A Six‐Crossing Doubly Interlocked [2]Catenane with Twisted Rings, and a Molecular Granny Knot

Abstract A molecular 623 link (a six crossing, doubly interlocked, [2]catenane with twisted rings) and a 31#31 granny knot (a composite knot made up of two trefoil tangles of the same handedness) were constructed by ring‐closing olefin metathesis of an iron(II)‐coordinated 2×2 interwoven grid. The connections were directed by pendant phenyl groups to be between proximal ligand ends on the same faces of the grid. The 623 link was separated from the topoisomeric granny knot by recycling size‐exclusion chromatography. The identity of each topoisomer was determined by tandem mass spectrometry and the structure of the 623 link confirmed by X‐ray crystallography, which revealed two 82‐membered macrocycles, each in figure‐of‐eight conformations, linked through both pairs of loops.

6-(hydroxymethyl)picolinaldehyde was synthesized following literature procedure. [S1] 5 A solution of 5-chloro-2-fluoronitrobenzene (5.0 g, 28.5 mmol), ethylamine hydrochloride (23.2 g, 285 mmol) and potassium carbonate (43.2 g, 313 mmol) in DMSO (170 mL) was heated at 90°C for 2 hours. Upon cooling, water (200 mL) and diethyl ether (200 mL) were added, giving two phases. The organic layer was washed with brine (5 x 50 mL). The organic layer was dried over MgSO4 and the solvent removed under reduced pressure. The desired product 5 was obtained as an orange solid (5.54 g, 97%), and was deemed sufficiently pure to use in the following step without further purification. 1  6-(Hydroxymethyl)picolinaldehyde (4.16 g, 30.4 mmol) and 5 (5.54 g, 27.6 mmol) were added into a mixture of ethanol (220 mL) and water (80 mL). The solution was degassed by bubbling argon for 1 hour and subsequently heated to 80°C until complete dissolution of the solids. Sodium dithionite (17.0 g, 82.8 mmol) was added in one portion under positive pressure of nitrogen, yielding a bright yellow suspension. The mixture was stirred under nitrogen at 80°C for 16 hours. After cooling to room temperature, most of the solvent was removed under reduced pressure. The residue was partitioned between CH2Cl2 (150 mL) and saturated aqueous Na2CO3 (100 mL), and after separation the aqueous layer was extracted with more CH2Cl2 (2 x 50 mL). The combined organic layers were washed with brine (50 mL) before drying over MgSO4 and the solvent was removed under reduced pressure. Recrystallization from toluene yielded 6 as a yellow solid (4.15 g, 52%

9
To a solution of 8 (1.08 g, 2.45 mmol) in chloroform (60 mL) was added MnO2 (2.37 g, 24.5 mmol) and the suspension was stirred under reflux for 17 hours. Upon cooling, the mixture was filtered over Celite® and eluted with a 9:1 CHCl3/MeOH mixture. The solvent from the filtrate was removed under reduced pressure. The desired product 9 was obtained as a yellow oil (1.03 g, 96%), and was deemed sufficiently pure to use in the following step without further purification.

2/3
[Fe42/3](BF4)8 (306 mg, 66 µmol) was dissolved in acetonitrile (15 mL) and a saturated aqueous solution of Na4EDTA (60 mL), H2O (100 mL) and CHCl3 (100 mL) were added. The mixture was vigorously shaken in a extraction funnel, and the phases were separated. The aqueous layer was further extracted with CHCl3 (4 x 30 mL), and the combined organic layers were dried over Na2SO4 and the solvent removed under reduced pressure. The crude organic material was purified by recycling GPC, affording 2/3 ( Figure S5, green fraction) (55 mg, 18%) as a brownish yellow powder. Fractions containing non-interlocked macrocycle ( Figure S5, blue fraction) and non-ring-closed compounds ( Figure S5, magenta fraction) were also collected. All fractions were analysed by MALDI-TOF MS in order to identify the isolated species and to assess the purity of the fractions.

GPC separation of topoisomers 2 and 3
Approximately 80 mg of the pure mixture of organic link 2 and knot 3 were subjected to purification by recycling GPC. The mixture, which appeared as a single sharp peak in the early cycles, required 6-7 cycles to show a noticeable splitting into two different peaks. After a minimum of approximately 15-17 cycles, the two peaks became sufficiently separated to be collected in different fractions ( Figure S14). The recycling was interrupted between each pass of the peak in order to discard any smearing of the previous pass into the upcoming peak from the recycling operation. This procedure, although necessary, results in some loss of material. The isolated fractions, each enriched in one of the topoisomers, were resubmitted to GPC separation following the procedure described above ( Figure S24 and S25) to ensure the removal of any traces of the other topoisomer. After exhaustive recycling, fractions containing pure link 2 (1.5 mg) and pure knot 3 (1.8 mg) were finally obtained. Both fractions were characterised by 1 H and 13 C NMR, where they afforded distinct but complex spectra ( Figures S33-S35).
ESI-MS of the collected fractions ( Figure S26 and S27) showed identical peaks to those of the mixture prior to separation ( Figure  S15). The starting mixture of topoisomers, as well as the separated fractions of link 2 ( Figure S24) and knot 3 ( Figure S25), were analysed through tandem ESI-MS by fragmentation of the triply charged [M+3H] 3+ peak. In the mixture ESI-MS/MS spectrum ( Figure  S16), a number of peaks from fragmentation of the base peak were observed, which were assigned to molecular fragments arising from both the link and the knot topologies based on the observed isotopic patterns for the detected peaks ( Figures S17-S23). The fraction containing pure knot 3 gave a series of low intensity ESI-MS/MS peaks ( Figure S30) that correspond to linear fragments that can only arise from the composite knot (fragmentation of the same bonds in link 2 would lead to dethreading and lower masses corresponding to non-interlocked macrocycle). Under the same conditions, fragmentation of the equivalent peak for the pure link (2) fraction afforded high intensity peaks coresponding to singly-and doubly-charged macrocycle ( Figure S28) without the appearance of any higher mass fragments in significant amount, behaviour consistent with a link topology. Figure S14. Representative recycling GPC UV-trace for the separation of link 2 (blue fraction) and knot 3 (green fraction). The red regions indicate the intervals of recycling during the elution. Both obtained fractions, which were each enriched in one of the desired topologies, were resubmitted to GPC separation to ensure purity ( Figures S24 and S25), followed by the identification of the topoisomer present in each fraction by ESI-MS/MS experiments ( Figures S28-S30).

11
6-(hydroxymethyl)picolinaldehyde (44 mg, 0.32 mmol) and 10 (79 mg, 0.29 mmol) were added into a mixture of ethanol (2 mL) and water (0.75 mL). The solution was degassed by bubbling argon and subsequently heated to 80°C until complete dissolution of the solids. Finally, sodium dithionite (152 mg, 0.88 mmol) was added in one portion under positive pressure of nitrogen, yielding a bright yellow suspension. The mixture was then stirred under nitrogen at 80°C for 16 hours. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was partitioned between CH2Cl2 and saturated aqueous Na2CO3, and, after separation, the aqueous layer was extracted with more CH2Cl2. The combined organic layers were then washed with brine before drying over MgSO4 and removal of the solvent under reduced pressure. Flash column chromatography (SiO2, DCM:MeOH 100:0 to 98:2) yielded 11 as a yellow solid (75 mg, 72%  1H,H e ),1H,H g ),1H,H c ),4.89 (s,2H,H v ),4.80 (q,J = 7.2 Hz,2H,H o

X-ray crystal structures
Data Collection. X-Ray data for compounds [Fe4134](BF4)8 and [Zn4134](BF4)8 were collected at a temperature of 100 K using a microfocused Bruker X8 Prospector diffractometer with Cu-kα (1.54178) equipped with a CCD detector and an Oxford Cryosystems nitrogen flow gas system. Data were measured using Bruker Apex2 suite of programs. X-Ray data for compound 2 were collected at a temperature of 100 K using a synchrotron radiation at single crystal X-ray diffraction beamline I19 in Diamond light Source, [S2] equipped with an Pilatus 2M detector and an Oxford Cryosystems nitrogen flow gas system. Data were measured using GDA suite of programs.
Crystal structure determinations and refinements. X-Ray data were processed and reduced using CrysAlisPro suite of programmes. Absorption correction was performed using empirical methods (SCALE3 ABSPACK) based upon symmetry-equivalent reflections combined with measurements at different azimuthal angles. [S3] The crystal structure was solved and refined against all F 2 values using the SHELXL and Olex 2 suite of programmes. [S4] Crystals of [Fe4134](BF4)8 and 2 present a diffraction limit of 1.1 and 1.0 Å, while crystal of [Zn4134](BF4)8 diffracted to 0.83 Å of resolution. All atoms were refined anisotropically except solvent molecules, BF4 anions and disordered moieties, which were refined isotropically. Hydrogen atoms were placed in the calculated positions. The aromatic moieties were heavily disordered and modelled over two positions were possible. The C-N, C-C and C-O distances in the disordered moieties were restrained using DFIX and SADI and constrained using AFIX SHELXL commands. No restrains were applied to force the configuration of the double bonds of the chains. The atomic displacement parameters (adp) have been restrained using RIGU and SIMU SHELXL commands. A BF4 anions were found disordered and modelled over two positions where possible. The B-F distances and F-B-F angles were restrained using DFIX SHELXL commands. The adps were also restrained using SIMU and RIGU SHELXL commands.
Compounds [Fe4134](BF4)8 and [Zn4134](BF4)8 presents large voids filled with a lot of scattered electron density, the solvent mask protocol inside Olex 2 software was used to account for the void electron density corresponding to the disordered solvent molecules placed in the intermolecular space in the crystal structure. Crystals of [Zn4134](BF4)8 present 309.3 electrons that were accounted for in a volume of 1352 Å 3 . There are 4 grid molecules per unit cell, so there were 77 electrons uncounted per knot, which may correspond to one molecule of disordered BF4 anions and 1.5 disordered molecules of acetonitrile. Crystals of [Fe4134](BF4)8 present 2806.3 electrons that were accounted for in a volume of 8979 Å 3 . There are 8 grid molecules per unit cell, so there were 701 electrons uncounted per knot, which may correspond to one molecule of disordered BF4 anions and 30 disordered molecules of acetonitrile.
A number of A alerts were found in crystals of [Fe4134](BF4)8 and 2 due to the poor resolution data obtained (1.1 and 1.0 Å). This resolution is common in big molecules with large intermolecular spaces filled with disordered anions and/or solvent molecules. In order to refine the crystal structure, different moieties were heavily restrained (using DIFX and SADI commands) and constrained (using AFIX commands) to have idealized geometries. Also A-alerts were found in compounds [Fe4134](BF4)8, [Zn4134](BF4)8 and 2 due to the isotropic refinement of the disordered atoms. Disorder alkyl chains in crystal structure 2 were unsolved in order to maximize the data/parameter ratio.
CCDC 1849964-1849966 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44)1223-336-033; or deposit@ccdc.cam.ac.uk). Crystal size/mm Figure S41. X-ray crystal structure of [Zn4134](BF4)8 (left) and [Fe4134](BF4)8 (right), showing the formation of the expected interwoven 2 × 2 grids. Disorder is observed around some of the pendant phenyl groups. Both structures contain a BF4 anion located in the central cavity of the grid, a behaviour previously observed for this type of system. [S5] Solvent molecules and all other anions have been ommited for clarity. C, grey; N, blue; O, red; S, yellow; B, pink; F, green; Zn, grey-blue; Fe, purple.