Directed C(sp3)–H arylation of tryptophan: transformation of the directing group into an activated amide

The aminoquinoline-directed C–H activation was used to synthezise unnatural tryptophans for solid phase peptide synthesis for the first time.


Table of Contents 1) List of compounds S2
2) General considerations S3

2) General considerations
The chemicals used herein were purchased from Sigma-Aldrich and TCI and used without further purification. SPPS building blocks and materials were purchased from Iris Biotech. Starting materials were synthesized according to the attached reference. Reaction solvents were degassed via freezepump-thaw cycles or by extrusion with argon. THF was distilled over sodium and stored under inert gas conditions with activated 4 Å molecular sieves. Reactions were monitored using crude NMR or TLC. TLC was performed using Merck silica gel TLC 60 F 254 plates. Staining was performed using UV light and Hannessian´s stain or ninhydrine stain, respectively. Purification of crude products was achieved by column chromatography using silica gel by Macherey-Nagel 60 M (0.040-0.063 mm/ 230-400 mesh). NMR spectra were recorded on Bruker AV II 300, Bruker HD-500, Bruker AV III 500 or Bruker AV II 600 spectrometers. Chemical shifts are reported in ppm using the signal of solvent (CDCl 3 : 1 H: 7.26 ppm, 13 C: 77.16 ppm; DMSO-d 6 : 1 H: 2.50 ppm, 13 C: 39.52 ppm) as reference or d 4trimethylsilylpropanoic acid ( 1 H: 0.00 ppm) for measurements in aqueous media. Multiplicity of signals are described as d = dublet, t = triplet, q = quartet, quint = quintet, oct = octet, br = broadened signal. 13 C shifts for peptides were extracted from the HSQC spectrum. Mass spectra (ESI+) were acquired on a Thermo Fisher Scientific LTQ-FT. Peptides were synthesized automatically on a Liberty Blue peptide synthesizer. Analytical HPLC was performed on a Thermo Scientific Dionex UltiMate 3000 system with an ACE UltraCore 2.5 SuperC18 column (150 x 2.1 mm). As eluent with a flow rate of 0.45 mL/min were used the following solvents: A: H 2 O + 0,1% TFA and B: MeCN + 0,085% TFA. Semi preparative HPLC was performed on a Thermo Scientific Dionex UltiMate 3000 with a Macherey-Nagel VP Nucleodur C18 Gravity column with a flow rate of 15.0 mL/min. Diastereomeric ratios (dr) were determined using NMR spectroscopy, enantiomeric excess (ee) was determined using a JASCO HPLC system with Chiralpak IA column and n-hexane/iPrOH (HPLC grade) as eluent.
Optical rotations were determined with an A:KRÜSS Optronic P8000-T polarimeter.
S5 A pressure flask was charged with 6.00 g (14.2 mmol, 1.0 eq) Phth-Phe-8AQ (97% ee) (Org. Lett. 2006, 8 (15), 3391-3394), 24.4 g (71.2 mmol, 5.0 eq) N-Boc-3-iodoindole (Eur. J. Org. Chem. 2013, 4564-4569), 639 mg (2.85 mmol, 20 mol%) Pd(OAc) 2 and 3.56 g (21.4 mmol, 1.5 eq) AgOAc. The flask was flushed with Ar. To this was added 12.0 mL of anhydrous toluene and the mixture was vigorously stirred at 80 °C for 72 h. After completion, the mixture was diluted with DCM and filtered through a pad of celite. The residue was concentrated and purified by column chromatography on silica using toluene/ethyl acetate to furnish 5.86 g (9.20 mmol, 65%) of compound 4 (dr > 25:1, 97% ee) as a white solid among with 19.4 g (56.5 mmol, 79%) of reisolated aryl halide as a pale brown oil. A pressure flask was charged with 5.00 g (8.92 mmol, 1.0 eq) Phth-Trp(Boc)-8AQ (7), 4.0 mL (35.7 mmol, 4.0 eq) iodobenzene, 200 mg (0.89 mmol, 10 mol%) Pd(OAc) 2 and 2.23 g (13.4 mmol, 1.5 eq) AgOAc. The flask was flushed with Ar. To this was added 5.0 mL of anhydrous toluene and the mixture was vigorously stirred at 80 °C for 16 h. After completion, the mixture was diluted with DCM and filtered through a pad of celite. The residue was concentrated and purified by column chromatography on silica using toluene/ethyl acetate to furnish 4.04 g (6.35 mmol, 71%) of compound 8 (dr > 25:1) as a white solid. 1  A pressure flask was charged with 2.00 g (3.59 mmol, 1.0 eq) Phth-Trp(Boc)-8AQ, 3.34 g (14.3 mmol, 4.0 eq) 4-iodoanisole, 80.1 mg (0.36 mmol, 10 mol%) Pd(OAc) 2 and 0.89 g (5.35 mmol, 1.5 eq) AgOAc. The flask was flushed with Ar. To this was added 2.0 mL of anhydrous toluene and the mixture was vigorously stirred at 80 °C for 16 h. After completion, the mixture was diluted with DCM and filtered through a pad of celite. The residue was concentrated and purified by column chromatography on silica using toluene/ethyl acetate to furnish 1.79 g (2.81 mmol, 79%) of compound 14 (dr > 25:1) as a white solid. In a pressure flask, the phthalimide-protected amino acid (1.0 eq) was dissolved in dichloromethane/methanol (1:1, final concentration 0.1 M) and ethylenediamine (5.0 eq) was added. The flask was tightly sealed and stirred at 40 °C for 16 h. After completion, the mixture was allowed to cool to rt and volatiles were removed under reduced pressure. The residue was purified by column chromatography on silica using dichloromethane/methanol to give the free amine in high yield (>82%). The amine (1.0 eq) was dissolved in dichloromethane (3/4 of final volume) and cooled to 0 °C. DIPEA (2.0 eq) was added and a solution of FmocCl (1.05 eq) in dichloromethane (1/4 of final volume, final concentration 0.1 M) was added dropwise. After complete addition, the mixture was stirred at 0 °C for five minutes to reach completion. Saturated NH 4 Cl solution was added and the mixture was extracted with dichloromethane. The organic layer was washed with brine, dried over MgSO 4 and filtered. Volatiles were removed under reduced pressure and the residue was purified by column chromatography on silica using toluene/ethyl acetate to give the respective Fmoc-carbamate in high yield (>93%).  1.15 g (2.14 mmol, 1.0 eq) of the free amine was dissolved in 20 mL dichloromethane and the solution was cooled to 0 °C. 0.92 mL (4.29 mmol, 2.0 eq) DIPEA was added and 0.73 mL (4.29 mmol, 2.0 eq) Boc 2 O was added dropwise. After complete addition the mixture was allowed to warm to rt and stirring was continued for 16 h. Upon completion, the mixture was concentrated, and the crude product was purified by column chromatography on silica using toluene/ethyl acetate to give 1.28 g (2.01 mmol, 94%) of the respective Boc carbamate as a white solid. To a flame-dried pressure flask containing anhydrous LiBr (1.0 eq) was added 8-aminoquinoline amide (1.0 eq) and Hantzsch´s tert-butyl ester (2.6 eq). The flask was flushed with Ar. The solids were dissolved in anhydrous THF (final concentration 0.1 M). After the solids were completely dissolved, formic acid (1.0 eq) was added in a single portion and the flask was tighly sealed and heated at 40 °C for 16 h. Upon completion, volatiles were removed under reduced pressure and the residue was taken up in ethyl acetate. The organic layer was washed with 1 M aq. NaHCO 3 and brine. The organic layer was dried over MgSO 4 , filtered and concentrated. The crude product was purified by column chromatography on silica to give the respective 8-amido tetrahydroquinolines in yields >57%.      Under inert atmosphere, 400 mg (1.58 mmol) of Hantzsch´s ethyl ester and 25.8 mg (0.08 mmol) diphenyl phosphate were suspended in 10 mL toluene and heated to 60° C for 12 h. After complete conversion, volatiles were removed under reduced pressure and the crude residue was purified by column chromatography on silica using toluene/ethyl acetate to give 224 mg (0.89 mmol, 56%) of Hantzsch pyridine and 158 mg (0.62 mmol, 39%) of a 2:1 cis/trans-mixture of diastereomers (stereochemistry not assigned) of tetrahydropyridine 6 as a yellow oil. The tetrahydroquinoline amide (1.0 eq) was dissolved in dichloromethane (5/6 of final volume), cooled to 0 °C and DIPEA (4.0 eq) was added. Triphosgene (0.5 eq) was dissolved in dichloromethane (1/6 of final volume, final concentration 0.1 M) and added dropwise to the reaction mixture. After complete addition, stirring was continued for five minutes. Upon completion, water was added and the mixture was extracted with dichloromethane and the organic layer was washed with brine. The organic layer was dried over MgSO 4 , filtered and concentrated. The crude product was purified by column chromatography on silica using toluene/ethyl acetate to give the respective acyl urea in yields >63%.  The acyl urea compound (1.0 eq) was dissolved in THF (4/5 of final volume) and cooled to 0 °C. Lithium hydroxide monohydrate (1.1 eq) was dissolved in water (1/5 of final volume, final concentration 0.1 M) and 50% aq. hydrogen peroxide (8.8 eq) was added. The aqueous solution of in situ generated lithium hydroperoxide was added dropwise to the solution containing the acyl urea and the mixture was stirred at 0 °C for five minutes. Upon completion, 1 M aq. sodium sulfite (12 eq) was added dropwise, ensuring that the temperature does not exceed 10 °C. After complete addition, the mixture was acidified to pH = 2 using 6 M aq. HCl by dropwise addition. The ice bath was removed and the mixture was extracted two times with ethyl acetate. The combined organic layer was washed with brine, dried over MgSO 4 , filtered and concentrated. The crude product was purified by column chromatography on silica using cyclohexane/ethyl acetate/acetic acid to give the free carboxylic acids in high yields (>96%).  The Leu-enkephalin derivative was synthesized in 0.1 mmol scale. Preloaded 2-Chlorotrityl-resin with C-terminal amino acid (loading approx. 0.6 mmol/g) was suspended in DMF and shaken for 30 min. The slurry was transferred to the reaction chamber of the peptide synthesizer and the excess DMF was removed. DMF was used as solvent for washing, coupling and Fmoc removal steps. Fmocremoval was achieved using 20 vol% piperidine in DMF for 7 minutes at rt. Microwave assisted single coupling of the Fmoc-protected amino acids was achieved using 5 eq of Fmoc-Xaa-OH, DIC and Oxyma for 5 minutes at 50 °C. After complete synthesis of the resin-bound peptide 19, the resin was portioned in half. 0.5 mmol of the resin were treated with TFA for 1 h, filtered and volatiles were removed under reduced pressure to give a crude tetrapeptide, which was dissolved in water and freeze-dried. The crude peptide was analyzed by rp-HPLC to serve as an authentic sample for reaction monitoring. The other half (0.5 mmol) of the resin were suspended in 375 L DMF and  50.0 mg (75 mol, 1.5 eq) Boc-Wsy(Boc,Me)-Nbz cyc (15) and 17 L (2 eq, 0.1 mmol) DIPEA were added and rotated at 50 °C using a RotaVap. The progress was monitored using rp-HPLC. After completion, the residue was transferred to a frit using DCM. The resin was washed three times with DMF and three times with DCM. The resin was dried under reduced pressure. Peptide cleavage was achieved by treatment of the resin with TFA and filtration. Volatiles were removed under reduced pressure and the crude peptide was purified via rp-HPLC using H 2 O with 0.085% TFA and MeCN with 0.085% TFA as the solvent system. Product containing fractions were combined and freeze-dried to give the target peptide 20 in >98% purity.  The Trp cage mutants were synthesized in 0.1 mmol scale. Preloaded 2-Chlorotrityl-resin with Cterminal amino acid (loading approx. 0.6 mmol/g) was suspended in DMF and shaken for 30 min. The slurry was transferred to the reaction chamber of the peptide synthesizer and the excess DMF was removed. DMF was used as solvent for washing, coupling and Fmoc removal steps. Fmoc-removal was achieved using 20 vol% piperidine in DMF for 7 minutes at rt. Microwave assisted single coupling of the Fmoc-protected amino acids was achieved using 5 eq of Fmoc-Xaa-OH, DIC and Oxyma for 5 minutes at 50 °C.     Single-crystal X-ray analysis of LN513. Data was collected with an STOE STADIVARI diffractometer eqipped with with CuKα radiation, a graded multilayer mirror monochromator (λ = 1.54186 Å) and a DECTRIS PILATUS 300K detector using an oil-coated schock-cooled crystal at 100(2) K. Absorption effects were corrected semiempirical using multiscanned reflexions ( X-Area LANA 1.68.2.0 (STOE, 2016)). Cell constants were refined using 18991 of observed reflections of the data collection. The structure was solved by direct methods by using the program XT V2014/1 (Bruker AXS Inc., 2014) and refined by full matrix least squares procedures on F 2 using SHELXL-2017/1 (Sheldrick, 2017). The non-hydrogen atoms have been refined anisotropically, carbon bonded hydrogen atoms were included at calculated positions and refined using the 'riding model' with isotropic temperature factors at 1.2 times (for CH 3 groups 1.5 times) that of the preceding carbon atom. CH 3 groups were allowed to rotate about the bond to their next atom to fit the electron density The Flack parameter refined to 0.09 (9). The absolute configuration could be determined.  [6] SHELXL-2017/1 (Sheldrick, 2017) [7] DIAMOND (Crystal Impact) [8] ShelXle (Hübschle, Sheldrick, Dittrich, 2011) [

11) Computational methods
The calculations were performed using Gaussian 09 version C.01 suite of programs. [1] The geometry optimization for the urea compound started from the crystal structure of compound 13. For the aminoquinoline amide, the x-ray structure of compound rac-4 was used, which was then modified using pymol [2] to get the initial structure in analogue to compound 13. We used B3LYP/6-311++G(d,p) [3,4] level of theory for the optimization in the gas phase. Extensive studies by various authors have showed that this level is accurate enough for the prediction of energetics of amides.
Furthermore, the optimized structure showed excellent agreement to the x-ray structure.
For the calculation of the resonance energies we employed the COSNAR method [5] following the depiction. The ketone, amine and hydrocarbon model compounds were built using pymol to alter the parent amide structure. This initial conformation was used to generate multiple structures through Frog2. [6] The lowest 20 energy conformers were then subjected to minimization in Gaussian 09. The absence of imaginary frequencies was used to characterize the structures as minima on the PES. ZPE and thermal energies were calculated under standard conditions (1atm and 298.15 K).
To compare compound 13 to recently developed amides for organic synthesis, we employed N-acetylglutarimide developed by Szostak et al. [7] to the COSNAR method.  [8] Coordinates of the calculated structures used in the COSNAR method.