The Molecular Monorail

RADBOUD UNIVERSITY NIJMEGEN

RESEARCHPROPOSAL

HONOURS ACADEMY FNWI

The molecular monorail

Authors: Robert BECKER Nadia ERKAMP Supervisor: Marieke GLAZENBURG Prof. Thomas BOLTJE Evert-Jan HEKKELMAN Lisanne SELLIES

May 2017 Contents

1 Details 2 1.1 Applicants ...... 2 1.2 Supervisor ...... 2 1.3 Keywords ...... 2 1.4 Field of research ...... 2

2 Summaries 3 2.1 Scientiﬁc summary ...... 3 2.2 Public summary ...... 3 2.3 Samenvatting voor algemeen publiek ...... 3

3 Introduction 5

4 Background 8 4.1 The Feringa motor ...... 8 4.2 The ring molecule ...... 9 4.3 Threading of the track ...... 11 4.4 The track polymer ...... 12

5 Methods 14 5.1 Optical trapping ...... 14 5.2 Detection ...... 15 5.3 Synthesis ...... 16 5.3.1 Ring...... 16 5.3.2 Track ...... 17

6 Future applications 18 6.1 Further shrink down lab-on-a-chip approach ...... 18 6.2 Lab-on-a-chip approach in health care: Point-of-care testing (POCT) . 18

1 1 Details

1.1 Applicants

Robert Becker - Biology Evert-Jan Hekkelman - Physics & Nadia Erkamp - Chemistry Mathematics Marieke Glazenburg - Physics Lisanne Sellies - Chemistry

1.2 Supervisor Name: Thomas Boltje Telephone: 024-3652331 Email: [email protected] Institute: Synthetic Organic Chemistry Radboud University Nijmegen

1.3 Keywords Nanomachine, Feringa, polymer, porphyrin, FRET

1.4 Field of research NWO division: Chemical Sciences [CW]

Code Main ﬁeld of research 13.20.00 Macromolecular chemistry, polymer chemistry Other ﬁelds of research 13.30.00 Organic chemistry 13.50.00 Physical chemistry 12.20.00 Nanophysics/technology 14.80.00 Nanotechnology

2 2 Summaries

2.1 Scientific summary In this proposal we present the design for a new kind of nanomachine. A nanomachine is an assembly of a distinct number of molecular components that are designed to perform machinelike movements as a result of an appropriate external stimulation. The currently existing nanomachines have at least one of the following disad- vantages: they are either slow, non-autonomous or move in an unpredictable direction. Some even suffer from a combination of these. In our design we couple an autonomous motor to a ring that moves along a track. The motor of the Nobel Prize winner Feringa is used to make the nanomachine move. A ring-shaped molecule will be designed and the motor of Feringa will be bound to it in such a manner that it produces a propulsive force. Furthermore, the ring consists of two porphyrin rings and some bulky groups to prevent it from collapsing or folding. The ring can move along a track made of alternating benzene rings and triple bonds, containing some fluorescent BODIPY groups. A BODIPY group is also present on the ring, opening up the possibility for FRET as detection method. In the future, the proposed nanomachine may be used for the active transport of cargo. This in turn may boost the development of a microfluidic lab-on-a chip system, where it would be used to transfer molecules from one fluid stream to another. Some microfluidic lab-on-a chip systems find application in health care.

2.2 Public summary Imagine an everyday utensil like a car or a switch and make it a billion times smaller: you now have a nanomachine. In the past years, there has been impressive process in the development of these tiny molecular devices, among which, perhaps the most appealing to the imagination, an actual four-wheel drive nanocar. Current molecular machines however still struggle with basic issues like speed and controllability. This research proposes the design of a new nanomachine to solve exactly these issues: a molecular ‘monorail’ consisting of a ring, driven by rotating propellers, sliding along a track in one direction. In the future, this design may be useful in medical applications, e.g. the transport of substances in miniature laboratoria.

2.3 Samenvatting voor algemeen publiek Neem een alledaags gebruiksvoorwerp zoals een auto of een schakelaar en maak dit een miljard keer kleiner: dit is het terrein van de nanomachines. De laatste jaren is er een indrukwekkende vooruitgang zichtbaar in de ontwikkeling van deze minis- cule moleculaire apparaatjes, met als meest tot de verbeelding sprekende doorbraak

3 het nano-autootje van de Groningse Ben Feringa. Toch hebben huidige moleculaire machines nog vaak problemen met onder andere snelheid en bestuurbaarheid. Dit onderzoek stelt het ontwerp voor van een nieuwe nanomachine die deze problemen oplost: een ‘moleculaire monorail’, bestaande uit een ring, aangedreven door draaiende propellers, die in één richting over een spoor glijdt. In de toekomst zou dit ontwerp toepassingen kunnen vinden in de medische wereld, bijvoorbeeld voor het transporteren van stoffen in miniatuur laboratoria.

4 3 Introduction

In 2016, the Nobel Prize in chemistry was awarded to three independently operat- ing researchers, Jean-Pierre Sauvage, Sir J. Fraser Stoddart and Bernard L. Feringa [1]. Each of them made a contribution to a relatively new area of research that has an enormous potential: the development of molecular motors. A molecular motor can be briefly defined as “an assembly of a distinct number of molecular components that are designed to perform machinelike movements (out- put) as a result of an appropriate external stimulation (input)” [2]. The Nobel Prize winner Feringa designed and built such a device: a molecular propeller. This molecule consists of a rigid stator, an axle and a rotor blade. When exposed to UV light, the rotor will perform consistent and unidirectional rotational movement [3]. Combining four of these motors led to perhaps even his biggest achievement: his design of a ‘nanocar’, illustrated in figure 1 [4]. One can clearly see the similarities with regular sized cars; Feringa’s nanocar is essentially just a very basic downscale of the macroscopic world. Section 4.1 later on in this proposal will pay elaborate at- tention to his work. Aside from Feringa’s motor, various other kinds of molecular motors have been developed over the years. An example of this the recent development of autonomously moving microparticles [5]. These particles, or stomatocytes, were able to move through a liquid due to a reaction with hydrogen peroxide, catalysed by the platinum they were loaded with. The oxygen bubbles produced in the process deliver the propulsive force. The stomatocytes are capable of reaching relatively high velocities. However, they move rather randomly through the fluid. Motors that do have this directionality are synthetic walkers, DNA walkers for example. DNA walkers are made en- Figure 1: Operation of Feringa’s nanocar [4] tirely of DNA and consist of little more . than two legs propagating along a DNA track by hybridization with DNA fuel strands [6]. This ensures the directional move-

5 ment, but the walkers are often extremely slow in their progress. Furthermore, most DNA walkers require periodic addition of fuel strands, whereas a more autonomous approach (i.e. no need for active chemical fuel addition) would be preferable.

The proposed research is aimed at ﬁnding solutions to exactly the three issues illustrated by the examples above: directionality, speed and autonomy. To achieve this, existing elements will be combined and improved, inspired by the achievements mentioned in the section above. Together this will form an attempt at solving the important challenges in the area mentioned above and coming a step closer to fully functioning, autonomous nanomachines.

Figure 2: Spatial impression of the proposed nanomachine. The motor molecules are indicated in green, the ring in blue, the track in purple, the bulky groups by the black circles and the ﬂuorescent BODIPY groups by the red circles.

6 All of the above led to the following research question:

How to build an autonomous and unidirectional nanomachine with a high speed?

A sketch of the proposed design is illustrated in ﬁgure 2. In this design, the motor molecules (green) are incorporated in a ring (blue) that will be threaded onto a track (purple polymer), allowing it to move in only one dimension. Propulsion by the motor molecules will yield the desired speed and autonomy.

7 4 Background

4.1 The Feringa motor No nanomachine is complete without a means of doing work, and Feringa’s motor is one of the most effective ways to achieve work in a rotational fashion. Its spectacular properties and successful track record has made this the motor of choice in this proposal. The motor consists of a so-called ‘stator’ and a ‘rotor’, connected by a double carbon bond. While different versions do exist, they all operate in the same manner (see ﬁgure 3). An absorbed photon causes a cis-trans isomerization in the rotor, after which the molecule ﬁnds itself in an unstable form. The motor reaches a stable form by thermal helix inversion, which results in a 180◦ rotation. When another photon hits the molecule, this process repeats itself. That way the motor keeps rotating in the same direction when illuminated constantly [7] [8]. The motion can only be performed in one direction because the chiral carbon atom at the back of the rotor dictates which way the rotor turns if a photon is absorbed. Changing this atom’s chi- rality will cause the motor to turn the other way around. [10]

Figure 3: All steps in the process of turning on which the Feringa motor is based.

The difference between various versions of this particular machine is the structure of the stator. A conformational change in the stator greatly inﬂuences the rate at which the thermal helix inversion takes place, researched in depth by the Feringa group [9] They found that a certain version of Feringa’s motor can achieve a rotational speed of 3 MHz [9]. This version is depicted in ﬁgure 4, including how the attachment to the ring will look.

Not only is this motor molecule fast, reliable and still quite small, the only source of energy it needs to move is light. Speciﬁcally, any light with a wavelength larger

8 Figure 4: Structure of the used Feringa motor, including how the motor will be attached to the ring. than 280 nm sufﬁces [11]. This means the solution will not be polluted by any fuel, catalysts or possible waste products.

The use of Feringa’s molecule as a propeller instead of as a wheel has so far not been explored. Because the motor turns so often every second, even the slightest blade pitch should result in a force parallel to its rotational axis. We intend to use this force to propel our machine forwards. If this project is realised, it will be the ﬁrst time a propeller is used for directional movement on a nanoscale.

4.2 The ring molecule Designing a specific new ring allowed for correct placement of the motor. The ‘stator’ of the motor is incorporated in the designed ring, as illustrated in figure 5. Incorpora- tion of the stator as a structural element has been examined before in for example a fourwheeled nanocar [4]. However, it has never been studied in a ring before. Incor- poration of the stator may help to place the motor at the correct angle with respect to the direction of movement for efficient movement. For this the bond between the stator and rotor should be in the direction along the track. Estimating which angle is reached for the current design of the nanomachine poses a challenge. When experimentally is determined that the direction of the stator causes inefficient movement the bond between the stator and benzene connected to the porphyrin can be substituted. Furthermore, rotation about the connection between the porphyrins and motor may be possible, through which the motor ends up in the interior of the ring. This problem can also be solved by using another more rigid connection. A very important aspect of the design of the ring is the diameter. This has been chosen to fit the width of the track. Besides the motors, porphyrins have been placed to form the ring. These groups are multifunctional for the complex. Firstly, they increase the rigidity of the ring, ensuring the ring does not collapse before being placed on the

9 Figure 5: Design of the ring molecule with incorporated Feringa motors (green), porphyrin rings (blue), bulky groups (black) and a BODIPY molecule (red). track. Furthermore, they enhance the hydrophobicity of the complex. In combination with a hydrophobic track and a hydrophilic solution this may lead to an energet- ically favourable threading reaction. This effect will later be further discussed. Lastly, porphyrins give rise to multiple applications for our nanomachine. A metal ion can be placed in the porphyrins. In addition of the bonds to the porphyrin an extra bond could be formed depending on the oxidation state of the ion and the ion that is chosen. Increasing the oxidation state may allow attachment and decreasing it may allow for detachment of an extra group. This effect has widely been examined within the ﬁeld of porphyrin chemistry [12].

A risk in the placement of the porphyrins in the ring is that the porphyrins might rotate to align with the aromatic groups in the track. This alignment could be en- ergetically favourable by π-π-stacking or increased hydrophobic interactions [13]. An increase of resistance could decrease the speed of the nanomachine. To prevent rotation of the porphyrin, bulky groups have been placed to form a roof over the porphyrin. To be able to detect the speed of the nanomachine one of these groups

10 has been chosen to be the ﬂuorescent group BODIPY (boron-dipyrromethene). The other groups have been chosen to be similar groups to prevent major disturbance of the symmetry of the system. For future applications, other functional groups can also be attached at this side.

The designed ring has never been built before. Very little is known from literature about placing the motor under the correct angle, ensuring the width of the ring is suf- ﬁcient for the track and the treading and making rotation of the porphyrins unattrac- tive. These subjects, although thoroughly examined with computer models as well as scale models, will be among the greatest challenges of this research.

4.3 Threading of the track One essential step in the process of creating this nanomachine is the so-called threading of the ring onto our chosen track. A lot of research has been done on the many aspects of this process in general, and it is a phenomenon that is used extensively.[14] [15] [16] [17] However, it is difficult to say beforehand if the threading in this particular case will go smoothly by itself. In general, it is not necessary for the ring and polymer to have any special interactions, since purely statistically threading may happen to some of the rings [18]. This can be improved by hydrophobic/hydrophilic interactions. The ring that needs to be threaded in this proposal has a hydrophobic interior. If the solution consists mainly of water or other hydrophilic substances, this may influence the threading rate positively given that the used track is hydrophobic as well [19]. We propose to measure if the threading rates in water are acceptable as is for continuation of the study. There is a possibility that the track and ring will aggregate in a hydrophilic solution. A way to solve this problem, if it rears its head, can be to make the bulky groups mentioned in the section above hydrophilic. This way the ring will be soluble in water. If these ‘natural’ rates are deemed too low, several steps can be taken to improve this. Changing the solution from H2O to MeCN or acetone can change the threading constants, for better or for worse [20] [14]. In the case that even more measures have to be taken, it is possible to look into increasing the attraction between track and ring. This could be achieved involving electrostatic forces by, for example, making sure the ring has a negative charge while giving, preferentially the ends of the polymer, positive charges. While possible, it has to be checked if the machine’s force is large enough to overcome this bound state. These charges could encourage the rings to find the threading locations. If the whole polymer is charged, the risk exists that the rings will simply stick to the polymer without threading, however this can be tried as well if the motors get stuck on the charges at the ends of the polymer [20]. Since threading is a common occurrence in papers on chemistry, we have full confidence that any problems can be handled swiftly.

11 4.4 The track polymer There are a number of requirements the track has to fulfil. Firstly, the polarity of the track should match the polarity of the interior of the ring. This way the probability of threading is increased. Because the interior of the ring is hydrophobic the track should also be hydrophobic. Secondly, to detect if the ring moves along the track, fluorescent groups need to be incorporated into the track. If the fluorescent groups and the track differ in polarity, the ring may stick onto the fluorescent groups. Con- sequently, the body of the track should be similar to the fluorescent groups. Thirdly, by using a relatively linear track, coiling of the track will be reduced. The track will consist of benzene groups with alternating triple bonds and BODIPY groups. The combination of BODIPY groups with triple bonds gives rise to a red coloured track when illuminated by light [21]. This opens up the possibility to use FRET for detection. This method is based on the interaction between BODIPYs on the track and the ring. To prevent interaction of BODIPYs with each other within the track, the BOD- IPY molecules need to be placed at a certain distance. BODIPY has a Förster radius of 57 Å, which is the distance at which energy transfer in FRET is 50% efficient [21]. Because the efficiency decreases with 1/1 ( R )6) [22], doubling the distance makes + R0 the efficiency decrease to 1.5%, Translating this distance to the length of atoms in the track, 1 in 22 benzene rings can be replaced with BODIPY groups to generate a fluorescent track. For measuring the speed of the ring sliding along the track, a linear track is required. To generate a linear track optical trapping of beads at the end of the track is used (see section 5.1). These polystyrene beads are placed onto the track after the threading of the ring. The complete design of the track with the optical beads is illustrated in figure 6. In polar solvents the track may form aggregates. If this problem is encountered, bulky groups can be incorporated into the design of the track. These bulky groups should not be too large, because they must fit in the interior of the ring.

Figure 6: Structure of the track with alternating benzene and triple bonds and with 1 in 22 benzene rings substituted with a BODIPY group.

The length of the track depends on the observed speed of the nanomachine and the maximum length of the track that can be synthesized. These can be experimentally determined, after which the track of the correct size can be synthesized. The length

12 of the track can be inﬂuenced by the amount of starting product or addition of ter- mination groups.

13 5 Methods

5.1 Optical trapping For the motor to slide over the track it is obviously necessary for the polymers to be as rigid and stationary as possible. We can take preventive measures to make sure this becomes a reality. It would be easiest if one was able to pick up both ends of a polymer and just stretch it out. In fact, this is done quite often already by a technique called ‘optical trapping’. The only thing needed to use it are two beads attached to the ends of the polymer.

Figure 7: Optical trapping, visualized by two light beams that create a force (FA) which moves the bead. A highly focused laser beam set up like in ﬁgure 7 creates a three-dimensional trap for the polystyrene beads. Since light carries momentum, and the refractive index of the beads result in a change of the direction of this momentum, the beads will experience a transfer of momentum from the light to the beads. By making sure the intensity of the laser is highest in the middle of the beam and lowest at the edges, as well as focusing the laser with a convex lens, the beads will always experience a force in the direction of the lasers’ focal point [23]. This effect can be seen more clearly in ﬁgure 7 [23]. Moving the light beam makes the trapped beads move as well. This way the polymer can be stretched to the point that it resembles a reasonably straight

14 thread. The use of this effect is common in DNA and protein research, so we can be conﬁdent in its effectivity (For example, in a paper by B. Jagannathan et al. [24] or even in combination with ﬂuorescence microscopy done by G.A. King et al. [25]).

5.2 Detection After synthesizing the molecule and incorporating the track, the next major step is to detect and verify the behaviour of the system. The movement of the motor along the track cannot easily be identiﬁed using regular optical microscopic techniques, because the scale of the molecules is beneath the diffraction limit of visible light. This means other methods of molecular detection need to be addressed.

Different detection methods were considered to evaluate if the nanomachine is placed correctly on the track and to determine its speed. The latter is important to rule out the possibility that the motion of the motor is only Brownian motion. For this, a ref- erence experiment is performed during which Feringa’s motors are not shined upon with light of the correct wavelength.

To test if the motor is placed correctly on the track and is not hanging against the side, NMR is used. If this effect occurs, hydrophilic groups can be attached to the outside of the ring to prevent this. To determine what the speed of the motor is AFM and STM were considered initially. However, both these methods require the fixation of the object on a surface. This might heavily influence the movement of the nanomachine and is not suitable for that reason. A more convenient way is the usage of fluorescent groups to identify the track and the nanomachine. As discussed earlier, both the track and the ring molecule will be equipped with a fluorescent BODIPY group. This setup enables the application of a detection method based on a phenomena called Förster resonance energy transfer (FRET).

Using FRET, one can achieve measurements of proximity on molecular length scales [26] not available through any other method [27]. The donor fluorophore on the track can be excited by using light. Hereby energy is transferred via induced dipole-dipole interaction to the acceptor on the ring. As mentioned before, the efficiency of the transfer is inversely proportional to the sixth power of the distance between the two chromophores [26]. This means FRET has an approximate resolution of 1 to 10 nm, which is sufficient to track the movement of the motor. BODIPY is known to have this specific interaction with itself [21], meaning it will be used both as the donor and the acceptor fluorophore. In this case, FRET can be detected by the resulting fluorescence depolarization, which has successfully been achieved in the past [28].

The idea is to let the nanomachine move freely along the track. As the ﬂuorophores come close to each other, it will be visible through the detection of FRET. Several

15 successive detections indicate the desired movement of the ring along the polymer. For the distance between the BODIPY groups is known, this yields rather detailed information about the molecule movement.

5.3 Synthesis 5.3.1 Ring

The ring is built up of several components, see ﬁgure 8.

Figure 8: Retrosynthetical scheme for the synthesis of the ring.

These components are synthesized with side groups that can be used for assembly of the ring. Using pinacolatoboron (Bpin) groups on one component and bromine atoms on the other, a Suzuki reaction can be used to couple the components. In this way similar rings are assembled [38]. Bis(pinacolato)diboron adds to alkenes

16 [29], therefore first 2-ethenyl groups are connected to the motor. Starting off with Feringa’s motor, firstly the stator is synthesized with 2-ethenyl groups [30] [31] [32]. Secondly in two successive reactions [33] the rotor is modified such that it can easily be placed onto the stator. Then the stator and rotor are combined and the double bond between these components is formed [33]. The synthesis of this motor has been researched extensively in the past. The only difference is the attachment of the 2-ethenyl side groups. To finish the motor the 2-ethenyl groups are replaced by Bpin groups [37].

Then the ring is formed following previous research [38] with some adjustments. Firstly, the bromine atom is shifted one place, to prevent the bulky groups from rotating into the interior of the ring. Secondly, iodine atoms are included in the design, to create a way to connect bulky and ﬂuorescent groups to the porphyrin ring [34]. Zinc atoms are added to the porphyrins to occupy the free centre in the middle [35]. These zinc atoms can easily be removed at the end and prevent other metals required for the coupling reactions from entering the porphyrin rings [36]. Then to half of the porphyrin rings a bulky and a ﬂuorescent group are coupled and to the other half only bulky groups are coupled.

Finally, the porphyrin rings (one with and one without a ﬂuorescent group) are coupled with the motor by using a Suzuki reaction to form the ring [38].

5.3.2 Track

The first step in the synthesis of the track is the synthesis of the polymers between the BODIPY’s (appendix 1) [39]. Shorter versions of this polymer have already been synthesised [39]. Hypothesized is that by mixing the starting compounds in a determined ratio the average length can be altered. The desired ratio will be determined experimentally since the reaction constants of the multiple reactions are unknown. The polymers with the average length of 21 repetitive units will be obtained by the use of column chromatography. If separating the fractions turns out to be difficult these polymers can be synthesized bottum-up to only have a specific length. Next, the BODIPY groups are synthesised [40]. This can be performed according to literature except for the addition of extra side groups, which are hypothesized to have little effect on the synthesis. The obtained polymers and BODIPY groups are mixed in 1:1 ratio to form the track [39] [40]. When no significant amount of lengthening occurs an excess of polymers is added to the mixture to secure the terminal group of the track is the terminal triple bond of the polymer and not the BODIPY. After the threading has taken place polystyrene beads can be attached to the terminals of the track by a click reaction [41].

17 6 Future applications

6.1 Further shrink down lab-on-a-chip approach The nanomachine suggested in this proposal could be a possible tool to further downscale the volumes of the microfluidic lab-on-a-chip approach. The term lab-on- a-chip describes the approach to conduct chemical reactions in volumes 5-9 times smaller than in conventional laboratories [42]. To process a reaction of two or more substances that are initially present in two different fluid streams, first the substances need to get in contact with each other [42]. At very small fluid volumes however, flow of the fluid streams is no longer tubular but laminar [42]. A laminar fluent stream can be regarded as a big stream consisting of several different parallel small streams. In contrast to tubular streams, mixing takes place only via diffusion [42]. Considering that diffusion only occurs if there is an in- terface present between the two streams, the necessity of mixing becomes obvious. As already mentioned a metal ion can be placed in the porphyrins. Depending on the oxidation state of the metal an additional group could be bound. Changing the oxidation state could allow attachment and detachment of this extra group. This effect has been described within the field of porphyrin chemistry. [43]. The usage of the nanomachine as carrier of single molecules could make it possible to circumvent the obstacle of mixing two fluid streams before a reaction between two substances can occur. By using nanomachines as carrier, it might be possible to reduce the amount of substrate within the microfluidic system. Theoretically the amount of product could be regulated by varying the amount of substrates carried by the nanomachine. However, to use the nanomachine for such a purpose, the ends of the polymer would have to be fixated in the separated fluids.

6.2 Lab-on-a-chip approach in health care: Point-of-care testing (POCT) The microﬂuidic lab-on-a-chip approach is of growing importance in health-care. Point-of-care testing devices provide both trained and untrained staff with diagnos- tic results at the patient room [44]. The most obvious advantage of POCT is that it is much faster than conventional laboratory analysis of specimens [45]. One of the most established microﬂuidic lab-on-a-chip devices is a glucose meter to monitor the glucose concentration in blood of patients. The glucose measuring is based on enzymatically reactions. The reactions in such devices are conducted with glucose- 1-dehydrogenase (GDH) which has lower accuracy than other enzymes [45]. Maybe, the nanomachine suggested in this proposal could transport essential co-factors, such as NAD, which are necessary for GDH to work as a catalyst. In that way the whole process of the glucose measurement could be regulated more exactly. How-

18 ever, as mentioned above, ﬁrst of all one would have to succeed in ﬁxing the ends of the polymer in separate sections on the chip, without impairing the function of the nanomachine.

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24 Appendices

Synthesis of the track Synthesis of the polymer and BODIPY

25 Coupling the polymer and BODIPY

26 Coupling polystyrene beads to the track

27 Synthesis of the ring Synthesis of the motor

28 Synthesis of the components of the ring

29 Synthesis of the phorphyrin ring with BODIPY

30 Synthesis of the phorphyrin ring without BODIPY

31 Coupling the motor and the porphyrin rings

32 Removal of the Zn