* 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.
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.
(S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid[ No CAS ]
[ 58-85-5 ]
[ 198561-07-8 ]
C66H94N20O21S[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 4.1.1. Peptide synthesis; 4.1.2; Solid-phase peptide synthesis (SPPS) was performed with standardFmoc chemistry on rink amide resin using an automated peptidesynthesizer (Syro I, Multisyntech). The resin was loaded into a5 mL reactor with a frit at the bottom. Swelling was performed bydispensing 1 mL DMF and incubating for 15 min (2) with 10 sshaking every minute. Fmoc deprotection was achieved by treatmentwith 40percent piperidine DMF for 3 min and 20percent piperidine inDMF for 12 min (10 s/min shaking). Peptide couplings were carriedout by double couplings with Fmoc-protected amino acids(5 equiv), HBTU (5 equiv), HOBt (5 equiv) and DIPEA (10 equiv) inDMF for 40 min (10 s/min shaking). At the respective position,Fmoc-F2Pmp-OH (3 equiv) was coupled in DMF (1 mL) by manualaddition using TBTU (3 equiv), HOBt (3 equiv) and DIPEA (6 equiv)for 3 h, after 3 min preactivation. In case of the sequences for which side-chain labeling with biotinor carboxyfluorescein was planned, an additional 4-methyltrityl-(Mtt-) protected lysine was coupled to the N-terminus. Toselectively remove the Mtt group the resin was washed for 1 minwith DCM (3), deprotection was then achieved by treatment with1.8percent TFA in DCM for 3 min (10). During the deprotection the DCMsolution turned yellow.For fluorescein-labeling of the amine side-chain 5(6)-carboxyfluorescein(3 equiv), HATU (3 equiv), HOAt (3 equiv) andDIPEA (6 equiv) were dissolved in DMF and pre-activated for3 min. The solution was aspirated and coupling was allowed toproceed for 1 h. This step was repeated 4 times.For biotin-labeling of the amine side-chain the resin waswashed for 1 min in NMP (3). D-(+)-Biotin (3 equiv), HATU(3 equiv), HOAt (3 equiv) and DIPEA (6 equiv) were dissolved inNMP and pre-activated for 3 min. The solution was aspirated andcoupling was allowed to proceed for 2 h. This step was repeated2 times. N-terminal acetylation (where applicable) was achieved by dispensing800 lL of a mixture of acetic anhydride/pyridine (1:9) andreaction twice for 5 min (10 s/min shaking). After each deprotection,coupling or acetylation step, 5 washings (1 min each) withDMF were performed (10 s/min shaking).After synthesis the resin was transferred in a 5 mL syringeequipped with a frit, washed with DCM for 1 min (3) and driedin high vacuum for at least 30 min. For cleavage 1 mL of a mixtureof TFA and TIS (20:1) was added. The syringe with the mixture waskept on a shaker for 3 h. Then the liquid phase was filtered into20 mL of ice-cold Et2O. Formed precipitate was centrifuged,washed with ice-cold Et2O (2 20 mL) and purified by HPLC. 4.1.2. Azide functionalization of the N-terminus; To the peptides with the longer carbon linker, 6-azidohexanoicacid was coupled (with standard coupling conditions) to the Nterminalamine.The N-terminal amine of the peptides with the shorter linkerwas converted to an azide functionality directly on solid support.Using the compound imidazole-1-sulfonyl-azide*HCl (synthesissee beneath) and modified conditions, which were reported forsolution phase chemistry from Goddard?Borger and Stick:8 Theresin was washed for 1 min each with DCM (2), DCM/MeOH(2) and MeOH (3). Then (for 40 mg resin, loading= 0.62 mmole/g) 1.4 equiv of imidazole-1-sulfonyl-azide*HClin 1 mL MeOH and 100 ll of a saturated and centrifuged solutionof CuSO4*5H2O was added. After 1 min, DIPEA (1.8 equiv) wasadded and the coupling was allowed to proceed for 1 h andrepeated once more with an intermediate washing with MeOH(3 1 min).
(S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid[ No CAS ]
[ 58-85-5 ]
[ 198561-07-8 ]
C72H105N21O22S[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 4.1.1. Peptide synthesis; 4.1.2; Solid-phase peptide synthesis (SPPS) was performed with standardFmoc chemistry on rink amide resin using an automated peptidesynthesizer (Syro I, Multisyntech). The resin was loaded into a5 mL reactor with a frit at the bottom. Swelling was performed bydispensing 1 mL DMF and incubating for 15 min (2) with 10 sshaking every minute. Fmoc deprotection was achieved by treatmentwith 40percent piperidine DMF for 3 min and 20percent piperidine inDMF for 12 min (10 s/min shaking). Peptide couplings were carriedout by double couplings with Fmoc-protected amino acids(5 equiv), HBTU (5 equiv), HOBt (5 equiv) and DIPEA (10 equiv) inDMF for 40 min (10 s/min shaking). At the respective position,Fmoc-F2Pmp-OH (3 equiv) was coupled in DMF (1 mL) by manualaddition using TBTU (3 equiv), HOBt (3 equiv) and DIPEA (6 equiv)for 3 h, after 3 min preactivation. In case of the sequences for which side-chain labeling with biotinor carboxyfluorescein was planned, an additional 4-methyltrityl-(Mtt-) protected lysine was coupled to the N-terminus. Toselectively remove the Mtt group the resin was washed for 1 minwith DCM (3), deprotection was then achieved by treatment with1.8percent TFA in DCM for 3 min (10). During the deprotection the DCMsolution turned yellow.For fluorescein-labeling of the amine side-chain 5(6)-carboxyfluorescein(3 equiv), HATU (3 equiv), HOAt (3 equiv) andDIPEA (6 equiv) were dissolved in DMF and pre-activated for3 min. The solution was aspirated and coupling was allowed toproceed for 1 h. This step was repeated 4 times.For biotin-labeling of the amine side-chain the resin waswashed for 1 min in NMP (3). D-(+)-Biotin (3 equiv), HATU(3 equiv), HOAt (3 equiv) and DIPEA (6 equiv) were dissolved inNMP and pre-activated for 3 min. The solution was aspirated andcoupling was allowed to proceed for 2 h. This step was repeated2 times. N-terminal acetylation (where applicable) was achieved by dispensing800 lL of a mixture of acetic anhydride/pyridine (1:9) andreaction twice for 5 min (10 s/min shaking). After each deprotection,coupling or acetylation step, 5 washings (1 min each) withDMF were performed (10 s/min shaking).After synthesis the resin was transferred in a 5 mL syringeequipped with a frit, washed with DCM for 1 min (3) and driedin high vacuum for at least 30 min. For cleavage 1 mL of a mixtureof TFA and TIS (20:1) was added. The syringe with the mixture waskept on a shaker for 3 h. Then the liquid phase was filtered into20 mL of ice-cold Et2O. Formed precipitate was centrifuged,washed with ice-cold Et2O (2 20 mL) and purified by HPLC. 4.1.2. Azide functionalization of the N-terminus; To the peptides with the longer carbon linker, 6-azidohexanoicacid was coupled (with standard coupling conditions) to the Nterminalamine.The N-terminal amine of the peptides with the shorter linkerwas converted to an azide functionality directly on solid support.Using the compound imidazole-1-sulfonyl-azide*HCl (synthesissee beneath) and modified conditions, which were reported forsolution phase chemistry from Goddard?Borger and Stick:8 Theresin was washed for 1 min each with DCM (2), DCM/MeOH(2) and MeOH (3). Then (for 40 mg resin, loading= 0.62 mmole/g) 1.4 equiv of imidazole-1-sulfonyl-azide*HClin 1 mL MeOH and 100 ll of a saturated and centrifuged solutionof CuSO4*5H2O was added. After 1 min, DIPEA (1.8 equiv) wasadded and the coupling was allowed to proceed for 1 h andrepeated once more with an intermediate washing with MeOH(3 1 min).
(S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid[ No CAS ]
[ 58-85-5 ]
[ 198561-07-8 ]
C67H95F2N20O23PS[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 4.1.1. Peptide synthesis; 4.1.2; Solid-phase peptide synthesis (SPPS) was performed with standardFmoc chemistry on rink amide resin using an automated peptidesynthesizer (Syro I, Multisyntech). The resin was loaded into a5 mL reactor with a frit at the bottom. Swelling was performed bydispensing 1 mL DMF and incubating for 15 min (2) with 10 sshaking every minute. Fmoc deprotection was achieved by treatmentwith 40percent piperidine DMF for 3 min and 20percent piperidine inDMF for 12 min (10 s/min shaking). Peptide couplings were carriedout by double couplings with Fmoc-protected amino acids(5 equiv), HBTU (5 equiv), HOBt (5 equiv) and DIPEA (10 equiv) inDMF for 40 min (10 s/min shaking). At the respective position,Fmoc-F2Pmp-OH (3 equiv) was coupled in DMF (1 mL) by manualaddition using TBTU (3 equiv), HOBt (3 equiv) and DIPEA (6 equiv)for 3 h, after 3 min preactivation. In case of the sequences for which side-chain labeling with biotinor carboxyfluorescein was planned, an additional 4-methyltrityl-(Mtt-) protected lysine was coupled to the N-terminus. Toselectively remove the Mtt group the resin was washed for 1 minwith DCM (3), deprotection was then achieved by treatment with1.8percent TFA in DCM for 3 min (10). During the deprotection the DCMsolution turned yellow.For fluorescein-labeling of the amine side-chain 5(6)-carboxyfluorescein(3 equiv), HATU (3 equiv), HOAt (3 equiv) andDIPEA (6 equiv) were dissolved in DMF and pre-activated for3 min. The solution was aspirated and coupling was allowed toproceed for 1 h. This step was repeated 4 times.For biotin-labeling of the amine side-chain the resin waswashed for 1 min in NMP (3). D-(+)-Biotin (3 equiv), HATU(3 equiv), HOAt (3 equiv) and DIPEA (6 equiv) were dissolved inNMP and pre-activated for 3 min. The solution was aspirated andcoupling was allowed to proceed for 2 h. This step was repeated2 times. N-terminal acetylation (where applicable) was achieved by dispensing800 lL of a mixture of acetic anhydride/pyridine (1:9) andreaction twice for 5 min (10 s/min shaking). After each deprotection,coupling or acetylation step, 5 washings (1 min each) withDMF were performed (10 s/min shaking).After synthesis the resin was transferred in a 5 mL syringeequipped with a frit, washed with DCM for 1 min (3) and driedin high vacuum for at least 30 min. For cleavage 1 mL of a mixtureof TFA and TIS (20:1) was added. The syringe with the mixture waskept on a shaker for 3 h. Then the liquid phase was filtered into20 mL of ice-cold Et2O. Formed precipitate was centrifuged,washed with ice-cold Et2O (2 20 mL) and purified by HPLC. 4.1.2. Azide functionalization of the N-terminus; To the peptides with the longer carbon linker, 6-azidohexanoicacid was coupled (with standard coupling conditions) to the Nterminalamine.The N-terminal amine of the peptides with the shorter linkerwas converted to an azide functionality directly on solid support.Using the compound imidazole-1-sulfonyl-azide*HCl (synthesissee beneath) and modified conditions, which were reported forsolution phase chemistry from Goddard?Borger and Stick:8 Theresin was washed for 1 min each with DCM (2), DCM/MeOH(2) and MeOH (3). Then (for 40 mg resin, loading= 0.62 mmole/g) 1.4 equiv of imidazole-1-sulfonyl-azide*HClin 1 mL MeOH and 100 ll of a saturated and centrifuged solutionof CuSO4*5H2O was added. After 1 min, DIPEA (1.8 equiv) wasadded and the coupling was allowed to proceed for 1 h andrepeated once more with an intermediate washing with MeOH(3 1 min).
Chemistry
Pharmaceutical Intermediates
Inhibitors/Agonists
Material Science
For Research Only
120K+ Compounds
Competitive Price
1-2 Day Shipping
