Effects of turn-structure on folding and entanglement in artificial molecular overhand knots

The length and constitution of spacers linking three 2,6-pyridinedicarboxamide units in a molecular strand influence the tightness of the resulting overhand (open-trefoil) knot that the strand folds into in the presence of lanthanide(iii) ions. The use of β-hairpin forming motifs as linkers enables a metal-coordinated pseudopeptide with a knotted tertiary structure to be generated. The resulting pseudopeptide knot has one of the highest backbone-to-crossing ratios (BCR)—a measure of knot tightness (a high value corresponding to looseness)—for a synthetic molecular knot to date. Preorganization in the crossing-free turn section of the knot affects aromatic stacking interactions close to the crossing region. The metal-coordinated pseudopeptide knot is compared to overhand knots with other linkers of varying tightness and turn preorganization, and the entangled architectures characterized by NMR spectroscopy, ESI-MS, CD spectroscopy and, in one case, X-ray crystallography. The results show how it is possible to program specific conformational properties into different key regions of synthetic molecular knots, opening the way to systems where knotting can be systematically incorporated into peptide-like chains through design.


S2. GENERAL EXPERIMENTAL
Unless stated otherwise, reagents were obtained from commercial sources and used without purification. Reactions were carried out in anhydrous solvents under an N2 atmosphere.
Anhydrous solvents were obtained by passing the solvent through an activated alumina column on a Phoenix SDS (solvent drying system; JC Meyer Solvent Systems, CA, USA).

and 2•[Lu]
Microwave-assisted reactions were carried out using the CEM Focused Microwave™ Synthesis System, Discover® SP (CEM, North Carolina, USA). Flash column chromatography was carried out using Silica 60 Å (particle size 40-63 μm, Sigma Aldrich, UK) as the stationary phase. Size exclusion chromatography was carried out using Sephadex LH-20 (MeOH) and Bio-Beads SX-1 (CH2Cl2) as the stationary phase. TLC was performed on precoated silica gel plates (0.25 mm thick, 60 F254, Merck, Germany) and visualized using both short and long wave ultraviolet light in combination with standard laboratory stains (basic potassium permanganate, acidic ammonium molybdate and ninhydrin). Low resolution ESI mass spectrometry was performed on a Thermo Scientific LCQ Fleet Ion Trap Mass Spectrometer or an Agilent Technologies 1200 LC system with either an Agilent 6130 single quadrupole MS detector or an Advion Expression LCMS single quadrupole MS detector. CD and UV/Vis S12

S4.1. Synthetic procedures and characterization
General method A for deprotection of tert-butoxycarbonyl (Boc) group: The Boc-protected starting material was dissolved in a solution of hydrochloric acid in 1,4dioxane (4.0 M) and the resulting mixture was left stirring at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure after diluting with dichloromethane and the crude product was carried through to the next step without further purification.

General method B for amino acid coupling:
A mixture of carboxylic acid and appropriate coupling reagents was stirred in CH2Cl2 or DMF for 10 min, after which the amine in CH2Cl2 was added. The resulting solution was stirred for 10 min followed by the dropwise addition of di-isopropylethylamine (DIPEA). The reaction mixture was left stirring at room temperature for the appropriate amount of time. The reaction mixture was diluted by CH2Cl2 and washed successively with citric acid (aq, 10%), sodium bicarbonate (sat. aq.) and brine. The combined organic phases were dried over sodium sulphate and concentrated under reduced pressure to give the crude product. Flash column chromatography, eluting with hexane and EtOAc in the appropriate ratio, was used to yield the pure products.

S6
A solution of N-Boc-L-leucine (

S11
Using the general method A, the compound S10 (2.40 g, 4.77 mmol) was converted into the title compound as a white foam. The crude compound was carried through to the next step without further purification.

S12
Using the general method B, the compound S11 (

S13
Using the general method A, the compound S12 (1.01 g, 4.88 mmol) was converted into the title compound as a white foam. The crude compound was carried through to the next step without further purification.
The reaction mixture was stirred at room temperature for 4 hours, after which the reaction mixture was diluted with EtOAc (10 mL). The solution was washed with lithium chloride (aq., 5%, 5 mL) and extracted with EtOAc (3 ×10 mL). The combined organics were washed with lithium chloride (aq., 5%, 5 mL), dried over sodium sulphate and concentrated under reduced pressure to give the crude product as a yellow oil. Flash column chromatography (CH2Cl2/EtOAc 4:1) yielded the pure compound A5 as a colourless solid (161 mg, 67%

S4.2. Ring-closing metathesis for covalent capture of 8•[Lu]
All alkene-terminated molecular overhand knots can be ring-closed as previously described by us. 6

S48
Spectrum S15. 1    K using a synchrotron radiation at single crystal X-ray diffraction beamline I19 in Diamond light Source, 9 equipped with a Pilatus 2M detector and an Oxford Cryosystems Cobra nitrogen flow gas system. Data was 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. 10 The crystal structures were solved and refined against all F 2 values using the SHELX and Olex 2 suite of programmes. 11 All atoms except hydrogens were refined anisotropically. Hydrogen atoms were placed in the calculated positions. Naphthalene and the aliphatic groups were found and modelled over two positions. The C-C and C-O 1,2 and 1,3 distances in the aliphatic chains were restrained using distance restrains (SHELX; DFIX and SADI). The atomic displacement parameters (adp) of the ligands were restrained using rigid body restrains (SHELX RIGU and SIMU commands).
The triflate anions were constrained to have the ideal structure. The adps were also restrained using rigid body restrains (SIMU and RIGU commands).
Compound Λ-4•[Lu] present medium size voids filled with a lot of scattered electron density.

S9. BCR CALCULATION
Backbone-to-crossing (BCR) ratios provide a measure of the tightness of a given knot. BCRs can also be used for open entanglements such as the overhand knots in this study (as is commonly done for knots in biology). [12] However, in such a case it is important to define what is the core part of the entanglement. For proteins, this is generally defined per amino acidthe moment when an amino acid is removed so that the minimized projection of the protein knot is no longer entangled is the minimum entanglement degree. For this study, we used a precise atomic definition and defined the peripheral naphthol oxygens as the endpoints of the entangled region, see Figure S11 for full numbering of an example ligand. This definition generated the approximate BCR values represented in Table S2 below. Figure S11. Example definition of numbering of ligand 3 to calculate BCR ratios.