Chemical Physics. 131 (21), 214109 (2009).</li><li>Yang, M., Yang, L., Gao, Y., Hu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combine+umbrella+sampling+with+integrated+tempering+method+for+efficient+and+accurate+calculation+of+free+energy+changes+of+complex+energy+surface.">Combine umbrella sampling with integrated tempering method for efficient and accurate calculation of free energy changes of complex energy surface.</a> The Journal of Chemical Physics. 141 (4), 044108 (2014).</li><li>Yang, Y. I., Zhang, J., Che, X., Yang, L., Gao, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+sampling+over+rough+energy+landscapes+with+high+barriers:+A+combination+of+metadynamics+with+integrated+tempering+sampling.">Efficient sampling over rough energy landscapes with high barriers: A combination of metadynamics with integrated tempering sampling.</a> The Journal of Chemical Physics. 144 (9), 094105 (2016).</li><li>Xie, L., Shen, L., Chen, Z. N., Yang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+free+energy+calculations+by+combining+two+complementary+tempering+sampling+methods.">Efficient free energy calculations by combining two complementary tempering sampling methods.</a> The Journal of Chemical Physics. 146 (2), 024103 (2017).</li><li>Xie, L., Cheng, H., Fang, D., Chen, Z. N., Yang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+QM/MM+sampling+for+free+energy+calculation+of+chemical+reactions:+A+case+study+of+double+proton+transfer.">Enhanced QM/MM sampling for free energy calculation of chemical reactions: A case study of double proton transfer.</a> The Journal of Chemical Physics. 150 (4), 044111 (2019).</li><li>Hu, X., Hu, H., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QM4D: an integrated and versatile quantum mechanical/molecular mechanical simulation package (http://www.qm4d.info/). , (2016).</li><li>Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., Klein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+simple+potential+functions+for+simulating+liquid+water.">Comparison of simple potential functions for simulating liquid water.</a> The Journal of Chemical Physics. 79 (2), 926-935 (1983).</li><li>Walewski, L., 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=Scc-dftb+energy+barriers+for+single+and+double+proton+transfer+processes+in+the+model+molecular+systems+malonaldehyde+and+porphycene.">Scc-dftb energy barriers for single and double proton transfer processes in the model molecular systems malonaldehyde and porphycene.</a> International Journal of Quantum Chemistry. 106 (3), 636-640 (2006).</li><li>Bakowies, D., Thiel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+models+for+combined+quantum+mechanical+and+molecular+mechanical+approaches.">Hybrid models for combined quantum mechanical and molecular mechanical approaches.</a> The Journal of Physical Chemistry. 100 (25), 10580-10594 (1996).</li><li>Hu, H., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+ab+initio+QM/MM+methods+for+mechanistic+simulation+of+reactions+in+solution+and+in+enzymes.">Development and application of ab initio QM/MM methods for mechanistic simulation of reactions in solution and in enzymes.</a> Journal of Molecular Structure: THEOCHEM. 898 (1-3), 17-30 (2009).</li><li>Xie, L., Yang, M., Chen, Z. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+entropic+effect+in+chorismate+mutase+reaction+catalyzed+by+isochorismate-pyruvate+lyase+from+pseudomonas+aeruginosa+(PchB).">Understanding the entropic effect in chorismate mutase reaction catalyzed by isochorismate-pyruvate lyase from pseudomonas aeruginosa (PchB).</a> Catalysis Science, Technology. 9 (4), 957-965 (2019).</li><li>Shibl, M. F., Pietrzak, M., Limbach, H. H., K&#252;hn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+H/D+isotope+effects+and+cooperativity+of+the+hydrogen+bonds+in+porphycene.">Geometric H/D isotope effects and cooperativity of the hydrogen bonds in porphycene.</a> ChemPhysChem. 8 (2), 315-321 (2007).</li></ol>

The authors have nothing to disclose.

The structure of porphycene was shown in Figure 1. The electrostatic embedding QM/MM hybrid potential with SITS method was used to describe the chemical reactions in water23,24. The proton transfer occurs within porphycene3 and thus porphycene is set as QM region and the reminding water is set as MM region. Herein we adopted DFTB/MIO as our QM method to treat the porphycene by balancing the efficiency and accu...

The proton transfer process in porphycenes holds potential applications in developing molecular switches, transistors and information storage devices1,2. In particular, tautomerization in porphycenes through double proton transfer process has attracted wide interest in the fields of spectroscopy and photophysics2. The inner hydrogen atoms of porphycene can migrate from one trans isomer to the other equivalent trans isomer through double proton transfer process as shown in Figure 1. Two mechanisms have been proposed for the double proton transfer process: the concerted and the stepwise mechanism3,4. In the concerted double proton transfer process, both proton atoms move to the transition state synchronously in a symmetric way, whereas one proton completes the transfer before the other proton in a stepwise process. Two hydrogen atoms can transfer simultaneously or stepwise depending on the correlation strength between two hydrogen atoms5.
Isotopic substitution has been used to detect the structural properties of molecules and rate constants of reaction kinetics6. Single deuterium substitution in the inner hydrogen of porphycene leads to an asymmetric shape of the molecule. The hydrogen bond may expand or contract because of mass difference between the hydrogen and deuterium atoms. The isotopic substitution introduces a perturbation in the scaffold of porphycene. The question arises that whether asymmetric structure would affect the proton transfer process. Limbach and coworkers reported that the replacement of hydrogen with deuterium will compress both hydrogen bonds, and the cooperative coupling of two hydrogen bonds in porphycene may favor concerted mechanism7, whereas Yoshikawa stated the deuteration would make the stepwise mechanism contribute more than the concerted mechanism8. Experimental techniques, such as force spectroscopy, have been developed to capture tautomerization details in a single porphycene9. However, it is still challenging to determine the atomic details of proton transfer experimentally because of its transient nature.
Theoretical calculations and simulations can act as complementary tools in elucidating the reaction mechanisms of proton transfer. Among different theoretical methods, molecular dynamics (MD) simulations can monitor dynamic motions of each atom, and has been widely used to reveal complex mechanisms in chemical and enzymatic reactions. However, regular MD simulations tend to suffer from insufficient sampling issue, especially when high energy barrier exists in the process of interest. Therefore, enhanced sampling methods have been developed, which include transition path sampling10,11, umbrella sampling (US)12,13, and integrated tempering sampling (ITS)14,15. Combination of different enhanced sampling methods can further increase sampling efficiency16,17,18. To harness the enhanced sampling algorithms in simulating chemical reactions, we have implemented the selective integrated tempering sampling (SITS) method with quantum mechanical and molecular mechanical (QM/MM) potentials recently19. The proposed SITS-QM/MM method combines the advantages from both methods: the SITS method accelerates the sampling and can explore all possible reaction channels without prior knowledge of the reaction mechanism, and QM/MM provides more accurate description of the bond forming and bond breaking process, which cannot be simulated by MM methods solely. The implemented SITS-QM/MM approach has successfully uncovered concerted double proton transfer, uncorrelated and correlated stepwise double proton transfer mechanism in different systems, without pre-defining reaction coordinates19. For porphycene, the stepwise but correlated proton transfer character has been reported19. The hybrid SITS-QM/MM method was used to investigate the isotopic effect in porphycene in our study, and below are the detailed descriptions of the algorithm and protocol of our method.
We have implemented SITS method with hybrid QM/MM potentials. The effective potential of SITS was defined to include the potential energy at different temperatures with the weighting factors nk to cover wider temperature ranges,
<img alt="Equation 1" src="/files/ftp_upload/60040/60040equ01.jpg" />
where, N is the number of canonical terms, &#946;k is the inverse temperature, and nk is the corresponding weighting factor for each canonical component. UE(R) and&#160;UN(R) represent the enhanced and non-enhanced terms in SITS and are defined as,
<img alt="Equation 2" src="/files/ftp_upload/60040/60040equ02.jpg" />
Us, Use and Ue are the potential energy of sub-system, the interaction between sub-system and the environment, and the potential energy of environment. QM/MM potential is expressed as a hybrid summation of three components,
<img alt="Equation 3" src="/files/ftp_upload/60040/60040equ03.jpg" />
where Uqm, Uqm/mm, and&#160;Umm are the internal energy term of the QM subsystem, the interaction energy between the QM and MM regions, and the interaction energy within the MM subsystem, respectively. The&#160;Uqm/mm term can be further divided into three components, which include the electrostatic, van der Waals, and covalent interaction energy terms between the QM and MM atoms,
<img alt="Equation 4" src="/files/ftp_upload/60040/60040equ04.jpg" />
We assign <img alt="Equation 5" src="/files/ftp_upload/60040/60040equ05.jpg" />, and <img alt="Equation 6" src="/files/ftp_upload/60040/60040equ06.jpg" /> into one&#160;Us term in SITS,
<img alt="Equation 7" src="/files/ftp_upload/60040/60040equ07.jpg" />
The full potential of the system was then decomposed into the energy of subsystem Us, the interaction energy between the subsystem and the environment Use, and the energy of environment Ue. For instance, in the system of the present work, the subsystem is the porphycence, and the environment the water.
The PMF profile along a collective variable&#160;&#964;(R) is derived as,
<img alt="Equation 8" src="/files/ftp_upload/60040/60040equ08.jpg" />
The generally used reaction coordinates for each hydrogen transfer of N1&#8722;H1&#183;&#183;&#183; N2 are q1 = (r1&#8722;r2)/2 and q2 = r1 + r2, where r1 is the distance of N1-H1 and r2 is the distance of H1-N2.
The method has been implemented in the QM/MM MD simulation package QM4D20. The complete source code and documentation can be found here: http://www.qm4d.info/.
Generally, the SITS-QM/MM MD simulations involve four steps: pre-equilibrium (pre-sits); optimization nk (opt-sits); production simulation and data analysis.

<table><tbody><tr><td>operating system</td><td>CentOS Linux release 6.0</td><td></td><td></td></tr><tr><td>QM4D software</td><td>http://www.qm4d.info/</td><td></td><td>in-house program</td></tr><tr><td>Computer desktop</td><td>HP</td><td></td><td></td></tr></tbody></table>

isotopic effect in double proton transfer process of porphycene investigated by enhanced qm/mm method

The single deuterium substitution in porphycene leads to an asymmetric molecular geometry, which may affect the double proton transfer process in the porphycene molecule. In this study, we applied an enhanced QM/MM method called SITS-QM/MM to investigate hydrogen/deuterium (H/D) isotope effects on the double proton transfer in porphycene. Distance changes in SITS-QM/MM molecular dynamics simulations suggested that the deuterium substituted porphycene adopted the stepwise double proton transfer mechanism. The structural analysis and the free energy shifts of double proton transfer process indicated that the asymmetric isotopic substitution subtly compressed the covalent hydrogen bonds and may alter the original transition state location.

The single deuterium substitution effect on double proton transfer process in porphycene was examined in the current protocol (Figure 1). The potential energy of QM sub-system and the water during pre-equilibrium and optimization step were checked to make sure the energy has been broadened to a wider energy range (Figure 2). The representative distance and angle changes (Figure 3 and Figure 4), and the ...

Watch this Scientific Journal Video about Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method at JoVE.com

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

A protocol that uses enhanced QM/MM method to investigate the isotopic effect on the double proton transfer process in porphycene is presented here.

The single deuterium substitution in porphycene leads to an asymmetric molecular geometry, which may affect the double proton transfer process in the ...

isotopic-effect-double-proton-transfer-process-porphycene

Research

JoVE Journal

Chemistry

6.2K Views.  Chinese Academy of Sciences. Een verbeterde QM/MM-methode overwint eenvoudige problemen in de normale QM/MM-simulatie. De Six QM/MM-methode versnelt de geselecteerde assemblage voor het QM-gebied en kan de chemische reactietrajecten vastleggen die resulteren in het definiëren van reactiecoördinaten. Met dit protocol hebben we met succes een chemische reactietrajecten van dubbele protonoverdracht en een onthult een deuterium substitutie-effect op een overdrachtsmechanisme van herkomst in water.Het protocol kan wo...

Video: Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

Engineering

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we demonstrate an optical spectroscopy method that is capable of resolving spectral features beyond that of the spin fine structure and homogeneous linewidth of single quantum dots (QDs) using a standard, easy-to-use spectrometer setup. This method incorporates both laser and photoluminescence spectroscopy, combining the advantage of laser line-width limited resolution with multi-channel photoluminescence detection. Such a scheme allows for considerable improvement of resolution over that of a common single-stage spectrometer. The method uses phonons to assist in the measurement of the photoluminescence of a single quantum dot after resonant excitation of its ground state transition. The phonon&#39;s energy difference allows one to separate and filter out the laser light exciting the quantum dot. An advantageous feature of this method is its straight forward integration into standard spectroscopy setups, which are accessible to most researchers.

The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup.

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we ...

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

Measurement of Ultrafast Vibrational Coherences in Polyatomic Radical Cations with Strong-Field Adiabatic Ionization

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the wavelength of the strong-field ionizing pump pulse from the commonly used 800 nm into the near-infrared (1200-1600 nm), the contribution of adiabatic electron tunneling to the ionization process increases relative to multiphoton absorption. Adiabatic ionization results in predominant population of the ground electronic state of the ion upon electron removal, which effectively prepares a coherent vibrational state (&#34;wave packet&#34;) amenable to subsequent excitation. In our experiments, the coherent vibrational dynamics are probed with a weak-field 800 nm pulse and the time-dependent yields of dissociation products measured in a time-of-flight mass spectrometer. We present the measurements on the molecule dimethyl methylphosphonate (DMMP) to illustrate how using 1500 nm pulses for excitation enhances the amplitude of coherent oscillations in ion yields by a factor of 10 as compared to 800 nm pulses. This protocol may be implemented in existing pump-probe setups through the incorporation of an optical parametric amplifier (OPA) for wavelength conversion.

We present a protocol for probing ultrafast vibrational coherences in polyatomic radical cations that result in molecular dissociation.

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the ...

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they are utilized in a wide variety of applications. In most studies employing photoswitches, several important photophysical properties such as maximum wavelengths of absorption and emission, molar attenuation coefficient, fluorescence lifetime, and photoisomerization quantum yield are carefully determined to investigate their electronic states and transition processes. However, measurement of the photoisomerization quantum yield, the efficiency of photoisomerization with respect to the absorbed photons, in a typical laboratory setting is often complicated and prone to error because it requires the implementation of rigorous spectroscopic measurements and calculations based on an appropriate integration method. This article introduces a set of procedures to measure the photoisomerization quantum yield of a bistable photoswitch using a photochromic hydrazone. We anticipate that this article will be a useful guide for the investigation of bistable photoswitches that are being increasingly developed.

Photoisomerization quantum yield is a fundamental photophysical property that should be accurately determined in the investigation of newly developed photoswitches. Here, we describe a set of procedures to measure the photoisomerization quantum yield of a photochromic hydrazone as a model bistable photoswitch.

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they ...

A Technical Guide for Performing Spectroscopic Measurements on Metal-Organic Frameworks

Metal-organic frameworks (MOFs) offer a unique platform to understand light-driven processes in solid-state materials, given their high structural tunability. However, the progression of MOF-based photochemistry has been hindered by the difficulty in spectrally characterizing these materials. Given that MOFs are typically larger than 100 nm in size, they are prone to excessive light scatter, thereby rendering data from valuable analytical tools like transient absorption and emission spectroscopy nearly uninterpretable. To gain meaningful insights of MOF-based photo-chemical and physical processes, special consideration must be taken toward properly preparing MOFs for spectroscopic measurements, as well as the experimental setups that garner higher quality data. With these considerations in mind, the present guide provides a general approach and set of guidelines for the spectroscopic investigation of MOFs. The guide addresses the following key topics: (1) sample preparation methods, (2) spectroscopic techniques/measurements with MOFs, (3) experimental setups, (3) control experiments, and (4) post-run stability characterization. With appropriate sample preparation and experimental approaches, pioneering advancements toward the fundamental understanding of light-MOF interactions are significantly more attainable.

Here, we use a polymer stabilizer to prepare metal-organic framework (MOF) suspensions that exhibit markedly decreased scatter in their ground-state and transient absorption spectra. With these MOF suspensions, the protocol provides various guidelines to characterize the MOFs spectroscopically to yield interpretable data.

Metal-organic frameworks (MOFs) offer a unique platform to understand light-driven processes in solid-state materials, given their high structural ...

Molecular Orbital (MO) Theory

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes featuring the ligand 1,1&#39;-bis(diphenylphosphino)ferrocene (dppf): M(dppf)Cl2, where M = Ni or Pd. While both of these transition metal complexes are 4-coordinate, they exhibit different geometries at the metal center. Using molecular orbital (MO) theory in conjunction with 1H NMR and Evans method, we will determine the geometry of these two compounds.

Molecular Orbital MO Theory: Predicting Geometry of Transition Metal Complexes

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes ...

Education

JoVE Science Education

Inorganic Chemistry

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Source: Winkelaar, G., et al. <a href="https://app.jove.com/v/58537">Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST).</a> J. Vis. Exp. (2018).
This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between 15N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.

Source: Winkelaar, G., et al. Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

Double Resonance Techniques: Overview

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or radiofrequency pulses to manipulate and observe two distinct nuclear spins. One important application of double resonance is spin decoupling, which selectively suppresses coupling with one type of nucleus while observing the NMR signal from another nucleus, simplifying the spectrum and enhancing resolution.
Spin decoupling is usually achieved by irradiating the sample atoms with a suitable radio frequency (rf) pulse sequence, effectively eliminating all the coupling with one nuclide. This simplifies the observed spectrum, making it easier to analyze and understand the relationships between different nuclei.
Double resonance experiments can be classified as heteronuclear or homonuclear, depending on whether the two sets of nuclei are of different isotopes or the same isotope, respectively. They can also be selective or nonselective, depending on whether the irradiating frequency covers only a portion or all of the resonance frequencies.
In nonselective heteronuclear decoupling, samples are exposed to an appropriate radiofrequency range to remove all coupling with one nuclide. However, increased external field strengths necessitate more powerful irradiation across broader frequency ranges. In certain instruments, continuous irradiation creates enough heat to cause damage to thermally sensitive samples.
To overcome these problems, modern methods using a series of pulses and exact timing delays, like J-modulated spin echo pulse sequences, can be employed to eliminate or adjust coupling effects within the spectrum.
For example, the attached proton transfer (APT) experiment applies a J-modulated spin echo pulse sequence to focus on the phase of detected carbon signals. Carbon atoms attached to an even number of protons exhibit positive signals in the spectrum, while those bonded to an odd number of protons appear as negative signals.
It utilizes a combination of a 180&#176; proton pulse and broadband decoupling to simplify spectral interpretation and assign multiplicities. The 180&#176; pulse is applied to the protons during the APT experiment. Its role is to refocus the spin coupling evolution that naturally occurs due to interactions between protons and carbons.
Specifically, after the 90&#176; pulse creates transverse magnetization, the 180&#176; pulse ensures that the effects of coupling are frozen during the initial period where the decoupler is turned off. This means the chemical shifts evolve only during the time 1/J&#8203; (the coupling constant period) after the first 180&#176; pulse.
By carefully timing this pulse sequence, the experiment isolates carbon multiplicities by utilizing the J-coupling between carbons and their attached protons. After the coupling evolution period (1/J&#8203;), broadband decoupling is turned on. This removes the J-coupling signals, collapsing multiplets into single peaks for each carbon resonance.
The decoupling ensures that carbon signals are free of splitting, allowing for a clear differentiation of signals based on phase (positive or negative) rather than splitting patterns.
The APT technique selectively enhances carbon signals, providing valuable information about carbon-proton connectivity, the number of attached hydrogen atoms, and the overall molecular structure.

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or ...

JoVE Core

Analytical Chemistry

Unit 1

Advanced Nuclear Magnetic Resonance Spectroscopy

KEY TERMS AND CONCEPTS

&sup1;H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first. However, the splittings can be applied in any order, and the same result is obtained.
In propyl bromide, the protons M are coupled to three methyl and two methylene protons, with the coupling constants JAM and JMX. The splitting diagram predicts a quartet of triplets with 12 lines, which would all be visible in the spectrum if JAM &#62;&#62; JMX. However, if JAM &#8773; 2 JMX, some peaks overlap, their intensities are added, and 9 peaks are seen. When JAM &#8773; JMX, as is the case in propyl bromide, 6 peaks are observed. As a result, the relative magnitudes of the J values can cause deviations from the number of peaks and relative intensities predicted by the n+1 rule and Pascal&#39;s triangle.

1H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one ...

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video