* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Reference：
[1] Journal of Physical Chemistry B, 2010, vol. 114, # 19, p. 6751 - 6762
2
[ 50-00-0 ]
[ 35661-39-3 ]
[ 84000-07-7 ]
Reference：
[1] Bioorganic and Medicinal Chemistry, 2018, vol. 26, # 6, p. 1232 - 1238
3
[ 35661-39-3 ]
[ 84000-07-7 ]
Reference：
[1] European Journal of Organic Chemistry, 2013, # 21, p. 4509 - 4513
[2] Bioorganic and Medicinal Chemistry, 2016, vol. 24, # 6, p. 1163 - 1170
4
[ 503-74-2 ]
[ 68858-20-8 ]
[ 35661-39-3 ]
[ 158257-40-0 ]
[ 26305-03-3 ]
Yield
Reaction Conditions
Operation in experiment
46%
Stage #1: With N-ethyl-N,N-diisopropylamine In dichloromethane for 3 h; Stage #2: With piperidine In N,N-dimethyl-formamide for 0.1 h;
General procedure: 4.2 General procedure A: resin loading (0026) Solid phase peptide synthesis was conducted manually in a sinter-fitted polypropylene syringe. 2-Chlorotritylchloride (CTC) resin was preswelled in DCM (mL) for 15min and drained. The first amino acid in 0.4M DIPEA/DCM was added and the mixture was agitated for 3h. After draining the solvent, any free 2-CTC resin linkers were capped by treatment of the resin with a solution of 17:2:1 DCM/MeOH/DIPEA (3×3mL×5min), and subsequently with a solution of 8:1:1 DMF/DIPEA/acetic anhydride (2×3mL×10min). The resin was finally washed with DCM (2×3mL×1min), DMF (2×3mL×1min), DCM (2×3mL×1min) and DMF (2×3mL×1min). 4.3 General procedure B: Fmoc deprotection (0027) The resin was agitated with a solution of 10percent piperidine inDMF (2×3mL×3min) and subsequently washed with DMF(3×3mL×1min), DCM (3×3mL×1min), DMF (5×3mL×1min). The deprotected solutions were combined and diluted appropriately (100-fold for 0.05mmol resin loading). The resin loading was estimated by measuring the absorbance of the piperidine-fulvene adduct with 10percent piperidine in DMF as a reference (λ=301nm; ε=7800M−1cm−1). 4.4 General procedure C: peptide coupling with HBTU (0028) A solution was prepared of the appropriate Fmoc-protected amino acid (3 equiv. relative to resin loading) and HBTU (2.9 equiv. relative to resin loading) in minimum amount of DMF. DIPEA (6 equiv. relative to resin loading) was added and the resin was agitated for 1.5h. The resin was then drained and washed with DMF (3×3mL×1min), DCM (3×3mL×1min) and DMF (5×3mL×1min). 4.5 General procedure D: peptide coupling with HATU (0029) A solution was prepared of the appropriate Fmoc-protected amino acid (3 equiv. relative to resin loading) and HATU (2.9 equiv. relation to resin loading) in minimal DMF. DIPEA (6 equiv. relation to resin loading) was added to the solution and the mixture was immediately added to the resin and agitated. Reaction times were altered based on the residue being coupled: Phe(NMe) and Ala (2×2h); Thr and Sta (1×2h); Asn, Leu, D-Val and L-Val (2×2h); DMVal (3×3h). Once the reaction was complete, the resin was drained and washed with DMF (3×3mL×1min), DCM (3×3mL×1min) and DMF (3×3mL×1min). 4.6 General procedure E: peptide coupling with DIC (0030) A solution was prepared of the appropriate Fmoc-protected amino acid (1.5 equiv. relative to resin loading), HOBt (1.5 equiv. relative to resin loading) and DIC (1.5 equiv. relative to resin loading) in minimal DMF. This solution was stirred for 20min, then added to the resin and agitated overnight. The resin was drained and washed with DMF (3×3mL×1min), DCM (3×3mL×1min) and DMF (5×3mL×1min). Double coupling of the next amino acid after the coupling of the fluorinated amino acid was applied. 4.7 General procedure F: resin cleavage (0031) After the last Fmoc deprotection, the resin was washed with DMF (3×3mL×1min) and DCM (3×3mL×1min) then dried in vacuo. The resin was agitated with a solution of 95:2.5:2.5 TFA/TIS/H2O (3mL) for 2h. The resin was drained and washed with the same TFA mixture above (2×3mL×1min). The combined cleavage solutions were concentrated under a stream of nitrogen. Diethyl ether was added and the supernatant was decanted (3×). The residue was then dried in vacuo to provide the crude linear peptide
Reference：
[1] Tetrahedron, 2007, vol. 63, # 28, p. 6577 - 6586
[2] Journal of Organic Chemistry, 2001, vol. 66, # 3, p. 697 - 706
[3] Journal of Organic Chemistry, 1993, vol. 58, # 8, p. 2313 - 2316
[4] Organic and Biomolecular Chemistry, 2011, vol. 9, # 19, p. 6566 - 6574
[5] Bioorganic and Medicinal Chemistry Letters, 2012, vol. 22, # 2, p. 1187 - 1188
[6] Angewandte Chemie - International Edition, 2013, vol. 52, # 23, p. 6006 - 6010[7] Angew. Chem., 2013, vol. 125, # 23, p. 6122 - 6126
11
[ 35661-39-3 ]
[ 161529-13-1 ]
Yield
Reaction Conditions
Operation in experiment
88%
Stage #1: With 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-trioxide; N-ethyl-N,N-diisopropylamine In ethyl acetate at 0℃; for 0.166667 h; Stage #2: With sodium tetrahydroborate In water; ethyl acetate at 0℃; for 0.416667 h;
General procedure: To a solution of carboxylic acid (10 mmol) in THF (10 mL), DIPEA (11 mmol, 1.42 mL) and 50percent T3P in EtOAc (20 mmol, 6.36 mL) were added at 0 °C and the solution was stirred for about 10 min. Then aqueous solution of NaBH4 (10 mmol, 388 mg in 0.3 mL of H2O) was added to the reaction mixture at the same temperature and the reaction was allowed to stir till the completion of the reaction as indicated by TLC. After the completion of the reaction, the solvent was evaporated and the crude alcohol was extracted into EtOAc and the organic phase was washed with 5percent citric acid (10 mL .x. 2), 5percent Na2CO3 (10 mL .x. 2), water, and brine solution. The product was isolated after the evaporation of solvent under reduced pressure and dried over anhydrous Na2SO4.
88%
Stage #1: With 1-[(1-(cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino)]-uronium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 0℃; for 0.333333 h; Stage #2: With sodium tetrahydroborate In water; N,N-dimethyl-formamide at 0℃; for 0.0833333 h;
To an ice cold solution of Fmoc-Ala-OH (1 mmol) and DIPEA (1.2 mmol) in DMF (5 mL) at 0 °C, COMU (1.1 mmol) was added. The reaction mixture was stirred for 20 min. Then, NaBH4 (1 mmol) in water was added to the reaction mixture and stirred at the same temperature for 5 min. The reaction mixture was taken into ethyl acetate (15 mL). The organic layer was successively washed with 10percent HCl (2 * 10 mL), 10percent aqueous sodium carbonate (3 * 10 mL) and brine (3 * 10 mL). Then the product was dried over anhydrous Na2SO4, and the solvent was evaporated under a reduced pressure. The residue was purified by column chromatography using ethyl acetate-hexane (30:70) as an eluent.
Reference：
[1] Journal of Organic Chemistry, 2001, vol. 66, # 25, p. 8454 - 8462
[2] Tetrahedron Letters, 2000, vol. 41, # 32, p. 6131 - 6135
[3] Tetrahedron Letters, 2012, vol. 53, # 38, p. 5059 - 5063
[4] Journal of Molecular Liquids, 2014, vol. 198, p. 94 - 100
[5] RSC Advances, 2014, vol. 4, # 87, p. 46947 - 46950
[6] European Journal of Organic Chemistry, 2015, vol. 2015, # 28, p. 6341 - 6350
[7] Organic and Biomolecular Chemistry, 2011, vol. 9, # 11, p. 4182 - 4187
[8] Journal of Organic Chemistry, 2009, vol. 74, # 15, p. 5260 - 5266
[9] Synthetic Communications, 2009, vol. 39, # 19, p. 3555 - 3566
[10] Organic and Biomolecular Chemistry, 2011, vol. 9, # 11, p. 4182 - 4187
[11] ACS Combinatorial Science, 2017, vol. 19, # 3, p. 131 - 136
12
[ 35661-39-3 ]
[ 456-47-3 ]
[ 211617-68-4 ]
[ 133865-89-1 ]
Reference：
[1] Journal of Medicinal Chemistry, 2007, vol. 50, # 20, p. 4909 - 4916
Stage #1: With N-ethyl-N,N-diisopropylamine In dichloromethane at 20℃; for 2.73333 h; Stage #2: With piperidine In N,N-dimethyl-formamide at 20℃;
A solution of Fmoc-l-Ala-OH (0.93 g, 3.0 mmol) in CH2Cl2 (25 mL) was added to 2-chlorotrityl chloride resin (6 g, loading at 0.5 mmol/g), followed by DIPEA (0.52 mL, 3.0 mmol). After the resin mixture was agitated for 14 min, DIPEA (0.78 mL, 4.5 mmol) was added and the mixture was further shaken for 2.5 h. The solution was drained off and the resin was washed with DMF (20 mL). A solution of CH2Cl2/MeOH/DIPEA (40 mL, 80:15:5) was added to the mixture and shaken for 30 min. The solution was drained and the procedure was repeated. The resin was then washed with DMF (20 mL). Piperidine in DMF (15.0 mL, 1:4) was added to the resin mixture and shaken for 10 min. The liquid was drained off and piperidine washing was repeated for another 40 min. The amino acid-loaded resin was thoroughly washed with DMF (35 mL), isopropanol (35 mL) and n-hexane (35 mL). The resin was then dried under vacuum for 30 min and placed in a desiccator overnight. CH2Cl2 (50 mL) was added to the resin and left for 1 h. The solution was drained and a solution of HBTU (4.27 g, 11.25 mmol), HOBt (1.72 g, 11.25 mmol), Fmoc-l-Ala-OH (2.34 g, 7.5 mmol) and DIPEA (2.61 mL, 15.0 mmol) in DMF (11.3 mL) was added. The amino acid resin mixture was agitated for 4 h. The solution was then drained and washed with DMF (30 mL), isopropanol (30 mL) and n-hexane (30 mL). 2,2,2-Trifluroethanol in CH2Cl2 (15 mL, 1:4) was added to the amino acid-loaded resin and agitated for 2 h. The solution was drained and the organic solvent removed in vacuo to afford 22 (0.90 g, 78percent) as a yellow foam. m.p. 195-196 °C; Rƒ 0.53 (CH2Cl2/MeOH 9:1); [α]D20 -23.9 (c 0.14, MeOH); IR (ATR) νmax 3297, 2918, 1692, 1650, 1533, 1450, 1318, 1229 cm-1; 1H NMR (CDCl3, 500 MHz) δ 8.09 (1H, d, J = 7.2 Hz, NH-5), 7.89 (2H, d, J = 7.4 Hz, 2H-FmocAr), 7.74 (1H, d, J = 7.4 Hz, 1H-FmocAr), 7.72 (1H, d, J = 7.4 Hz, 1H-FmocAr), 7.50 (1H, d, J = 7.5 Hz, NH-1), 7.41 (2H, t, J = 7.4 Hz, H-FmocAr), 7.33 (2H, td, J = 7.4, 0.9 Hz, 2H-FmocAr), 4.27-4.15 (4H, m, CO2CH2CH, CO2CH2CH, H-6), 4.08 (1H, d, J = 7.5, 7.2 Hz, H-2), 1.27 (3H, d, J = 7.3 Hz, H3-7), 1.22 (3H, d, J = 7.2 Hz, H3-3); 13C NMR (CDCl3, 125 MHz) δ 174.2 (C-8), 172.2 (C-4), 155.6 (CO2CH2CH), 143.9 (C-FmocAr), 143.8 (C-FmocAr), 140.7 (2C-FmocAr), 127.6 (2C-FmocAr), 127.1 (2C-FmocAr), 125.3 (2C-FmocAr), 120.1 (2C-FmocAr), 65.6 (CO2CH2CH), 49.7 (C-2), 47.5 (C-6), 46.7 (CO2CH2CH), 18.2 (C-3), 17.3 (C-7); (+)-HRESIMS [M+Na]+ 405.1424 (calcd. for C21H22NaN2O5, 405.1421).
Reference：
[1] Tetrahedron, 2018, vol. 74, # 48, p. 6929 - 6938
[2] Journal of the American Chemical Society, 2014, vol. 136, # 10, p. 3919 - 3927
[3] Patent: WO2015/187540, 2015, A1,
[4] Patent: EP3184540, 2017, A1,
[5] Angewandte Chemie - International Edition, 2017, vol. 56, # 50, p. 15984 - 15988[6] Angew. Chem., 2017, vol. 129, # 50, p. 16200 - 16204,5
[(R)-1-((R)-1-{(R)-2-Benzyloxy-1-[(R)-1-((S)-1-carbamoyl-ethylcarbamoyl)-2-(1H-indol-3-yl)-ethylcarbamoyl]-ethylcarbamoyl}-ethylcarbamoyl)-2-(1H-imidazol-4-yl)-ethyl]-carbamic acid benzyl ester[ No CAS ]
[(R)-1-[(R)-1-{(R)-2-Benzyloxy-1-[(R)-1-((S)-1-carbamoyl-ethylcarbamoyl)-ethylcarbamoyl]-ethylcarbamoyl}-2-(4-benzyloxy-phenyl)-ethylcarbamoyl]-2-(1H-imidazol-4-yl)-ethyl]-carbamic acid benzyl ester[ No CAS ]
[(R)-1-[(R)-1-{(R)-2-Benzyloxy-1-[(R)-1-((R)-1-carbamoyl-ethylcarbamoyl)-2-(1H-indol-3-yl)-ethylcarbamoyl]-ethylcarbamoyl}-2-(4-benzyloxy-phenyl)-ethylcarbamoyl]-2-(1H-imidazol-4-yl)-ethyl]-carbamic acid benzyl ester[ No CAS ]
Chelmical synthesis: Peptides were synthesized on a Rink amide resin, 0.45 mmol/g [Fmoc-Cys(Trityl)-Wang; Novabiochem, San Diego, Calif.] usinig N-(9-fluorenyl)methoxycarboxyl chemistry and standard side chain protection except on cysteine residues. Cysteine residues were protected in pairs with either S-trityl on the first and third cysteines or S-acetamidomethyl on the second and fourth cysteines. Amino acid derivatives were from Advanced Chemtech (Louisville, Ky.). The peptides were removed from the resin and precipitated, and a two-step oxidation protocol was used to selectively fold the peptides as described previously (Luo et al., 1999). Briefly, the first disulfide bridge was closed by dripping the peptide into an equal volume of 20 mM potassium feliicyanide and 0.1 M Tris, pH 7.5. The solution was allowed to react for 30 min, and the monocyclic peptide was purified by reverse-phase HPLC. Simultaneous removal of the S-acetamidomethyl groups and closure of the second disulfide bridge was carried out by iodine oxidation. The monocyclic peptide and HPLC eluent was dripped into an equal volume of iodine (10 mM) in H20/trifluoroacetic acid/acetonitrile (78:2:20 by volume) and allowed to react for 10 min. The reaction was terminated by the addition of ascorbic acid diluted 20-fold with 0.1percent trifluoroacetic acid and the bicyclic product purified by HPLC. Mass Spectrometry: Measurements were performed at the Salk Institute for Biological Studies (San Diego, Calif.) under the direction of Jean Rivier. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid secondary ionization mass spectrometry were used.
Lys(Boc)-Pra-Asn(Trt)-Thr(tBu)-Ala-Thr(tBu)-Ala(N3)-Ala-Pal-PEG resin (16) (0.498 g, 0.09 mmol) was cleaved with TFA/iPr3SiH/H2O (v/v/v; 95/2.5/2.5, 5.0 mL) for 2 h, peptide was isolated as described in the general section to afford 47.3 mg of crude peptide 17. The crude peptide 17 (16.3 mg) was purified by RP-HPLC on a preparative Phenomenex Gemini C18, column at a flow rate of 5 mL min-1, using a linear gradient of 1percentB to 61percentB over 60 min (ca. 1percentB per minute) and lyophilised to give the title compound 17 as a white amorphous solid (4.9 mg, 18percent).
Lys(Boc)-Pra-Asn(Trt)-Thr(tBu)-Ala-Thr(tBu)-Ala(N3)-Ala-Pal-PEG resin (16) (0.498 g, 0.09 mmol) was cleaved with TFA/iPr3SiH/H2O (v/v/v; 95/2.5/2.5, 5.0 mL) for 2 h, peptide was isolated as described in the general section to afford 47.3 mg of crude peptide 17. The crude peptide 17 (16.3 mg) was purified by RP-HPLC on a preparative Phenomenex Gemini C18, column at a flow rate of 5 mL min-1, using a linear gradient of 1percentB to 61percentB over 60 min (ca. 1percentB per minute) and lyophilised to give the title compound 17 as a white amorphous solid (4.9 mg, 18percent). Purified Lys-Pra-Asn-Thr-Ala-Thr-Ala(N3)-Ala-NH2 (17) (4.4 mg, 5.42 x 10-3 mmol) was dissolved in a mixture of water and tert-butyl alcohol (1 : 2.5, 3.5 mL in total). A stock solution of CuSO4 (0.87 mg, 5.42 x 10-3 mmol) and sodium ascorbate (2.68 mg, 13.6 x 10-3 mmol) in water (1.5 mL) was added and the mixture was microwaved for 20 min at 80 °C in a sealed reaction vessel (120 W max) to afford crude peptide (8), containing inseparable dehydroalanine by-product. The crude peptide (8) was purified by RP-HPLC on a preparative Waters XTerra.(R). Prep. C18 column at a flow rate of 10 mL/min, using a linear gradient of 1percentB to 51percentB over 50 min (ca. 1percentB per minute). Fractions were lyophilised to give the title compound 8 as a white amorphous solid (0.7 mg, 18percent), containing inseparable dehydroalanine by-product.
General procedure: Fmoc SPPS was performed on a Liberty Microwave Peptide Synthesiser (CEM Corporation, Mathews, NC) using the Fmoc/tBu strategy as previously described35 or manually starting from PAL-PEG polystyrene resin (0.21 mmol/g). For manual synthesis the following steps were undertaken: (a) Fmoc deprotection with 20percent piperidine for 5 min, then 15 min, washing with DMF 5.x.; (b) coupling of the Fmoc amino acid (5 equiv) in the presence of HBTU in DMF (4.9 equiv) and iPr2NEt (10 equiv) for 1 h and washing with DMF 5.x.. For coupling of Fmoc-Pra (1.5 equiv) and Fmoc-N3Ala (1.5 equiv), 1.45 equiv of HBTU and 4.5 equiv of iPr2NEt were used. The progress of the acylation step was monitored by the Kaiser test. A minimum amount of DMF was used for dissolution of the Fmoc amino acid. The resulting peptides were cleaved from the resin with simultaneous side chain protecting group removal by treatment with either TFA/iPr3SiH/DODT/H2O (v/v/v/v; 94/1/2.5/2.5), or with TFA/iPr3SiH/H2O (v/v/v; 95/2.5/2.5) for 2 h at room temperature. Crude peptides were precipitated and triturated with cold diethyl ether, isolated (centrifugation), dissolved in 20percent acetonitrile (aq) containing 0.1percent TFA and lyophilized. Analytical RP-HPLC was performed using a Dionex P680 (flow rate of 1 mL/min), or Dionex Ultimate U3000 system (flow rate of 0.5 mL/min or 0.2 mL/min) using Waters XTerra.(R). column (MS C18, 150 mm .x. 4.6 mm; 5 mum) or, Phenomenex Aqua column (C18, 250 mm .x. 4.6 mm; 5mu), or Phenomenex, Gemini column (C18, 50 mm .x. 2.0 mm, 5mu), using gradient systems as indicated in the Supplementary data. The solvent system used was A (0.1percent TFA in H2O) and B (0.1percent TFA in acetonitrile) with detection at 210 nm, 254 nm, and 280 nm****. The ratio of products was determined by integration of spectra recorded at 210 nm. Peptide masses were confirmed by an inline Thermo Finnegan MSQ mass spectrometer using ESI in the positive mode. When appropriate, a Bruker micrOTOF-Q II mass spectrometer was used for ESI-MS analysis (positive mode). Infrared spectra were obtained using a Perkin Elmer Spectrum One Fourier Transform infrared spectrometer with a universal ATR sampling accessory. Peptide purification was performed using a Waters 600E or Dionex Ultimate U3000 system using a Waters XTerra.(R). column (C18, 300 mm .x. 19 mm; 10 mum), or Phenomenex Gemini C18, 250 mm .x. 10 mm; 5 mum column. Gradient systems were adjusted according to the elution profiles and peak profiles obtained from the analytical RP-HPLC chromatograms. Fractions were collected, analysed by either RP-HPLC or ESI-MS, pooled and lyophilised three times from 10 mM aq HCl.
General procedure: Fmoc SPPS was performed on a Liberty Microwave Peptide Synthesiser (CEM Corporation, Mathews, NC) using the Fmoc/tBu strategy as previously described35 or manually starting from PAL-PEG polystyrene resin (0.21 mmol/g). For manual synthesis the following steps were undertaken: (a) Fmoc deprotection with 20percent piperidine for 5 min, then 15 min, washing with DMF 5.x.; (b) coupling of the Fmoc amino acid (5 equiv) in the presence of HBTU in DMF (4.9 equiv) and iPr2NEt (10 equiv) for 1 h and washing with DMF 5.x.. For coupling of Fmoc-Pra (1.5 equiv) and Fmoc-N3Ala (1.5 equiv), 1.45 equiv of HBTU and 4.5 equiv of iPr2NEt were used. The progress of the acylation step was monitored by the Kaiser test. A minimum amount of DMF was used for dissolution of the Fmoc amino acid. The resulting peptides were cleaved from the resin with simultaneous side chain protecting group removal by treatment with either TFA/iPr3SiH/DODT/H2O (v/v/v/v; 94/1/2.5/2.5), or with TFA/iPr3SiH/H2O (v/v/v; 95/2.5/2.5) for 2 h at room temperature. Crude peptides were precipitated and triturated with cold diethyl ether, isolated (centrifugation), dissolved in 20percent acetonitrile (aq) containing 0.1percent TFA and lyophilized. Analytical RP-HPLC was performed using a Dionex P680 (flow rate of 1 mL/min), or Dionex Ultimate U3000 system (flow rate of 0.5 mL/min or 0.2 mL/min) using Waters XTerra.(R). column (MS C18, 150 mm .x. 4.6 mm; 5 mum) or, Phenomenex Aqua column (C18, 250 mm .x. 4.6 mm; 5mu), or Phenomenex, Gemini column (C18, 50 mm .x. 2.0 mm, 5mu), using gradient systems as indicated in the Supplementary data. The solvent system used was A (0.1percent TFA in H2O) and B (0.1percent TFA in acetonitrile) with detection at 210 nm, 254 nm, and 280 nm****. The ratio of products was determined by integration of spectra recorded at 210 nm. Peptide masses were confirmed by an inline Thermo Finnegan MSQ mass spectrometer using ESI in the positive mode. When appropriate, a Bruker micrOTOF-Q II mass spectrometer was used for ESI-MS analysis (positive mode). Infrared spectra were obtained using a Perkin Elmer Spectrum One Fourier Transform infrared spectrometer with a universal ATR sampling accessory. Peptide purification was performed using a Waters 600E or Dionex Ultimate U3000 system using a Waters XTerra.(R). column (C18, 300 mm .x. 19 mm; 10 mum), or Phenomenex Gemini C18, 250 mm .x. 10 mm; 5 mum column. Gradient systems were adjusted according to the elution profiles and peak profiles obtained from the analytical RP-HPLC chromatograms. Fractions were collected, analysed by either RP-HPLC or ESI-MS, pooled and lyophilised three times from 10 mM aq HCl.
(3S,7S,10S,13S)-3-((3aR,6S,6aR)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-13-(benzyloxymethyl)-10-sec-butyl-7-methyl-5,8,11-trioxo-1-phenyl-2,6,9,12-tetraazatetradecan-14-oic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
79%
General procedure: The peptides were synthesized on an activated [44] 2-chlorotrityl chloride resin (1 g) which had been swelled in dry DCM for 30 min. The first Fmoc amino acid (4 equiv) was coupled to the resin manually using dry DCM (10 mL) and DIPEA (6 equiv) for 2 h under a N2 atmosphere. Resin substitution was then determined using the Fmoc UV assay. On 0.1 mM scale subsequent amino acids were also coupled manually using amino acid (0.20 mM, 2.5 mL), DIPEA (1 mM, 1.0 mL) and HBTU (0.50 mM, 1.0 mL) in DMF. The Fmoc group of amino acid was removed using 20percent piperidine?DMF (3 × 10 mL) for 30 min and the next amino acid and sugar amino acid 1a/b were coupled on resin using the same condition. The excess reagents were washed with DMF (2 × 7 mL) and DCM (2 × 7 mL). Cleavage from resin was performed manually, using a cleavage mixture of TFA?DCM [5:95percent (v/v), 3 × 10 mL] for 30 min. The crude compound was then purified using semi-preparative HPLC. All compounds were obtained in good yields.
(2S)-2-((2S,3S)-2-((2S)-2-((3S)-3-amino-3-((3aR,6S,6aR)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propanamido)propanamido)-3-methylpentanamido)-3-(benzyloxy)propanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
80%
General procedure: The peptides were synthesized on an activated [44] 2-chlorotrityl chloride resin (1 g) which had been swelled in dry DCM for 30 min. The first Fmoc amino acid (4 equiv) was coupled to the resin manually using dry DCM (10 mL) and DIPEA (6 equiv) for 2 h under a N2 atmosphere. Resin substitution was then determined using the Fmoc UV assay. On 0.1 mM scale subsequent amino acids were also coupled manually using amino acid (0.20 mM, 2.5 mL), DIPEA (1 mM, 1.0 mL) and HBTU (0.50 mM, 1.0 mL) in DMF. The Fmoc group of amino acid was removed using 20percent piperidine?DMF (3 × 10 mL) for 30 min and the next amino acid and sugar amino acid 1a/b were coupled on resin using the same condition. The excess reagents were washed with DMF (2 × 7 mL) and DCM (2 × 7 mL). Cleavage from resin was performed manually, using a cleavage mixture of TFA?DCM [5:95percent (v/v), 3 × 10 mL] for 30 min. The crude compound was then purified using semi-preparative HPLC. All compounds were obtained in good yields.
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 268209-75-2 ]
(Boc-Ser(tBu))-Lys(Boc)-Glu(OtBu)-Pro-Leu-Arg(pbf)-Pro-Arg(pbf)-(S-(acetylaminomethyl)Cys)-Arg(pbf)-Pro-Ile-(O-(2-(isopropyl)phenyl)Asp)-Ala-(N,O-isopropylidene-Thr)-Leu-Ala-Val-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Gly-OH[ No CAS ]
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 268209-75-2 ]
(Boc-Ser(tBu))-Lys(Boc)-Glu(OtBu)-Pro-Leu-Arg(pbf)-(S-(acetylaminomethyl)Cys)-Arg(pbf)-Pro-Ile-(O-(2-(isopropyl)phenyl)Asp)-Ala-(N,O-isopropylidene-Thr)-Leu-Ala-Val-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Gly-(S-(acetylaminomethyl)Cys)-Pro-Val-(S-(acetylaminomethyl)Cys)-Ile-(N,O-isopropylidene-Thr)-Val-(O-(2-(isopropyl)phenyl)Asp)-Thr(tBu)-Thr(tBu)-Ile-(S-(acetylaminomethyl)Cys)-Ala-Gly-OH[ No CAS ]
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 268209-75-2 ]
Ser(Boc)-Arg(pbf)-Glu(OtBu)-Pro-Leu-Arg(pbf)-Pro-Trp(Boc)-(S-(acetylaminomethyl)Cys)-His(Trt)-Pro-Ile-Asn(Trt)-Ala-Ile-Leu-Ala-Val-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Gly-(S-(acetylaminomethyl)Cys)-Pro-Val-(S-(acetylaminomethyl)Cys)-Ile-(N,O-isopropylidene-Thr)-Val-(O-(2-(isopropyl)phenyl)Asp)-Thr(tBu)-Thr(tBu)-Ile-(S-(acetylaminomethyl)Cys)-Ala-Gly-OH[ No CAS ]
(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-carboxyethyl)pyridine 1-oxide[ No CAS ]
Ac-(4-PyrAla N-oxide)-Ala-Leu-Ala-Dab-NH<SUB>2</SUB>[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Knorr amide MBHA resin (0.50 g, 0.15 mmol) was swelled in DMF for 2 h. The suspension was filtered. 20percent piperidine in DMF (3 resinvolumes) was added into the resin to cleave the Fmoc group. The suspension was kept at room temperature for 0.5 h while a streamof nitrogen was bubbled through it. After 0.5 h, the suspension was filtered and washed with DMF (5 x 3 resin volumes). Thesuspension was filtered, and Fmoc-Dab(Boc)-OH (263 mg, 0.6 mmol), HBTU (216 mg, 0.57 mmol), NMM (132 L, 1.2 mmol) and DMF(about 1.2 resin vol) were added to the resin. The reaction was carried out under a nitrogen atmosphere. Kaiser ninhydrin test wasused to indicate reaction completion. After the reaction was complete, the suspension was filtered and washed with DMF (5 x 3 resinvolumes). Then, 20percent piperidine in DMF (3 resin volumes) was added into the resin to cleave the Fmoc group. The suspension waskept at room temperature for 0.5 h while a stream of nitrogen was bubbled through it. After 0.5 h, the suspension was filtered andthe resin was washed with DMF (5 x 3 resin volumes). Subsequent amide couplings followed the generic procedure. 4 eq of Fmoc-Xaa,3.8eq of HBTU, and 8 eq of NMM in DMF were added to the resin. The reaction mixture was agitated under nitrogen until theamidation went to completion based on the Kaiser ninhydrin test. After the reaction was deemed complete, the suspension wasfiltered, and the resin was washed with DMF (5 x 3 resin volumes). Then, 20percent piperidine in DMF (3 resin volumes) was added to theresin bound peptide. The suspension was kept at room temperature for 0.5 h while a stream of nitrogen was bubbled through it.After 0.5 h, the suspension was filtered, and the resin was washed with DMF (5 x 3 resin volumes). This procedure was repeated until4-PyrAla N-oxide-Ala-Leu-Ala-Dab-Knorr amide MBHA resin bound peptide was delivered.A mixture of acetic anhydride:NMM:DMF (1.7:1:14, 3 resin volumes) was then added to 4-PyrAla-N-oxide-Ala-Leu-Ala-Dab-Knorramide MBHA resin bound peptide, and the reaction mixture was kept under nitrogen. The reaction was deemed complete based onthe Kaiser ninhydrin test to deliver Ac-(4-PyrAla-N-oxide)-Ala-Leu-Ala-Dab-Knorr amide MBHA resin bound peptide. The peptidylresin was then washed with DMF (5x10 mL) and methanol (5x10 mL) and dried in vacuo overnight. The peptidyl resin was then treated with a mixture of TFA:water (95:5), and the mixture was kept at rt for 3 h, and then filtered. Ether (7-8 filtrate volumes) wasadded to the filtrate which resulted in a precipitate. The mixture was centrifuged, and the supernatant was decanted. The resultingpellet was then washed with ether (3 x 7-8 filtrate volume) and dried under vacuum overnight to afford crude Ac-(4-PyrAla N-oxide)-Ala-Leu-Ala-Dab-NH2 (5). THF (5 mL) was added to the crude peptide followed by PyBroP (200 mg, 0.45 mmol) and DIPEA (130 muL,0.75 mmol). The reaction mixture was then stirred at room temperature for 5 h, whereupon it was concentrated under reducedpressure to give crude 9a.The crude peptide (pellet) was purified by reverse phase HPLC described in the purification details section (Purification Method 1)using a solvent gradient of A:B (90:10 to 30:70) over 60 minutes at a flow rate of 20 mL/min. Like fractions were combined, and thepeptide was repurified by eluting with a solvent gradient of A:B (85:15 to 65:35) over 60 minutes at a flow rate of 20 mL/min . Likefractions were once again combined and lyophilized to deliver 8 mg (9.5percent) of 9a as a white solid. UV purity (220 nm) = 96percent (retentiontime = 8.86 min; solvent gradient A:C, 82:18 to 72:28); ESI-MS (m/z) = 1121.2 (M+H)+, 561.4 (M/2+H)+.
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C77H118N20O19[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C73H113N21O17S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C71H111N21O16S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. (0997) The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedur...
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. (0997) The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedur...
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-fluorophenyl)propanoic acid[ No CAS ]
C34H54FN5O10[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
N-[(9-fluorenyl)methoxycarbonyl]-4-chloro-L-phenylalanine[ No CAS ]
C34H54ClN5O10[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(p-tolyl)propanoic acid[ No CAS ]
C35H57N5O10[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
S-(((9H-fluoren-9-yl)methoxy)carbonyl)cyclohexylglycine[ No CAS ]
C33H59N5O10[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 58-85-5 ]
NH2-NK(Biotin)VPNLRGDLQVLAQ[diaminobutyric acid]VART-OH[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
29%
(0234) To enable investigation of introducing non-proteinogenic amino acids (listed in Figure 1) on A20FMDV2 binding activity, biotinylated peptides 1-15 (see Table 1), except for peptide 6, were synthesised by standard Fmoc SPPS on the acid liable (0235) hydroxymethylphenoxypropionic acid linker (HMPP) which delivers a C- terminal carboxylic acid using to the conditions depicted in Scheme 1. The desired peptide sequences were assembled using 20percent (0236) piperidine/DMF to remove the Fmoc protecting group and 0- (0237) (benzotriazol-l-yl) -N, N, N ' , N '-tetramethyluronium hexafluorophosphate (HBTU) / DIPEA as coupling reagents. (0238) Since specific binding to the nubetabeta integrin was to be studied by flow cytometry, the native alanine at the second residue in A20FMDV2 (1) and all analogues thereof, were substituted with a biotinylated lysine residue. This substitution has previously been shown to be well tolerated [24,25] . We chose to install the D-biotin moiety by selective deprotection of a 1- ( 4 , 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene ) ethyl (Dde) [19] group on the side chain group followed by condensation with D-biotin using HBTU/DIPEA. (0239) Trifluoroacetic acid (TFA) /H2O/3, 6-dioxa-l , 8-octanedithiol (0240) (DODT) /triisopropylsilane (TIPS) (94:2.5:2.5:1.0, v/v/v/v) effected cleavage of the synthesised peptides from the corresponding (0241) peptidyl-resins . Peptides 1-15 were obtained in good yields ranging from 2percent-50percent and purity exceeding 99percent (see peptide characterization data) . (0242) For the synthesis of peptide 6 containing an i\7-L-methyllysine modification we employed an on-resin i\7-methylation protocol [22] which furnished peptide 6 in good yield (30percent) following TFA-mediated peptide cleavage and RP-HPLC purification. (0243) The lead peptide, A20FMDV2, which contains all naturally-occurring amino acids would be susceptible to degradation by exopeptidases which act on the amino- and carboxy terminuses. To mitigate this, six N- and/or C-terminus-modified and biotinylated A20FDMV2 mimics were prepared wherein we systematically modified the amino and carboxy ends (peptides 16-18) and the N-terminal and C-terminal amino acids (Asnl and Thr20, respectively, peptides 19-21) . N- terminal/C-terminal modified peptides 16-18 were obtained by capping of the N-terminus with acetic anhydride (16) or by employing the Rink amide linker to afford the C-terminal carboxamide (17) or a combination of both (peptide 18) . (0244) Peptide 19, bearing the unnatural D-Asnl in place of the native Asnl at the N-terminus of biotinylated A20FMDV2 (1) was obtained using the synthetic route outlined in Scheme 1 except that the Fmoc-D- Asn(Trt)-OH building block was incorporated into the synthesis as the N-terminal residue. For the preparation of peptides 20 and 21, which contains the unnatural D-Thr at the C-terminus, HMP-anchored resin 27 (see Scheme 1, HMP = hydroxymethylphenoxyacetic acid) was first esterified with Fmoc-D-Thr (tBu) -OH using DIC/DMAP and the sequence then elongated by Fmoc SPPS . (0245) Table 1. List of prepared synthetic peptides [N-term] - XiK (Biotin) VPNLRGDLQVX2AQX3VARX4- [C-term] containing substitutions for the native Lysl6 (peptides 2-6) or Leul3 (peptides 7-15), C- terminal/N-terminal variants (peptides 16-21) and DTPA-modified peptides (22-26) . NB: nomenclature, particularly X position (0246) numbering used in this table is not the same as that used in the claims . (0247) Compound N- Xl X2 X3 X4 C- term. term. (0248) 1 NH2 Asn Leu Lys Thr C02H (0249) 2 NH2 Asn Leu D-Lys Thr C02H (0250) 3 NH2 Asn Leu L-Orn Thr C02H (0251) 1-2,4- (0252) 4 NH2 Asn Leu diaminobutyric Thr C02H acid (0253) 1-2,3- (0254) 5 NH2 Asn Leu diaminopropionic Thr C02H acid (0255) 6 NH2 Asn Leu ZV-L-meth llysine Thr C02H (0256) 7 NH2 Asn aminoisobutyric Lys Thr C02H acid (0257) 8 NH2 Asn L-norvaline Lys Thr C02H (0258) 9 NH2 Asn L-norleucine Lys Thr C02H (0259) 10 NH2 Asn L-allylglycine Lys Thr C02H (0260) L-tert- (0261) 11 NH2 Asn Lys Thr C02H butylalanine (0262) 12 NH2 Asn L-homoleucine Lys Thr C02H (0263) L-2-amino-3- (0264) 13 NH2 Asn ethylpentanoic Lys Thr C02H acid (0265) L- (0266) 14 NH2 Asn Lys Thr C02H cyclohexylalanine (0267) 15 L- (0268) NH2 Asn Lys Thr C02H adamantylglycine (0269) 16 Ac-NH Asn Leu Lys Thr C02H (0270) 17 NH2 Asn Leu Lys Thr CONH2 (0271) 18 Ac-NH Asn Leu Lys Thr CONH2 (0272) 19 D- (0273) NH2 Leu Lys Thr C02H (0274) Asn (0275) 20 D- (0276) NH2 Asn Leu Lys C02H (0277) Thr (0278) 21 D- D- (0279) NH2 Leu Lys C02H (0280) Asn Thr (0281) 22 DTPA- (0282) Asn Leu Lys Thr C02H NH (0283) 23 DTPA- (0284) Asn Leu Lys Thr C02H Gly-NH (0285) 24 DTPA- (0286) Asn Leu Lys Thr CONH2 NH (0287) 25 DTPA- D- (0288) Leu Lys Thr C02H NH Asn (0289) 26 DTPA- D- D- (0290) Leu Lys C02H NH Asn Thr (0291) 1 D -yrpercent \?Q ^ (0292) ? -^H Q ? (0293) (0294) Scheme 1. Synthetic protocol for the preparation of the biotinylated A20FMDV2 peptide variants. (0295) The results obtained from th...
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