Guillaume Erbland

Seifallah Abid

Yohan Gisbert

Nathalie Saffon-Merceron

Yuichiro Hashimoto

Leonardo Andreoni

Théo Guérin

Claire Kammerer

Gwénaël Rapenne
email: rapenne@cemes.fr

Star-Shaped Ruthenium Complexes as Prototypes of Molecular Gears

Keywords: cross-coupling, cyclopentadienyl ligands, molecular gears, molecular machines, ruthenium Preprint

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

For four decades, molecular gears have stimulated the imagination of synthetic chemists. In 1980, Iwamura [1] and Mislow [START_REF] Hounshell | Proc. Natl. Acad. Sci[END_REF] were the first to independently report molecular gears consisting of two triptycene subunits close enough to interact with each other in a bevel gear fashion. In 1997, C.J. Richards published an organometallic metallocene molecular gear with a cobalt ion gathering together a tetraphenylcyclobutadiene wheel in close contact with a triptycene, therefore displaying 3 x 4 gearing ratio. [START_REF] Stevens | [END_REF] In 2012 Siegel et al. again used triptycene units in a different design mimicking spur gears with a special focus on the optimal interaxle distance leading to correlated disrotatory gearing motion. [4] Five years later, Shionoya et al. reported a Pt(II)-centered molecular gear, in which the two azaphosphatriptycene cogwheels were very elegantly engaged or disengaged via cis-trans isomerization processes around the platinum center. [5] In all these examples, coordinated motions have been evidenced in solution, which limits the number of cogwheels possibly inter-digitated and where unidirectionality is not achievable due to the second law of thermodynamics and Brownian motion. The drawbacks of these studies in solution strongly limit the potentialities of such molecular machines.

While Nature uses the anisotropy of the cell's membrane to overcome the second law of thermodynamics, our strategy is to orient molecules for deposition of molecular gears on surfaces. Thus, it becomes possible to deal with multimolecular gearing systems, each molecule being considered as a single gear. After deposition, one could build a large variety of trains of gears with very good control over the intermolecular distance between neighbouring molecules. Dealing with linear assemblies of molecules allows us to follow the transmission of motion from one molecule to another, which is impossible in solution where gearing mechanisms are restricted to intramolecular correlated rotations.

The first demonstration of intermolecular gearing motion on a surface was achieved by Moresco et al. in a rack and pinion device. [6] They used a star-shaped molecule of penta(p-tert-butylphenyl)-(p-tertbutylpyrimido)benzene which was moved laterally with the tip of a Scanning Tunneling Microscope (STM) along the edge of an island of the same molecules on Cu (111). The rotation of this single molecule was evidenced on successive STM images by following the motion of the contrasting nitrogen atoms. Subsequent studies on Au(111) revealed that it is possible to achieve stepwise rotation of this star-shaped molecule at the single molecular level when mounted on a defect used as rotation axis. [7] Unfortunately it has not been possible to transfer the rotary motion to another molecule in close proximity due to the preferred diffusion of the molecules on the surface. This seminal work highlighted the prime importance of anchoring molecular gears, thus precluding diffusion and providing a defined axis of rotation, as in the macroscopic world, to ultimately control intermolecular gearing on a surface.

Our strategy to avoid diffusion of a series of rotary molecular machines deposited on metallic surfaces relies on a hydrotris(indazolyl)borate scorpionate ligand [8] functionalized with three thioether groups connected at the 6-position of the indazoles. This tripodal platform provides on one side strong anchoring on the metallic surface through the three S-Au bonds, and on the other side coordination to a ruthenium center which acts as a ball bearing, allowing the rotation of the upper ligand. This architecture has been used for the ruthenium-based molecular motor 1 (Figure 1), [9] in which the rotating platform is a dissymmetric pentaarylcyclopentadienyl ligand, displaying four identical ferrocenyl-terminated arms and one shorter tolyl arm. Once deposited on Au(111), it has been shown using STM experiments that lateral manipulation of such molecular machine is possible at 77 K whilst molecular diffusion is totally frozen at 5 K. [9] The hydrotris(indazolyl)borate ligand embedded in a ruthenium complex is therefore very attractive towards building a train of gears since it would allow the control and fine tuning of the intermolecular distances between gears at 77 K when the subsequent cooling to 5 K would lock the spatial arrangement of molecules during gearing experiments. In our targeted experiments, the STM tip will then be used both as a source of mechanical energy to induce the rotary motion of the first cogwheel by pushing on one of the teeth (Figure 2), and also as a probe to monitor a possible transmission of motion by imaging the final state.

Figure 2. Expected gearing effect between two star-shaped ruthenium complexes in close proximity on a metallic surface, using a STM tip as a source of mechanical energy to rotate the first cogwheel.

For the design of the molecular gears involved in such on-surface experiments, the overall scaffold of the piano-stool ruthenium complex used in motor 1 was preserved but the structure of rotating platform was modified to favor the transmission of rotary motion (Figure 1, right). Indeed, a careful choice of the cyclopentadiene (Cp) substituents is critical since intermolecular interactions of neighbouring teeth are key to successful gearing. Given the lack of precedence in gearing systems with a tunable intermolecular distance and with a cogwheel decoupled from the surface, it is difficult to anticipate the requirements for an effective transfer of rotary motion. It was thus decided to prepare a series of ruthenium-based molecular gears, incorporating five identical teeth of various lengths, shapes and structures. A divergent synthetic approach was devised, allowing for all of the symmetrical molecular gears to stem from the same precursor, namely the penta(p-bromophenyl)cyclopentadienyl ruthenium(II) complex 4a, incorporating the thioether-functionalized scorpionate ligand (Scheme 1). The optimized synthesis of pentaaryl bromide 4a has been recently reported [10] and it has been shown that this key intermediate is prone to undergo five-fold Suzuki-Miyaura cross-coupling reactions. To expand the scope of crosscoupling reactions and access structurally different teeth in a straightforward manner, it appeared interesting to prepare the analogous pentaaryl iodide precursor 4b since aryl iodides are known to be more reactive in most cross-coupling reactions as compared to the corresponding aryl bromides. [START_REF] Negishi | Handbook of Organopalladium Chemistry for Organic Synthesis[END_REF] Herein we report the preparation of penta(p-iodophenyl)cyclopentadienyl ruthenium(II) complex 4b, and the use of pentahalogenated ruthenium complexes 4a and 4b as key precursors of molecular gears through a divergent synthetic approach. We also present the synthesis and characterization of two families of molecular gears prototypes, respectively incorporating pseudo 1-dimensional aryl teeth or large planar 2-dimensional paddles of various nature.

Results and Discussion

The synthesis of the pentaaryl bromide precursor 4a was recently optimized, with a particular emphasis on the preparation of the thallium hydrotris(indazolyl)borate intermediate 3, functionalized with thioether anchoring groups (Scheme 1). [10] The latter is now obtained in 4 steps from commerciallyavailable 3-amino-4-methylbenzoic acid, via a newly developed "N-deprotection / reductive sulfidation" one-pot sequence mediated by indium(III). The bromidodicarbonyl[ 5 -1,2,3,4,5-penta(pbromophenyl)]cyclopentadienyl ruthenium(II) complex 2a stems from pentaphenylcyclopentadiene, that first undergoes bromination on the phenyl para-positions and on the Cp ring. The resulting hexabrominated compound then adds oxidatively onto the ruthenium(0) cluster Ru3(CO)12 to yield the ruthenium(II) complex 2a, bearing a  5 -cyclopentadienyl ligand surrounded by five p-bromophenyl arms. The final ligand exchange step in the presence of the thallium tripod 3 under microwave activation gives rise to the desired pentabrominated ruthenium complex 4a, in 31% yield over the longest linear sequence (5 steps).

We recently reported an important reactivity difference between pentabromo-and pentaiodo-derivatives of the same family of ruthenium complexes bearing bare hydrotris(indazolyl)borate tripodal ligands (without S-based anchoring groups). [START_REF] Carella | [END_REF] While the pentabrominated derivative did not react with ethynylferrocene under Sonogashira conditions, it proved possible to achieve the five-fold Sonogashira coupling using the pentaiodinated precursor. It is inferred that the high electron density of the aryl bromide moieties within the ruthenium cyclopentadienyl complex might preclude the oxidative addition on the [Pd(PPh3)4] catalyst in this reaction. This tendency is also confirmed by the restricted set of catalysts which are active even for Suzuki-Miyaura cross-couplings on the brominated precursor 4a.

In our efforts to synthesize a variety of molecular gears, the implementation of various types of crosscoupling reactions will diversify the nature of the teeth, and/or the length of the linkers. The possibility to use either the brominated or iodinated precursors 4a and 4b, respectively, will thus result in a higher flexibility in cross-coupling reactions.

Preparation of pentaiodinated ruthenium complex 4b

In order to prepare the pentaiodinated precursor 4b, bearing five iodines instead of bromines at the phenyl para-positions, it was first envisioned to follow a synthetic route involving the iodinated analogue of complex 2a, which would be obtained by iodination of pentaphenylcyclopentadiene followed by oxidative addition onto ruthenium(0). Unfortunately, in our hands, the iodination step proved unsuccessful. Scheme 1. Synthesis of pentaiodinated ruthenium complex 4b starting from precursor 2a via two different routes, each involving a Cu-catalyzed aromatic Finkelstein-type reaction and a ligand exchange step.

Alternatively, it was decided to introduce the iodine centers later in the synthetic sequence, via aromatic Finkelstein-type Br/I halogen exchange reactions. As reported earlier, [START_REF] Carella | [END_REF] hexabrominated ruthenium(II) complex 2a was treated with sodium iodide in the presence of copper iodide combined with a diamine ligand as the catalytic system to afford the corresponding hexaiodinated complex 2b. This exchange process was initially observed by Klapars and Buchwald as a side reaction of copper-catalyzed Narylations, using aryl bromides as substrates and copper iodide as precatalyst. [13] It is important to note that the Br/I exchange process is an equilibrium and that the solubility difference between the halide salts has an impact on the position of this equilibrium. Under optimized conditions, the authors reported yields higher than 93% for the exchange in a variety of (hetero)aryl bromides. In our initial attempt to perform this reaction on ruthenium(II) complex 2a, the five-fold copper-catalyzed Br/I exchange on the phenyl para-positions combined with a Br/I exchange on the ruthenium center led to compound 2b in 45% yield. [START_REF] Carella | [END_REF] So as to reach a higher efficiency, the reaction conditions were optimized: complex 2a was refluxed in 1,4-dioxane in the presence of sodium iodide (2 equiv. / Br), copper iodide (10 mol-% / Br) and ()-N,N'-dimethyl-trans-cyclohexane-1,2-diamine L1 (20 mol-% / Br) (Scheme 1). After 36 h, the 1 H NMR spectrum of the crude mixture showed a very clean Br/I exchange process but only a partial overall conversion of the bromophenyl to iodophenyl moieties (see Fig. S1 in the Supporting Information). Indeed, following a fast substitution of the bromides located on the ruthenium center, the crude material composed of a mixture of mono-to penta(p-iodophenyl) derivatives, indicating that the copper-catalyzed Br/I exchange on the aromatic positions was not quantitative. After removal of the inorganic residues by passing the mixture of partially and totally iodinated compounds through a plug of silica, the obtained mixture was resubmitted to the same reaction conditions in order to drive the equilibrium towards aryl iodides. Complete Br/I exchange was observed after another 36 h (see Fig. S1 in the Supporting Information) and the desired iodinated complex 2b isolated in 83% yield. Ligand exchange on complex 2b in the presence of the thallium hydrotris(indazolyl)borate 3 in acetonitrile under microwave irradiation afforded the desired pentaiodinated precursor 4b in 52% yield.

Interestingly, with the new optimized conditions for the copper-catalyzed Br/I exchange, the pentabrominated precursor 4a incorporating the thioether-functionalized scorpionate ligand undergoes halogen exchange in spite of the steric hindrance to give its iodinated counterpart 4b in 44% yield (85% per Br/I exchange). It must be noted that a previous attempt of Br/I exchange on the analogue complex lacking thioether functional groups under non-optimized conditions led to a complex mixture of partially substituted products. [START_REF] Carella | [END_REF] These new exchange conditions highlight the reactivity of the pentabrominated precursor 4a in copper catalysis, in spite of the functional complexity and steric hindrance within this ruthenium complex.

The pentaiodinated key intermediate 4b can be obtained from ruthenium complex 2a by two routes, involving first the halogen exchange step followed by coordination of the tripodal ligand, or the reverse sequence, in comparable overall yields of 42% and 36%, respectively. Our previous work on the improvement of the synthesis of thallium hydrotris(indazolyl) borate 3 combined with our efforts to optimize the five-fold aromatic Finkelstein-type Br/I exchange allowed the preparation of the desired pentabrominated and pentaiodinated ruthenium complexes 4a and 4b, in 31% and 19% overall yields on the longest linear sequence (5 steps), respectively. Both complexes were further engaged in multiple cross-coupling reactions according to a divergent approach, to vary the structure and the length of the teeth in molecular gears prototypes.

Prototypes of molecular gears with pseudo 1D-teeth

A first family of molecular gears prototypes was prepared, bearing five substituted 4,4'-biphenyl groups connected to the central cyclopentadienyl ring. The pentaaryl bromide precursor 4a was engaged in Suzuki-Miyaura cross-coupling reactions with appropriately substituted phenylboronic acids (Scheme 2). This type of five-fold aryl-aryl cross-coupling reaction was already exploited in our group for the synthesis of ruthenium-based molecular motors, incorporating ester-or thioether-functionalized scorpionate ligands. [8,10] Under optimized conditions, pentaaryl bromide 4a was treated with a large excess of 4-tert-butylphenylboronic acid (4 equiv. / Br) in carefully degassed anhydrous toluene, using palladium diacetate (6 mol-%/Br) with SPhos (12 mol-% / Br) as catalytic system and potassium phosphate (2 equiv./Br) as base. The resulting mixture was refluxed for 24 h and the same amounts of catalyst and reagents were added again. After another 24 h at reflux, the desired product 5 resulting from five consecutive Suzuki-Miyaura couplings was obtained in 82% yield.

Scheme 2. Five-fold Suzuki-Miyaura cross-coupling reactions of pentabrominated precursor 4a to yield molecular gears prototypes 5-7 incorporating pseudo-1D teeth.

Compound 5 was fully characterized by 1 H and 13 C NMR spectroscopy, as well as mass spectrometry, thereby confirming the incorporation of the five 4-tert-butylphenyl peripheral groups. In particular, 1 H NMR clearly shows two AA'BB' patterns integrating each for 20 protons and corresponding to the five

Pd(OAc) 2 (0.6 equiv.) SPhos (1.2 equiv.) K 3 PO 4 (20 equiv.) Toluene, reflux, 48 h R 2 R 1 R 2 (HO) 2 B (40 equiv.) 4a 5 R 1 = t Bu 6 R 1 = H 7 R 1 = H R 2 = H R 2 = Me R 2 = t Bu 82% & 52% 65% Ru N N N N B H N N SEt EtS EtS R 1 R 2 R 2 R 1 R 2 R 2 R 1 R 2 R 2 R 1 R 2 R 2 R 1 R 2 R 2 Ru Br Br N N N N B H N N SEt EtS EtS
Br Br Br 4,4'-biphenyl systems surrounding the central cyclopentadienyl ring and substituted by a tert-butyl group in the outer para-position. Moreover, careful analysis of the spectra revealed that the three indazole moieties are chemically equivalent, which shows that this molecular gear prototype undergoes free rotation at room temperature.

It was possible to grow crystals of compound 5 suitable for X-ray diffraction by slow diffusion of methanol into a solution of complex 5 in toluene. Molecular views of 5, shown in Figure 3, confirmed the piano-stool structure of the ruthenium complex, with the hydrotris(indazolyl)borate ligand binding in a facial tripodal  3 -N,N',N'' mode. The average distance between the three coordinating nitrogen atoms and the ruthenium center is 2.15 Å, while the distance between the cyclopentadienyl ligand and the ruthenium center is 1.80 Å. These values are fully consistent with those obtained previously for parent ruthenium complexes. [START_REF] Carella | [END_REF] Strikingly, all the 4,4'-biphenyl teeth are located out of the central Cp mean plane, pointing away from the hydrotris(indazolyl)borate ligand, with the Cipso of the inner phenylene groups undergoing deviation of 0.10 Å to 0.36 Å compared to the Cp mean plane (see Supporting Information). This effect can be ascribed to steric hindrance within the ruthenium complex. For such piano-stool systems combining a C3-and a C5-symmetric ligand, a staggered and an eclipsed conformation are observed with, respectively, minimal dihedral angles of 12° and 0° between one Cp substituent and the closest indazole ring. [START_REF] Carella | [END_REF] With a torsion angle of 9° between the biphenyl tooth located on cyclopentadienyl C4 and the indazole core underneath, it appears that complex 5 crystallizes in a close to staggered conformation. The five Cp biphenyl substituents are thus inequivalent, as confirmed by the different tilt angles of their inner phenylene units. Indeed, the phenylene group connected to C1 is almost perpendicular to the Cp plane (87°) while the one on C4 exhibits the smallest angle of 39°, due to its interaction with the indazole core. The three remaining phenylenes have tilting angles of 51° to 59°. Finally, as expected, the 4,4'-biphenyl systems are all twisted with the magnitude of the torsion between consecutive para-phenylene units is variable, with values ranging from 16° to 44° in the solid state.

The molecular structure of complex 5 highlights the bifunctional role of the scorpionate ligand, with one face dedicated to ruthenium coordination while the other one will allow on-surface adsorption (Figure 3a). As anticipated in the design, this ligand will act both as anchor and rotation axle. The star-shaped cogwheel, based on the pentaaryl cyclopentadienyl ligand, will in turn be lifted up from the surface and allow intermeshing of the teeth of neighbouring molecules (Figure 3b).

To increase the steric hindrance of the teeth, 3,5-disubstituted phenylboronic acids were next selected and following the same approach, the analogues 6 and 7 carrying methyl or tert-butyl groups on the peripheral meta positions were obtained in 52% and 65% yield, respectively (Scheme 2). Even if the efficiency of the five-fold Suzuki-Miyaura couplings with 3,5-disubstituted phenylboronic acids seems substantially lower than with 4-tert-butylphenylboronic acid, it must be noted that the yield per coupling remains in the same range (96%, 88% and 92% for 5, 6 and 7, respectively).

A first family of molecular gears with substituted 4,4'-biphenyl moieties as teeth was thus obtained via multiple aryl-aryl cross-coupling reactions, starting from the same pentaaryl bromide precursor 4a. These three prototypes exhibit a similar size, with a diameter in the 2-2.5 nm range, and incorporate pseudo one-dimensional teeth. These first prototypes will be studied by STM on Au(111) by physicists in the near future. The deposition conditions as well as the possibility to assemble trains of gears by STM tip lateral manipulation will be assessed, and the first investigations of intermolecular transfer of rotary motion will be performed. However, preliminary theoretical studies suggest a possible vertical deformation of the teeth due to their relative flexibility, thus preventing gearing.

To avoid potential gear slippage and favor mechanical transmission of motion, it was envisioned to increase the size of the teeth, moving from pseudo 1D-biphenyl teeth to larger 2D paddles. Several appropriate candidates such as carbazoles, BODIPYs and porphyrins were identified to possibly act as paddles, since they display flat structures and large areas. Importantly, the incorporation of such bidimensional fragments in molecular gears prototypes might lead to solubility issues during the synthesis and to enhanced -stacking between neighbouring molecules on surface, which could be detrimental for gearing processes. Bulky alkyl groups were thus systematically integrated in the design of the paddles.

Prototypes of molecular gears with 2D-paddles

A carbazole core carrying two tert-butyl groups in the 3,6-positions was first chosen to act as a 2Dpaddle, since the diameter of the resulting molecular gear would be similar to the diameter of complexes 5-7. This would allow a direct comparison of the influence of the structure of the teeth, while keeping their length constant.

A first attempt of palladium-catalyzed Buchwald-Hartwig reaction [14] between pentabrominated precursor 4a and commercially-available 3,6-di-tert-butyl-9H-carbazole under standard coupling conditions failed to afford the expected product. However, we remembered that the copper-catalyzed Finkelstein-type halogen exchange in aryl halides had been developed by exploiting a side-reaction observed during copper-catalyzed N-arylation reactions, [15] as mentioned by Klapars and Buchwald. [13] Given the reactivity of the pentaaryl bromide precursor 4a under Br/I exchange conditions, it was envisioned to perform direct functionalization under copper catalysis using carbazole as the nucleophile.

Precursor 4a was thus treated with 3,6-di-tert-butyl-9H-carbazole (2 equiv./Br) in the presence of potassium phosphate as base, combined with copper iodide and ()-trans-cyclohexane-1,2-diamine L2 as the catalytic system (Scheme 3). After 2 days in refluxing 1,4-dioxane, the pentasubstituted gear 8 was obtained in 19% yield. It has been shown that such copper-catalyzed N-arylation reactions are more efficient using aryl iodides rather than bromides. [16] The same reaction was subsequently tested with the iodinated precursor 4b and the product 8 was isolated in a higher yield of 28%, corresponding to 78% per C-N coupling. Mass spectrometry and NMR spectroscopy confirmed the presence of five carbazole units. The aromatic region of the 1 H NMR spectrum displays the 30 protons corresponding to the five 3,6-disubstituted carbazole moieties, as compared to 20 protons for the AA'BB' pattern of the five phenylene groups surrounding the Cp ring, and the 12 characteristic protons of the indazole cores. Free rotation at room temperature was still observed, in spite of the enhanced steric hindrance of the teeth.

Scheme 3.

Five-fold Cu-catalyzed N-arylation of precursors 4a and 4b to yield molecular gear prototype 8 incorporating carbazole-based paddles.

To increase the diameter of the cogwheel and vary the nature of the paddles, a BODIPY fragment was selected to prepare the next prototype of molecular gear, with a 4,4'-biphenyl linkage to the central cyclopentadienyl ring. For enhanced stability and solubility, the pyrrole cores were peralkylated with methyl and ethyl groups. The appropriate BODIPY-substituted phenyl boronic acid pinacol ester 9 was prepared [17] and subsequently engaged in a Suzuki-Miyaura cross-coupling with the pentaaryl bromide precursor 4a (Scheme 4, right). Under the conditions previously used for the synthesis of bis-aryl gears 5-7, the desired product 10 was detected by mass spectrometry analysis of the crude mixture, along with by-products of mono-, di-, tri-and tetrafunctionalization. Unfortunately, only traces amounts of compound 10 were obtained after purification. A biphasic solvent system combining toluene/ethanol/water (2:2:1) [18] was then employed leading to an increase of the reaction efficiency, and the desired penta-BODIPY product 10 was isolated in 36% yield. This new complex was characterized by mass spectrometry as well as 1 H, 13 C, [START_REF] Negishi | Handbook of Organopalladium Chemistry for Organic Synthesis[END_REF] B and 19 F NMR spectroscopy. On the 1 H NMR spectrum, integration of the two AA'BB' systems corresponding to the 4,4'-biphenyl linkers and of the methyl and ethyl groups located on the pyrroles as compared to protons belonging to the equivalent indazole rings show that five BODIPY moieties have indeed been coupled. In the 19 F NMR, a quadruplet is observed at -145.7 ppm with a coupling constant J ( 11 B-19 F) of 33.3 Hz, proving the coupling with the neighbouring boron atom. The same pattern was observed in the BODIPY-substituted phenylboronic acid pinacol ester precursor 9, indicating that both fluorine atoms are equivalent on each of the BODIPY teeth, which are themselves all equivalent, due to combined free rotation along the Cp-Ru-B axis and each 4,4'-biphenyl axis. This is further confirmed by [START_REF] Negishi | Handbook of Organopalladium Chemistry for Organic Synthesis[END_REF] B NMR, exhibiting one triplet at 0.7 ppm with a coupling constant J ( 11 B-19 F) of 33.3 Hz. A rigid planar porphyrin scaffold was also used for the next generation of paddles to further increase the size and the surface of the teeth. The design of these paddles involves 3,5-di-tert-butylphenyl groups at three of the porphyrin meso positions, for higher stability, higher solubility and limited -stacking. Moreover, to avoid any undesired metallation of the porphyrins once deposited on the metallic surface, [19] we planned to use metallated porphyrins with nickel(II) selected as the metallic center. Using this core structure for paddles, it is possible to modulate the diameter of the gears by variation of the nature of the linker between the cyclopentadienyl platform and the porphyrin. Indeed, as mentioned earlier, Suzuki-Miyaura cross-coupling with a phenyl boronic acid derivative carrying the porphyrin core will give rise to a gear prototype with 4,4'-biphenyl linkers, whereas a Sonogashira cross-coupling will afford the corresponding extended gear with longer phenylethynylphenyl linkers.

The A3B-type porphyrinic coupling precursor 11 was synthesized following Lindsey's method [20] under statistical conditions (Scheme 4, left). Stoichiometric amounts of pyrrole, 3,5-di-tert-butylbenzaldehyde and 4-(tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde were treated with BF3OEt2 in chloroform, and subsequent oxidation with chloranil yielded the corresponding A3B porphyrin after purification. Metallation was then performed in the presence of excess nickel(II) acetylacetonate and the desired nickel porphyrin 11 was isolated in 8% overall yield over two steps. The same strategy was followed for the synthesis of the second porphyrin 13, carrying an ethynylphenyl group at one meso position (Scheme 5). The reaction of pyrrole, 3,5-di-tert-butylbenzaldehyde and 4-[(trimethylsilyl)ethynyl]benzaldehyde 15 under Lindsey's conditions, followed by alkyne deprotection and subsequent metallation gave the desired porphyrinic precursor 13 in 7% yield over 3 steps.

With both precursors in hand, the palladium-catalyzed cross-coupling reactions were tackled. The Suzuki-Miyaura reaction of brominated precursor 4a with A3B porphyrin-derived boronic acid pinacol ester 11 required some optimization. The desired gear 12 carrying five porphyrin paddles was isolated in 34% yield after 48 h at 100 °C in a DMF/water system (99:1), using only 7.5 equiv. of boronic ester 11 (i.e. 1.5 equiv./aryl bromide) in the presence of Pd(dppf)Cl2 (10 mol-%/Br) as catalyst and excess cesium carbonate as base (Scheme 4, left). In contrast, the pentaiodinated precursor 4b was selected for the Sonogashira reaction since aryl iodides are known to react under very mild conditions in such couplings. [21] Pentaaryl iodide 4b was thus reacted with alkynylporphyrin 13 in the presence of Pd(PPh3)2Cl2 (10 mol-%/I) and CuI (6 mol%/I) as catalysts in triethylamine (Scheme 5). After 24 h at 45 °C, product 14 resulting from five consecutive C-C couplings was isolated in 62% yield. Scheme 5. Five-fold Sonogashira cross-coupling reaction of pentaiodinated precursor 4b to yield molecular gear prototype 14, incorporating extended porphyrin-based paddles.

Both complexes were fully characterized by mass spectrometry as well as NMR and UV-visible spectroscopies. The lighter compound 12, incorporating 4,4'-biphenyl linkers, has a molar weight of 6162 g.mol -1 . This consists to a total 870 atoms, among which 405 carbon atoms. MALDI-TOF mass spectrometry was thus run on both samples and confirmed the penta-functionalization of the cyclopentadienyl ruthenium(II) core with nickel(II) porphyrins. Moreover, the presence of 40 -pyrrolic protons and 40 phenylene protons (in AA'BB' patterns) compared to a total of 12 protons on the indazole cores corroborated the expected structures for 12 and 14. Due to the high degree of symmetry within these structures and to free rotation at room temperature, the 1 H and 13 C NMR spectra could be fully assigned. Interestingly, two distinct sets of signals corresponding to the 3,5-di-tert-butylphenyl groups located on the 5,10,15 meso positions of the porphyrins were detected. Indeed, the ten 3,5-di-tertbutylphenyl substituents on the 5 and 15 meso positions are all equivalent, and can be distinguished from the five aryl groups located on the outer meso positions of the trans-A2BC porphyrins. It is important to note that this is observed for both complexes 12 and 14, meaning that the porphyrins carried by the smaller gear prototype 12 still undergo unrestricted rotation around the 4,4'-biphenyl axes, in addition to the free rotation around the Cp-Ru-B axis. Finally, a comparison of the UV-visible spectra of porphyrin precursors 11 and 13 with those of the corresponding penta-porphyrin complexes 12 and 14, respectively, showed that no shift of the Soret and Q bands occurs as a consequence of the porphyrins' connection. Incidentally, the molar extinction coefficients  associated to these bands are proportional to the number of porphyrin cores in the studied molecular species, revealing an additivity of the absorption properties of the chromophores in ruthenium complexes 12 and 14.

Conclusions

In this work, a series of prototype star-shaped molecular gears were synthesized with the aim of assembling them into trains of gears on surface and ultimately achieving controlled intermolecular gearing motion. The design of such rotary molecular machines is based on a piano-stool architecture, with the ruthenium center acting as ball bearing between the pentaarylcyclopentadienyl cogwheel and a hydrotris(indazolyl) borate ligand as rotation axle. The latter is specifically functionalized to provide tight anchoring on metallic surfaces, thus preventing diffusion of the molecular gears at 5 K and offering control over intermolecular distances in linear assemblies.

Given the lack of precedence in intermolecular transfer of motion between complexes, the star-shaped cogwheel were equipped with various types of teeth, ranging from substituted 4,4'-biphenyl groups to rigid nickel(II) porphyrins, in order to study the influence of their structure on intermeshing and gearing phenomena. Two distinct families of molecular gear prototypes were prepared, incorporating five identical pseudo 1-dimensional aryl teeth or large planar 2-dimensional paddles of various natures and lengths.

To efficiently reach such structural diversity, a divergent synthetic approach was followed starting from a key precursor, penta(p-bromophenyl)cyclopentadienyl ruthenium(II) complex and implementing various types of transition metal-catalyzed cross-couplings. Five-fold Suzuki-Miyaura reactions with different partners such as substituted phenylboronic acids, as well as BODIPY-and porphyrin-derived boronic esters gave a series of molecular gear prototypes featuring teeth of increasing size and length. To expand the scope of cross-coupling reactions, the pentaaryl iodide counterpart of the key precursor was prepared via an aromatic Finkelstein-type Br/I exchange. Subsequently, two extra gears incorporating carbazoles and porphyrins as 2D-paddles were obtained by copper-catalyzed N-arylation and Sonogashira coupling, respectively. All these compounds were fully characterized and were shown to undergo free rotation at room temperature, even in the case of the most sterically-hindered penta-porphyrin derivatives. Additionally, an X-ray crystal structure of the penta(p-tert-butylbiphenyl)cyclopentadienyl ruthenium complex was obtained, thus allowing validation of the expected 3D structural features of the molecular gears in anticipation of their on-surface study by STM.

In the absence of time-resolution, controlled motion at the single molecule scale is evidenced by comparison of STM images acquired before and after the stimulus. It thus appears highly desirable to include a dissymmetry element in cogwheels, in order to precisely monitor by STM a possible gearing effect within trains of gears. The synthesis of such dissymmetric molecular gears, including one tagged tooth, is currently underway.

Experimental Section

General methods: All commercially available chemicals were of reagent grade and were used without further purification. Anhydrous 1,2-dichloroethane, anhydrous THF, anhydrous acetonitrile, anhydrous toluene, anhydrous 1,4-dioxane, anhydrous N,N-dimethylformamide, methanol, chloroform, anhydrous triethylamine, 2,3dichloro-5,6-dicyano-p-benzoquinone (DDQ), 4-bromobenzaldehyde, chloranil, 3,5-bis(tertbutyl)benzaldehyde, 3,5-dimethylphenylboronic acid , 3,5-bis(tert-butylphenyl)boronic acid, bis(triphenylphosphine)palladium(II) dichloride, [1,1′-bis(diphenylphosphino)ferrocene]dichloro palladium(II), potassium phosphate, nickel(II) acetylacetonate, sodium iodide and (±)-trans-N,N′dimethylcyclohexane-1,2-diamine were purchased from Aldrich. Boron trifluoride diethyl etherate, pyrrole, 4-tert-butylphenylboronic acid, potassium carbonate, copper iodide and [1,1′bis(diphenylphosphino) ferrocene]dichloropalladium(II) (dichloromethane adduct) were purchased from Acros.

3,6-Di-tert-butyl-9H-carbazole, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)benzaldehyde, and palladium(II) acetate were purchased from ABCR. 2,4-Dimethyl-3-ethylpyrrole, trifluoroacetic acid, 2-dicyclohexylphosphino-2′-6′-dimethoxybiphenyl (SPhos) and (±)-trans-1,2diaminocyclohexane from TCI. Compounds 2a, 3 and 4a were synthesized according to the procedures recently reported by our group. [10] All reactions were carried out using standard Schlenk techniques under an argon atmosphere. Column chromatography was carried out on 230-400 mesh silica gel (Aldrich) unless otherwise stated. Microwave reactions were carried out using CEM Discover LabMate. Thin layer chromatography (TLC) was performed on pre-coated aluminum-backed silica gel 60 UV254 plates (Macherey-Nagel) with visualization effected using ultraviolet irradiation (λ = 254, 366 nm). NMR, IR and mass spectra were recorded by the appropriate services of the Toulouse Institute of Chemistry (ICT -FR2599). 1 H and 13 C NMR spectra were recorded on Bruker Avance III HD 500 MHz (cryoprobe Prodigy 5mm BBO, 1H ATMA), Avance 500 MHz (cryoprobe 5mm 1 H, 13 C), and Avance 300 MHz (probe 5mm BBO BB-1H Z-GRD) spectrometers. [START_REF] Negishi | Handbook of Organopalladium Chemistry for Organic Synthesis[END_REF] B and 19 F NMR spectra were recorded on an Avance 300 MHz (probe 5mm BBO BB-1H Z-GRD) spectrometer. Residual solvent signals were used as internal reference for 1 H and 13 C NMR, and 11 B and 19 F NMR spectra were referenced according to the solvent. Chemical shifts (δ) are reported in ppm. Coupling constants (J) are given in Hz and the following abbreviations have been used to describe the signals: singlet (s); broad singlet (br. s); doublet (d); triplet (t); quadruplet (q); quintuplet (quint); multiplet (m). Full assignments of 1 H and 13 C NMR spectra were made with the assistance of COSY, HMBC, and HSQC spectra when necessary. The numbering system used for the assignment of signals in new compounds is provided in the supporting information document, along with the corresponding spectra. IR spectra were recorded with a Nicolet 6700 FTIR-ATR. Only selected characteristic peaks are recorded. High-resolution mass spectra (HRMS) were performed with a Waters GCT Premier spectrometer for desorption chemical ionization (DCI/CH4), with a Waters Xevo G2 QTof spectrometer for electrospray ionization (ESI), and with a Waters MALDI micro MX spectrometer for matrix-assisted laser desorption ionization (MALDI) (matrix: trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile DTCB;  = 337 nm). UV/Vis spectra were recorded with a Shimadzu UV-26000 spectrometer (sh = shoulder, ε [mol -1 dm 3 cm - 1 ] is reported in parentheses). Cyclic voltammograms were obtained with an Autolab system (PGSTAT100) in CH2Cl2 (0.1 M tetrabutylammonium hexafluorophosphate nBu4NPF6 as the supporting electrolyte, scan rate of 0.1 V s -1 ) at 25 °C. A three-electrode cell was used comprised of a 1 mm platinum disk working electrode, a platinum wire auxiliary electrode, and an aqueous SCE reference electrode. Crystallographic data for compound 5 were collected at 193(2) K on a Bruker-AXS Kappa APEX II Quazar diffractometer equipped with a 30W air-cooled microfocus source using Mo Kα radiation (λ=0.71073 Å). Phi-and omega-scans were used. Space group was determined on the basis of systematic absences and intensity statistics. Semi-empirical absorption correction was employed. [22] The structure was solved using an intrinsic phasing method (SHELXT), [23] and refined using the leastsquares method on F 2 . [24] All non-H atoms were refined with anisotropic displacement parameters. Hydrogen atoms were refined isotropically at calculated positions using a riding model with their isotropic displacement parameters constrained to be equal to 1.5 times the equivalent isotropic displacement parameters of their pivot atoms for terminal sp 3 carbon and 1.2 times for all other carbon atoms. Hydrogen on Boron atom was located by difference Fourier map.

Iodo  5 -1,2,3,4,5-penta-(p-iodophenyl)cyclopentadienyl dicarbonyl ruthenium(II) (2b):

In a dry Schlenk tube were successively added a magnetic stirrer bar, NaI (700 mg, 4.67 mmol, 10 equiv.), bromo  5 -1,2,3,4,5-penta-(4-bromophenyl)cyclopentadienyl dicarbonyl ruthenium (II) 2a (500 mg, 0.46 mmol, 1.0 equiv.) and CuI (44 mg, 0.23 mmol, 0.5 equiv.). It was briefly evacuated and backfilled with argon. Degassed dry 1,4-dioxane (3 mL) and ()-trans-N,N'-dimethylcyclohexane-1,2diamine (75 μL, 0.48 mmol, 1.02 equiv.) were then added under argon. The reaction mixture was heated at 110 °C for 36 h in the dark. It was then allowed to cool down to room temperature, filtered through a celite pad (using CH2Cl2) and the solvent was removed under vacuum. The crude mixture was purified by column chromatography (SiO2, CH2Cl2/cyclohexane 1:1) to collect all the fractions containing partially and totally iodinated compounds. After evaporation of the solvents, the mixture of iodinated compounds was reengaged under the same conditions as above. After 36 h at 110 °C, the reaction mixture was filtered through a celite pad (using CH2Cl2) and the solvent was removed under vacuum. The crude mixture was purified by column chromatography (SiO2, CH2Cl2/cyclohexane 10:90) to yield the iodinated compound 2b as a yellow/orange solid (521.8 mg, 0.38 mmol, 83%). Rf = 0.45 (CH2Cl2/cyclohexane, 50:50); 1 H NMR (300 MHz, CD2Cl2, 25 °C): δ = 7.50 (AA'BB' pattern, 3 J = 8.7 Hz, 10H, Hb), 6.77 (AA'BB' pattern, 3 J = 8.7 Hz, 10H, Ha) ppm. 13 C{ 1 H} NMR (75 MHz, CD2Cl2, 25 °C): δ = 196.1 (C 1 ), 137.8 (C 5 ), 134.5 (C 4 ), 128.9 (C 3 ), 105.9 (C 2 ), 95.6 (C 6 ) ppm. MS (DCI/CH4): m/z: 1303.05 [M+H-2CO] + . The data match those reported in the literature. [START_REF] Carella | [END_REF]  5 -1,2,3,4,5-Penta-(p-iodophenyl)cyclopentadienyl hydrotris{6-[(ethylsulfanyl)methyl]indazol-1-yl}borate ruthenium(II) (4b): Conditions A (via ligand exchange on complex 2b): In a dry tube designed for microwave irradiation and under argon, compound 3 (160 mg, 0.20 mmol, 2.0 equiv.) and iodo  5 -1,2,3,4,5-penta-(4iodophenyl)cyclopentadienyl dicarbonyl ruthenium(II) 2b (140 mg, 0.10 mmol, 1.0 equiv.) were introduced followed by degassed anhydrous acetonitrile (4 mL). The tube was sealed and the reaction mixture was heated under microwave irradiation at 100 °C for 3 × 10 min. A pressure of 5 bar was achieved and it was released between heating cycles. The completion of the reaction was monitored by TLC. The resulting mixture was diluted with CH2Cl2 and filtered through a pad of silica gel. The solvents were removed under reduced pressure and the residue was purified by column chromatography (SiO2, CH2Cl2/cyclohexane 50:50) to afford compound 4b (94.7 mg, 0.05 mmol, 52 %) as an orange solid. Conditions B (via Br/I exchange on complex 4a): In a dry Schlenk tube were successively added a magnetic stirrer bar, NaI (99.8 mg, 0.66 mmol, 10 equiv.), complex 4a (100 mg, 65 µmol, 1.0 equiv.) and CuI (6.2 mg, 33 µmol, 0.5 equiv.). It was briefly evacuated and backfilled with argon. Degassed dry 1,4-dioxane (390 µL) and ()-trans-N,N'-dimethyl-1,2-cyclohexanediamine (10 µL, 66 µmol, 1.0 equiv.) were then added under argon. The reaction mixture was heated at 110 °C for 36 h in the dark. The reaction mixture was then filtered through a celite pad (using CH2Cl2) and the solvent was removed under vacuum. The crude mixture was quickly purified by column chromatography (SiO2, CH2Cl2/cyclohexane 1:1) to collect all the fractions containing iodinated compounds. After evaporation of the solvents, the mixture of iodinated compounds was reengaged under the same conditions as above. After 36 h at 110 °C, the reaction mixture was filtered on a celite pad (using CH2Cl2) and the solvent was removed under vacuum. The crude mixture was purified by column chromatography (SiO2, CH2Cl2/cyclohexane gradient from 10:90 up to 50:50) to yield the iodinated compound 4b as an orange solid (51.3 mg, 29 µmol, 44 %). Rf = 0.75 (CH2Cl2/cyclohexane, 50:50); 1 H NMR (300 MHz, CD2Cl2, 25 °C): δ = 7.87 (br. s, 3H, Hd), 7.78 (d, 4 J = 0.6 Hz, 3H, Ha), 7.39 (AA'BB' pattern, 3 J = 8.5 Hz, 10H, Hi), 7.35 (dd, 3 J = 8.4 Hz, 4 J = 0.6 Hz, 3H, Hc), 7.08 (AA'BB' pattern, 3 J = 8.5 Hz, 10H, Hh), 7.04 (dd, 3 J = 8.4 Hz, 4 J = 1.4 Hz, 3H, Hb), 3.90 (s, 6H, He), 2.46 (q, 3 J = 7.4 Hz, 6H, Hf), 1.27 (t, 3 J = 7.4 Hz, 9H, Hg) ppm. 13

General procedure for the synthesis of gears 5, 6 and 7 via Suzuki-Miyaura cross-couplings:

In a dry Schlenk tube under argon, pentaaryl bromide 4a (25 mg, 16 µmol, 1.0 equiv.), palladium(II) acetate (1.1 mg, 4.9 µmol, 0.3 equiv.), K3PO4 (35 mg, 170 µmol, 10.6 equiv.), the appropriate arylboronic acid (330 µmol, 20 equiv.) and 2-dicyclohexylphosphino-2′-6′-dimethoxybiphenyl (SPhos) (4 mg, 10 µmol, 0.6 equiv.) were successively introduced and degassed anhydrous toluene (2 mL) was added. The resulting suspension was stirred at 100 °C for 24 h and a second amount of palladium(II) acetate (1.1 mg, 4.9 µmol, 0.3 equiv.), K3PO4 (35 mg, 170 µmol, 10.6 equiv.), the appropriate arylboronic acid (330 µmol, 20 equiv.) and SPhos (4 mg, 10 µmol, 0.6 equiv.) were introduced. The reaction mixture was further stirred at 100 °C for 24 h and the completion of the reaction was monitored by TLC. The reaction mixture was cooled to room temperature, filtered through a celite pad (using CH2Cl2) and the solvents evaporated in vacuo. Purification by column chromatography yielded the corresponding product 5, 6 or 7.

 5 -1,2,3,4,5-Penta-(p-tert-butylbiphenyl)cyclopentadienyl hydrotris {6-[(ethylsulfanyl)methyl] indazol-1-yl}borate ruthenium(II) (5):

4-tert-Butylphenylboronic acid (2 x 60 mg) was used as coupling partner. The crude product was purified by column chromatography (SiO2, cyclohexane/CH2Cl2 70:30) followed by a precipitation in a CH2Cl2/CH3OH mixture (1:1). Complex 5 (24 mg, 13 µmol, 82 %) was obtained as a yellow-orange solid. Rf = 0.50 (cyclohexane/CH2Cl2, 50:50); 1 H NMR (500 MHz, CD2Cl2, 25 °C): δ = 8.03 (d, 3 J = 0.8 Hz, 3H, Ha), 7.92 (br. s, 3H, Hd), 7.56 (AA'BB' pattern, 3 J = 8.7 Hz, 10H, Hh), 7.47 (AA'BB' pattern, 3 J = 8.8 Hz, 10H, Hj), 7.39 (AA'BB' pattern, 3 J = 8.8 Hz, 10H, Hk), 7.34 (AA'BB' pattern, 3 J = 8.7 Hz, 10H, Hi), 7.31 (dd, 3 J = 8.3 Hz, 4 J = 0.8 Hz, 3H, Hb), 7.00 (dd, 3 J = 8.4 Hz, 4 J = 1.4 Hz, 3H, Hc), 3.91 (s, 6H, He), 2.48 (q, 3 J = 7.4 Hz, 6H, Hf), 1.30 (s, 45H, Hl), 1.28 (t, 3 J = 7.4 Hz, 9H, Hg) ppm. 13 3,5-Dimethylphenylboronic acid (2 x 49 mg) was used as coupling partner. The crude product was purified by column chromatography (SiO2, cyclohexane/CH2Cl2 0-50%) to yield complex 6 (14 mg, 9 µmol, 52 %) as a yellow-orange solid. Rf = 0.50 (cyclohexane/CH2Cl2, 50:50); 1 H NMR (500 MHz, CD2Cl2, 25 °C): δ = 8.03 (br. s, 3H, Ha), 7.92 (br. s, 3H, Hd), 7.56 (AA'BB' pattern, 3 J = 8.5 Hz, 10H, Hh), 7.33 (AA'BB' pattern, 3 J = 8.5 Hz, 10H, Hi), 7.32 (d, 3 J = 8.4 Hz, 3H, Hb), 7.16 (s, 10H, Hj), 7.01 (dd, 3 J = 8.4 Hz, 4 J = 1.3 Hz, 3H, Hc), 6.94 (s, 5H, Hk), 3.91 (s, 6H, He), 2.48 (q, 3 J = 7.4 Hz, 6H, Hf), 2.30 (s, 30H, Hl), 1.28 (t, 3 J = 7.4 Hz, 9H, Hg) ppm. 13 Conditions A (starting from pentaaryl bromide 4a): In a dry Schlenk tube under argon, pentaarylbromide 4a (25 mg, 16 µmol, 1.0 equiv.), 3,6-di-tert-butyl-9H-carbazole (46 mg, 160 µmol, 10 equiv.), CuI (3 mg, 16 µmol, 1.0 equiv.), K3PO4 (36.5 mg, 160 µmol, 10 equiv.) and ()-trans-1,2cyclohexanediamine (4 µL, 33 µmol, 2.0 equiv.) were successively introduced and degassed anhydrous 1,4-dioxane (2 mL) was added. The resulting suspension was stirred at 110 °C for 48 h in the dark and the completion of the reaction was monitored by TLC. The reaction mixture was cooled to room temperature, through a celite pad (using CH2Cl2) and the solvents evaporated in vacuo. The residue was purified by column chromatography (SiO2, CH2Cl2/cyclohexane 20:80) to afford compound 8 as an orange-red solid (8 mg, 3.2 µmol, 19 %). Conditions B (starting from pentaaryl iodide 4b): In a dry Schlenk tube under argon, penta-aryliodide 4b (29 mg, 16 µmol, 1.0 equiv.), 3,6-di-tert-butyl-9H-carbazole (46 mg, 160 µmol, 10 equiv.), CuI (3 mg, 16 µmol, 1.0 equiv.), K3PO4 (36.5 mg, 160 µmol, 10 equiv.) and ()-trans-1,2-cyclohexanediamine (4 µL, 33 µmol, 2 equiv.) were successively introduced and degassed anhydrous 1,4-dioxane (2 mL) was added. The resulting suspension was stirred at 110 °C for 48 h in the dark and the completion of the reaction was monitored by TLC. The reaction mixture was cooled to room temperature, filtered through a celite pad (using CH2Cl2) and the solvents evaporated in vacuo. The residue was purified by column chromatography (SiO2, CH2Cl2/cyclohexane 20:80) to afford compound 8 as an orange-red solid (11.7 mg, 4.6 µmol, 28 %). Rf = 0.55 (cyclohexane/CH2Cl2, 70:30); 1 H NMR (500 MHz, CD2Cl2, 25 °C): δ = 8.28 (br. s, 3H, Ha), 8.12 (dd, 4 J = 1.6 Hz, 5 J = 0.9 Hz, 10H, Hl), 7.96 (br. s, 3H, Hd), 7.84 (AA'BB' pattern, 3 J = 8.6 Hz, 10H, Hh), 7.48 (d, 3 J = 8.5 Hz, 3H, Hb), 7.44 (AA'BB' pattern, 3 J = 8.6 Hz, 10H, Hi), 7.37 (m, 20H, Hj and Hk), 7.09 (dd, 3 J = 8.5 Hz, 4 J = 1.4 Hz, 3H, Hc), 3.92 (s, 6H, He), 2.47 (q, 3 J = 7.4 Hz, 6H, Hf), 1.41 (s, 90H, Hm), 1.27 (t, 3 J = 7.4 Hz, 9H, Hg) ppm. 13 To a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (1 g, 4.31 mmol, 1.0 equiv.) and 3-ethyl-2,4-dimethyl-1H-pyrrole (1.2 mL, 8.89 mmol, 2.0 equiv.) in CH2Cl2 (360 mL) under argon, trifluoroacetic acid (30 µL, 0.404 mmol, 0.1 equiv.) was added. After 3 h at room temperature in the dark, DDQ (0.98 g, 4.31 mmol, 1.0 equiv.) was added. The solution immediately turned dark purple and was stirred for an additional 30 min at room temperature. Triethylamine (13 mL, 93.5 mmol, 21 equiv.) was introduced and BF3•OEt2 was then added dropwise until green reflects appeared in the solution (added volume: 9 mL, i.e. 71.7 mmol, 16.6 equiv.). The resulting mixture was washed with water (3 x 500 mL), dried over Na2SO4, filtered and evaporated. The crude product was purified by column chromatography (SiO2, CH2Cl2/cyclohexane 1:1) to afford compound 9 (1.13 g, 2.23 mmol, 52 %) as a dark red solid. 1 H NMR (300 MHz, CD2Cl2, 25 °C): δ = 7.88 (AA'BB' pattern, 3 J = 8.2 Hz, 2H), 7.31 (AA'BB' pattern, 3 J = 8.2 Hz, 2H), 2.49 (s, 6H), 2.32 (q, 3 J = 7.5 Hz, 4H), 1.38 (s, 12H), 1.27 (s, 6H), 0.99 (t, 3 J = 7.5 Hz, 6H) ppm. 13 C{ 1 H} NMR (126 MHz, CD2Cl2, 25 °C): δ = 154.0, 140.7, 139.0, 138.9, 135.6, 133.3, 130.9, 128.1, 84.5, 25.2, 17.4, 14.8, 12.7 (t, J ( 13 C-19 F) = 2.3 Hz), 12.0 ppm. 19 F NMR (282 MHz, CD2Cl2, 25 °C): δ = -145.7 (q, J ( 11 B-19 F) = 33.3 Hz) ppm. [START_REF] Negishi | Handbook of Organopalladium Chemistry for Organic Synthesis[END_REF] B NMR (96 MHz, CD2Cl2,

Figure 1 .

 1 Figure 1. Structure of molecular motor 1 (left) and general design of ruthenium-based star-shaped molecular gears (right).

Figure 3 .

 3 Figure 3. Side view (a) and top view (b) of the molecular structure of ruthenium complex 5. Thermal ellipsoids drawn at 50% probability, hydrogen atoms, solvent molecules and disordered atoms are omitted for clarity.

Scheme 4 .

 4 Scheme 4. Five-fold Suzuki-Miyaura cross-coupling reactions of pentabrominated precursor 4a to yield molecular gears prototypes 10 and 12, incorporating BODIPY-and porphyrin-based paddles, respectively.

  C{ 1 H} NMR (126 MHz, CD2Cl2, 25 °C): δ = 144.0 (C 2 ), 140.7 (C 1 ), 138.2 (C 5 ), 137.0 (C 13 ), 135.7 (C 14 ), 133.2 (C 12 ), 122.7 (C 4 ), 122.4 (C 7 ), 120.4 (C 3 ), 111.3 (C 6 ), 94.1 (C 15 ), 87.5 (C 11 ), 36.9 (C 8 ), 25.7 (C 9 ), 14.7 (C 10 ) ppm. HRMS (ESI + ): calcd. for C65H54BI5N6RuS3 [M] + :1761.7955, found 1761.7992.

 5 - 1 , 2 , 3 , 4 , 5 -

 512345 Penta-(3,5-dimethylbiphenyl)cyclopentadienyl hydrotris {6-[(ethylsulfanyl)methyl] indazol-1-yl}borate ruthenium(II) (6):

5 -

 5 C{ 1 H} NMR (126 MHz, CD2Cl2, 25 °C): δ = 144.1 (C 2 ), 141.0 (C 1 ), 140.4 (C 16 ), 140.3 (C 15 ), 138.7 (C 18 ), 137.7 (C 5 ), 134.6 (C 13 ), 133.3 (C 12 ), 129.3 (C 19 ), 126.0 (C 14 ), 125.0 (C 17 ), 122.6 (C 7 ), 122.4 (C 4 ), 120.4 (C 3 ), 111.4 (C 6 ), 88.3 (C 11 ), 36.9 (C 8 ), 25.7 (C 9 ), 21.5 (C 20 ), 14.8 (C 10 ) ppm. HRMS (MALDI): calcd. for C105H99BN6RuS3 [M] + :1653.6278, found 1653.6239. Di-tert-butylphenylboronic acid (2 x 77 mg) was used as coupling partner. The crude product was purified by column chromatography (SiO2, cyclohexane/CH2Cl2 70:30) to yield complex 7 (22 mg, 11 µmol, 65 %) as a yellow-orange solid. Rf = 0.55 (cyclohexane/CH2Cl2, 50:50); 1 H NMR (500 MHz, CD2Cl2, 25 °C): δ = 8.06 (br. s, 3H, Ha), 7.93 (br. s, 3H, Hd), 7.62 (AA'BB' pattern, 3 J = 8.5 Hz, 10H, Hh), 7.39 (m, 25H, Hi, Hj and Hk), 7.35 (d, 3 J = 8.4 Hz, 3H, Hb), 7.01 (dd, 3 J = 8.4 Hz, 4 J = 1.4 Hz, 3H, Hc), 3.91 (s, 6H, He), 2.48 (q, 3 J = 7.4 Hz, 6H, Hf), 1.32 (s, 90H, Hl), 1.29 (t, 3 J = 7.4 Hz, 9H, Hg) ppm. 13 C{ 1 H} NMR (126 MHz, CD2Cl2, 25 °C): δ = 151.7 (C 18 ), 144.1 (C 2 ), 141.3 (C 15 ), 140.9 (C 1 ), 140.0 (C 16 ), 137.7 (C 5 ), 134.7 (C 13 ), 132.3 (C 12 ), 126.4 (C 14 ), 122.6 (C 7 ), 122.4 (C 4 ), 121.9 (C 19 ), 121.7 (C 17 ), 120.5 (C 3 ), 111.4 (C 6 ), 88.4 (C 11 ), 37.0 (C 8 ), 35.3 (C 20 ), 31.7 (C 21 ), 25.7 (C 9 ), 14.8 (C 10 ) ppm. HRMS (MALDI): calcd. for C135H160BN6RuS3 [MH] + :2074.1047, found 2074.1011.

  C{ 1 H} NMR (126 MHz, CD2Cl2, 25 °C): δ = 144.2 (C 2 ), 143.5 (C 19 ), 140.5 (C 1 ), 139.3 (C 16 ), 138.1 (C 5 ), 137.8 (C 15 ), 135.3 (C 13 ), 133.2 (C 12 ), 125.8 (C 14 ), 124.1 (C 18 ), 123.8 (C 21 ), 122.7 (C 7 ), 122.7 (C 4 ), 120.5 (C 3 ), 116.7 (C 20 ), 111.5 (C 6 ), 109.6 (C 17 ), 87.8 (C 11 ), 36.9 (C 8 ), 35.0 (C 22 ), 32.1 (C 23 ), 25.7 (C 9 ), 14.8 (C 10 ) ppm. HRMS (MALDI): calcd. for C165H174BN11RuS3 [M] + :2519.2314, found 2519.2317. 2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)phenyl]-4-bora-3a,4a-diaza-s-indacene (9):

  C{1 H} NMR (126 MHz, CD2Cl2, 25 °C): δ = 150.9 (C19 ), 144.0 (C 2 ), 140.9 (C 1 ), 139.8 (C15 ), 137.7 (C 5 ), 137.6 (C 16 ), 134.6 (C 13 ), 133.3 (C 12 ), 126.7 (C 17 ), 126.1 (C 18 ), 125.9 (C 14 ), 122.6 7 ), 122.4 (C 4 ), 120.4 (C 3 ), 111.4 (C 6 ), 88.3 (C 11 ), 36.9 (C 8 ), 34.8 (C 20 ), 31.4 (C 21 ), 25.7 (C 9 ), 14.8 (C 10 ) ppm. HRMS (MALDI): calcd. for C115H120BN6RuS3 [MH] + :1793.7913, found 1793.7889. Crystal data: Crystals suitable for X-Ray diffraction were obtained from the slow diffusion of methanol in a toluene solution of complex 5. CCDC-1943538 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Selected data for 5: C115H119BN6RuS3, 2 C7H8 M=1977.48, triclinic, space group P1, a=17.6038(8) Å, b=8.0649(7) Å, c=19.7291(9) Å, =109.049(2)°, =92.140(3)°, =96.350(2)° V=5876.1(4) Å 3 , Z=2, crystal size 0.24 x 0.22 x 0.18 mm 3 , 150409 reflections collected (23944 independent, Rint = 0.0874), 1742 parameters, 2402 restraints, R1[I>2(I)]=0.0694, wR2[all data]=0.2199, largest diff. peak and hole: 1.271 and -0.825 eÅ -3 .

Acknowledgements

This work was supported by the University Paul Sabatier (Toulouse, France) and the Centre National de la Recherche Scientifique (CNRS). It has received funding from the Agence Nationale de la Recherche (ANR) (ACTION project ANR-15-CE29-0005) and from the European Union's Horizon 2020 research and innovation programme under the project MEMO, grant agreement No 766864. This research was also partly supported by the JSPS KAKENHI grant in aid for Scientific Research on Innovative Areas "Molecular Engine (No.8006)" 18H05419. Y.G. thanks the French Ministry of National Education for a PhD Fellowship. Dr. Colin Martin is warmly acknowledged for his careful reading and improving of our manuscript and Dr. Jacques Bonvoisin is acknowledged for his expertise regarding cyclic voltammetry experiments.

mol -1 dm 3 cm -1 ). HRMS (ESI + ): calcd. for C29H39B2F2N2O2 [MH] + :507.3166, found 507.3168. The data match those reported ( 1 H NMR and MS) in the literature. [17]  5 -1,2,3,4,5-Penta-{p-[2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl]-4,4'-biphenyl}cyclopentadienyl hydrotris{6-[(ethylsulfanyl)methyl]indazol-1-yl}borate ruthenium(II) (10): In a dry Schlenk tube under argon, pentaaryl bromide 4a (50 mg, 33 µmol, 1.0 equiv.), palladium(II) acetate (2.5 mg, 11 µmol, 0.3 equiv.), K3PO4 (70 mg, 330 µmol, 10 equiv.), the arylboronic acid pinacol ester 9 (166 mg, 328 µmol, 10 equiv.) and 2-dicyclohexylphosphino-2′-6′-dimethoxybiphenyl (SPhos) (8 mg, 19.5 µmol, 0.6 equiv.) were successively introduced in a degassed mixture of toluene/ethanol/water 2:2:1 (10 mL). The resulting suspension was stirred at 100 °C for 48 h and the completion of the reaction was monitored by TLC. The reaction mixture was cooled to room temperature, filtered through a celite pad (using ethyl acetate) and the solvents evaporated in vacuo. The crude product was purified by column chromatography (SiO2, CH2Cl2/MeOH 0-5%) followed by precipitation in a CH2Cl2/MeOH mixture (1:1) to afford complex 10 (35.7 mg, 11.8 µmol, 36%) as a red solid. Rf = 0.60 (CH2Cl2/CH3OH, 95:5); 1 H NMR (500 MHz, CD2Cl2, 25 °C): δ = 8.09 (s, 3H, Ha), 7.97 (br. s, 3H, Hd), 7.76-7.67 (m, 20H, Hh and Hj), 7.52 (AA'BB' pattern, 3 J = 8.6 Hz, 10H, Hi), 7.39 (d, 3 J = 8.4 Hz, 3H, Hb), 7.32 (AA'BB' pattern, 3 J = 8.7 Hz, 10H, Hk), 7.06 (dd, 3 J = 8.4 Hz, 4 J = 1.4 Hz, 3H, Hc), 3.94 (s, 6H, He), 2.50 (q, 3 J = 7.4 Hz, 6H, Hf), 2.47 (s, 30H, Ho), 2.29 (q, 3 J = 7.5 Hz, 20H, Hm), 1.31 (s, 30H, Hl), 1.30 (t, 3 J = 7.4 Hz, 9H, Hg), 0.96 (t, 3 J = 7.5 Hz, 30H, Hn) ppm. 13 To a solution of freshly distilled pyrrole (1.0 mL, 14.31 mmol, 4.0 equiv.) in anhydrous CHCl3 (1.5 L), 3,5-di-tert-butylbenzaldehyde (2.34 g, 10.73 mmol, 3.0 equiv.), 4-(tetramethyl-1,3,2-dioxaborolan-2yl)benzaldehyde (0.83 g, 3.58 mmol, 1.0 equiv.) and BF3OEt2 (0.54 mL, 4.30 mmol, 1.2 equiv.) were added under argon. After stirring the reaction mixture for 1 h at room temperature, chloranil (3.82 g, 15.55 mmol, 4.35 equiv.) was added and the mixture was refluxed for 30 min. After cooling to room temperature, the crude product was washed with water and evaporated to dryness. Then, the residue was purified by column chromatography (SiO2, cyclohexane/CH2Cl2 gradient from 4:1 to 1:1) to yield 5,10,15-tris (3,5-

porphyrin (500 mg, 0.46 mmol, 13%) as a purple solid. In a dry Schlenk tube under argon, a solution of this free-base porphyrin (226 mg, 0.21 mmol, 1.0 equiv.) and nickel(II) acetylacetonate Ni(acac)2 (216 mg, 0.84 mmol, 4.0 equiv.) in dry toluene (20 mL) was heated at 110 °C overnight. The mixture was cooled to room temperature and volatiles were evaporated. The crude product was purified by column chromatography (SiO2, cyclohexane/CH2Cl2 2:1) to yield nickel porphyrin 11 (152 mg, 0.13 mmol, 64%) as a red solid. Rf = 0.60 (cyclohexane/CH2Cl2, 2:1); 1 H NMR (500 MHz, CDCl3, 25 °C): δ = 8.80 (m, 4H, Hβ), 8.77 (d, 3 J = 5.0 Hz, 2H, Hβ), 8.72 (d, 3 J = 5.0 Hz, 2H, Hβ), 8.11 (AA'BB' pattern, 3 J = 8.0 Hz, 2H, H11), 8.04 (AA'BB' pattern, 3 J = 8.0 Hz, 2H, H12), 7.88-7.85 (m, 6H, H5 and H5'), 7.72-7.69 (m, 3H, H4 and H4'), 1.47 (s, 66H, H1 and H1' and H15) ppm. 13 In a dry Schlenk tube under argon, pentaaryl bromide 4a (10 mg, 6.6 μmol, 1.0 equiv.), nickel porphyrin 11 (56 mg, 49.1 μmol, 7.5 equiv.), Pd(dppf)Cl2CH2Cl2 (2.7 mg, 3.3 μmol, 0.5 equiv.) and Cs2CO3 (26 mg, 78.6 μmol, 12 equiv.) were successively introduced in 2 mL of a preliminary degassed solution of DMF and water (99:1). The resulting mixture was heated for 48 h at 100 °C. The reaction mixture was then allowed to cool to room temperature and the solvents were evaporated in vacuo. The residue was adsorbed onto silica and further purified by column chromatography (SiO2, heptane/CH2Cl2 4:1) to afford molecular gear 12 (13.8 mg, 2.2 µmol, 34%) as a red solid. Rf = 0.50 (heptane/CH2Cl2, 1:1); 1 H NMR (500 MHz, CD2Cl2, 25 °C): δ = 8.83-8.74 (m, 40H, Hβ), 8.36 (s, 3H, H1), 8.15 (br. s, 3H, H6), 8.11 (AA'BB' pattern, 3 J = 8.3 Hz, 10H, H11'), 8.02 (AA'BB' pattern, 3 J = 8.3 Hz, 10H, H12'), 7.95 (AA'BB' pattern, 3 J = 8.5 Hz, 10H, H13), 7.88 (d, 4 J = 1.9 Hz, 10H, H5''), 7.85 (d, 4 J = 1.9 Hz, 20H, H5'), 7.83 (AA'BB' pattern, 3 J = 8.5 Hz, 10H, H14), 7.77 (t, 4 J = 1.9 Hz, 5H, H4''), 7.70 (t, 4 J = 1.9 Hz, 10H, H4'), 7.57 (d, 3 J = 8.5 Hz, 3H, H3), 7.12 (d, 3 J = 8.5 Hz, 3H, H4), 4.01 (s, 6H, H8), 2.56 (q, 3 J = 7.4 Hz, 6H, H9), 1.48 (s, 90H, H1''), 1.41 (s, 180H, H1'), 1.34 (t, 3 J = 7.4 Hz, 9H, H10) ppm. 13

Nickel(II) 5-(4-ethynylphenyl)-10,15,20-tris(3,5-di-tert-butylphenyl) porphyrin (13):

To a solution of freshly distilled pyrrole (0.8 mL, 12.1 mmol, 4.0 equiv.) in anhydrous CHCl3 (1.2 L), 3,5-di-tert-butylbenzaldehyde (2.0 g, 9.17 mmol, 3.0 equiv.), 4-[(trimethylsilyl)ethynyl]benzaldehyde 15 (0.617 g, 3.04 mmol, 1.0 equiv.) and BF3OEt2 (1.19 mL, 9.47 mmol, 3.1 equiv.) were added under argon. After stirring the reaction mixture for 1 h at room temperature, DDQ (2.07 g, 9.11 mmol, 3.0 equiv.) was added and the mixture again stirred for 1 h at room temperature. In order to neutralize the Lewis acid, Et3N (0.5 mL) was added and the solution was stirred for 10 min. The solvent was evaporated and the residue was purified by column chromatography (SiO2, cyclohexane/CH2Cl2 5:1). The second band was collected to afford 5-[4-(trimethylsilylethynyl)phenyl]-10,15,20-tris(3,5-di-tertbutylphenyl)porphyrin in 8% yield (264 mg, 0.25 mmol). This porphyrin (264 mg, 0.25 mmol, 1.0 equiv.) was then dissolved in a MeOH/CH2Cl2 (3:1) solution (10 mL) and K2CO3 (104 mg, 0.76 mmol, 3.0 equiv.) was added. After stirring for 12 h at room temperature, the solution was quenched with H2O and the aqueous layer was extracted with CH2Cl2. The combined organic layers were then washed with brine, dried on Na2SO4, filtered and the solvent was removed in vacuum affording 5-(4-ethynylphenyl)-10,15,20-tris(3,5-di-tert-butylphenyl)porphyrin (241 mg, 0.24 mmol, 98%) as a purple solid. In a dry Schlenk tube under argon, a solution of this free-base porphyrin (104 mg, 0.107 mmol, 1.0 equiv.) and nickel(II) acetylacetonate Ni(acac)2 (110 mg, 0.426 mmol, 4.0 equiv.) in dry toluene (20 mL) was heated at 110 °C overnight. Then the mixture was cooled to room temperature and volatiles were evaporated.

The crude product was purified by column chromatography (SiO2, cyclohexane/CH2Cl2 7:3) to yield nickel porphyrin 13 (104 mg, 0.102 mmol, 95%) as a red solid. Rf = 0.80 (cyclohexane/CH2Cl2, 2:1); 1 H NMR (500 MHz, CDCl3, 25 °C): δ = 8.81 (m, 4H, Hβ), 8.80 (d, 3 J = 4.9 Hz, 2H, Hβ), 8.71 (d, 3 J = 4.9 Hz, 2H, Hβ), 8.00 (AA'BB' pattern, 3 J = 8.4 Hz, 2H, H11), 7.89-7.86 (m, 6H, H5 and H5'), 7.81 (AA'BB' pattern, 3 J = 8.4 Hz, 2H, H12), 7.74-7.71 (m, 3H, H4 and H4'), 3.27 (s, 1H, H15), 1.48 (s, 54H, H1 and H1') ppm. 13 ) ppm. UV/Vis (CH2Cl2): λmax (ε) = 417 (248 000), 528 nm (18 000 mol -1 dm 3 cm -1 ). HRMS (ESI + ): calcd. for C70H77N4Ni [MH] + : 1031.5502, found 1031.5498. The data match those reported in the literature. [25]  In a dry Schlenk tube under argon, pentaaryl iodide 4b (10 mg, 5.7 μmol, 1.0 equiv.), nickel porphyrin 13 (44 mg, 42.6 μmol, 7.5 equiv.), Pd(PPh3)2Cl2 (2 mg, 2.85 μmol, 0.5 equiv.) and CuI (0.3 mg, 1.7 μmol, 0.3 equiv.) were successively introduced and dry NEt3 (2 mL) was added. The resulting mixture was degassed for 15 min and then heated for 24 h at 45 °C. After evaporation, the residue was adsorbed onto silica and further purified by column chromatography (SiO2, cyclohexane/CH2Cl2 4:1) to afford molecular gear 14 (22 mg, 3.5 µmol, 62%) as a red solid. Rf = 0.50 (cyclohexane/CH2Cl2, 2:1); 1 H NMR (500 MHz, CD2Cl2, 25 °C): δ = 8.80-8.75 (m, 40H, Hβ), 8.29 (br. s, 3H, H6), 8.13 (s, 3H, H1), 8.04 (AA'BB' pattern, 3 J = 8.4 Hz, 10H, H11'), 7.90-7.85 (m, 40H, H12' and H5' and H5''), 7.77 (t, 4 J = 1.8 Hz, 5H, H4''), 7.73 (t, 4 J = 1.8 Hz, 10H, H4'), 7.63 (AA'BB' pattern, 3 J = 8.3 Hz, 10H, H13), 7.54 (d, 3 J = 8.6 Hz, 3H, H3), 7.50 (AA'BB' pattern, 3 J = 8.3 Hz, 10H, H14), 7.10 (d, 3 J = 8.6 Hz, 3H, H4), 4.07 (s, 6H, H8), 2.61 (q, 3 J = 7.4 Hz, 6H, H9), 1.48 (s, 90H, H1''), 1.44 (s, 180H, H1'), 1.36 (t, 3 J = 7.4 Hz, 9H, H10) ppm. 13 ), 129.1 (C 5' ), 129.1 (C 5'' ), 123.0 (C 13' ), 122.9 (C 7 and C 4 ), 122.8 (C 15 ), 121.8 (C 4' and C 4'' ), 120.9 (C 8' ), 120.8 (C 7' ), 120.5 (C 3 ), 118.3 (C 9' ), 112.1 (C 6 ), 90.7 (C 15' ), 90.5 (C 14' ), 88.3 (C 11 ), 37.5 (C 8 ), 35.3 (C 2' and C 2'' ), 31.8 (C 1' and C 1'' ), 26.6 (C 9 ), 14.7 (C 10 ) ppm. UV/Vis (CH2Cl2): λmax (ε) = 418 (1 390 000), 529 nm (103 000 mol -1 dm 3 cm -1 ). CV (CH2Cl2, nBu4NPF6, 0.1 V s -1 ), E1/2 (V/SCE): +0.78 rev (1e), +0.97 rev (10e). HRMS (MALDI): calcd. for C415H430BN26Ni5RuS3 [MH] + : 6275.9507, found 6275.9233.

4-[2-(Trimethylsilyl)ethynyl]benzaldehyde (15):

In a dry Schlenk tube under argon, 4-bromobenzaldehyde (3.5 g, 19 mmol, 1.0 equiv.), Pd(PPh3)2Cl2 (80 mg, 0.11 mmol, 0.6 mol%) and CuI (11 mg, 0.06 mmol, 0.3 mol%) were successively introduced, followed by NEt3 (10 mL), THF (10 mL) and ethynyltrimethylsilane (3.24 mL, 22.8 mmol, 1.2 equiv.). The system was then degassed and heated overnight at 45 °C. After evaporation, the residue was adsorbed onto silica and purified by column chromatography (SiO2, heptane/CH2Cl2 2:1) to afford the desired product 15 (3.7 g, 18.3 mmol, 97%) as a white powder. Rf = 0.40 (heptane/CH2Cl2, 2:1); 1 H NMR (300 MHz, CDCl3, 25 °C): δ = 10.00 (s, 1H, CHO), 7.81 (AA'BB' pattern, 3 J = 8.5 Hz, 2H, CHAr), 7.60 (AA'BB' pattern, 3 J = 8.5 Hz, 2H, CHAr), 0.26 (s, 9H, CH3) ppm. The data match those reported in the literature. [26]


Maren Preuss
email: maren142@web.de

Narongrit Muangmai

Wendy A Nelson

Marie-Laure Guillemin

John A West

Giuseppe C Zuccarello

from Australia and New Zealand

Keywords: cox1, ITS1, Phylogeny, rbcL, Rhodophyta, Systematics, Taxonomy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The Gracilariaceae is one of the most species-rich and commercially important red algal families. Some Gracilariaceae members are known for for their phenotypic plasticity, relatively simple and convergent morphologies. Due to their commercial importance and classification challenge, numerous taxonomic studies have been done on the group (e.g., [START_REF] Abbott | Taxonomy of economic seaweeds: with reference to the Pacific and other locations[END_REF]). Some genera have been proposed and later discarded, especially lineages related to Gracilaria. The stability and utility of the genus Hydropuntia has been controversial, with authors both accepting [START_REF] Gurgel | Systematics of the Gracilariaceae (Gracilariales, Rhodophyta): A critical assessment based on rbcL sequence analyses[END_REF][START_REF] Gurgel | Systematics of the Gracilariales (Rhodophyta) including new subfamilies, tribes, subgenera, and two new genera, Agarophyton gen. nov. and Crassa gen[END_REF] and not accepting this change [START_REF] Iha | Organellar genomics: a useful tool to study evolutionary relationships and molecular evolution in Gracilariaceae (Rhodophyta)[END_REF][START_REF] Lyra | Phylogeny of Gracilariaceae (Rhodophyta): Evidence from plastid and mitochondrial nucleotide sequences[END_REF], plus a name was suggested, but not formally proposed, for a lineage containing one of the most important commercial Gracilaria's, Gracilaria chilensis C.J. Bird, McLachlan & E.C.Oliveira, and a well-known invasive alga, Gracilaria vermiculophylla Ohmi [START_REF] Gurgel | Systematics of the Gracilariaceae (Gracilariales, Rhodophyta): A critical assessment based on rbcL sequence analyses[END_REF]. A recent taxonomy has accepted both Hydropuntia, and formally described a new genus, Agarophyton, for the lineage containing Gracilaria chilensis [START_REF] Guiry | nom. inval. (Gracilariaceae, Rhodophyta), with some additional nomenclatural notes[END_REF][START_REF] Gurgel | Systematics of the Gracilariales (Rhodophyta) including new subfamilies, tribes, subgenera, and two new genera, Agarophyton gen. nov. and Crassa gen[END_REF]. & Fredericq, are the second most intensively cultivated red algal group world-wide with 3.9 million tonnes harvested per year (after Eucheuma with 10.2 million tonnes, [START_REF] Ferdouse | The global status of seaweed production, trade and utilization. Food and agriculture organization of the United Nations[END_REF]. Plus, the invasive nature of A. vermiculophyllum (Ohmi) Gurgel, J.N.Norris & Fredericq has been well studied [START_REF] Bippus | Palatability of an introduced seaweed does not differ between native and non-native populations[END_REF][START_REF] Hu | Adaptation mechanisms and ecological consequences of seaweed invasions: A review case of agarophyte Gracilaria vermiculophylla[END_REF][START_REF] Krueger-Hadfield | Invasion of novel habitats uncouples haplo-diplontic life cycles[END_REF][START_REF] Krueger-Hadfield | Genetic identification of source and likely vector of a widespread marine invader[END_REF]. Many members of the Gracilariaceae have remarkable applications including for human and animal consumption, in the pharmaceutical industry, and as medical agar [START_REF] Ferdouse | The global status of seaweed production, trade and utilization. Food and agriculture organization of the United Nations[END_REF]. Also some species are being cultivated for bioremediation in polyculture as part of multi-trophic aquaculture [START_REF] Pereira | Mass production of marine macroalgae[END_REF].

The similar morphological characters of some Gracilariaceae species has led to problems in their taxonomy. Gracilaria chilensis (now Agarophyton chilense) in New Zealand and Chile was initially described as Gracilaria sordida [START_REF] Nelson | The New Zealand species of Gracilaria Greville (Rhodophyta, Gigartinales)[END_REF]) in New Zealand. Gracilaria sordida was later synonymised with G. chilensis, a species that was described a year earlier [START_REF] Bird | Gracilaria chilensis sp. nov. (Rhodophyta, Gigartinales), from Pacific South America[END_REF], based on similarities in morphology and molecular data [START_REF] Bird | A critical comparison of Gracilaria chilensis and G. sordida (Rhodophyta, Gracilariales)[END_REF]. Later a molecular analysis of a sample, incorrectly, identified as A.

chilense from the Manukau Harbour, Auckland, was grouped as sister to A. tenuistipitatum [START_REF] Goff | Molecular delineation of species and species relationships in the red algal agarophytes Gracilariopsis and Gracilaria (Gracilariales)[END_REF]. ITS restriction fragment length polymorphism demonstrated that while A. chilense is present in New Zealand, another species was found in Manukau Harbour [START_REF] Candia | Comparison of ITS RFLP patterns of Gracilaria (Rhodophyceae, Gracilariales) populations from Chile and New Zealand and an examination of interfertility of Chilean morphotypes[END_REF]. The difference between these two species in New Zealand was further demonstrated by the presence of gigartinine (5-(3-amidinoureido)-2-aminovaleric acid) [START_REF] Ito | Gigartinine: A new amino-acid in red algae[END_REF] in the Manukau Harbour samples and not in A. chilense (Wilcox et al. 2001(Wilcox et al. , 2007)). Further phylogenetic studies demonstrated that the specimen sequenced by [START_REF] Goff | Molecular delineation of species and species relationships in the red algal agarophytes Gracilariopsis and Gracilaria (Gracilariales)[END_REF] was identical with material from Australia [START_REF] Byrne | Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content[END_REF] and therefore was also placed erroneously in A. chilense. A phylogenetic study incorporating specimens from Australia, Chile and New Zealand clearly showed that there are two genetically different species incorrectly placed under the same name: A. chilense from Chile and New Zealand, and an undescribed species from Australia and New Zealand (Clade B) [START_REF] Cohen | Molecular identification of two sibling species under the name Gracilaria chilensis (Rhodophyta, Gracilariales)[END_REF].

The aim of this study is to formally describe the vegetative and reproductive morphology of this new species, currently known as A. sp. clade B from New Zealand and Australia, and compare its morphological characteristics and phylogenetic position to A. chilense and the other two Agarophyton species, A. tenuistipitatum and A. vermiculophyllum. This present work extends our understanding of the genus Agarophyton in New Zealand providing a number of opportunities for future research in their phylogeography, ecophysiology and biochemistry.

MATERIALS AND METHODS

Algal sampling. Algae were collected during low tide from Australia and New Zealand (Table S1). Algal samples were pressed onto herbarium sheets and subsamples stored in silica gel. Type and voucher herbarium specimens have been deposited in the herbarium of the Museum of New Zealand Te Papa Tongarewa, Wellington, New Zealand (WELT -Thiers 2019). For morphological observation, sections were made by hand using a stainless-steel razor blade and stained with 1% aniline blue in 40% Karo® corn syrup. Photographs were obtained by microscope digital camera Olympus DP-70 (Olympus, Tokyo, Japan).

Molecular analyses. DNA extraction followed the Chelex-based technique (Zuccarello et al. 1999). A mitochondrial (the cytochrome-c-oxidase subunit I, cox1), a nuclear (internal transcript spacer 1, ITS1) marker and a plastid (the ribulose 1,5-bisphosphate carboxylase/oxygenase gene, rbcL) marker were chosen according to previous systematic studies of Gracilariaceae [START_REF] Cohen | Molecular identification of two sibling species under the name Gracilaria chilensis (Rhodophyta, Gracilariales)[END_REF][START_REF] Gurgel | Systematics of the Gracilariales (Rhodophyta) including new subfamilies, tribes, subgenera, and two new genera, Agarophyton gen. nov. and Crassa gen[END_REF]. Genomic DNA was amplified by PCR using primer combinations for cox1 [START_REF] Saunders | Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications[END_REF], for ITS1 [START_REF] White | Amplification and direct sequencing of fungal 1 This study[END_REF], and for rbcL [START_REF] Freshwater | Phylogenetic relationships of some European Gelidium (Gelidiales, Rhodophyta) species, based on rbcL nucleotide sequence analysis[END_REF]Kim et al. 2010). The PCR conditions for ITS1 amplification were as follows: initial denaturation at 94°C for 5 mins, followed by 16 cycles of 94°C for 1 min, 62.5°C for 30sec and 72°C for 1 min, followed by 25 cycles of 94°C/55°C/72°C for 1 min each and a final step at 72°C for 10 min. The PCR profile for cox1 and rbcL were as follows: 94°C for 5 min; 36 cycles of 94°C/45°C/72°C for 1 min each and then a final step of 5 min for extension at 72°C. The PCR amplifications were examined by electrophoresis in 1% agarose gel, and PCR products were cleaned with ExoSAP-IT (USB product, Affymetrix, Santa Clara, California, USA). Purified PCR products were Sanger sequenced commercially (Macrogen Inc., Seoul, South Korea).

All sequences were assembled, edited and aligned using Geneious 10.2.6 (https://www.geneious.com). Additional sequences of Gracilariaceae species included in the cox1, ITS1 and rbcL dataset are presented in Table S2. TCS statistical parsimony network in PopArt 1.7 (http://popart.otago.ac.nz) was used to analyze genetic diversity of ITS1.

Maximum likelihood (ML) and Bayesian phylogenetic analyses were performed with all three codons partitioned for cox1 and rbcL sequences. ML analysis was performed using the General Time Reversible + gamma model and hill-climbing algorithm. 1000 non-parametric bootstrap replicates were inferred from 10 distinct alternative runs [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF] in RAxML 7.2.8 [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF]. Bayesian phylogenetic analysis was conducted using MrBayes 3.2.0 [START_REF] Ronquist | MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF]. Two independent sets of four Markov chain Monte Carlo (MCMC) chains were run for three million generations with sampling every 1000 generations. The two independent MCMC chains were converged when split frequency was below 0.01. The first 25% of sampled trees as a 'burn-in' was discarded and 25,000 trees were saved to construct the consensus tree.

RESULTS

Molecular phylogeny. The 1467 bp rbcL alignment contained 37 taxa representing the new species and all known Agarophyton species. This dataset strongly supported the new species as sister to A. tenuistipitatum with high bootstrap (100%) and posterior probability (1.0) and as member of the genus Agarophyton (Fig. 1).

The cox1 alignment, with a length of 654 bp, contained the holotype of the new species and representatives of all other Agarophyton species. The cox1 alignment grouped the new species as sister to A. tenuistipitatum with strong support (Fig S1).

The 383 bp ITS1 alignment contained 13 samples of the new species and no indels were detected. Five different ribotypes were found that differed by a maximum of 2.9% (11 bp; A3 and A5). Lineage A1 (n=7, including Genbank Accession no. AY131297) was the most common ribotype and was found in Australian and New Zealand. Ribotypes A2 (AY131295) and A3 (AY131295) occurred in Australia. Lineage A4 (n=3) was the most common ribotype exclusive to Australia. Ribotype A5 (AF034265) was only present in New Zealand (Fig. 2).

The cox1 and rbcL markers showed that the new species belongs within the genus Agarophyton and is genetically distinct from A. chilense and A. tenuistipitatum. In addition, the ITS1 marker showed that samples from Australia previously identified as A. chilense, were similar to New Zealand sequences and, therefore, belong to this new species. We DESCRIPTION: Plants attached to rocks and shells or unattached, and usually occurring in the intertidal zone in estuaries. Plants were slender, 5-17 cm tall, and cylindrical, 0.5-0.7 mm in diameter, growing in tufts or solitary , and dark brownish red to black in colour.

Branches were numerous, alternate, mostly irregular, up to 4 or 5 orders (Fig. 3). Short filiform branchlets distributed around axes and branches (Figs. 34). Branches and branchlets were not, or only slightly, basally constricted. Axes and branches were tapered toward the tips, and branch tips were often hooked . In transverse section the thallus consisted of 1 or 2 layers of pigmented cortical cells, and 6-8 layers of medullary cells, 50-100 μm in diameter (Fig. 5). There was an abrupt transition in cell size from the medulla to the cortex (Fig. 5).

Male gametophytes were more branched and larger than female thalli (Figs. 34).

Spermatangial conceptacles were scattered throughout the thallus (Fig. 6), forming shallow pits (textorii-type), 20-25 μm deep, 20-40 μm wide (Figs. 78). Spermatangia formed solitary (Fig. 7) to confluent conceptacles (Fig. 8). Cystocarps were scattered over the thallus surface.

Mature cystocarps were depressed spherical, 700-900 μm in diameter, with a rostrum and constricted base (Fig. 9). Gonimoblast filaments consisted of ovoid to elongate cells, and produced outer unbranched chains of carposporangia, 5-25 μm in diameter (Fig. 10).

Nutritive filaments connected to the base of the gonimoblasts (Fig. 11). Pericarps were thin, 70-110 μm thick, and consisted of 6-10 cell layers (Fig. 12). Pericarp tissue was an outer layer of 1-3 elongate cells grading into an inner layer of 4-8 oblate to elongate branched cells (Fig. 12).

DISCUSSION

This study used morphological and molecular data to distinguish this new species, from New Zealand and Australia. Molecular data (cox1, rbcL) clearly indicate that our samples from New Zealand and Australia are distinct from all other Agarophyton species, and thus we propose a new species, Agarophyton transtasmanicum sp. nov. In addition, our molecular data (ITS1) have shown that our recently collected samples are conspecific with samples previously sequenced from Australia and New Zealand [START_REF] Byrne | Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content[END_REF][START_REF] Goff | Molecular delineation of species and species relationships in the red algal agarophytes Gracilariopsis and Gracilaria (Gracilariales)[END_REF], indicating that these sequences had been misidentified when placed in Agarophyton chilense, as previously suggested by [START_REF] Cohen | Molecular identification of two sibling species under the name Gracilaria chilensis (Rhodophyta, Gracilariales)[END_REF].

Within the genus Agarophyton, A. transtasmanicum and A. chilense are morphologically very similar, and their similarity has led to the confusion of the two species in the field [START_REF] Bird | Gracilaria chilensis sp. nov. (Rhodophyta, Gigartinales), from Pacific South America[END_REF][START_REF] Byrne | Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content[END_REF][START_REF] Nelson | The New Zealand species of Gracilaria Greville (Rhodophyta, Gigartinales)[END_REF], especially in the region of overlapping distribution range (Wilcox et al. 2007). Agarophyton transtasmanicum shares several characteristics with A. chilense, including branching pattern, spermatangial conceptacles, cystocarp shape, the position of nutritive filaments and pericarp arrangement (Table 1), but they may be distinguished by the number of medullary layers (6-8 cells in A. transtasmanicum and 8-10 cells in A. chilense), and cortex-to-medulla transition (abrupt in A.

transtasmanicum, but relatively gradual in A. chilense). In the field, A. transtasmanicum is generally shorter than A. chilense and has hooked apices on several branch tips [START_REF] Byrne | Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content[END_REF]. Despite their morphological similarity, molecular data (rbcL) clearly distinguish between A. transtasmanicum and A. chilense. The phylogenetic relationship of A. transtasmanicum and A. tenuistipitatum as sister species was also shown using the cox2-3 spacer and RUBISCO spacer [START_REF] Cohen | Molecular identification of two sibling species under the name Gracilaria chilensis (Rhodophyta, Gracilariales)[END_REF]). Both species, A. transtasmanicum and A. chilense, can be found in the same locations in New Zealand, and based on currently available data only A. transtasmanicum is present in Australia.

Our phylogenetic analyses of rbcL data demonstrated that A. transtasmanicum is closely related to A. tenuistipitatum. Morphologically, both A. transtasmanicum and A. tenuistipitatum possess often an irregular branching pattern with several branchlets and abrupt cell transition [START_REF] Chang | [END_REF][START_REF] Chang | [END_REF][START_REF] Lewmanomont | The species of Gracilaria from Thailand[END_REF][START_REF] Ohno | The species of Gracilaria from Vietnam[END_REF]). The differences between the two are thallus length, the number of medullary cells and pericarp thickness (Table 1). Thalli of A. transtasmanicum are smaller in length and diameter than A. tenuistipitatum [START_REF] Chang | [END_REF][START_REF] Lewmanomont | The species of Gracilaria from Thailand[END_REF]. The main axis of A. transtasmanicum contains more medullary cell layers (6-8 cells) than A. tenuistipitatum (4-6 cells, [START_REF] Chang | [END_REF][START_REF] Lewmanomont | The species of Gracilaria from Thailand[END_REF]. The mature cystocarps of A.

transtasmanicum have more pericarp cell layers (6-10 cells) than A. tenuistipitatum (4-5 cells, [START_REF] Chang | [END_REF][START_REF] Lewmanomont | The species of Gracilaria from Thailand[END_REF]. The distribution range of the two do not overlap.

Agarophyton transtasmanicum is currently known to occur in southeastern Australia and New Zealand whereas A. tenuistipitatum is mostly found in China, Southeast Asia and North America [START_REF] Chang | [END_REF][START_REF] Lewmanomont | The species of Gracilaria from Thailand[END_REF][START_REF] Ohno | The species of Gracilaria from Vietnam[END_REF].

Agarophyton transtasmanicum also shares a variety of common morphological features with A. vermiculophyllum, for example, thallus shape, branching pattern, cystocarp shape and anatomy (Table 1). The greatest differences between these two species is that the spermatangial conceptacle is of the textorii-type in A. transtasmanicum, and the verrucosatype (deep pot-shaped) in A. vermiculophyllum [START_REF] Rueness | Life history and molecular sequences of Gracilaria vermiculophylla (Gracilariales, Rhodophyta), a new introduction to European waters[END_REF]Terada & Yamamoto 2002). These species do not overlap in distributional ranges: A. vermiculophyllum is native to China, Korea and Japan, and introduced to the North Atlantic and north west Pacific [START_REF] Bellorin | Gracilaria vermiculophylla: A western Pacific species of Gracilariaceae (Rhodophyta) first recorded from the eastern Pacific[END_REF]Kim et al. 2010;[START_REF] Krueger-Hadfield | Everywhere you look, everywhere you go, there's an estuary invaded by the red seaweed Gracilaria vermiculophylla (Ohmi) Papenfuss[END_REF]Terada & Yamamoto 2002).

The genus Agarophyton contains four species, including our new species, A. transtasmanicum. The genus was originally established on the basis of several characters, including the abrupt transition between cortex and medulla [START_REF] Gurgel | Systematics of the Gracilariales (Rhodophyta) including new subfamilies, tribes, subgenera, and two new genera, Agarophyton gen. nov. and Crassa gen[END_REF]. However, the significance of the characteristic transition between cortex-medulla cells is uncertain at the genus level as it occurs as abrupt transition in A. transtasmanicum and A. tenusitipitatum, and gradual transition in A. chilense and A. vermiculophyllum (Table 1). Accordingly, we conclude that the diagnosis of cortex-medulla cell transition is flawed as a genus level character, but appears to be useful at the species level.

Our knowledge of A. transtasmanicum is still limited. As a result, further studies on the genetic diversity and population connectivity across Tasman Sea of A. transtasmanicum are needed to shed light on the species history, and demographic patterns. In addition, the ability of Agarophyton transtasmanicum to produce gigartinine, serving as a means of nitrogen storage, has raised interest in the possible commercial attributes of this species (Wilcox et al. 2007). Therefore A. transtasmanicum could be an ideal candidate for use in bioremediation in nitrogen loaded environments such as polluted rivers and in polyculture as Pericarp tissue consisting of outer elongate cell and inner roundish cells. Scale bar = 40 μm.

  Agarophyton chilense (C.J.Bird, McLachlan & E.C.Oliveira) Gurgel, J.N.Norris & Fredericq and a sister species, A. tenuistipitatum (C.F.Chang & B.-M.Xia) Gurgel, J.N.Norris

  Figs. 3-12

  part of multi-trophic aquaculture. Future physiological studies are needed to understand more about the responses of A. transtasmanicum to environmental conditions. ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications (Ed. M.A. Innis, D.H. Gelfand, J.J. Snin-sky & T.J. White), pp. 315-22. Academic Press, Orlando, Florida. Wilcox S.J., Barr N., Broom J., Furneaux R.H. & Nelson W.A. 2007. Using gigartinine to track the distribution of an alien species of Gracilaria in New Zealand. Journal of Applied Phycology 19:313-23. DOI: 10.1007/s10811-006-9138-3. Wilcox S.J., Bloor S.J., Hemmingson J.A., Furneaux R.H. & Nelson W.A. 2001. The presence of gigartinine in a New Zealand Gracilaria species. Journal of Applied Phycology 13:409-13. DOI: 10.1007/s10811-006-9138-3. Zuccarello G.C., West J.A., Kamiya M. & King R.J. 1999. A rapid method to score plastid haplotypes in red seaweeds and its use in determining parental inheritance of plastids in the red alga Bostrychia (Ceramiales). Hydrobiologia 401:207-14. DOI: 10.1023/A:1003706931897.

Table 1 .

 1 Comparison of some morphological characteristics of Agarophyton transtasmanicium sp. nov. with all other Agarophyton species.

Fig. 1 .

 1 Fig. 1. Bayesian topology of rbcL sequences for Agarophyton transtasmanicum sp. nov., A.

Fig. 2 .

 2 Fig. 2. ITS1 ribotype network of Agarophyton transtasmanicum sp. nov. with five different

Figs. 3 - 4 . 4 .

 344 Figs. 3-4. Habits of Agarophyton transtasmanicum sp. nov. from Whanganui River Mouth,

Figs. 5 - 12 .

 512 Figs. 5-12. Detailed morphological feature of Agarophyton transtasmanicum sp. nov. Fig. 5.

Fig. 9 .

 9 Fig. 9. Cross-section of mature cystocarp, with rostrum and constricted base, showing

ACKNOWLEDGEMENTS

This work was supported by Victoria University of Wellington (MP, GZ), NIWA SSIF funding (Coasts and Oceans Research Programme 2, Marine Biological Resources) (WAN) The staff of the herbarium of the Museum of New Zealand Te Papa Tongarewa (WELT) are thanked for their assistance. MLG and WAN were supported by FONDECYT Regular Grant#1170541 (CONICYT, Chile). Special thanks goes to the GCRF GlobalSeaweedSTAR program for providing travel funds (GSS/TF/006) to NM.

Species