Evaluation of synthetic linear motor-moleculeactuation energeticsBranden Brough†‡, Brian H. Northrop§¶, Jacob J. Schmidt�, Hsian-Rong Tseng††, Kendall N. Houk§‡‡,J. Fraser Stoddart§¶‡‡, and Chih-Ming Ho†‡,‡‡

Departments of †Mechanical and Aerospace Engineering, §Chemistry and Biochemistry, �Bioengineering, and ††Molecular and Medical Pharmacology,‡Institute for Cell Mimetic Space Exploration, and ¶California Nanosystems Institute, University of California, Los Angeles, CA 90095

Edited by Julius Rebek, Jr., The Scripps Research Institute, La Jolla, CA, and approved April 21, 2006 (received for review November 14, 2005)

By applying atomic force microscope (AFM)-based force spectros-copy together with computational modeling in the form of mo-lecular force-field simulations, we have determined quantitativelythe actuation energetics of a synthetic motor-molecule. This mul-tidisciplinary approach was performed on specifically designed,bistable, redox-controllable [2]rotaxanes to probe the steric andelectrostatic interactions that dictate their mechanical switching atthe single-molecule level. The fusion of experimental force spec-troscopy and theoretical computational modeling has revealedthat the repulsive electrostatic interaction, which is responsible forthe molecular actuation, is as high as 65 kcal�mol�1, a result that issupported by ab initio calculations.

computational modeling � force spectroscopy � molecular motors �switchable rotaxanes

Molecular motors have recently garnered considerable in-terest within the domains of microsciences and nano-

sciences (1, 2). Harnessing the ability to selectively, coopera-tively, and repeatedly induce structural changes in moleculesmay hold the promise of engineered systems that operate withthe same complexity, elegance, and efficiency as biologicalmotors function in the human body. In natural systems, it hasbecome apparent that both macro and micro processes areinitiated and controlled by nanoscale molecular motors (3). Forexample, myosin and kinesin are associated with muscle con-traction and intracellular trafficking, respectively, and haverecently found their ways into engineered devices (4, 5). More-over, initial work has been performed that demonstrates theability of natural nanoscale molecular motors to power micro-fabricated systems (6).

Synthetic motor-molecules (7–11), which are designed to excelwhere their biological counterparts fall short, also have beeninvestigated. Whereas devices powered by biological moleculesrequire (4–6) chemical diffusion for actuation stimulus, syn-thetic molecules have been shown (2, 9) to operate with a varietyof different stimuli, thereby lending much greater flexibility toa particular system’s design. Moreover, a synthetic nanoscaleactuating molecule carries with it an inherent ability to bemodified and optimized precisely for a specific task.

Switchable, bistable rotaxanes (2, 9), compounds comprised ofa dumbbell-shaped component containing two different recog-nition sites for an encircling ring-shaped component, showparticular promise as molecular actuators, given their ability toundergo controllable, reversible mechanical switching with theappropriate chemical, electrochemical, or photochemical stim-ulus in solution. Toward the goal of device applications, switch-ing has been shown to operate in condensed phases such as in apolymer electrolyte gel (12), on a self-assembled monolayer(SAM) (13), on the solid supports of engineered systems (14),and in molecular switch tunnel junctions (15). Bistable rotaxanesbenefit from their synthesis being highly modular, a virtue thatallows for a considerable degree of flexibility in their design.Recently, the use of linearly actuating rotaxanes in microfabri-cated molecular devices has been demonstrated (16) where the

redox-controlled switching of palindromic, doubly bistable [3]ro-taxane molecules self-assembled on gold-coated microcantile-vers was shown to generate enough surface stress to bend themreversibly and repeatedly. The successful operation of thesefundamental devices demonstrates the potential for such rotax-anes to form the foundation on which a bottom-up fabricatedactuator paradigm based at the nanoscale could be built.

In addition to the actuation characteristics of syntheticmotor-molecules and their potential to be redesigned, thepossibility of producing forces greater than their naturalcounterparts (16, 17) is a distinct benefit. Not only is thisassertion critical to the pursuit of artificial motor-moleculeengineering, but its verification is essential to any system’sdesign. Force spectroscopy using an atomic force microscope(AFM) has been demonstrated to be an effective probe (18) ofthe strengths of various covalent bonds (19) as well as thenoncovalent bonding associated with the biotin–streptavidincomplex (20) and hydrophobic cyclodextrin-based complexesin water (21). In addition, Gaub and coworkers (22) have usedAFM force spectroscopy to investigate the force generated bypolyazobenzenes operating as molecular motors.

Along that same vein, we have expanded this field ofresearch to include the probing of steric and electrostaticinteractions within highly complex synthetic molecules. In sodoing, we have been able to quantify the power stroke of theswitchable, bistable [2]rotaxane R4� (Fig. 1), which can func-tion as a linear motor-molecule by harnessing the redox-controlled mechanical shuttling of its ring along its dumbbell.Although it features the same actuation mechanism as thedoubly bistable [3]rotaxane recently used to drive a set ofmicrocantilevers (16), R4� was specifically designed (i) with athioctic acid tethered to its ring component for attachment toa gold AFM tip and (ii) with a hydroxymethyl group on one ofthe stoppers of its dumbbell for attachment (Scheme 1b) toSiO2 via covalently bound monolayers of isocyanatopropyllinkers (23). This dual functionality allows for AFM forcespectroscopy studies to probe the steric and electrostaticinteractions present in the ground (Fig. 1a) and oxidized (Fig.1b) states of R4�. AFM probing of molecules in neat ethanol(EtOH) will measure the repulsive steric interactions (Fig. 2a)between the cyclobis(paraquat-p-phenylene) (CBPQT4�) ringand the diisopropylphenyl ether stopper and serves as acontrol. The probing of the R6� molecules in an oxidizingsolution will measure the repulsive force (Fig. 2b) between theCBPQT4� ring and an oxidized tetrathiafulvalene (TTF2�),which is responsible for molecular actuation. In conjunction

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: AFM, atomic force microscope�microscopy; CBPQT4�, cyclobis(paraquat-p-phenylene); TTF, tetrathiafulvalene; DNP, 1,5-dioxynaphthalene; SAM, self-assembledmonolayer.

‡‡To whom correspondence may be addressed. E-mail: houk@chem.ucla.edu, stoddart@chem.ucla.edu, or chihming@seas.ucla.edu.

© 2006 by The National Academy of Sciences of the USA

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with computational chemistry, these experiments enable athorough quantitative evaluation of the actuating energetics ofa single-molecule motor. For further details, see SupportingMaterials and Methods, Tables 1 and 2, Figs. 5–16, and Scheme2, which are published as supporting information on the PNASweb site.

Results and DiscussionThe routes used to synthesize the dumbbell-shaped compound3 and the bifunctional [2]rotaxane R4� are shown in Scheme1a, and that used to covalently attach [2]rotaxane R4� to SiO2wafers is shown in Scheme 1b. The concentration of R4� waskept dilute, �1 per 100 nm2, to promote the observation ofsingle-molecule events. Compiled AFM force probe data aresummarized (Fig. 3) in the form of histograms, which wereanalyzed by using commercial peak-finding and curve-fittingalgorithms. The first histogram (Fig. 3a) summarizes thecontrol experiments on unoxidized single molecules and re-veals that the most probable molecular rupture force occurs at74 pN. In these control experiments, any measured force peakstems from rupture either within the molecule or its linkagesto the AFM tip or the substrate. As in any structural system,failure will occur at the weakest point. Published reportsindicate that this ruptured 74-nN mechanical barrier is con-siderably weaker than any of the covalent bonds in the system,revealing that the failure occurs as the ring passes over thestopper, a process referred to as ‘‘deslipping’’ (24). To ourknowledge, all experimental and theoretical data at compa-rable experimental conditions indicate that the rupture of anycovalent bond requires �1 nN (see Table 2).

The second histogram (Fig. 3b) summarizes the results offorce probing in the presence of a 10�4 M Fe(ClO4)3 oxidizingenthanolic solution, which has been shown to induce ringtranslation to the 1,5-dioxynaphthalene (DNP) site as a resultof electrostatic repulsion from the TTF2� site (14). Whereasin the unoxidized situation the histogram reveals a singularforce, this histogram indicates that there are two distinct forcebarriers, one at 66 pN (O1, solid blue curve) and the other at145 pN (O2, solid green curve). We believe that the lower forceregime is caused by the probing of single molecules that havenot undergone redox-controlled switching despite the oxidiz-ing environment. This belief is supported by comparing thepeak value from the unoxidized histogram (U, dashed bluecurve) with those from the oxidized histogram (Fig. 3b). Thepeak of the curve for the unoxidized molecules coincides wellwith the lower force peak curve from the oxidation experi-ments. This interpretation also is supported by earlier studies

Fig. 1. Structural formula and schematic representation of bistable [2]ro-taxane R4�, in which an electron-poor cyclobis(paraquat-p-phenylene) (CB-PQT4�) ring is confined to a dumbbell containing two electron-rich recogni-tion sites, tetrathiafulvalene (TTF) and 1,5-dioxynaphthalene (DNP), by thepresence of bulky 2,6-diisopropylphenyl ether stoppers at each end. One ofthese stoppers, the one closer to DNP, carries a hydroxymethyl group on its4-position for subsequent attachment to silicon wafers. (a) The CBPQT4� ring,which carries a tether terminated by a thioctic acid ester for attachment to agold-coated AFM tip, displays a stronger interaction with TTF than with DNPand thus resides selectively (16) on the former. (b) Chemical oxidation of TTFto TTF2� results in a strong charge–charge repulsion between the CBPQT4�

ring and TTF2�, a situation that causes the CBPQT4� ring to shuttle to DNP (9,12–16) in the oxidized [2]rotaxane R6�. The mechanical movement of theCBPQT4� ring from TTF2� to DNP resembles the power stroke of a linear motor.Reduction of the TTF2� to its neutral state (TTF) prompts the ring to shuttleback thermally from a metastable state to its ground state where it encirclesTTF in a manner reminiscent of the diffusive stroke of a linear motor. Thisexternally controllable shuttling process and bistability allows [2]rotaxane R4�

to function as a nanoscale linear motor given its ability to contract and expandreversibly in the presence of a chemical oxidant or reductant, respectively.

Scheme 1. Reaction schemes illustrating the synthesis of dual recognition [2]rotaxane R4� (a) and the attachment of R4� to SiO2 wafers via covalently boundisocyanatopropyl tethers (b).

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at a lower concentration of oxidant (10�6 M), which yieldedresults that were not easily distinguishable from those obtainedfor the unoxidized molecule with respect to peak-force mag-nitude and well defined peak distribution (see Fig. 16). Analternative explanation could be that electron transfer be-tween the Fe(ClO4)3 oxidant and TTF occurs within the timeframe of individual pulling experiments, resulting in an equi-librium of neutral and oxidized TTF, which would give rise toboth unoxidized as well as oxidized force data. We believe,however, that results of probing an oxidant concentration of10�6 M support the former explanation over the latter.

Although the second force regime at 145 pN has clearlyemerged as a result of the presence of oxidant, it is moreimportant to examine how these results enhance our understand-ing of the bistable rotaxane’s mechanical switching behavior.Previous experimental results have revealed that Fe(ClO4)3

oxidizes the TTF unit to TTF2�, resulting in concomitant

shuttling of the CBPQT4� ring to the DNP unit (9, 12–16) evenwhen immobilized on a surface or between surfaces in a mannernot dissimilar to that present in the AFM-based system. There-fore, the new higher-energy barrier can be ascribed to theelectrostatic repulsion between TTF2� and CBPQT4� or cova-lent bond rupture. Given that the measured peak rupture forcein the presence of the oxidant is still an order of magnitude lowerthan those associated with the rupture of relevant covalent bonds(see Table 2), it is clear that the second force regime present inthe experiments on the oxidized molecules emerges as a result ofthe newly generated repulsive electrostatic barrier, which isultimately responsible for the molecule’s actuation.

Although the results indicate that the 145-pN force peak is ameasurement of the electrostatic repulsion between the bistablerotaxane’s CBPQT4� ring at TTF2� dication, and therefore agood indication of the maximum switching force producibleunder load by the oxidized molecule, force spectroscopy mea-

Fig. 2. Schematics of AFM force spectroscopy of [2]rotaxane R4�. (a) Ground-state probing in the absence of oxidant. (i) [2]Rotaxane R4� confined to asilicon surface and attached to a gold AFM tip via two gold–sulfur bonds. (ii)Retraction of the AFM tip from the surface pulls the CBPQT4� ring away fromthe TTF unit and toward the bulky stopper. (iii) Continued retraction causesthe CBPQT4� ring to pass over the stopper (deslip) at which point the forcerequired to overcome this physical barrier is measured. (b) Oxidized-stateprobing performed in the presence of 10�4 M Fe(ClO4)3 in EtOH. (iv) Theattachment of a gold AFM tip to [2]rotaxane R6� whose ring is now located onthe lower DNP unit as a result of the chemically induced oxidation of the TTFunit to TTF2� and subsequent electrostatic repulsion of CBPQT4�. (v) Retrac-tion of the AFM tip forces the tetracationic CBPQT4� ring toward the dicationicTTF2� despite the electrostatic repulsion between the two moieties. The AFMwill measure the repulsive force between the ring and the TTF2� that wasoriginally responsible for the molecule’s actuation. (vi) Continued retractionof the AFM tip enables the CBPQT4� ring to overcome the electrostatic barrierimposed by TTF2�, resulting in rupture of the mechanical bond posed by thestopper. When measuring the higher-energy electrostatic repulsion of theoxidized molecule, the subsequent deslipping event will not be measuredbecause of the dynamic instability of the AFM probe.

Fig. 3. Force probe data obtained during single-molecule investigations ofR4� and R6�. (a) Histogram of compiled AFM force spectroscopy data ofground-state [2]rotaxane R4� obtained in an EtOH solution with a loading rateof 6 pN�s�1. The dashed blue curve (U) is obtained from commercial peakfinding and curve-fitting algorithms and indicates a most probable ruptureforce of 74 pN. (b) Histogram of AFM force spectroscopy data obtained (forR6�) in an oxidizing solution of 10�4 M Fe(ClO4)3 in EtOH (loading rate of 6pN�s�1) displays two different force regimes, indicating two distinct forcebarriers having most probable rupture forces of 66 pN (O1, solid blue curve)and 145 pN (O2, solid green curve). The magnitude of the solid blue curvematches well with the dashed blue curve superimposed from a, indicating thatnot all molecules are oxidized. The solid green curve corresponds to theemergence of a new, larger barrier, which is a direct measure of the electro-static repulsion between the TTF2� dication and the CBPQT4� tetracationresponsible for molecular actuation.

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surements require loading-rate analysis for a complete under-standing of the energy barrier that was investigated. A directcorollary between force and energy through distance cannot beestablished because the work done by an AFM’s tip distorts thesystem’s energy landscape. According to Evans (25), forcesmeasured by force spectroscopy will be altered by thermalfluctuations as a function of the probe’s loading rate such thatthe most probable measured force, f*, is defined as

f* �kBTx�

�ln�k s�� � ln� toff x�

kBT � � , [1]

where kBT is the thermal energy, x� is the bond length, toff is thelifetime of the bond under no external force, and ks and � are thespring constant and retract velocity of the AFM probe, respec-tively. Previously, only with a detailed loading-rate analysis thatspans several orders of magnitude can interactions be described(20, 25) by x� and toff. Although rotaxanes do operate reversibly(2, 9, 12–16), the irreversible rupture of molecules required bythis experiment (see Loading Rate Analysis in Dynamic ForceSpectroscopy in Supporting Materials and Methods) prohibits sucha study. However, by augmenting the AFM data with moleculardynamics simulations (see Fig. 10), which describe (Fig. 4) theground-state energy profile of R4� as a function of the ring’sposition on the dumbbell, an accurate description of the physicalswitching mechanism can be formulated.

toff is the time at which a bond, described by an energy barrierwith Arrhenius dependence, spontaneously ruptures (25, 26).Conversely, the Arrhenius equation relates the rate, kr, of a

first-order chemical process to the activation energy, Ea, asso-ciated with that process

kr � Ae�� Ea

RT� �1

toff,

where A is a constant preexponential factor and R is the molargas constant. By substituting this relationship into Evans’ equa-tion, a new relationship arises that describes the energy differ-ence between two rupture events at a single loading rate

Ea1� Ea2

�RkB

� f1*x�1� f2* x�2

� kBT lnx�1

x�2

� .

By using the measured force spectroscopy values of 66 pN for thedeslipping barrier under nonoxidative conditions and 145 pN forthe oxidant-induced electrostatic repulsion barrier, along with x�

values, produced from molecular simulations, of 8.6 Å for thedeslipping of the CBPQT4� ring over the bulky stopper and 13.0Å as the distance necessary for the CBPQT4� ring to travel fromthe DNP recognition site to the TTF2� dication, the differencein interaction energies can be calculated as 19 kcal�mol�1.Although the value of 66 pN was generated in the presence ofoxidant, this value was used instead of the 74-pN value producedin the nonoxidizing environment because of the availability ofsignificantly more experimental data points. It should be notedthat the use of the 74-pN value only changes the results of thisstudy by 1 kcal�mol�1. By combining the 19 kcal�mol�1 differencein oxidative and nonoxidative interaction energies with thetheoretically determined value of 46 kcal�mol�1 for the energyassociated with ground-state deslipping (Fig. 4), a final value of65 kcal�mol�1 was obtained, representing the amount of repul-sive actuation energy between CBPQT4� and TTF2� producedby the oxidation of R4� (Fig. 4). As measured by this approach,65 kcal�mol�1 represents the upper limit of the total actuationenergy. In the experimental set-up, the retraction of the AFM tipforces the CBPQT4� ring into the most energetically unfavorableconformation possible with respect to the TTF2�, thus maxi-mizing the electrostatic repulsion between the tetracationic anddicationic moieties.

This value, although seemingly low for the repulsion of sixpositive charges confined within a radius of �5 Å, is stronglysupported by ab initio calculations (see Model System andCalculations of Electrostatic Repulsion in Supporting Materialsand Methods). Single-point quantum mechanical calculationswere performed in EtOH by using atomic coordinates from thecrystal structures of TTF, [TTF][ClO4]2, [CBPQT][PF6]4, and[CBPQT�TTF][PF6]4 as well as on five model systems for thehexacationic [CBPQT�TTF][Cl]6 complex (see Fig. 11). Therepulsive energy within this hexacationic complex was calculatedto be 70.6–76.0 kcal�mol�1. By using this value and that producedby the molecular force field simulations, computational analysispredicts that the electrostatic barrier resulting from the TTF2�

dication exceeds the steric barrier of the stopper by 25–30kcal�mol�1. This entirely theoretical value compares well withthe value of 19 kcal�mol�1 produced experimentally by means ofAFM force spectroscopy. The similarity between the purelyindependent ab initio calculations, which only take into accountthe interaction between CBPQT4� and TTF2�, and experimen-tal AFM results lends support to the conclusion that single-molecule rupture events are being measured accurately as 65kcal�mol�1. Perhaps even more significant is the fact that thisresult lends validity to the methods developed in this work usedto analyze force spectroscopy experiments at a single loadingrate with the help of molecular force-field simulations.

Employing a combination of AFM force spectroscopy andmolecular simulations, we have determined and verified that the

Fig. 4. The ground-state energy profile obtained from molecular dynamicsand mechanics simulations. The x axis indicates the position of the CBPQT4�

ring along the linear dumbbell (in Å), and the y axis displays relative energiesof the different CBPQT4� ring co-conformers (in units of kcal�mol�1). Thesimulations correctly predict the co-conformation of the CBPQT4� ring sta-tioned on TTF to be the lowest-energy state and therefore the most favorableone. The energy barrier that exists when the CBPQT4� ring passes over eitherof the bulky stoppering units (deslipping) is calculated to be 46 kcal�mol�1. Acombination of theoretical modeling and experimental AFM measurementspredict oxidation of [2]rotaxane R4� to result in a 65-kcal�mol�1 repulsionbetween the CBPQT4� tetracation and the TTF2� dication. This result is sup-ported by quantum mechanical calculations. The dashed green line is aschematic representation of what the full, oxidized energy profile may looklike, but it is not quantitative.

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energy output of a single synthetic motor-molecule operatingwithin an engineered environment is 65 kcal�mol�1. Assumingthat the total energy available to biological systems from thehydrolysis of ATP (14.4 kcal�mol�1 at physiological concentra-tions) (1) is 100% efficiently converted, rotaxanes still outper-form nature by �4.5 times. Furthermore, the synthetic origin ofswitchable, bistable rotaxanes allows them to be designed andredesigned according to a particular application’s requirementsas well as from the feedback obtained through characterizationstudies. Molecular-level characterization is a critical step in theiterative process that may eventually bring about viable engi-neered systems that extend far beyond mere scientific novelties.Meanwhile, the promise of up to 65 kcal�mol�1 of energyproduction from bistable rotaxanes and its family of syntheticmolecules will always headline systems that have the potential toexceed anything seen in nature.

Materials and MethodsGeneral Methods. Chemicals were purchased from Aldrich andused as received. Silicon wafers were purchased from SiliconQuest International (Santa Clara, CA). The TTF-containingtosylate 1 (27), the DNP-containing diol 2 (28), and the dication4�2PF6 (27) were all prepared according to procedures describedin the literature. Solvents were dried following methods de-scribed in the literature (29). All reactions were carried outunder an anhydrous argon�nitrogen atmosphere. Thin-layerchromatography (TLC) was performed on aluminum sheetscoated with silica-gel 60F (Merck 5554). The plates were in-spected by UV light and, if necessary, developed in I2 vapor.Column chromatography was carried out by using silica-gel 60(Merck 9385; 230–400 mesh). Melting points were determinedon a melting point apparatus (Model 9100, Electrothermal,Dubuque, IA) and are uncorrected. All 1H and 13C NMR spectrawere recorded on either (i) an ARX500 (500 and 125 MHz,respectively) or (ii) an Avance500 (500 and 125 MHz, respec-tively) (both from Bruker, Billerica, MA) by using residualsolvent as the internal standard. Samples were prepared by usingCD3COCD3 or CD3CN purchased from Cambridge IsotopeLaboratories (Cambridge, MA). All chemical shifts are quotedby using the � scale, and all coupling constants are expressed inhertz. Matrix-assisted laser desorption ionization spectra(MALDI) were recorded on a instrument from PerSeptiveBiosystems (Framingham, MA). Electrospray mass spectra(ESI) were measured on a ProSpec triple focusing mass spec-trometer (VG Analytical, Manchester, U.K.).

Synthesis of Dumbbell-Shaped Compound 3 and [2]Rotaxane R�4PF6. AMeCN solution of the tosylate 1 (27), the diol 2 (28), K2CO3,LiBr, and 18C6 was heated under reflux for 16 h. After work-up,the crude product was subjected to column chromatography(SiO2:EtOAc�hexane, 1:1) to give 3 as a yellow solid in 82%yield.

The synthesis of R4� was completed by stirring a solution of3, 1,4-bis(bromomethyl)benzene, and the dication 4�2PF6 (27) inanhydrous dimethylformamide (DMF) at room temperature for10 d. The reaction mixture then was subjected directly to columnchromatography (SiO2), and unreacted 3 was recovered withMe2CO, whereupon the eluent was changed to Me2CO:NH4PF6(1.0 g of NH4PF6 in 100 ml of Me2CO), and the green bandcontaining the [2]rotaxane R�4PF6 was collected. After removalof solvent, H2O was added, and the resulting precipitate wascollected by filtration to afford 57% yield of [2]rotaxane R�4PF6as a green solid.

Attachment of R4� to SiO2. A SAM of covalently bound isocyana-topropyl tethers (23) was formed by vapor deposition of 3-(triethoxysilyl)propyl isocyanate in PhMe onto BOE andpiranha-treated 1 cm2 SiO2 wafers. R4� then was covalently

linked to the tethers upon reaction of its terminal alcohol withthe isocyanate of the SAM. Unreacted isocyanates were cappedwith MeOH. Surface coverage and quality were evaluatedthrough hydrophilicity and homogeneity studies. Freshly pre-pared SAMs were stored under Ar(g) in glass vials until use.

Force Spectroscopy. AFM-based dynamic force spectroscopy stud-ies were performed at standard temperature and pressure witha Multimode scanning probe microscope with a Nanoscope 3Acontroller and Pico Force system (Digital Instruments�VeecoProbes, Santa Barbara, CA), both in degassed 200 proof EtOHand in the presence of an oxidizing solution of 10�4 M Fe(ClO4)3in EtOH, which is consistent with previous work that demon-strated mechanical molecular switching within highly packedrotaxane-based thin films (14). Samples were exposed to theoxidant for no less than 10 min to ensure oxidation of the surfacebound molecules but no more than 90 min to guarantee theintegrity of the SAM. Control experiments for ground-statemolecule investigation were conducted in the presence of pureEtOH. The nonaqueous solvent allowed the molecules to beexposed to a plasma-cleaned, Au-coated AFM tip for thiol bondformation. The bio-lever probe tip (Asylum Research, SantaBarbara, CA) was lowered to the surface and held at thatposition for 3 s at a force of 1.0–1.2 nN and then raised at a rateof 1.05 �m�s�1. This procedure resulted in an �10% probabilityof specific bond formation, thereby minimizing the probability ofmultiple molecule binding events. Specific bond formation wasassumed to have taken place when a measurable rupture forceoccurred while the tip was no longer in contact with thesubstrate. Each AFM probe had an average spring constant of 6pN�nm�1 leading to a loading rate of �6 nN�s�1. Individualspring constants were measured by using the on-board thermal-tuning program, and data points were analyzed with theirassociated probes’ spring constant values. Experiments wereconducted with new samples, fresh solution, and new probes andcontinued until the tip surface was saturated, as indicated by thelack of any binding events despite a change in probe position.Data were plotted in histograms prepared by using ORIGINPROsoftware (OriginLab, Northampton, MA). Different bin sizes didnot affect the analysis or conclusions drawn from the data. Peakswere identified and analyzed by using the Lorentzian functionthat best fit our data. In the case of the multiple peak data, theLevenberg–Marquardt algorithm was used in association withthe Lorentzian function.

Molecular Force-Field Simulations of R4�. A ground-state energyprofile representing the relative energies of the different co-conformations of R4� as the CBPQT4� ring is moved along thedumbbell was generated by using the program MAESTRO V3.0.038(30) with the AMBER* force field (31) and GB�SA solventmodel (32) for CHCl3. Electrostatic point charges were calcu-lated at the HF�6-31G* level by using the program GAUSSIAN 03(33) and fit to each atom. To model the ring’s movement alongthe dumbbell, a ‘‘dummy’’ atom was placed at a fixed distancefrom the model system and constraints were used to ‘‘pull’’ theCBPQT4� ring along the linear dumbbell in specified increments(see Fig. 10). A 50-ps molecular-dynamics simulation (1.5-fs timestep) at a simulation temperature of 500 K, followed by energyminimization of 200 randomly selected co-conformations, wasused for co-conformational searching at each fixed distancealong the dumbbell reaction coordinate. The output of each stepthen was used as the input for the next. Energies were normal-ized relative to the overall lower-energy co-conformer of R4�,which corresponds to the CBPQT4� ring encircling the TTF unit.The resulting energy differences (�E) plotted against CBPQT4�

ring position gave the ground-state energy profile of R4�. Thesame method also was applied to a series of single-station[2]rotaxanes containing stoppers of different sizes (see Fig. 8) as

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well as a degenerate [2]rotaxane containing two DNP recogni-tion units and compared with experimental results to access theviability of the molecular modeling procedure. In the case of thedegenerate [2]rotaxane, modeling predicted a shuttling barrierof 19 kcal�mol�1, which is within 3 kcal�mol�1 of experimentalvalue of 16 kcal�mol�1 (34).

Model System and Calculations of Electrostatic Repulsion BetweenCBPQT4� and TTF2�. Quantum mechanical calculations were usedto theoretically examine the electrostatic repulsion between theCBPQT4� tetracation and the TTF2� dication that is responsiblefor redox-controlled mechanical actuation of R4�. Single-pointquantum-mechanical calculations were performed at the HF�6-31�G* level by using the program GAUSSIAN 03 (33). Geom-etries of TTF (35), [TTF][ClO4]2 (36), [CBPQT][PF6]4 (37), andthe [CBPQT�TTF][PF6]4 complex (38) were taken from x-raycrystal data (see Fig. 11). To minimize computational cost,all counterions present in crystal structures of charged specieswere replaced with Cl� anions. As a result of its energeticinstability, no crystal data exists for the hexacationic[CBPQT�TTF][PF6]6 complex. Therefore, five model systems

for the hexacationic [CBPQT�TTF][Cl]6 complex were con-structed by using the crystal structure of [CBPQT�TTF][PF6]4(38), with PF6

� counterions replaced by Cl� and two additionalcounterions to balance the charge of oxidized TTF. The twoadditional counterions were positioned within the most elec-tropositive regions of the complex as determined by electrostaticpotential calculations (see Fig. 12 and 13). The five modelsystems differed slightly in their positioning of the two newcounterions to produce a range of values for the repulsionbetween [CBPQT][Cl]4 and [TTF][Cl]2. The CPCM solventmodel for EtOH was included to account for solvent effects.

We thank Tony Jun Huang, Paul A. Bonvallet, Amar H. Flood, MiguelGarcia-Garibay, and Dean Astumian for valuable discussions and tech-nical assistance. This work was supported in part by National ScienceFoundation (NSF) Nanoscale Interdisciplinary Research Teams GrantECS-0103559, NSF Integrative Graduate Education and ResearchTraineeship (Materials Creation Training Program) Fellowship DGE-0114443 (to B.H.N.), and by the Defense Advanced Research ProjectsAgency (DARPA) Biomolecular Motors program. Some of the com-pound characterizations are supported by NSF Equipment GrantsCHE-9974928 and CHE-0092036.

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