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CAS No. : | 132327-80-1 | MDL No. : | MFCD00077056 |
Formula : | C39H34N2O5 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | WDGICUODAOGOMO-DHUJRADRSA-N |
M.W : | 610.70 | Pubchem ID : | 10919157 |
Synonyms : |
|
Num. heavy atoms : | 46 |
Num. arom. heavy atoms : | 30 |
Fraction Csp3 : | 0.15 |
Num. rotatable bonds : | 14 |
Num. H-bond acceptors : | 5.0 |
Num. H-bond donors : | 3.0 |
Molar Refractivity : | 175.94 |
TPSA : | 104.73 Ų |
GI absorption : | Low |
BBB permeant : | No |
P-gp substrate : | No |
CYP1A2 inhibitor : | No |
CYP2C19 inhibitor : | Yes |
CYP2C9 inhibitor : | Yes |
CYP2D6 inhibitor : | Yes |
CYP3A4 inhibitor : | No |
Log Kp (skin permeation) : | -5.08 cm/s |
Log Po/w (iLOGP) : | 3.56 |
Log Po/w (XLOGP3) : | 6.97 |
Log Po/w (WLOGP) : | 6.76 |
Log Po/w (MLOGP) : | 4.57 |
Log Po/w (SILICOS-IT) : | 6.64 |
Consensus Log Po/w : | 5.7 |
Lipinski : | 2.0 |
Ghose : | None |
Veber : | 1.0 |
Egan : | 1.0 |
Muegge : | 2.0 |
Bioavailability Score : | 0.56 |
Log S (ESOL) : | -7.58 |
Solubility : | 0.0000162 mg/ml ; 0.0000000265 mol/l |
Class : | Poorly soluble |
Log S (Ali) : | -8.98 |
Solubility : | 0.000000635 mg/ml ; 0.000000001 mol/l |
Class : | Poorly soluble |
Log S (SILICOS-IT) : | -12.62 |
Solubility : | 0.0000000001 mg/ml ; 0.0 mol/l |
Class : | Insoluble |
PAINS : | 0.0 alert |
Brenk : | 0.0 alert |
Leadlikeness : | 3.0 |
Synthetic accessibility : | 5.18 |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P280 | UN#: | N/A |
Hazard Statements: | H317-H413 | Packing Group: | N/A |
GHS Pictogram: |
* 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.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
fully automated peptide synthesizer; | The resin used for solid synthesis of the peptide was a 2-chlorotrisyl chloride resin, which will not impair the protective groups on various amino acid residues and from which the peptide can be cleaved with a weak acid. A 0.25-mmol (368-mg) portion of the resin was weighed and used. The peptide synthesis was carried out according to the Fmoc (9-fluorenylmethoxycarbonyl) chemistry and a Fmoc-side chain-protected peptide-resin was obtained by starting the synthesis from the C terminus on a fully automated peptide synthesizer using the following Fmoc-side chain-protected amino acids 1) to 12) (1.0 mmol each).1) Fmoc-Gly-OH 1.0 mmol2) Fmoc-L-Arg(Pmc)-OH 1.0 mmolPmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl3) Fmoc-L-Asp(OtBu)-OH 1.0 mmolOtBu: O-t-butyl4) Fmoc-L-Asp(OtBu)-OH 1.0 mmol5) Fmoc-L-Ala-OH 1.0 mmol6) Fmoc-L-Glu(OtBu)-OH 1.0 mmol7) Fmoc-Gly-OH 1.0 mmol8) Fmoc-L-Glu(OtBu)-OH 1.0 mmol9) Fmoc-L-Lys(Boc)-OH 1.0 mmolBoc: benzyloxycarbonyl10) Fmoc-L-Gln(Trt)-OH 1.0 mmolTrt: trityl11) Fmoc-L-Ser(tBu)-OH 1.0 mmoltBu: t-butyl12) Fmoc-L-Asp(OBzl)-OH 1.0 mmolOBzl: O-benzyl |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
85% | The peptide (1) was synthesized using Fmoc strategy in manual solid- phase peptide synthesis, on Sieber Amide resin. Sieber resin is a 9-Fmoc- amino-xanten-3-yloxy-Merrifield resin (100-200 mesh, 1% 2,2,4,6,7- pentamethyldihydro benzo-furane, substitution level 0.52 mmol g"1), and it is an excellent support for the synthesis of protected peptide amides.The amino acids were inserted as Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)- OH, Fmoc-Arg(Pbf)-OH. The Lys residue in position 9 was inserted as <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong>. The Methoxytrityl protecting group (Mmt) is a protecting group that can be removed from the side chain of lysine in mild acid conditions (1% TFA in DCM or AcOH/TFE/DCM 1 :2:7 (v/v)); this behaviour allows the selective removal of Mmt group in the presence of other side-chain protecting groups, which require up to 95% TFA for removal. The selective removal of this group allows the coupling of a fully protected peptide, through the free epsilon-amino group of the Lys 9 side chain, with the porphyrin macrocycle.The synthesis was carried out on a 0.25 mmol scale. N-aFmoc deprotection was accomplished with a solution of 20% piperidine/DMF (v/v). Two separate treatments, of 3 and 7 minutes, were used for each cycle.Two coupling steps were performed for each amino acid (45 min coupling time). For the first coupling, 3 equivalents of Fmoc-amino acid, 3 equivalents of PyBop/HOBt, and 6 equivalents of DIEA in Nu,Nu-Dimethylformamide (DMF) were used; 2 equivalents of Fmoc-amino acid, 2 equivalents of HATU, and 4 equivalents of DIEA in DMF were employed for the second coupling, instead. Completeness of the reaction was checked after each coupling by the Kaiser test.After synthesis completion, the N-terminus was acetylated with 4.7% acetic anhydride and 4% pyridine in DMF for 15 min.Selective removal of Mmt group of Lysine 9 was accomplished by treatment of the peptidyl-resin with AcOH/TFE/DCM 1 :2:7 (v/v), in a sintered glass funnel. The resin was shaken for 10 min, and the solvent was removed under vacuum. This step was repeated 15 times. Finally, the resin was washed with isopropanol and DCM. After the clevage of the Mmt group, the fully protected peptide amide was cleaved from the resin by applying a 1% TFA/DCM (volume percentage) solution. The resin was shaken for 2 min, and the filtered solution was collected into an ice-cooled flask containing 5% pyridine/ methanol (volume percentage). This treatment was repeated many times. Finally, the resin was washed with DCM. The filtrates were checked by silica gel TLC in chloroform/methanol/acetic acid 80: 18:2 (v/v/v). The fractions containing the desired product were combined and evaporated under reduced pressure up to 5% of the volume.Ice-cold water was added to the residue and the mixture was cooled on ice to aid precipitation of the protected peptide. The product was filtered, washed several times with fresh water, and dried under vacuum to give the crude C-terminal decapeptide amide (1).Product homogeinity was ascertained by analytical RP-HPLC, on a C18 column, using a gradient of acetonitrile in 0.1% aqueous TFA, 50% to 95% over 30 min, flow rate 1 mLmin"1). Chromatogram showed a main peak at 23.8 min retention gtime. Peptide identity was checked via ESI-MS spectrometry that confirmed the expected molecular weight (2463 a.m.u.)The peptide was obtained with a 85% yield. |
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). |
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). |
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). |
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). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Amino acids, building blocks, coupling reagents and Wang resins were purchased from Novabiochem AG. The glycosylated amino acids were prepared in our lab following procedures as reported in our previous work. 1 All reagents used for synthesis were from analytical grade. Peptides were synthesized manually following standard solid-phase methods and Fmoc protocols on Wang resin using amino acids with orthogonal protections on lateral chains. Amide couplings were performed manually ina peptide synthesis column using DIC/HOBt in DMF under reciprocal oscillating agitation. Coupling efficiencies were monitored by Kaiser ninhydrin test. Fmoc groups were removed with a 20% piperidine in DMF solution. Peptides and glycopeptides were cleaved from the resin by shaking with a cleavage cocktail consisting of TFA:H2O:TIS(95:2.5:2.5) for 2 h. The filtrate was evaporated, washed several times with ice-cold tert-butyl methyl ether and concentrated under reduced pressure. The crude glycopeptides were precipitated with ice-cold tert-butyl methyl ether, filtered, redissolved in water and lyophilized. In the case of glycopeptides, the acetyl protecting groups on the sugar moieties were next hydrolyzed carefully adding sodium methoxide/MeOH solutions to methanol solutions of the protected glycopeptides (monitored by analytical reverse-phase HPLC). The glycopeptides were neutralized by addition of AcOH and concentrated under reduced pressure. Crude peptides and glycopeptides were purified by C-18 RP-HPLC (VersaFlashTM Flash Chromatograph ysystem) using a water-acetonitrile gradient and followed by lyophilization. The final pure peptides were characterized by HRMS. Analytical RP-HPLC were performed using the following solvents A (0.1% TFA in H2O) and B (0.1% TFA in acetonitrile) and the Nucleosil 100 RP-18 (5mum) C18 column (4x 250 mm). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Amino acids, building blocks, coupling reagents and Wang resins were purchased from Novabiochem AG. The glycosylated amino acids were prepared in our lab following procedures as reported in our previous work. 1 All reagents used for synthesis were from analytical grade. Peptides were synthesized manually following standard solid-phase methods and Fmoc protocols on Wang resin using amino acids with orthogonal protections on lateral chains. Amide couplings were performed manually ina peptide synthesis column using DIC/HOBt in DMF under reciprocal oscillating agitation. Coupling efficiencies were monitored by Kaiser ninhydrin test. Fmoc groups were removed with a 20% piperidine in DMF solution. Peptides and glycopeptides were cleaved from the resin by shaking with a cleavage cocktail consisting of TFA:H2O:TIS(95:2.5:2.5) for 2 h. The filtrate was evaporated, washed several times with ice-cold tert-butyl methyl ether and concentrated under reduced pressure. The crude glycopeptides were precipitated with ice-cold tert-butyl methyl ether, filtered, redissolved in water and lyophilized. In the case of glycopeptides, the acetyl protecting groups on the sugar moieties were next hydrolyzed carefully adding sodium methoxide/MeOH solutions to methanol solutions of the protected glycopeptides (monitored by analytical reverse-phase HPLC). The glycopeptides were neutralized by addition of AcOH and concentrated under reduced pressure. Crude peptides and glycopeptides were purified by C-18 RP-HPLC (VersaFlashTM Flash Chromatograph ysystem) using a water-acetonitrile gradient and followed by lyophilization. The final pure peptides were characterized by HRMS. Analytical RP-HPLC were performed using the following solvents A (0.1% TFA in H2O) and B (0.1% TFA in acetonitrile) and the Nucleosil 100 RP-18 (5mum) C18 column (4x 250 mm). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Example 1The following amino acids were used: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc- Dap(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH and mono-tBu succinate. MS (M+H+): expected 971.1; observed 971.2Peptide Synthesis:The peptide was synthesized using CEM Microwave technology with coupling times of 5 minutes per amino acid at elevated temperature (78 °C) and a 0.25mmol scale. The synthesis is carried out using the TentalGel-S RAM resin as a solid support (0.24 meq /g). All amino acids used were dissolved in DMF to 0.2 mol concentration. A mixture of HOBT/HBTU 1: 1 (0.5 mol /L) 4 eq. and DIPEA 4eq. was used to activate the amino acids. Fmoc-Cleavage was achieved with Piperidine in DMF (20 percent) for 3 min. Fmoc-cleavage was repeated.General Synthesis Description:The cyclic peptides can be generated either via on-bead cyclisation (Allyl/Aloc strategy METHODE A) or as fully deprotected linear peptides via solution phase cyclisation (METHODE B).Linear peptides were either synthesized manually or using microwave technology via state-of- the-art solid phase synthesis protocols (Fmoc-chemistry) as referenced by e.g.: Kates and Albericio, Eds., "Solid Phase Synthesis: A practical guide", Marcel Decker, New York, Basel, 2000. As a solid support TentaGel-S-RAM resin (0.24 meq /g) was used. All Fmoc-amino acids were added in a 4-fold excess after activation with HOBT/HBTU 1 : 1 (0.5 mol/L in DMF) and 4 eq of DIPEA (2 mol/L in NMP). Fmoc-cleavage was achieved with 20percent Piperidine in DMF.Allyl/Aloc- Cleavage & Lactam- Cyclisation:(METHODE A: The resin-linked peptides were treated manually with a solution of 20 eq phenylsilane in DCM and 0.05 eq of tetrakis triphenylphosphine palladium for 30 min at RT. This procedure was repeated. The resin was washed with a solution of 0.5percent sodium dithiocarbamate in DMF. For the on-bead lactam formation, again activation reagent was added to the resin and shaken for additional 8h at RT. Completion of cyclisation was verified via Ninhydrin-test.METHODE B: Peptides were cyclized in solution after deprotection and cleavage from the resin and standard work-up. Crude peptides were treated with standard peptide activation regents in DMF. The cyclisation was monitored via HPLC.Cleavage & work-up:A cleavage-cocktail of trifluoroacetic acid, triisopropylsilane and water (95/2.5/2.5) was added to the resin and shaken for lh at RT. Cleaved peptides were precipitated in cold Ether (-18°C ). The peptides were centrifuged and the residue washed twice with cold ether. The residues were again dissolved in water/ acetonitrile and lyophilized.Purification :Peptides were purified using reversed phase high performance liquid chromatography (RP- HPLC) using a Reprospher 100 C18-T Columm (100 x 4.6 mm, 5u particle size) as a stationary phase and water/acetonitrile as eluent (0053) (Gradient 1-50 percent MeCN over 30 min). Fractions were collected and analyzed by LC/MS. Pure product samples were combined and lyophilized. All peptides were obtained as white powders with a purity >85 percent. Product identification was obtained via mass spectrometry. All standard amino acids were purchased from CEM. Fmoc-SAR-OH and Fmoc-N-Cyclopropyl- Glycine were purchased from Bachem and Enamine respectively. Mono-tBu-Succinate were purchased from Sigma- Aldrich |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The solid phase peptide syntheses were performed on a Prelude Peptide Synthesizer (Protein Technologies Inc) using standard Fmoc chemistry and HBTU/DIPEA activation. DMF was used as the solvent. Deprotection: 20percent piperidine/DMF for 2×2.5 min. Washes: 7×DMF. Coupling 2:5:10 200 mM AA/500 mM HBTU/2M DIPEA in DMF 2× for 20 min. Washes: 5×DMF. In cases where a Lys-side-chain was modified, Fmoc-L-Lys(ivDde)-OH was used in the corresponding position. After completion of the synthesis, the ivDde group was removed according to a literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). The following acylations were carried out by treating the resin with the N-hydroxy succinimide esters of the desired acid or using coupling reagents like HBTU/DIPEA or HOBt/DIC. (0270) All the peptides that had been synthesized were cleaved from the resin with King's cleavage cocktail consisting of 82.5percent TFA, 5percent phenol, 5percent water, 5percent thioanisole, 2.5percent EDT. The crude peptides were then precipitated in diethyl or diisopropyl ether, centrifuged, and lyophilized. Peptides were analyzed by analytical HPLC and checked by ESI mass spectrometry. Crude peptides were purified by a conventional preparative HPLC purification procedure. Example 1 The solid phase synthesis was carried out on Rink-resin with a loading of 0.38 mmol/g, 75-150 mum from the company Agilent Technologies. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18, 5 muM) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). (0296) Finally, the molecular mass of the purified peptide was confirmed by LC-MS. M.W. (calculated)=4188.5 g/mol; M.W. (found)=4188.6 g/mol. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The conjugates were built up by solid-phase peptide synthesis on Rink amide MBHA resin using Fmoc/tBu strategy. The side chain of amino acids were protected by 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf, in Arg), by trityl (Trt, in Asn, Gln), by tert-butyl group (tBu, in Glu) and by tert-butyloxycarbonyl group (Boc, in Lys). The N-terminal Fmoc protecting group was removed with 2% piperidine in the presence of 2% 1.8-diazabicyclo [5.4.0]undec-7-ene (DBU) in DMF (2+2+5+10min). This reagent was removed by washing with DMF (8×0.5min). In the coupling reaction, amino acid derivatives, which were used in 3M excess to the resin capacity, were activated by N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) dissolved in DMF. The reaction proceeded at RT for 60min. Then, the resin was washed with DMF (2×0.5min) and dichloromethane (DCM) (3×0.5min). Ninhydrin assay was used to monitor the efficiency of the coupling reaction [36]. After the removal of the terminal Nalpha-Fmoc group, methotrexate (MTX) or 5 (6)-carboxyfluorescein (cf) were attached to the peptide using the HOBt-DIC coupling reagents. The free compounds were obtained by cleavage from the resin with 10mL TFA containing 0.75g phenol, 0.5mL distilled water, 0.5mL thioanisole and 0.25mL 1,2-ethandithiol (EDT) as scavengers. Crude products were precipitated by dry diethyl-ether, dissolved in 10% acetic acid and freeze-dried. The crude peptide was purified by RP-HPLC as described below. The purified compounds were characterized by analytical RP-HPLC and ESI-MS (Table. 1). The purity of the compounds was higher than 95%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The conjugates were built up by solid-phase peptide synthesis on Rink amide MBHA resin using Fmoc/tBu strategy. The side chain of amino acids were protected by 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf, in Arg), by trityl (Trt, in Asn, Gln), by tert-butyl group (tBu, in Glu) and by tert-butyloxycarbonyl group (Boc, in Lys). The N-terminal Fmoc protecting group was removed with 2% piperidine in the presence of 2% 1.8-diazabicyclo [5.4.0]undec-7-ene (DBU) in DMF (2+2+5+10min). This reagent was removed by washing with DMF (8×0.5min). In the coupling reaction, amino acid derivatives, which were used in 3M excess to the resin capacity, were activated by N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) dissolved in DMF. The reaction proceeded at RT for 60min. Then, the resin was washed with DMF (2×0.5min) and dichloromethane (DCM) (3×0.5min). Ninhydrin assay was used to monitor the efficiency of the coupling reaction [36]. After the removal of the terminal Nalpha-Fmoc group, methotrexate (MTX) or 5 (6)-carboxyfluorescein (cf) were attached to the peptide using the HOBt-DIC coupling reagents. The free compounds were obtained by cleavage from the resin with 10mL TFA containing 0.75g phenol, 0.5mL distilled water, 0.5mL thioanisole and 0.25mL 1,2-ethandithiol (EDT) as scavengers. Crude products were precipitated by dry diethyl-ether, dissolved in 10% acetic acid and freeze-dried. The crude peptide was purified by RP-HPLC as described below. The purified compounds were characterized by analytical RP-HPLC and ESI-MS (Table. 1). The purity of the compounds was higher than 95%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The conjugates were built up by solid-phase peptide synthesis on Rink amide MBHA resin using Fmoc/tBu strategy. The side chain of amino acids were protected by 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf, in Arg), by trityl (Trt, in Asn, Gln), by tert-butyl group (tBu, in Glu) and by tert-butyloxycarbonyl group (Boc, in Lys). The N-terminal Fmoc protecting group was removed with 2% piperidine in the presence of 2% 1.8-diazabicyclo [5.4.0]undec-7-ene (DBU) in DMF (2+2+5+10min). This reagent was removed by washing with DMF (8×0.5min). In the coupling reaction, amino acid derivatives, which were used in 3M excess to the resin capacity, were activated by N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) dissolved in DMF. The reaction proceeded at RT for 60min. Then, the resin was washed with DMF (2×0.5min) and dichloromethane (DCM) (3×0.5min). Ninhydrin assay was used to monitor the efficiency of the coupling reaction [36]. After the removal of the terminal Nalpha-Fmoc group, methotrexate (MTX) or 5 (6)-carboxyfluorescein (cf) were attached to the peptide using the HOBt-DIC coupling reagents. The free compounds were obtained by cleavage from the resin with 10mL TFA containing 0.75g phenol, 0.5mL distilled water, 0.5mL thioanisole and 0.25mL 1,2-ethandithiol (EDT) as scavengers. Crude products were precipitated by dry diethyl-ether, dissolved in 10% acetic acid and freeze-dried. The crude peptide was purified by RP-HPLC as described below. The purified compounds were characterized by analytical RP-HPLC and ESI-MS (Table. 1). The purity of the compounds was higher than 95%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
7.4 g | With N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate; In N,N-dimethyl-formamide; at 0 - 20℃; for 2h; | To a solution of Fmoc-Gln(Trt)-OH (5.0 g, 8.2 mmol) in DMF (25.0 mL) HATU(3.4 g, 9.0 mmol), H-Thr(OtBu)-OtBu (1.9 g, 8.2 mmol) and DIPEA (2.9 mL, 16.4mmol) were added at 0 C and stirred at room temperature for 2 h. The completion of the reaction was confirmed by TLC analysis. The reaction mixture was quenched with water, the resulting solid was filtered, washed with hexane and dried to yield 7.4 g of compound6a. LCMS: 824.1 (M+H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide Synthesis: (0036) The peptide was synthesized using CEM Microwave technology with coupling times of 5 minutes per amino acid at elevated temperature (78° C.) and a 0.25 mmol scale. The synthesis is carried out using the TentalGel-S RAM resin as a solid support (0.24 meq/g). All amino acids used were dissolved in NMP to 0.2 mol concentration. A solution of 4 eq. COMU in DMF (0.5 mol/L) and DIPEA was used to activate the amino acids. Fmoc-Cleavage was achieved with Piperidine in DMF (20percent) for 3 min. Fmoc-cleavage was repeated. Cleavage from Resin: (0037) 10 ml of a cleavage-cocktail consisting of 95/2.5/2.5 Trifluoroacetic acid, Triisopropylsilane, and water was added to the resin and shaken for 3 h at RT. Cleaved peptide was precipitated in cold Et2O (?18° C.). The peptide was centrifuged 2×50 ml polypropylene tubes. The precipitates were washed two times with cold ether. Afterwards the precipitate was dissolved in H2O/Acetonitrile and lyophilizied to yield 88 mg white powder. Cyclization: (0038) Crude peptide was dissolved in DMF (15 ml). 1 eq of coupling reagents PyoAP (0.5 mol/L) in DMF and DIPEA in NMP (2 mol/1) were added. The reaction mixture was stirred at RT for 1 h. After the reaction was completed (LCMS control) the DMF content was concentrated down to approximately 2 ml. The residue was precipitated in cold (?18° C.) diethyl ether (40 ml). The peptide was centrifuged and the precipitate washed with cold ether. Purification: (0039) The crude peptide was purified by preparative HPLC on a Reprospher 100 C18-T Column (100×4.6 mm, 5 um particle size). As eluent system a mixture of 0.1percent TFA/water/acetonitrile was used with a gradient of 0-100percent acetonitrile within 0-75 min. The fractions were collected and checked by analytical HPLC. Fractions containing pure product were combined and lyophilized. 27 mg of white powder were obtained. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The peptide component of Compound 1 is synthesized by automated solid-phase synthesis using Fluorenylmethyloxycarbonyl (Fmoc)/tert-Butyl (t-Bu) chemistry on a Symphony 12-channel multiplex peptide synthesizer (Protein Technologies, Inc. Tucson, Ariz.). The synthesis resin consists of 1% DVB cross-linked polystyrene (Fmoc-Rink-MBHA Low Loading resin, 100-200 mesh, EMD Millipore, Temecula, Calif.) at a substitution 0.3-0.4 meq/g. Standard side-chain protecting groups are as follows: tert-butyloxycarbonyl (Boc) for Trp and Lys; tert-butyl ester (OtBu) for Asp and Glu; tBu for Ser, Thr and Tyr; and triphenylmethyl (Trt) for Gln; N-alpha-Fmoc-N--4-methyltrityl-L-lysine (Fmoc-Lys(Mtt)-OH) was used for the lysine at position 20 of SEQ ID NO: 3 and Nalpha,N(im)-di-Boc-L-histidine (Boc-His(Boc)-OH) was used for the histidine at position 1. Fmoc groups were removed prior to each coupling step (2×7 minutes) using 20% piperidine in dimethylformamide (DMF). All standard amino acid couplings are performed for 1 hour, using an equal molar ratio of Fmoc amino acid (EMD Millipore, Temecula, Calif.), diisopropylcarbodiimide (DIC)(Sigma-Aldrich, St. Louis, Mo.) and Oxyma (Oxyma Pure, Iris Biotech, Marktredwitz, Germany), at a 9-fold molar excess over the theoretical peptide loading and at a final concentration of 0.18 M in DMF. Two exceptions are the glutamine residue at position 3 of SEQ ID NO: 5, which is double-coupled (2×1 hour), and the histidine residue at position 1 of SEQ ID NO: 5, which was coupled at a 6-fold molar excess using 1-Hydroxy-7-azabenzotriazole (HOAt) instead of Oxyma for 18 hours. After completion of the synthesis of the linear peptide, the resin was transferred to a disposable fritted 25 mL polypropylene syringe (Torviq, Niles, Mich.) equipped with a polytetrafluoroethylene (PTFE) stopcock (Biotage, Charlotte, N.C.) and the 4-Methyltrityl (Mtt) protecting group on the lysine at position 20 of SEQ ID NO: 5 was selectively removed from the peptide resin using three treatments with 20% hexafluoroisopropanol (Oakwood Chemicals, West Columbia, S.C.) in DCM (2×10 minutes and 1×45 minutes) to expose the free epsilon amine of the lysine at position 20 and make it available for further reaction. Subsequent attachment of the fatty acid-linker moiety is accomplished by performing two succeeding couplings of [2-(2-(Fmoc-amino)ethoxy)ethoxy]acetic acid (Fmoc-AEEA-OH) (ChemPep, Inc. Wellington, Fla.; 3-fold excess of amino acid (AA):1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU): N,N-diisopropylethylamine (DIPEA) [1:1:5 mol/mol] for a 3 hour coupling time), followed by coupling of Fmoc-glutamic acid alpha-t-butyl ester (Fmoc-Glu-OtBu)(Ark Pharm, Inc. Libertyville Ill., 3-fold excess of AA:HATU:DIPEA [1:1:5 mol/mol] for a 3 hour coupling time). In each case, the Fmoc moiety is removed as described above. Finally, mono-OtBu-octadecanedioic acid (WuXi AppTec, Shanghai, China) is coupled to the resin over 18 hours using a 3-fold excess of acid:HATU:DIPEA (1:1:5 mol/mol). After the synthesis is complete, the peptide resin is washed with dichloromethane (DCM), diethyl ether and thoroughly air dried by applying vacuum suction to the syringe for 5 minutes. The dry resin is treated with a cleavage cocktail (trifluoroacetic acid (TFA): anisole: water: triisopropylsilane, 88:5:5:2 v/v) for 2 hours at room temperature to release the peptide from the solid support and remove all side-chain protecting groups. The resin is filtered off, washed twice with neat TFA, and the combined filtrates are treated with cold diethyl ether to precipitate the crude peptide. The peptide/ether suspension is then centrifuged at 4000 rpm to form a solid pellet, the supernatant is decanted, and the solid pellet is triturated with ether two additional times and dried in vacuo. The crude peptide is solubilized in 20% acetonitrile/water and purified by RP-HPLC on a C8 preparative column (Luna 21×250 mm, Phenomenex, Torrance, Calif.) with linear gradients of acetonitrile and water using three different buffer systems: 1) 0.1 M ammonium acetate in water, pH 5.0; 2) 0.1% TFA in water; and 3) 5% acetic acid in water. Subsequent lyophilization of the final main product pool yields the lyophilized peptide acetate salt. In a synthesis performed essentially as described above, the purity of Compound 1 is assessed using analytical RP-HPLC and found to be >97%. The molecular weight is determined by analytical electrospray MS. The molecular weight of Compound 1 is calculated to be 4535.0 Daltons while the observed deconvoluted averaged molecular weight was determined to be 4535.0 Daltons and the following ions were observed: 1512.3 (M+3H), 1134.3 (M+4H), 908 (M+5H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The Wang resin (0.3 -0.6 mmol/g, loading capacity) was loaded to peptide synthesis vessel, washed twice with 10 v of MDC, decanted the washings, added 10 v of MDC and kept for swelling for 1 h. Fmoc-Gly-OH (3.0 - 5.0 eq.) was dissolved in MDC, added minimum quantity of DMF to obtain clear solution and the mixture was transferred to reaction vessel. Added DIPC (3.0 - 6.0 eq.) followed by DMAP (0.01- 0.1 eq.) to the reaction vessel and stirred for 1.0? 3.0 h, at rt. Drained the reaction mass and washed the amino acid loaded resin twice with MDC followed by DMF. Capping of the unreacted functional sites were carried out using acetic anhydride and DIPEA. Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 15-25 percent piperidine in DMF two times for 5 and 10 min. followed by the resin was washed with 3-5*8 v 0.01? 0.1 M HOBt in DMF. The Fmoc-Arg(Pbf)-OH (2.0? 4.0 eq.), was coupled using coupling agents such as HBTU, COMU, DEPBT, and DIC, preferably DEPBT (2.0 - 4.0 eq.) and oxymapure, HOBt, preferably oxymapure (2.0? 4.0 eq.) and DIPEA, NMM, TMP, preferably DIPEA (5.0 -8.0 eq.) and MgCl2, ZnCl2, preferably MgCl2 (0.01? 0.1 eq) and DMF/NMP mixture as solvent. The reaction was performed in nitrogen atmosphere and r.t. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin with 3 x 10 v DMF |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The Wang resin (0.3 -0.6 mmol/g, loading capacity) was loaded to peptide synthesis vessel, washed twice with 10 v of MDC, decanted the washings, added 10 v of MDC and kept for swelling for 1 h. Fmoc-Gly-OH (3.0 - 5.0 eq.) was dissolved in MDC, added minimum quantity of DMF to obtain clear solution and the mixture was transferred to reaction vessel. Added DIPC (3.0 - 6.0 eq.) followed by DMAP (0.01- 0.1 eq.) to the reaction vessel and stirred for 1.0? 3.0 h, at rt. Drained the reaction mass and washed the amino acid loaded resin twice with MDC followed by DMF. Capping of the unreacted functional sites were carried out using acetic anhydride and DIPEA. Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 15-25 percent piperidine in DMF two times for 5 and 10 min. followed by the resin was washed with 3-5*8 v 0.01? 0.1 M HOBt in DMF. The Fmoc-Arg(Pbf)-OH (2.0? 4.0 eq.), was coupled using coupling agents such as HBTU, COMU, DEPBT, and DIC, preferably DEPBT (2.0 - 4.0 eq.) and oxymapure, HOBt, preferably oxymapure (2.0? 4.0 eq.) and DIPEA, NMM, TMP, preferably DIPEA (5.0 -8.0 eq.) and MgCl2, ZnCl2, preferably MgCl2 (0.01? 0.1 eq) and DMF/NMP mixture as solvent. The reaction was performed in nitrogen atmosphere and r.t. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin with 3 x 10 v DMF |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. In an analogous way, the other peptides listed in Table 3 were synthesized and characterized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0374) Deconvolution of the mass signals found under the peak with retention time 9.935 min revealed the peptide mass 4853.73 which is in line with the expected value of 4853.67. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
8% | (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 20% (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 2%-50% and purity exceeding 99% (see peptide characterization data) . (0242) For the synthesis of peptide 6 containing an i7-L-methyllysine modification we employed an on-resin i7-methylation protocol [22] which furnished peptide 6 in good yield (30%) 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 -yr% -Q ^ (0292) - -^H Q - (0293) (0294) Scheme 1. Synthetic protocol for the preparation of the biotinylated A20FMDV2 peptide variants. (0295) The results obtained from the binding assays (Table 2, see later) s... |
Yield | Reaction Conditions | Operation in experiment |
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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... |
Yield | Reaction Conditions | Operation in experiment |
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In many embodiments, Arg10-teixobactin and other homologues were synthesized by SPPS on 2-chlorotrityl chloride resin, followed by solution-phase macrolactamization to form the Arg10-Ile11 amide bond (FIG. 25). Fmoc protecting groups were used to construct all of the amide bonds and carried D-Thr8 through the entire synthesis without side chain protection. All homologues were prepared and studied as the trifluoroacetic acid (TFA) salts. The synthesis began by attaching Fmoc-Arg(Pbf)-OH to 2-chlorotrityl chloride resin. Residues 9 through 1 were then introduced by standard Fmoc-based SPPS using HCTU as the coupling reagent. D-Thr8 was introduced without a protecting group at the hydroxy position. No O-acylation of D-Thr8 was observed in the subsequent rounds of SPPS. D-Thr8 was then O-acylated with Fmoc-Ile-OH using DIC and DMAP. Fmoc-deprotection, followed by cleavage from the resin with 20% hexafluoroisopropanol (HFIP) in CH2Cl2 afforded the linear precursor. Macrolactamization with HBTU and HOBt, followed by global deprotection with trifluoroacetic acid (TFA) and RP-HPLC purification afforded Arg10-teixobactin. A series of homologues were also prepared using similar procedures. The details of the Arg10-teixobactin synthesis are described in the subsequent paragraphs. Resin Loading. 2-Chlorotrityl chloride resin (300 mg, 1.2 mmol/g) was added to a 10 mL Bio-Rad Poly-Prep chromatography column. The resin was suspended in dry CH2Cl2 (10 mL) and allowed to swell for 30 min. The resin was loaded with a solution of Fmoc-Arg(Pbf)-OH (117 mg, 0.18 mmol, 0.50 equiv) and 2,4,6-collidine (300 muL) in dry CH2Cl2 (5 mL). The suspension was agitated for 12 h. The solution was drained, and the resin was washed with dry CH2Cl2 (3×). A mixture of CH2Cl2/MeOH/DIPEA (17:2:1, 8 mL) was added to the resin and agitated for 1 h to cap any unreacted resin sites. The solution was drained, and the resin was washed with dry CH2Cl2 (3×). The resin loading was determined to be 0.09 mmol [0.29 mmol/g, 48% loading] through UV analysis of the Fmoc cleavage product. Peptide Coupling. The loaded resin was suspended in dry DMF and transferred to a solid-phase peptide synthesis reaction vessel for automated peptide coupling with Fmoc-protected amino acid building blocks. The linear peptide was synthesized through the following cycles: i. Fmoc deprotection with 20% (v/v) piperidine in dry DMF (3 mL) for 10 min, ii. resin washing with dry DMF (3×), iii. coupling of amino acid (0.36 mmol, 4 equiv) with HCTU (142 mg, 0.36 mmol, 4 equiv) in 20% (v/v) 2,4,6-collidine in dry DMF (3 mL) for 20 min, and iv. resin washing with dry DMF (6×). For D-to-L and L-to-D amino acid couplings, the reaction time in step iii was increased to 1 h. After completing the linear synthesis, the resin was transferred to a 10 mL Bio-Rad Poly-Prep chromatography column. The resin was then washed with dry DMF (3×) and dry CH2Cl2 (3×). Esterification. In a test tube, Fmoc-Ile-OH (303 mg, 0.90 mmol, 10 equiv) and diisopropylcarbodiimide (140 muL, 0.90 mmol, 10 equiv) were dissolved in dry CH2Cl2 (5 mL). The resulting solution was filtered through 0.20 mum nylon filter, and then 4-dimethylaminopyridine (11 mg, 0.09 mmol, 1 equiv) was added to the filtrate. The resulting solution was transferred to the resin and was gently agitated for 1 h. The solution was drained and the resin was washed with dry CH2Cl2 (3×) and DMF (3×). Fmoc Deprotection and Cleavage of the Linear from the Resin. The Fmoc protecting group on Ile11 was removed by adding 20% piperidine in dry DMF (5 mL) for 30 min. The solution was drained, and the resin was washed with dry DMF (3×) and then with dry CH2Cl2 (3×). To cleave the peptide, the resin was treated with 20% hexafluoroisopropanol in dry CH2Cl2 (6 mL) followed by gentle agitation for 1 h. The filtrate was collected in a round-bottomed flask. The resin was washed with a second aliquot of 20% hexafluoroisopropanol (6 mL) and then washed with dry CH2Cl2 (3×). The filtrates were combined and concentrated under reduced pressure to afford a clear oil. The oil was placed under vacuum (10 mTorr) to remove any residual solvents. |
Yield | Reaction Conditions | Operation in experiment |
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576 g | The active substance Lixisenatide is a polypeptide amide composed of 44 amino acids; acetate functions as counterion. In the one-letter code, the amino acid sequence of Lixisenatide is as follows: H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-l-E-W-L-K-N-G-G-P-S-S- G-A-P-P-S-K-K-K-K-K-K-N H2 The peptide chain was constructed by means of linear solid-phase synthesis, starting from the C-terminus, Lys-44. The method of synthesis is Fmoc solid-phase peptide synthesis, in which a Rink amide resin was used in order to obtain a peptide amide. The reactions were carried out in DMF at room temperature. Between the reactions, washing was carried out repeatedly, mostly with DMF, with one of the middle washing steps being carried out with isopropanol. The synthesis of Lixisenatide on the polymeric support can be broken down into the following steps: ? Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin ? Capping of the un reacted amino group ? Cleavage of the temporary protecting group Fmoc ? Coupling of the further Fmoc-amino acids or Fmoc-dipeptides ? Capping of the un reacted amino group ? Final Fmoc cleavage ? Cleavage of Lixisenatide from the resin and simultaneous removal of the side chain protecting groups The synthesis cycle is illustrated in Figure 1. 1.1 Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin Before the synthesis began, the Rink amide resin was swollen in DMF. The swelling was carried out for 2-15 h. Subsequently, the temporary protecting group Fmoc was cleaved from the Rink amide resin using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. The coupling of the first Fmoc-amino acid, Fmoc-Lys(Boc)-OFI, was carried out in an excess of 2.4 eq, in order to load the resin. HOBt hydrate, HBTU and DIPEA served as coupling reagents. The coupling time was 60-120 min. In order to completely load the Rink resin with Fmoc-Lys(Boc)-OFI, a further loading was carried out with the coupling reagents FIOBt hydrate and DIC. The coupling time was 6-18 h. The mixture was stirred while step 1.1 was carried out. The capping was subsequently carried out. 1.2 Capping of the unreacted amino group The consequence of incomplete loading of the resin is that as yet unreacted amino groups are found on the resin. These were inactivated, and hence made unavailable for further coupling, by adding a mixture of acetic anhydride/DIPEA/DMF (10:5:85). The capping mixture remained on the resin for 20 minutes while stirring. The remaining free amino group is acylated. Subsequently, the resin was washed repeatedly with DMF and once with isopropanol. A capping method according to the invention at least at 5 positions of a Lixisenatide synthesis is described in examples 4 and 5. 1.3. Cleavage of the temporary protecting group Fmoc The temporary protecting group Fmoc was cleaved using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. 1.4 Coupling of the further Fmoc-amino acids or Fmoc-dipeptides The next Fmoc-amino acid was coupled to the deprotected amino group on the resin. The coupling was carried out in DMF at different equivalents. The coupling times were between 2 h and 18 h. HOBt/DIC, and also HBTU/DIPEA, were used as coupling reagents. The following derivatives were used as Fmoc-amino acids: ? Fmoc-Lys(Boc)-OFI ? Fmoc-Ser(tBu)-OFI ? Fmoc-Pro-OFI ? Fmoc-Ala-OH x FI2O ? Fmoc-Gly-OH ? Fmoc-Asn(Trt)-OH ? Fmoc-Leu-OH ? Fmoc-Trp(Boc)-OH ? Fmoc-Glu(OtBu)-OFI x H2O ? Fmoc-lle-OFI ? Fmoc-Phe-OFI ? Fmoc-Arg(Pbf)-OFI ? Fmoc-Val-OH ? Fmoc-Met-OH ? Fmoc-Gln(Trt)-OFI ? Fmoc-Asp(OtBu)-OH ? Fmoc-Thr(tBu)-OH ? Fmoc-His(Trt)-OH Alternatively, it was also possible to use Fmoc-dipeptides (method according to the invention): · <strong>[129223-22-9]Fmoc-Pro-Pro-OH</strong> (CAS 129223-22-9) ? Fmoc-Ala-Pro-OH (CAS 186023-44-9) ? Fmoc-Ser(tBu)-Gly-OH (CAS 113247-80-6) ? Fmoc-Gly-Pro-OH (CAS 212651-48-4) ? Fmoc-Gly-Gly-OH (CAS 35665-38-4) · Fmoc-Asn(Trt)-Gly-OH (from Bachem B-3630) ? Fmoc-Glu(OtBu)-Gly-OH (CAS 866044-63-5) ? Fmoc-His(T rt)-Gly-OH If the coupling was found to be incomplete according to the Kaiser test (E. Kaiser et al, Anal. Biochem. 34, 1970, 595), further coupling was possible. For this purpose, the Fmoc-amino acid was coupled again, together with HBTU/DIPEA/HOBt hydrate. 1.5 Capping of the unreacted amino group See description under point 1.2. 1.6 Final Fmoc cleavage The final Fmoc cleavage was carried out as described under point 1.3. The resin was finally washed again with diisopropyl ether and dried under reduced pressure. 1.7 Cleavage of Lixisenatide from the resin and simultaneous removal of the side chain protecting groups The cleavage of Lixisenatide from the Rink resin was carried out as described in example 6. 1.8 Synthesis of ... | |
576 g | The active substance Lixisenatide is a polypeptide amide composed of 44 amino acids; acetate functions as counterion. In the one-letter code, the amino acid sequence of Lixisenatide is as follows: H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-l-E-W-L-K-N-G-G-P-S-S- G-A-P-P-S-K-K-K-K-K-K-N H2 The peptide chain was constructed by means of linear solid-phase synthesis, starting from the C-terminus, Lys-44. The method of synthesis is Fmoc solid-phase peptide synthesis, in which a Rink amide resin was used in order to obtain a peptide amide. The reactions were carried out in DMF at room temperature. Between the reactions, washing was carried out repeatedly, mostly with DMF, with one of the middle washing steps being carried out with isopropanol. The synthesis of Lixisenatide on the polymeric support can be broken down into the following steps: ? Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin ? Capping of the un reacted amino group ? Cleavage of the temporary protecting group Fmoc ? Coupling of the further Fmoc-amino acids or Fmoc-dipeptides ? Capping of the un reacted amino group ? Final Fmoc cleavage ? Cleavage of Lixisenatide from the resin and simultaneous removal of the side chain protecting groups The synthesis cycle is illustrated in Figure 1. 1.1 Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin Before the synthesis began, the Rink amide resin was swollen in DMF. The swelling was carried out for 2-15 h. Subsequently, the temporary protecting group Fmoc was cleaved from the Rink amide resin using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. The coupling of the first Fmoc-amino acid, Fmoc-Lys(Boc)-OFI, was carried out in an excess of 2.4 eq, in order to load the resin. HOBt hydrate, HBTU and DIPEA served as coupling reagents. The coupling time was 60-120 min. In order to completely load the Rink resin with Fmoc-Lys(Boc)-OFI, a further loading was carried out with the coupling reagents FIOBt hydrate and DIC. The coupling time was 6-18 h. The mixture was stirred while step 1.1 was carried out. The capping was subsequently carried out. 1.2 Capping of the unreacted amino group The consequence of incomplete loading of the resin is that as yet unreacted amino groups are found on the resin. These were inactivated, and hence made unavailable for further coupling, by adding a mixture of acetic anhydride/DIPEA/DMF (10:5:85). The capping mixture remained on the resin for 20 minutes while stirring. The remaining free amino group is acylated. Subsequently, the resin was washed repeatedly with DMF and once with isopropanol. A capping method according to the invention at least at 5 positions of a Lixisenatide synthesis is described in examples 4 and 5. 1.3. Cleavage of the temporary protecting group Fmoc The temporary protecting group Fmoc was cleaved using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. 1.4 Coupling of the further Fmoc-amino acids or Fmoc-dipeptides The next Fmoc-amino acid was coupled to the deprotected amino group on the resin. The coupling was carried out in DMF at different equivalents. The coupling times were between 2 h and 18 h. HOBt/DIC, and also HBTU/DIPEA, were used as coupling reagents. The following derivatives were used as Fmoc-amino acids: ? Fmoc-Lys(Boc)-OFI ? Fmoc-Ser(tBu)-OFI ? Fmoc-Pro-OFI ? Fmoc-Ala-OH x FI2O ? Fmoc-Gly-OH ? Fmoc-Asn(Trt)-OH ? Fmoc-Leu-OH ? Fmoc-Trp(Boc)-OH ? Fmoc-Glu(OtBu)-OFI x H2O ? Fmoc-lle-OFI ? Fmoc-Phe-OFI ? Fmoc-Arg(Pbf)-OFI ? Fmoc-Val-OH ? Fmoc-Met-OH ? Fmoc-Gln(Trt)-OFI ? Fmoc-Asp(OtBu)-OH ? Fmoc-Thr(tBu)-OH ? Fmoc-His(Trt)-OH Alternatively, it was also possible to use Fmoc-dipeptides (method according to the invention): · <strong>[129223-22-9]Fmoc-Pro-Pro-OH</strong> (CAS 129223-22-9) ? Fmoc-Ala-Pro-OH (CAS 186023-44-9) ? Fmoc-Ser(tBu)-Gly-OH (CAS 113247-80-6) ? Fmoc-Gly-Pro-OH (CAS 212651-48-4) ? Fmoc-Gly-Gly-OH (CAS 35665-38-4) · Fmoc-Asn(Trt)-Gly-OH (from Bachem B-3630) ? Fmoc-Glu(OtBu)-Gly-OH (CAS 866044-63-5) ? Fmoc-His(T rt)-Gly-OH If the coupling was found to be incomplete according to the Kaiser test (E. Kaiser et al, Anal. Biochem. 34, 1970, 595), further coupling was possible. For this purpose, the Fmoc-amino acid was coupled again, together with HBTU/DIPEA/HOBt hydrate. 1.5 Capping of the unreacted amino group See description under point 1.2. 1.6 Final Fmoc cleavage The final Fmoc cleavage was carried out as described under point 1.3. The resin was finally washed again with diisopropyl ether and dried under reduced pressure. 1.7 Cleavage of Lixisenatide from the resin and simultaneous removal of the side chain protecting groups The cleavage of Lixisenatide from the Rink resin was carried out as described in example 6. 1.8 Synthesis of ... |
Yield | Reaction Conditions | Operation in experiment |
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General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General method for preparation of the peptide and introduction of the substituent The preparation of the peptide was carried out with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.). The Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc- Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. A Wang resin preloaded with an amino acid such as Fmoc-Phe-Wang resin or the like was used (for derivatives containing Nphe, 4-CI-Nphe, or alpha-Me-Phe, a 2-Chlorotrityl resin was used). Fmoc- deprotection was achieved with 20% piperidine in NMP. Peptide couplings were performed by using DIC/Oxyma Pure with collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a molar excess of 3-10 fold) was added to the resin followed by the same molar equivalent of DIC (3 M in NMP) followed by collidine (3 M in NMP). Fmoc-Cys(Trt)-OH was used for peptides prepared for methylene bridge introduction according to method A, while Fmoc-Cys(Mmt)-OH was used for peptides prepared for methylene bridge introduction according to method B. |
Tags: 132327-80-1 synthesis path| 132327-80-1 SDS| 132327-80-1 COA| 132327-80-1 purity| 132327-80-1 application| 132327-80-1 NMR| 132327-80-1 COA| 132327-80-1 structure
[ 198544-94-4 ]
(R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexanoic acid
Similarity: 0.96
Precautionary Statements-General | |
Code | Phrase |
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P283 | Wear fire/flame resistant/retardant clothing. |
P284 | Wear respiratory protection. |
P285 | In case of inadequate ventilation wear respiratory protection. |
P231 + P232 | Handle under inert gas. Protect from moisture. |
P235 + P410 | Keep cool. Protect from sunlight. |
Response | |
Code | Phrase |
P301 | IF SWALLOWED: |
P304 | IF INHALED: |
P305 | IF IN EYES: |
P306 | IF ON CLOTHING: |
P307 | IF exposed: |
P308 | IF exposed or concerned: |
P309 | IF exposed or if you feel unwell: |
P310 | Immediately call a POISON CENTER or doctor/physician. |
P311 | Call a POISON CENTER or doctor/physician. |
P312 | Call a POISON CENTER or doctor/physician if you feel unwell. |
P313 | Get medical advice/attention. |
P314 | Get medical advice/attention if you feel unwell. |
P315 | Get immediate medical advice/attention. |
P320 | |
P302 + P352 | IF ON SKIN: wash with plenty of soap and water. |
P321 | |
P322 | |
P330 | Rinse mouth. |
P331 | Do NOT induce vomiting. |
P332 | IF SKIN irritation occurs: |
P333 | If skin irritation or rash occurs: |
P334 | Immerse in cool water/wrap n wet bandages. |
P335 | Brush off loose particles from skin. |
P336 | Thaw frosted parts with lukewarm water. Do not rub affected area. |
P337 | If eye irritation persists: |
P338 | Remove contact lenses, if present and easy to do. Continue rinsing. |
P340 | Remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P341 | If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P342 | If experiencing respiratory symptoms: |
P350 | Gently wash with plenty of soap and water. |
P351 | Rinse cautiously with water for several minutes. |
P352 | Wash with plenty of soap and water. |
P353 | Rinse skin with water/shower. |
P360 | Rinse immediately contaminated clothing and skin with plenty of water before removing clothes. |
P361 | Remove/Take off immediately all contaminated clothing. |
P362 | Take off contaminated clothing and wash before reuse. |
P363 | Wash contaminated clothing before reuse. |
P370 | In case of fire: |
P371 | In case of major fire and large quantities: |
P372 | Explosion risk in case of fire. |
P373 | DO NOT fight fire when fire reaches explosives. |
P374 | Fight fire with normal precautions from a reasonable distance. |
P376 | Stop leak if safe to do so. Oxidising gases (section 2.4) 1 |
P377 | Leaking gas fire: Do not extinguish, unless leak can be stopped safely. |
P378 | |
P380 | Evacuate area. |
P381 | Eliminate all ignition sources if safe to do so. |
P390 | Absorb spillage to prevent material damage. |
P391 | Collect spillage. Hazardous to the aquatic environment |
P301 + P310 | IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician. |
P301 + P312 | IF SWALLOWED: call a POISON CENTER or doctor/physician IF you feel unwell. |
P301 + P330 + P331 | IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. |
P302 + P334 | IF ON SKIN: Immerse in cool water/wrap in wet bandages. |
P302 + P350 | IF ON SKIN: Gently wash with plenty of soap and water. |
P303 + P361 + P353 | IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower. |
P304 + P312 | IF INHALED: Call a POISON CENTER or doctor/physician if you feel unwell. |
P304 + P340 | IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for breathing. |
P304 + P341 | IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P305 + P351 + P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. |
P306 + P360 | IF ON CLOTHING: Rinse Immediately contaminated CLOTHING and SKIN with plenty of water before removing clothes. |
P307 + P311 | IF exposed: call a POISON CENTER or doctor/physician. |
P308 + P313 | IF exposed or concerned: Get medical advice/attention. |
P309 + P311 | IF exposed or if you feel unwell: call a POISON CENTER or doctor/physician. |
P332 + P313 | IF SKIN irritation occurs: Get medical advice/attention. |
P333 + P313 | IF SKIN irritation or rash occurs: Get medical advice/attention. |
P335 + P334 | Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages. |
P337 + P313 | IF eye irritation persists: Get medical advice/attention. |
P342 + P311 | IF experiencing respiratory symptoms: call a POISON CENTER or doctor/physician. |
P370 + P376 | In case of fire: Stop leak if safe to Do so. |
P370 + P378 | In case of fire: |
P370 + P380 | In case of fire: Evacuate area. |
P370 + P380 + P375 | In case of fire: Evacuate area. Fight fire remotely due to the risk of explosion. |
P371 + P380 + P375 | In case of major fire and large quantities: Evacuate area. Fight fire remotely due to the risk of explosion. |
Storage | |
Code | Phrase |
P401 | |
P402 | Store in a dry place. |
P403 | Store in a well-ventilated place. |
P404 | Store in a closed container. |
P405 | Store locked up. |
P406 | Store in corrosive resistant/ container with a resistant inner liner. |
P407 | Maintain air gap between stacks/pallets. |
P410 | Protect from sunlight. |
P411 | |
P412 | Do not expose to temperatures exceeding 50 oC/ 122 oF. |
P413 | |
P420 | Store away from other materials. |
P422 | |
P402 + P404 | Store in a dry place. Store in a closed container. |
P403 + P233 | Store in a well-ventilated place. Keep container tightly closed. |
P403 + P235 | Store in a well-ventilated place. Keep cool. |
P410 + P403 | Protect from sunlight. Store in a well-ventilated place. |
P410 + P412 | Protect from sunlight. Do not expose to temperatures exceeding 50 oC/122oF. |
P411 + P235 | Keep cool. |
Disposal | |
Code | Phrase |
P501 | Dispose of contents/container to ... |
P502 | Refer to manufacturer/supplier for information on recovery/recycling |
Physical hazards | |
Code | Phrase |
H200 | Unstable explosive |
H201 | Explosive; mass explosion hazard |
H202 | Explosive; severe projection hazard |
H203 | Explosive; fire, blast or projection hazard |
H204 | Fire or projection hazard |
H205 | May mass explode in fire |
H220 | Extremely flammable gas |
H221 | Flammable gas |
H222 | Extremely flammable aerosol |
H223 | Flammable aerosol |
H224 | Extremely flammable liquid and vapour |
H225 | Highly flammable liquid and vapour |
H226 | Flammable liquid and vapour |
H227 | Combustible liquid |
H228 | Flammable solid |
H229 | Pressurized container: may burst if heated |
H230 | May react explosively even in the absence of air |
H231 | May react explosively even in the absence of air at elevated pressure and/or temperature |
H240 | Heating may cause an explosion |
H241 | Heating may cause a fire or explosion |
H242 | Heating may cause a fire |
H250 | Catches fire spontaneously if exposed to air |
H251 | Self-heating; may catch fire |
H252 | Self-heating in large quantities; may catch fire |
H260 | In contact with water releases flammable gases which may ignite spontaneously |
H261 | In contact with water releases flammable gas |
H270 | May cause or intensify fire; oxidizer |
H271 | May cause fire or explosion; strong oxidizer |
H272 | May intensify fire; oxidizer |
H280 | Contains gas under pressure; may explode if heated |
H281 | Contains refrigerated gas; may cause cryogenic burns or injury |
H290 | May be corrosive to metals |
Health hazards | |
Code | Phrase |
H300 | Fatal if swallowed |
H301 | Toxic if swallowed |
H302 | Harmful if swallowed |
H303 | May be harmful if swallowed |
H304 | May be fatal if swallowed and enters airways |
H305 | May be harmful if swallowed and enters airways |
H310 | Fatal in contact with skin |
H311 | Toxic in contact with skin |
H312 | Harmful in contact with skin |
H313 | May be harmful in contact with skin |
H314 | Causes severe skin burns and eye damage |
H315 | Causes skin irritation |
H316 | Causes mild skin irritation |
H317 | May cause an allergic skin reaction |
H318 | Causes serious eye damage |
H319 | Causes serious eye irritation |
H320 | Causes eye irritation |
H330 | Fatal if inhaled |
H331 | Toxic if inhaled |
H332 | Harmful if inhaled |
H333 | May be harmful if inhaled |
H334 | May cause allergy or asthma symptoms or breathing difficulties if inhaled |
H335 | May cause respiratory irritation |
H336 | May cause drowsiness or dizziness |
H340 | May cause genetic defects |
H341 | Suspected of causing genetic defects |
H350 | May cause cancer |
H351 | Suspected of causing cancer |
H360 | May damage fertility or the unborn child |
H361 | Suspected of damaging fertility or the unborn child |
H361d | Suspected of damaging the unborn child |
H362 | May cause harm to breast-fed children |
H370 | Causes damage to organs |
H371 | May cause damage to organs |
H372 | Causes damage to organs through prolonged or repeated exposure |
H373 | May cause damage to organs through prolonged or repeated exposure |
Environmental hazards | |
Code | Phrase |
H400 | Very toxic to aquatic life |
H401 | Toxic to aquatic life |
H402 | Harmful to aquatic life |
H410 | Very toxic to aquatic life with long-lasting effects |
H411 | Toxic to aquatic life with long-lasting effects |
H412 | Harmful to aquatic life with long-lasting effects |
H413 | May cause long-lasting harmful effects to aquatic life |
H420 | Harms public health and the environment by destroying ozone in the upper atmosphere |
Sorry,this product has been discontinued.
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