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CAS No. : | 166108-71-0 | MDL No. : | MFCD01321015 |
Formula : | C21H23NO6 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | XQPYRJIMPDBGRW-UHFFFAOYSA-N |
M.W : | 385.41 | Pubchem ID : | 2756092 |
Synonyms : |
Fmoc-NH-PEG2-CH2COOH
|
Num. heavy atoms : | 28 |
Num. arom. heavy atoms : | 12 |
Fraction Csp3 : | 0.33 |
Num. rotatable bonds : | 12 |
Num. H-bond acceptors : | 6.0 |
Num. H-bond donors : | 2.0 |
Molar Refractivity : | 101.76 |
TPSA : | 94.09 Ų |
GI absorption : | High |
BBB permeant : | No |
P-gp substrate : | Yes |
CYP1A2 inhibitor : | No |
CYP2C19 inhibitor : | Yes |
CYP2C9 inhibitor : | Yes |
CYP2D6 inhibitor : | No |
CYP3A4 inhibitor : | Yes |
Log Kp (skin permeation) : | -6.98 cm/s |
Log Po/w (iLOGP) : | 3.04 |
Log Po/w (XLOGP3) : | 2.36 |
Log Po/w (WLOGP) : | 2.64 |
Log Po/w (MLOGP) : | 1.39 |
Log Po/w (SILICOS-IT) : | 3.0 |
Consensus Log Po/w : | 2.49 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 1.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.56 |
Log S (ESOL) : | -3.24 |
Solubility : | 0.221 mg/ml ; 0.000573 mol/l |
Class : | Soluble |
Log S (Ali) : | -3.98 |
Solubility : | 0.0407 mg/ml ; 0.000106 mol/l |
Class : | Soluble |
Log S (SILICOS-IT) : | -5.91 |
Solubility : | 0.00047 mg/ml ; 0.00000122 mol/l |
Class : | Moderately soluble |
PAINS : | 0.0 alert |
Brenk : | 0.0 alert |
Leadlikeness : | 2.0 |
Synthetic accessibility : | 3.93 |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P261-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | H302-H315-H319-H335 | 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 |
---|---|---|
200 g | With hydrogenchloride; In ethanol; water;pH 9.0; | In a 3000L three-necked flask equipped with mechanical stirring, condenser and constant pressure dropping funnel,330 g of crude product (Intermediate 3) was dissolved in 1000 ml of ethanol, and 350 g of 30% NaOH solution was added dropwise.After the completion of the dropwise addition, the mixture was heated to reflux, and monitored by TLC, the intermediate 3 was completely disappeared, and the temperature was lowered. The pH was adjusted to 9 with hydrochloric acid, and a solution of 337 g of Fmoc-Osu in 1000 ml of ethanol was added dropwise.If the pH has dropped, add sodium bicarbonate and keep it at around 8.TLC was monitored until Intermediate 4 disappeared. The pH was adjusted to 1-2 with hydrochloric acid, the ethanol was distilled off, 1000 ml of water was added, and the mixture was extracted three times with ethyl acetate 500*3, and washed twice with saturated brine.The ethyl acetate was concentrated to give 285 g of crude material.Recrystallization from 1000 ml of ethyl acetate gave 200 g of product[2-[1-(Fmoc-Amino)ethoxy]ethoxy]acetic acid, yield 51.9%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With PS-trisamine resin; In N,N-dimethyl-formamide; | The title compound was prepared by coupling of 4-[3-(2H-tetrazol-5-yl)carbazol-9- ylmethyl]benzoic acid 2,5-dioxopyrrolidin-1 -yl ester with [2-(2-aminoethoxy)ethoxy]acetic acid (prepared from [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid by treatment with PS-Trisamine resin in DMF). HPLC-MS (Method C): m/z: 515 (M+1 ); Rt = 3.10 min. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Synthesis of compound 10 [] 10 was obtained according to the standard solid-phase synthesis protocol. The amino acids Fmoc-Dpr(Boc)-OH, Fmoc-Dpr(Fmoc)-OH, Fmoc-Dpr(Fmoc)-OH, Fmoc-Ado-OH, and Fmoc-Dpr(Fmoc)-OH were coupled to TentaGel Sieber amide resin. After final finoc removal the resin was agitated with 5 eq maleimidopropionic acid and 5 eq DIC in relation to amino groups in DMF for 30 min. 10 was cleaved from resin with TFA/TES/water 95/3/2 (v/v/v). After evaporation of solvent, product 10 was purified by RP-HPLC. MS: [M+H]+ = 2494.6 (MW calculated = 2495.4 g/mol) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
37.9% | With 1-hydroxy-7-aza-benzotriazole; trifluoroacetic acid; In water; acetonitrile; | Synthesis of the (S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oic acid compound with 2,2,2-trifluoroacetic acid (1:1) 1-Chlorotrytyl chloride resin (1.55 mmol/g) (0.500 g, 0.775 mmol) in 100mL glassware was swollen in DCM (20 ml) for 30 min and it was drained. A suspension of 2-(aminooxy)acetic acid hemihydrochloride (0.338 g, 3.10 mmol) and DIPEA (1.354 ml, 7.75 mmol) in NMP (7 ml)/DCM (4 ml) was added to the resin, which was shaken for 5 h. Solvent was drained, resin was rinsed with DCM/MeOH/DIPEA (17/2/1, 40mL), DCM (50mL), NMP (50 mL) DCM (50mL) respectively, resulting resin was dried with KOH/NaOH overnight. Resin (0.775 mmol) in 100 mL glassware was swollen in DCM (20 ml) for 30 min and it was drained. Into a suspension of (9H-fluoren-9-yl)methyl 2-aminoethylcarbamate hydrochloride (0.081 g, 0.775 mmol), HOAt (0.422 g, 3.10 mmol) and DIPEA (1.354 ml, 7.75 mmol) in NMP (8 ml) was added HBTU (1.176 g, 3.10 mmol) in NMP (2.5 ml), which was shaken for 2 h at RT, solvent was drained, resin was rinsed with NMP (10mL), DCM (10 mL) respectively. The resulting resin was dried overnight. Resin (0.775 mmol) was charged into reaction vassel. 10mL of 20% PIPERIDINE/NMP was added into resin, which suspension was agitated at RT for 5 min. After solvent was drained, additional 10 mL of 20% PIPERIDINE/NMP was added and agitated for 20 min at RT. Solution of HOAt (0.316 g, 2.325 mmol) and 1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-oic acid (0.896 g, 2.325 mmol) in NMP (8 ml) was added into resin and DIC (0.362 ml, 2.325 mmol) in NMP (1 ml) was added. Reaction mixture was agitated for 2 h at RT. Resin was filtered off and rinsed with NMP (10 ml) four times. The resulting resin was dried overnight. Resin (0.775 mmol) was charged into reaction vassel. 10 mL of 20% PIPERIDINE/NMP was added into resin, which suspension was agitated at RT for 5 min. After solvent was drained, additional 10 mL of 20% PIPERIDINE/NMP was added and agitated for 20 min at RT. Solution of HOAt (0.316 g, 2.325 mmol) and Fmoc-Glu-OtBu (0.989 g, 2.325 mmol) in NMP (8 ml) was added into resin and DIC (0.362 ml, 2.325 mmol) in NMP (2.00 ml) was added. The reaction mixture was agitated for 2 h at RT. Resin was filtered off and rinsed with NMP (10 ml) four times. The resulting resin was dried overnight. Resin (0.775 mmol) was charged into reaction vassel. 10 mL of 20% PIPERIDINE/NMP (0.775 mmol) was added into resin, which suspension was agitated at RT for 5 min. after solvent was drained, additional 10 mL of 20% PIPERIDINE/NMP (0.775 mmol) (0.775 mmol) was added and agitated for 20 min at RT. Solution of <strong>[843666-40-0]18-tert-butoxy-18-oxooctadecanoic acid</strong> (0.862 g, 2.325 mmol) and HOAt (0.316 g, 2.325 mmol) in NMP (8 ml) was added into resin and DIC (0.362 ml, 2.325 mmol) in NMP (2.00 ml) was added. The reaction mixture was agitated for 4 h at RT. Resin was filtered off and rinsed with NMP (10 ml) four times. The resulting resin was dried overnight. Resin (0.775 mmol) was treated with 20 mL of cleavage cocktail (TFA/TIPS/water=95/2.5/2.5) for 1.5 h at RT. Resin was removed by filtration and rinsed with TFA. The filtrate was concentrated in vacuo. RP-HPLC with C18 column eluting 15-50% MeCN/water with 0.1% TFA gave (S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oic acid with 2,2,2-trifluoroacetic acid (1:1) (207 mg, 0.294 mmol, 37.9% yield). HRMS (method D) [M+1]; 704.4459 (observed), 704.4486 (expected). Retension time; 2.63 min. The polypeptides of Examples 1-66 can be purified and isolated as described supra and/or by a combination of conventional purification techniques such as solvent extraction, column chromatography, liquid chromatography and recrystallization. Where the polypeptide isolated in the above Examples is a free compound, it can be converted to a suitable salt by the known method. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
86% | 10541] 2-Chlorotrityl resin 100-200 mesh (42.6 g, 42.6 mmol) was lefi to swell in dry dichloromethane (205 mE) for 20 mm. A solution of {2-[2-(9H-fluoren-9-ylmethoxycarbo- nylamino)-ethoxy]-ethoxy}-acetic acid (13.7 g, 35.5 mmol) and N,N-diisopropylethylamine (23.5 mE, 135 mmol) in dry dichloromethane (30 mE) was added to resin and the mixture was shaken for 3 irs. Resin was filtered and treated with a solution of N,N-diisopropylethylamine (12.4 mE, 70.9 mmol) in methanol/dichloromethane mixture (4:1, 250 mE, 2x5 mm). Then resin was washed with N,N-dimethylformamide (2x1 50 mE), dichloromethane (3x1 50 mE) and N,Ndimethylformamide (3x150 mE). Fmoc group was removed by treatment with 20% piperidine in dimethylformamide (1x5 mi 1x30 mi 2x150 mE). Resin was washed with N,N-dimethylformamide (3x 150 mE), 2-propanol (2x1 50 mE) and dichloromethane (200 mE, 2x150 mE). Solution of {2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (20.5 g, 53.2 mmol), O-(6-chloro-benzo- triazol-1 -yl)-N,N,N?,N?-tetramethyluronium tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and N,Ndiisopropylethylamine (16.7 mE, 95.7 mmol) in N,Ndimethylformamide (100 mE) and dichloromethane (50 mE) was added to resin and mixture was shaken for 1 hr. Resin was filtered and washed with N,N-dimethylformamide (2x1 50 mE), dichloromethane (3x 150 mE) and N,N-dimethylformamide (155 mE). Fmoc group was removed by treatment with 20% piperidine in dimethylformamide (1x5 mi 1x30 mm, 2x1 SOmE). Resin was washed with N,N-dimethylformamide (3x 150 mE), 2-propanol (2x 150 mE) and dichioromethane (200 mE, 2x150 mE). Solution of Fmoc-Glu-OtBu (22.6 g, 53.2 mmol), O-(6-chloro-benzotriazol-1 -yl)-N,N,N?,N?-tetramethyluronium tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and N,N-diisopropylethylamine (16.7 mE, 95.7ethylamine (16.7 mE, 95.7 mmol) in N,Ndimethylformamide/dichloromethane mixture (1:4, 200 mE) was added to resin. Resin was shaken for 2 irs, filtered and washed with N,N-dimethylformamide (3x150 mE), dichloromethane (2x 150 mE), methanol (2x1 50 mE) and dichloromethane (300 mE, 6x 150 mE). The product was cleaved from resin by treatment with 2,2,2-trifluoethanol (200 mE) for 19 irs. Resin was filtered off and washed with dichloromethane (2x1 50 mE), 2-propanol/dichloromethane mixture (1:1, 2x150 mE), 2-propanol (150 mE) and dichloromethane (2x150 mE). Solutions were combined; solvent evaporated and crude product was purified by flash column chromatography (Silicagel 60, 0.040-0.060 mm; eluent:dichioromethane/methanol 1:0-9:1). Pure product was dried in vacuo and obtained as yellow oil. [0542] Yield: 25.85 g (86%) .[0543] RF(5i02, chioroformlmethanol 85:15): 0.25. [0544] ?H NMR spectrum (300 MHz, CDC13, H): 7.38 (bs,1H); 7.08 (bs, 1H); 6.61 (d, J=7.5 Hz, 1H); 4.43 (m, 1H); 4.15 (s, 2H); 4.01 (s, 2H); 3.78-3.39 (m, 16H); 2.31 (t, J=6.9 Hz, 2H); 2.27-2.09 (m, 5H); 2.01-1.84 (m, 1H); 1.69-1.50 (m, 4H); 1.46 (s, 9H); 1.43 (s, 9H); 1.24 (bs, 24H). [0545] EC-MS purity: 100%.10546] EC-MS Rt (Sunfire 4.6 mmx 100 mm, acetonitrile/water 60:40 to 0:100+0.1% FA): 7.89 mi EC-MS mlz: 846.6 (M+H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SPPS Method B, starting with low-load Fmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OH was used in position 26, and Boc-His(trt)-OH was used in position 7. The Mtt was removedwith HFIP, and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech),Fmoc-Glu-OtBu, and 3-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester were coupled using a double couplingmethod on the Liberty Peptide synthesiser.UPLC (method 04_A4_1): 10.01 minUPLC (method 08_B4_1): 8.81 minLCMS4: m/z = 978.5 (M+5H)5+, 1222.8 (M+4H)4+, 1630.1 (M+3H)3+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling.Preparation method: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wang resin.Fmoc-Lys(Mtt)-OH was used in position 26, and Boc-His(trt)-OH was used in position 7. The Mtt was removed withHFIP, and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech)and 4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared as described in Example 25, step 2 of WO2006/082204) were coupled using a double coupling method on the Liberty Peptide synthesiserUPLC (method 04_A3_1): 10.51 minLCMS4: m/z = 1085.2 (M+4H)4+, 1447.3 (M+3H)3+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OHwas used in position 26, and Boc-His(Trt)-OH was used in position 7. The Mtt was removed with HFIP, and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech), Fmoc-Glu-OtBu,and 4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared as described in Example 25, step 2 of WO2006/082204) were coupled using a double coupling method on the Liberty Peptide synthesiser.UPLC (method 04_A3_1): 7.19 minLCMS4: m/z = 978.5 (M+5H)5+, 1222.8 (M+4H)4+ 1630.1 (M+3H)3+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OHwas used in position 26, and Boc-His(Trt)-OH was used in position 7. The Mtt was removed with HFIP, and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech), Fmoc-Glu-OtBuand 4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared as described in Example 25, step 2 of WO2006/082204) were coupled using SPPS method D.UPLC (method 08_B4_1): Rt = 8.8 minUPLC (method 04_A3_1): Rt = 9.6 minLCMS4: 4598.0Calculated MW = 4598.2 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SPPS method BLCMS4: Rt = 2.12 min, m/z: 4916.0UPLC (method: 08_B2_1): Rt = 12.59 minUPLC (method: 04_A3_1): Rt = 10.57 min |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SSPS method BUPLC (method:08_B2_1): Rt = 13.193 minUPLC (method:05_B5_1): Rt = 6.685 minLCMS4: m/z: 4887; m/3:1630; m/4:1222; m/5:978 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SPPS Method B. The 3-(11-carboxy-undecyloxy)-benzoic acid tert-butyl ester was preparedin similar fashion as described for 3-(15-carboxy-pentadecyloxy)-benzoic acid tert-butyl ester, empoying 12-bromododecanoicacid. The final product was characterised by analytical UPLC and LC-MS with the exception that an aceticanhydride capping step was performed after the coupling of the following amino acids: Trp31, Ala25, Tyr19, Phe12 andAib8 (2© min, 65°C with 1 N Acetic acid anhydride in NMP)UPLC (method 08_B4_1): Rt = 9.449 minLCMS4: Rt = 2.37 min, m/z = m/z: 1011.88(m/4); 1264.32(m/3); 4942.24Calculated MW = 4944.608 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OH was usedin position 26, and Boc-His(trt)-OH was used in position 7. The Mtt was removed with HFIP manually, and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech), Fmoc-Glu-OtBu andtetradecanedioc were coupled using a double coupling method on the Liberty Peptide synthesiser. The theoreticalmolecular mass was confirmed by MALDI-MS.UPLC (method 08_B4_1): Rt = 8.6 minUPLC (method 04_A3_1): Rt = 9.7 minMALDI-MS: 4788 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation: SPPS method B, starting with low-load Fmoc-Lys(Mtt)-Wang resin. Fmoc-Lys(Mtt)-OH was used in position 26, and Boc-His(trt)-OH was used in position 7. The Mtt was removed with HFIP manually, and 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commercially available from Iris Biotech), Fmoc-Glu-OtBu andtetradecanedioc were coupled using a double coupling method on the Liberty Peptide synthesiser. The theoreticalmolecular mass was confirmed by MALDI-MS.UPLC (method 08_B4_1): Rt = 8.8 minUPLC (method 04_A3_1): Rt = 10 minMALDI-MS: 4787 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SPPS method BLCMS4: Rt: 1.93 min, m/z: 4832.4; M/4: 1208.5; M/3: 1611.0UPLC (method 09_B4_1): Rt = 8.10 minUPLC (method 04_A3_1): Rt = 8.15 minUPLC (method 05_B5_1): Rt = 5.30 min |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preperation method: SSPS method B. 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commerciallyavailable from Iris Biotech), Fmoc-Glu-OtBu, and 5-(12-Carboxy-dodecyl)-thiophene-2-carboxylic acid tertbutylester (prepared as described in Example 6 of WO07128815) were coupled using SSPS method D method on theLiberty synthesiser.UPLC (method 08_B4_1): Rt = 9.87 minLCMS4: m/z =1651 (m/3), 1239 (m/4), 991 (m/5) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation method: SPPS Method B. The final product was characterised by analytical UPLC and LC-MS withthe exception that an acetic anhydride capping step was performed after the coupling of the following amino acids:Trp31, Ala25, Tyr19, Phe12 and Aib8 (2© min, 65°C with 1 N Acetic acid anhydride in NMP). The 4-(15-carboxypentadecyloxy)benzoic acid tert-butyl ester can be prepared as decribed in Example 17 in WO07128817.UPLC (method 08_B4_1): Rt = 11.272 minUPLC (method 05_B10_1): Rt = 7.319 minLCMS4: Rt = 2.37 min, m/z = 5054.48 Calculated MW = 5056.82 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
SPPS method B refers to the synthesis of a protected peptidyl resin using Fmoc chemistry on a microwave based Liberty peptide synthesiser (CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wang resin, 0.35 mmol/g). Fmoc-deprotection was with 5percentpiperidine in NMP at up to 70 or 75°C. The coupling chemistry was DIC/HOAt in NMP. Amino acid/HOAt solutions (0.3M in NMP at a molar excess of 3-10 fold) were added to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAt solution were used per coupling for the followingscale reactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling times and temperatures weregenerally 5 minutes at up to 70 or 75°C. Longer coupling times were used for larger scale reactions, for example 10min. Histidine amino acids were double coupled at 50°C, or quadruple coupled if the previous amino acid was stericallyhindered (e.g. Aib). Arginine amino acids were coupled at RT for 25 min then heated to 70 or 75°C for 5 min. Someamino acids such as but not limited to Aib, were "double coupled", meaning that after the first coupling (e.g. 5 min at75°C), the resin is drained and more reagents are added (amino acid, HOAt and DIC), and the mixture in heated again(e.g. 5 min at 75°C). When a chemical modification of a lysine side chain was desired, the lysine was incorporated asLys(Mtt). The Mtt group was removed by washing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM and NMP. The chemical modification of the lysinewas performed either by manual synthesis (see SPPS method D) or by one or more automated steps on the Libertypeptide synthesiser as described above, using suitably protected building blocks (see General methods), optionallyincluding a manual coupling. Preparation: SPPS method B, 8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (commerciallyavailable from Iris Biotech), 4-(4-t-butylphenyl)butyric acid and Fmoc-Glu-OtBu were coupled using SPPS method D.UPLC (method 08_B4_1): Rt = 9.07 minLCMS4: Rt = 2.29 min, m/z = 943 (m/5) 1179 (m/4) 1571 (m/3) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Fmoc deprotection: In general, to the resin pre-swollen in DCM was added 20percent piperidine in DMF and stirred at rt (2x10 min). The solution was drained and the resin washed with DMF (3x10 mL), DCM (3x10 mL) and MeOH (3x10 mL). In the cases were Fmoc 38 SUBSTITUTE SHEET RULE 26 deprotection was done in Cy5 containing peptides, a solution of 2percent DBU in DMF (2 x 10 min, rt) was used instead. Aminoacid coupling: A solution of the appropriate D- or L-amino acid (3.0 eq per amine) and Oxyma (3.0 eq) in DMF (0.1 M) was stirred for 10 min. DIC (3.0 eq) was added and stirred for 1 min. The pre-activated mixture was then added to the resin pre-swollen in DCM and the reaction heated at 50°C for 30 min. The solution was drained and washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_). The completion of the coupling and deprotection reactions was monitored by Kaiser test or Chloranil test when secondary amines are involved. The side chain protecting group used was Boc for arginine, tryptophan and lysine. Fmoc-Lys(Dde)-OH was used as orthogonal reagent to introduce the dyes. Coupling of other carboxylic acids: Coupling of {2-[2-(Fmoc-amino)ethoxy]ethoxy}acetic acid (PEG), <strong>[76823-03-5]5-Carboxyfluorescein</strong> (FAM), Fmoc-Lys(N3)-OH and MethylRed-Lys-(4- pentynoyl)-OH was done following the same procedure described for Aminoacid coupling. Dde deprotection: (a) Dde deprotection in non-containing Fmoc peptides was done following the next procedure: to the resin pre-swollen in DCM was added 2percent hydrazine in DMF and stirred at rt (5x10 min). The solution was drained and the resin washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_). (b) Selective Dde deprotection in Fmoc- protected peptides was done with a solution containing Imidazole (1.35 mmol) and Hydroxylamine hydrochloride (1.80 mmol) in NMP (5 ml_). [Diaz-Mochon, J. J.; Bialy, L; Bradley, M. Org Lett 2004, 6 (7), 1127-1 129]. After complete dissolution 5 volumes of this solution were diluted with 1 volume of CH2CI2 and the resin was treated with the final mixture for 3h at room temperature. The solution was drained and the resin washed with DMF (3x10 ml_), DCM (3x10 ml_) and MeOH (3x10 ml_). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
64% | Fmoc group was removed by treatment with 20% piperidine in N,N- dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 50 mL). Resin was washed with N,N-dimethylformamide (3 x 50 mL), 2-propanol (3 x 50 mL) and dichloromethane (3 x 30 mL). Solution of <strong>[843666-40-0]octadecanedioic acid mono-tert-butyl ester</strong> (C18(OtBu)-OH, 0.85 g, 2.28 mmol), 0-(6-chloro-benzotriazol-l-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate (TCTU, 0.81 g, 2.28 mmol) and N,N-diisopropylethylamine (0.72 mL,4.11 mmol) in N,N-dimethylformamide (50 mL) was added to resin and mixture was shaken for 1.5 hour. Resin was filtered and washed with N,N-dimethylformamide (3 x 50 mL), dichloromethane (3 x 50 mL) and N,N-dimethylformamide (3 x 50 mL). Mtt group was removed by treatment with 80% l,l,l,3,3,3-hexafluoro-2-propanol indichloromethane (2 x 10 min, 2 x 30 min, 4 x 50 mL). Resin was washed withdichloromethane (6 x 50 mL). Solution of bromoacetic acid (4.24 g, 30.5 mmol) and N,N'-diisopropylcarbodiimide (DIC, 4.01 mL, 25.9 mmol) in N,N-dimethylformamide (50 mL) was added to resin and mixture was shaken for 45 minutes. Resin was filtered and washed with N,N-dimethylformamide (5 x 50 mL) and dichloromethane (10 x 50 mL). The product was cleaved from resin by treatment with trifluoroacetic acid (50 mL) for 1 hour. Resin was filtered off and washed with trifluoroacetic acid (1 x 25 mL) and dichloromethane (2 x 30 mL). Solutions were combined and solvents were evaporated to dryness giving the compound as thick brownish oil.Yield : 2.18 mg (64%).1H NMR spectrum (300 MHz, AcOD-d4, 80C, dH) : 4.72-4.55 (m, 2 H); 4.16 (s, 2 H);4.12 (s, 2 H); 3.80-3.62 (m, 12 H); 3.58-3.44 (m, 4 H); 3.32 (t, J = 6.8 Hz, 2 H); 3.15 (d, J = 6.8 Hz, 2 H); 2.51-2.07 (m, 8 H); 2.01-1.77 (m, 6 H); 1.72-1.44 (m, 11 H); 1.33(bs, 24 H); 1.13-0.95 (m, 2 H). LC-MS purity: 96%.LC-MS Rt (Kinetex 4.6 mm x 50 mm, acetonitrile/water 20: 80 to 100 : 0 + 0.1% FA) : 3.68 min.LC-MS m/z: 1124.1 (M + H) + . |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
98% | Example 4.17: Preparation of 20-[[4-[[(lS)-4-[2-[2-[2-[2-[2-[2-[[(lS)-5-[(2- bromoacetyl)amino]-l-carboxy-pentyl]amino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo- ethoxy]ethoxy]ethylamino]-l-carboxy-4-oxo-butyl]carbamoyl]cyclohexyl]methylamino]- 20-oxo-icosanoic acidSynthetic protocol :Wang Fmoc-Lys(Mtt) resin 0.26 mmol/g (1, 11.2 g, 2.90 mmol) was left to swell in dichloromethane (100 mL) for 45 minutes. Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 mL). Resin was washed with N,N-dimethylformamide (3 x 90 mL), 2-propanol (3 x 90 mL) and dichloromethane (3 x 90 mL). A solution of {2-[2-(9H-fluoren-9- ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-OEG-OH, 2.23 g, 5.80 mmol), 0-(6-chlorobenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate(TCTU, 2.06 g, 5.80 mmol) and N,N-diisopropylethylamine (2.02 mL, 11.6 mmol) in N,N- dimethylformamide (100 mL) was added to resin and the mixture was shaken for 1 hour. Resin was filtered and washed with N,N-dimethylformamide (3 x 90 mL),dichloromethane (3 x 90 mL) and N,N-dimethylformamide (3 x 90 mL). Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 mL). Resin was washed with N,N-dimethylformamide (3 x 90 mL), 2-propanol (3 x 90 mL) and dichloromethane (3 x 90 mL). Solution of {2-[2-(9H- fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-OEG-OH, 2.23 g, 5.80 mmol), 0-(6-chloro-benzotriazol-l-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate (TCTU, 2.06 g, 5.80 mmol) and N,N-diisopropylethylamine (2.02 mL, 11.6 mmol) in N,N-dimethylformamide (100 mL) was added to resin and mixture was shaken for 1.5 hour. Resin was filtered and washed with N,N-dimethylformamide (3 x 90 mL), dichioromethane (3 x 90 mL) and N,N-dimethylformamide (3 x 90 mL). Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 mL). Resin was washed with N,N-dimethylformamide (3 x 90 mL), 2-propanol (3 x 90 mL) and dichioromethane (3 x 90 mL). Solution of (S)-2-(9H- fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid 1-tert-butyl ester (Fmoc-LGIu- OtBu, 1.85 g, 4.35 mmol), 0-(6-chloro-benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU, 1.55 g, 4.35 mmol) and N,N-diisopropylethylamine (1.36 mL, 7.82 mmol) in N,N-dimethylformamide (100 mL) was added to resin and mixture was shaken for 1.5 hour. Resin was filtered and washed with N,N-dimethylformamide (3 x 90 mL), dichioromethane (3 x 90mL) and N,N-dimethylformamide (3 x 90 mL). Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 mL). Resin was washed with N,N-dimethylformamide (3 x 90 mL), 2-propanol (3 x 90 mL) and dichioromethane (3 x 90 mL). Solution of4-[(9H-fluoren-9-ylmethoxycarbonylamino)methyl]cyclohexanecarboxylic acid(Fmoc-Trx-OH, 1.65 g, 4.35 mmol), 0-(6-chloro-benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TCTU, 1.55 g, 4.35 mmol) and N,N- diisopropylethylamine (1.36 mL, 7.82 mmol) in N,N-dimethylformamide (100 mL) was added to resin and mixture was shaken for 2 hours. Resin was filtered and washed with N,N-dimethylformamide (3 x 90 mL), dichioromethane (3 x 90mL) and N,N- dimethylformamide (3 x 90 mL). Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 mL). Resin was washed with N,N-dimethylformamide (3 x 90 mL), 2-propanol (3 x 90 mL) and dichioromethane (3 x 90 mL). Solution of <strong>[683239-16-9]icosanedioic acid mono-tert-butyl ester</strong>(C20(OtBu)-OH, 1.73 g, 4.35 mmol), 0-(6-chloro-benzotriazol-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TCTU, 1.55 g, 4.35 mmol) and N,N- diisopropylethylamine (1.36 mL, 7.82 mmol) in N,N-dimethylformamide (100 mL) was added to resin and mixture was shaken for 2 hours. Resin was filtered and washed with N,N-dimethylformamide (3 x 90 mL), dichioromethane (3 x 90 mL), N,N- dimethylformamide (3 x 90 mL) and dichioromethane (3 x 90 mL). Mtt group was removed by treatment with 80% l, l,l,3,3,3-hexafluoro-2-propanol in dichioromethane (2 x 10 min, 2 x 30 min, 4 x 100 mL). Resin was washed with dichioromethane (6 x 90 mL) and N,N-dimethylformamide (3 x 90 mL). Solution of bromoacetic acid (8.06 g, 58.0 mmol) and N,N'-diisopropylcarbodiimide (DIC, 7.60 mL, 49.3 mmol) in N,N- dimethylformamide (100 mL) was added to resin and mixture was shaken for 40 minutes. Resin was filtered and washed with N,N-dimethylformamide (5 x 90 mL) and dichloromethane (12 x 90 mL). The product was cleaved from resin by treatment with trifluoroacetic acid (100 mL) for 1 hour. Resin was filtered off and washed with trifluoroacetic acid (1 x 50 mL) and dichloromethane (7 x 70 mL). Solutions were combined and solvents were evaporated to dryness giving a thick brownish oil.Yield : 3.28 g (98%).1H NMR spectrum (300 M... |
Yield | Reaction Conditions | Operation in experiment |
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65% | 2-Chlorotrityl resin 100-200 mesh 1.8 mmol/g (1, 41.7 g, 75.1 mmol) was left to swell in dry DCM (350 mL) for 20 mm. A solution of {2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-OEG-OH, 19.3 g, 50.1 mmol) and DIPEA (33.1 mL, 190 mmol) in dry DCM (250 mL) was added to resin and the mixture was shaken overnight. The resin was filtered and treated with a solution of DIPEA (17.4 mL, 100 mmol) in MeOH/DCM mixture (4:1, 5 mm, 200 mL). Then resin waswashed with DCM (2 x 250 mL) and DMF (2 x 250 mL). Fmoc group was removed by treatment with 20% piperidine in DMF (1 x 5 mm, i x 15 mm, 2 x 250 mL). The resin was washed with DMF (2 x 250 mL), 2-propanol (2 x 250 mL), DCM (2 x 250 mL) and DMF (2 x 250 mL). Solution of 2-(9H-fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid 1-tert-butyl ester (Fmoc-Glu-OtBu, 42.6 g, 100 mmol), O-(6-chloro-benzotriazol-1-yl)-N,N,N?,N?-tetramethyluronium tetrafluoroborate (TCTU, 35.6 g, 100 mmol) and DIPEA (31.4 mL, 180 mmol) in DMF (200 mL) was added to resin and mixture was shaken for 4 hours. Resin was filtered and washed with DMF (2 x 250 mL) and DCM (10 x 250 mL). The product was cleaved from resin by treatment with 2,2,2-trifluoroethanol (350 mL) overnight. Resin was filtered off and washed with DCM (2 x 200 mL), solvent wasevaporated and crude product was purified by flash column chromatography (Silicagel60, 0.040-063 mm; eluent: DCM/MeOH 95:5 to 85:15) giving (R)-4-[2-(2- carboxymethoxy-ethoxy) -ethylcarba moyl]-2- (9H-fl uoren-9-yl methoxycarbonyla mi no) - butyric acid tert-butyl ester (2) as yellowish waxy solid.Yield: 18.6 g (65%).1H NMR spectrum (300 MHz, CDCI3, oH). 7.77 (d, J=7.5 Hz, 2 H); 7.65-7.50 (m,2 H); 7.47-7.37 (m, 2 H); 7.37-6.72 (m, 2 H); 6.84-6.72 (m, 1 H); 5.92-5.81 (m, 1 H);4.58-4.30 (m, 2 H); 4.30-3.95 (m, 4 H); 3.79-3.51 (m, 6 H); 3.43 (q, J=4.6 Hz, 2 H);2.39-1.90 (m, 4 H); 1.54-1.39 (m, 9 H). LC-MS purity: lOO%.LC-MS Rt (Kinetex C18, 4.6 mm x 50 mm, MeCN/water 20:80 to 100:0 + 0.l%FA): 3.65 mm.LC-MS m/z: 570.6 (M+H). |
Yield | Reaction Conditions | Operation in experiment |
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86% | 2-Chlorotrityl resin 100-200 mesh (42.6 g, 42.6 mmol) was left to swell in dry dichloromethane (205 mL) for 20 mm. A solution of {2-[2-(9H-fluoren-9- ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (13.7 g, 35.5 mmol) and N, N-diisopropylethylamine (23.5 mL, 135 mmol) in dry dichloromethane (30 mL) was added to resin and the mixture was shaken for 3 hrs. Resin was filtered and treated with a solution of N,N-diisopropylethylamine (12.4 mL, 70.9 mmol) in methanol/dichloromethane mixture (4:1, 250 mL, 2 x 5 mm). Then resin was washed with N,N-dimethylformamide (2 x 150 mL), dichloromethane (3 x 150 mL) and N,N-dimethylformamide (3 x 150 mL). Fmoc group was removed by treatment with 20% piperidine in dimethylformamide (1 x 5 mm, i x 30 mm, 2 x150 mL). Resin was washed with N,N-dimethylformamide (3 x 150 mL), 2-propanol (2 x 150 mL) and dichloromethane (200 mL, 2 x 150 mL). Solution of {2-[2-(9H-fluoren-9- ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (20.5 g, 53.2 mmol), O-(6-chloro- benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol)and N,N-diisopropylethylamine (16.7 mL, 95.7 mmol) in N,N-dimethylformamide (100 mL) and dichloromethane (50 mL) was added to resin and mixture was shaken for 1 hr. Resinwas filtered and washed with N,N-dimethylformamide (2 x 150 mL), dichloromethane (3 x150 mL) and N,N-dimethylformamide (155 mL). Fmoc group was removed by treatment with20% piperidine in dimethylformamide (1 x 5 mm, i x 30 mm, 2 x 150 mL). Resin was washedwith N,N-dimethylformamide (3 x 150 mL), 2-propanol (2 x 150 mL) and dichloromethane (200 mL, 2 x 150 mL). Solution of Fmoc-Glu-OtBu (22.6 g, 53.2 mmol), O-(6-chloro- benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and N,N-diisopropylethylamine (16.7 mL, 95.7 mmol) in N,N-dimethylformamide (155 mL) was added to resin and mixture was shaken for 1 hr. Resin was filtered and washed withN,N-dimethylformamide (2 x 150 mL), dichloromethane (2 x 150 mL) and N,Ndimethylformamide (150 mL). Fmoc group was removed by treatment with 20% piperidine in dimethylformamide (1 x 5 mm, i x 30 mm, 2 x 150 mL). Resin was washed with N,Ndimethylformamide (3 x 150 mL), 2-propanol (2 x 150 mL) and dichloromethane (200 mL, 2 x 150 mL). Solution of <strong>[843666-40-0]octadecanedioic acid mono-tert-butyl ester</strong> (19.7 g, 53.2 mmol), O-(6-chloro-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and N,N-diisopropylethylamine (16.7 mL, 95.7 mmol) in N,Ndimethylformamide/dichloromethane mixture (1:4, 200 mL) was added to resin. Resin was shaken for 2 hrs, filtered and washed with N,N-dimethylformamide (3 x 150 mL), dichloromethane (2 x 150 mL), methanol (2 x 150 mL) and dichloromethane (300 mL, 6 x 150 mL). The product was cleaved from resin by treatment with 2,2,2-trifluoroethanol (200 mL) for 19 hrs. Resin was filtered off and washed with dichloromethane (2 x 150 mL), 2- propanol/dichloromethane mixture (1:1,2 x 150 mL), 2-propanol (150 mL) and dichloromethane (2 x 150 mL). Solutions were combined; solvent evaporated and crude product was purified by flash column chromatography (Silicagel 60, 0.040-0.060 mm; eluent:dichloromethane/methanol 1:0-9:1). Pure product was dried in vacuo and obtained as yellow oil.Yield of 1 7-{(S)- 1 -tert-B utoxycarbonyl-3-[2-(2-[2-(2-carboxymethoxy-ethoxy)- ethylcarbamoyl]-methoxy}-ethoxy)-ethylcarbamoyl]-propylcarbamoyl}-heptadecanoic acid tert-butyl ester: 25.85 g (86%).RE (5i02, chloroform/methanol 85:15): 0.25.1 H NMR spectrum (300 MHz, CDCI3, dH): 7.38 (bs, 1 H); 7.08 (bs, 1 H); 6.61 (d,J=7.5 Hz, 1 H); 4.43 (m, 1 H); 4.15 (s, 2 H); 4.01 (s, 2 H); 3.78-3.39 (m, 16 H); 2.31 (t, J=6.9Hz, 2 H); 2.27-2.09 (m, 5 H); 2.01-1.84 (m, 1 H); 1.69-1.50 (m, 4 H); 1.46 (s, 9 H); 1.43 (s, 9H); 1.24(bs, 24 H).LC-MS m/z: 846.6 (M+H)+. | |
1.1 g | Starting resin: 50 2-Chlorotrityl resin, 1.60 mmol/g (0516) 1.0 g of the resin was swelled for 30 min in 51 DCM (10 ml). 1. Acylation with Fmoc-?-amino-3,6-dioxaoctanoic acid: (0517) 0.39 g (0.63 eq, 1.0 mmol) of Fmoc-?-amino-3,6-dioxaoctanoic acid (Fmoc-OEG-OH) was dissolved in DCM (15 ml) and was added to the resin. N,N-Diisopropylethylamine (DIEA) (0.44 ml, 2.5 mmol) was added dropwise. The reaction mixture was vortexed for 30 min. and then methanol (2 ml) was added and the mixture was vortexed for additional 15 min. The resin was filtered and washed with NMP (2×8 ml) and DCM (8×8 ml). (0518) 20% piperidine/NMP (8 ml) was added, standing 10 min. repeated once. Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). A positive TNBS test gave red-coloured resins. 2. Acylation with Fmoc-?-amino-3,6-dioxaoctanoic acid: (0519) 0.78 g (2 eq, 2.0 mmol) of 54 Fmoc-?-55 amino-3,6-dioxaoctanoic acid was dissolved in NMP/51 DCM 1:1 (10 ml). 0.28 g (2.2 eq, 2.4 mmol) of HOSu was added followed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reaction mixture was allowed to stand for 1 hour and was then added to the resin and finally 0.407 ml (2.2 eq) of 56 DIEA was added. The mixture was vortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). A positive TNBS test gave colourless resins. (0520) 20% 57 piperidine/NMP (10 ml) was added, standing 10 min. repeated once. Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). A positive TNBS test gave red-coloured resins. Acylation with Fmoc-Glu-OtBu: (0521) 0.86 g (2 eq, 2.0 mmol) of 58 Fmoc-Glu-OtBu was dissolved in NMP/DCM 1:1 (10 ml). 0.32 g (2.2 eq, 2.4 mmol) of 59 HOBT was added followed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reaction mixture was allowed to stand for 20 min and was then transferred to the resin and finally 0.407 ml (2.2 eq) of DIEA was added. The mixture was vortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). A positive TNBS test gave colourless resins. (0522) 20% piperidine/NMP (10 ml) was added, standing 10 min. repeated once. Filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). A positive TNBS test gave red-coloured resins. Acylation with Octadecanedioic Acid Mono Tert-Butyl Ester: (0523) 0.75 g (2 eq, 2.0 mmol) 60 Octadecanedioic acid mono tert-butyl ester was dissolved NMP/DCM 1:1 (10 ml). 0.32 g (2.2 eq, 2.4 mmol) HOBT was added followed by addition of 0.37 ml (2.2 eq, 2.4 mmol) of DIC. The reaction mixture was allowed to stand for 20 min and was then transferred to the resin and finally 0.41 ml (2.2 eq) of DIEA was added. The mixture was vortexed for 16 hours, filtered and washed with NMP (2×8 ml), DCM (3×8 ml), and NMP (5×8 ml). Cleavage with TFA: (0524) 8 ml of 5% 61 TFA/DCM was added to the resin and the reaction mixture was vortexed for 2 hours, filtered and the filtrate was collected. More 5% TFA/DCM (8 ml) was added to the resin, and the mixture was vortexed for 10 min, filtered and the resin was washed with DCM (2×10 ml). The combined filtrates and washings were pH adjusted to basic using about 800 ul of DIEA. The mixture was evaporated in vacuo affording an oil (3.5 g). 62 Diethylether (30 ml) was added and the not dissolved oil was separated by decantation and evaporated in vacuo. This afforded 1.1 g of 63 17-{(S)-1-tert-butoxycarbonyl-3-[2-(2-[2-(2-carboxymethoxyethoxyl)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}heptadecanoic acid tert-butyl ester (alternative name: tert-butyl octadecandioyl-Glu(OEG-OEG-OH)-OTBU) as an oil. (0525) LC-MS (Sciex100 API): m/z=846.6 (M+1)+. |
Yield | Reaction Conditions | Operation in experiment |
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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 |
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In a similar way as described in Example 1 above and depicted below the following compound was prepared using Boc-Gly-PAM resin as starting material. TOF-MS: mass 1128.38 |
Yield | Reaction Conditions | Operation in experiment |
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7.5 mg | Sieber amide resin (0.71 meqg, 70.4 mg, 0.05 mmol) was added to a reaction tube, which was then set in a peptide synthesizer, and amino acids were sequentially extended according to the protocol using 20% piperidineNMP [reacted at 50C for 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids DIPCDIOxyma [reacted at 50C for 15 minutesj to condense the Fmoc-amino acids. The operation wherein the obtained BocMeTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tB u)-Iva-Ile-Ser(tB u)-Asp(OtBu)-Tyr(tBu)-Ser(t B u)-Ile-Aib-Leu-Asp(OtBu)-Arg(Pbf)-Lys(ivDde)-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Va l-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tB u)-Gly-Ala-P ro-Pro-Pro-Ser(tBu)-Sieber amide resin was suspended in a 2% hydrazine/NMP solution, the resulting suspension was stirred at room temperature for 3 hours, and then the solution was removed by filtration. After the filtration, the resin was suspended in a 2% hydrazineNMP solution and reacted at room temperature overnight to deprotect the ivDde group of Lys at position 14. Subsequently, the resin was washed with MeOH, and dried under reduced pressure to thereby obtain 388.8 mg of BocMeTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tB u)-Iva-Ile-Ser(tB u)-Asp(OtBu)-Tyr(tBu)-Ser(t B u)-Ile-Aib-Leu-Asp(OtBu)-Arg(Pbf)-Lys-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(T rt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tB u)-Gly-Ala-Pro-ProPro-Ser(tBu)-Sieber amide resin.38.9 mg (0.01 mmol) of the obtained resin was weighed into a reaction tube, which was then set in a peptide synthesizer. According to the protocol using 20% piperidine NMP [reacted at 50C for S minutesj to deprotect the Fmoc group, and using S equivalents of acid agent (Fmoc-amino acids or eicosanedioic acid mono-tert-butyl ester) and DIPCDIOxyma to condense, PEG(3), PEG(3), eicosanedioic acid monotert-butyl ester were sequentially introduced using the peptide synthesizer. The condensation reaction was carried out using the double coupling method was used in which after the reactions at 50C for 15 minutes, the solution was removed by filtration, and the same condensation reaction was repeated. After completion of solidphase synthesis, the resin was washed with MeOH, and dried under reduced pressure to thereby obtain 39.3 mg of the protected peptide resin of interest, BocMeTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tB u)-Iva-Ile-Ser(tB u)-Asp(OtBu)-Tyr(tBu)-Ser(t B u)-Ile-Aib-Leu-Asp(OtBu)-Arg(Pbf)-Lys( 1 9-tert-butoxycarbonyl-nonadedanoyl-PEG(3)-PEG(3)-)-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln (Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tB u)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin.Subsequently, to the total amount of the obtained resin, 0.5 mL of TFA:m-cresol:thioanisole:ethandithiol:H20:triisopropylsilane (80:5:5:5:2.5:2.5) was added and the resulting mixture was stirred for 1.5 hours. Diethyl ether was added to the reaction solution to obtain a precipitate and after centrifugation the supernatant was removed. This operation was repeated twice and the precipitate was washed. The residue was extracted with a 90% acetic acid aqueous solution and the resin was removed by filtration, and then the purification was carried out by preparative HPLC using YMC-Triart C8-S-10 lIm, 20 nm column (250 x 20 mm I.D.) by the linear concentration gradient elution (60 minutes) with solution A: 0.1% TFA-water and solution B: 0.1% TFA-containing acetonitrile at a flow rate of 8 mL/minute from A/B: 52/48 to 42/58, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 7.5 mg of a white powder.Mass spectrometry result: (M+H)+ 4846.10 (calculated 4845.61) HPLC elution time: 7.11 minutesElution conditions:Column: Kinetex 1.7 lIm C8 bOA, (100 x 2.1 mm I.D.)Eluents: Using solution A: 0.1% TFA-water, solution B: 0.1% TFA-containing acetonitrile, A/B: 80/20 to 30/70. Linear concentration gradient elution (10 minutes). Flow rate: 0.5 mL/minutesTemperature: 40C |
Yield | Reaction Conditions | Operation in experiment |
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72% | Compound A is generated by solid-phase peptide synthesis. Fluorenylmethyloxycarbonyl (Fmoc)-Lys(Hoc)OH (1430 mg, 3 mmol, 2 eq rd to resin; Novabiochem catalog 852012) is mixed with 1 -[His(dimethylamino) methylene]- 1 H-i ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (1150mg, 3 mmol, 2 eq rd to resin; Oakwood Chemical catalog 023926) and N,N-Diisopropylethylamine (DIEA) (1333 ul, 7.6 mmol, 5 eq.) in 10 mL DMF for 2 minutes and then transferred to the vessel containing H-AEEA-2-chiorotrityl-chloride resin (2.11 g, 0.72 mmol/g, 1.52 mmol; Peptides International catalog RHX-i 1074-PI), which is pre-swelled in dichloromethane (DCM) and pre-washed with dimethylformamide (DMF).The slurry is mixed for 1.5 h, filtered and the resin is washed well with DMF (Kaiser test is negative). The Fmoc protecting group is removed by treatment of the resin with 20% piperidine/DMF (10 mL, 30 mi. Afier a DMF wash of the resin (40 mL), the Kaiser test is positive to provide H2N-Lys(l3oc)-AEEA-2-chlorotrityl-chloride resin (1.52 mmol in theory). Fmoc-Glu-OtHu (1297 mg, 3.0 mmol, 2.0 eq, Ark Pharma catalog AK-48532) is pre-activated (2 mm) with HATU (1153 mg, 3 mmol, 2 eq) using DIEA as base (1333 pL, 7.7 mmol, 5.0 eq) in 10 mL DMF, then transferred to the resin. The slurry is mixed for 3 hours, filtered, and the resin washed well with DMF (Kaiser test is negative).The Fmoc protecting group is removed by treatment of the resin with 20% piperidine/DMF (10 mL, 30 mm) followed by a DMF wash of the resin (40 mL, Kaiser test is positive). A second Fmoc-Glu-OtHu is coupled to the resin by repeating the conditions above using a 1.5 hour coupling time.After removing the last Fmoc protecting group, <strong>[843666-40-0]18-tert-butoxy-18-oxo-octadecanedioic acid</strong> (1.13 g, 3.0 mmol, 2.0 eq) is pre-activated (2 mm) with HATU (1.15 g, 3.0 mmol, 2.0 eq) using DIEA as base (1333 pL, 7.7 mmol,5.0 eq) in 10 mL DMF, then transferred to the resin. The slurry is mixed for 3 hours, filtered, and the resin washed well first with DMF and then with DCM (Kaiser test is negative). The protected linker-fatty acid is cleaved from the resin by mixing with 30% HFIP/DCM (20 mE) for 1 hout The resin is filtered oil and rinsed well with DCM. The combined filtrates are evaporated to an oil in vacuo. The residual oil is diluted with acetonitrile (15-20 mE) and evaporated in vacuo again to an oil. The sample is again dissolved with acetonitrile (15-20 mE) and is evaporated in vacuo to form an oil. A gentle stream of nitrogen evaporates the residual acetonitrile to give 2.6 grams of crude, amorphous solid (theory=1 .7 grams).Purification begins with the crude sample being dissolved in 5 mE DMF and 20 mE acetonitrile (including washes of flask). Then water is added to give 40 mE of a hazy solution. 5 mE additional acetonitrile gives a clear solution (total volume equaled 45 mE (33% aqueous). Purification is performed by loading the sample onto a semi-prep cyano reversed phase HPEC column (SilaChrom XDB1-CN; 10 tm, 100 A; 2.1x25 cm).10034] The sample is then eluted using a 40-60% B gradient over 72 mm, 15 mE/mm, 60 C. (buffer A=0.1% TFA in water and buffer B=0. 1% TFA in acetonitrile). Fractions determined to contain the desired product by analytical RP-HPEC are pooled and lyophilized to give 1.22 grams of product (Compound A) as a white amorphous solid (72% of theory; 91% purity by RP-HPEC; obs MW=1 114.6 Daltons (Da); theo MW=1114.45 Da). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
203 g | With hydrogenchloride; In ethanol; water;pH 9.0; | In a 3000L three-necked flask equipped with mechanical stirring, condenser and constant pressure dropping funnel,330 g of crude product was dissolved in 1000 ml of ethanol, and 400 g of 30% NaOH solution was added dropwise.After the addition is completed, the mixture is heated to reflux, and the TLC is monitored. The etherification intermediate disappears completely and the temperature is lowered.The pH was adjusted to 9 with hydrochloric acid, and 259 g of Fmoc-Cl was added dropwise to dissolve in 1000 ml of ethanol solution.If the pH has dropped, add sodium bicarbonate and keep it at around 8.TLC was monitored until the free amino intermediate disappeared. Adjust the pH to 1-2 with hydrochloric acid,Distill the ethanol, add 1000 ml of water, and extract three times with ethyl acetate 500*3.Wash twice with saturated brine and concentrate ethyl acetate to give 288 g of crude material.Recrystallization from 1000 ml of ethyl acetate gave 203 g of product[2-[1-(Fmoc-Amino)ethoxy]ethoxy]acetic acid, yield 52.68%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
[0149] The synthetic scheme of SILY-DBCO is shown in FIGS. 1A and 1B. Fmoc-SILY-k was first synthesized by standard solid phase peptide synthesis (SSPS) approach, followed by DBCO coupling in solution phase with DBCO-OSu. Rink amide MBHA resin (1.0 g, 0.503 mmol, loading 0.503mmol/g) was swollen in DMF for 3 hours before Fmoc-deprotection. Protected Fmoc/Dde-SILY-k(Boc) beads were prepared with automated peptide synthesizer (CS-Bio). Five-fold excess of Fmoc-amino acids were used for the coupling in presence of HCTU (6 eq.)/DIEA (12 eq.). The coupling times for the first 10 and the latter couplings were 2 hours and 3 hours, respectively. The Fmoc was removed with 20% 4-methylpiperidine twice (5 minutes, 15 minutes). The following Fmoc-amino acids were coupled sequentially: Fmoc-D-Lys(Boc)-OH, Fmoc-AEEA linker, Fmoc-AEEA linker, Fmoc-Tyr(tBu)-OH, Fmoc- Leu-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ala-OH, Fmoc- Arg(pbf)-OH, Fmoc-Arg(Pbf)-OH. The beads were transferred to a 20-ml column from the synthesizer. The TV-terminal Fmoc and Dde groups were removed with 3% NH2NH2 in DMF (10 minutes, 15 minutes). After washing with DMF, MeOH, and DCM respectively, the free amino groups were re-protected with Fmoc using Fmoc-OSu (10 eq.) in presence of DIEA (20 eq.) in DCM. This step was repeated until a Kaiser test was negative. The resulting Fmoc-protected SILY-k(Boc) beads were thoroughly washed with DMF, MeOH and DCM and then dried under vacuum for 1 hour before adding a cleavage cocktail of 82.5% TFA: 5% phenol: 5% thioanisole: 5% water: 2.5% TIS. The beads were rotated at room temperature for 4 hours. The liquid was collected and concentrated by blowing with nitrogen gas. The crude product was precipitated with cold ether and purified by reverse-phase HPLC and lyophilized to give Fmoc-SILY-k in powder form. The identity of the compound was confirmed by MALDI-TOF MS, calculated for Ci55H2i8N32037 (m/z): 3119.61; Found: 3120.82 (MH+). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The activated fatty acid side chain, Moiety A-OSu was prepared by solid phase synthesis using 2-chlorotrityl chloride resin as schematically represented in FIG. 1A. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid (Intermediate-1) was attached to 2-chlorotrityl chloride resin in presence of N,N?-di-isopropylethylamine (DIPEA) which yielded 2-[2-(2-Fmoc-aminoethoxy)ethoxy] acetic acid-2-Cl-Trt-Resin. The intermediate-1 can be prepared by coupling of 2-[2-(2-amino ethoxy)-ethoxy] acetic acid with Fmoc N-hydroxysuccinimide ester. Alternatively, intermediate-1 is available commercially and can be procured as such. The Fmoc protecting group was removed by selective de-blocking of amino group of 2-[2-(2-Fmoc-aminoethoxy)ethoxy] acetic acid-2-Cl-Trt-Resin using piperidine and the free amino group was then coupled to 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid using 1-hydroxybenztriazole(HOBt) and N,N?-di-isopropylcarbodiimide (DIPC) which yielded 2-[2-[2-[[2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetyl] amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group was then removed by selective de-blocking of amino group of 2-[2-[2-[[2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetyl] amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin using piperidine and the free amino group was then coupled to Fmoc-Glu-OtBu using HOBt and DIPC to obtain 2-[2-[2-[[2-[2-[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy] acetic acid-2-Cl-Trt-Resin. The resultant 2-[2-[2-[[2-[2-[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]ethoxy] ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin was selectively deblocked using piperidine and then coupled with octadecanedioic acid mono tert-butyl ester to give intermediate-2 namely [2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy] acetic acid]-2-Cl-Trt-Resin. The intermediate 2 was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1). The resultant compound was then reacted with N-hydroxysuccinimide (HOSu) in presence of isobutyl chloroformate (IBCF) and N-methylmorpholine (NMM) followed by de-protection with trifluoroacetic acid to yield the title compound (Moiety A-OSu, intermediate-3). The whole process can also be depicted as schematically represented in FIG. 1B. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The activated fatty acid side chain, Moiety C-OSu was prepared using solid phase synthesis using 2-chlorotrityl chloride resin as schematically represented in FIG. 2. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine and the free amino group was then activated using p-nitrophenylchlroformate in THF and DIPEA followed by reaction with Fmoc-amino butylamine hydrochloride salt in THF: DMAc and DIPEA, which yielded 2-[2-[2-(4-Fmoc-aminobutylcarbamoylamino) ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blocking using piperidine and the free amino group was then coupled to Fmoc-Glu-OtBu using of HOBt and DIPC, which yielded 2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The resultant 2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-butylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin was selectively deblocked using piperidine and then coupled with octadecanedioic acid mono tert-butyl ester to give intermediate 2-[2-[2-[4-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1) to obtain 2-[2-[2-[4-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid (LCMS=m/z: 814.56 (M+H+)). The resultant was then reacted with HOSu in presence of dicyclohexyl carbodiimide (DCC) to yield succinimide protected intermediate, which was de-protected with trifluoroacetic acid to yield the title compound (Moiety C-OSu). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The fatty acid side chain was prepared using solid phase synthesis using 2-chlorotrityl chloride resin as schematically represented in FIG. 3. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine followed by coupling with Fmoc-Aib-OH in THF: DMAc using DIPC and HOBt which yielded 2-[2-[2-[(2-Fmoc-amino-2-methyl-propanoyl)amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blocking using piperidine and the free amino group was coupled with Fmoc-Glu-OtBu using HOBt and DIPC to yield 2-[2-[2-[[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-2-methyl-propanoyl] amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group of the resultant was selectively de-blocked using piperidine and the free amino group was then coupled with octadecanedioic acid mono tert butyl ester to give 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]-amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1) to obtain 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid (LCMS=m/z: 786.39 (M+H+)). The resultant was then reacted with HOSu in presence of DCC to yield succinimide protected intermediate, which was de-protected with trifluoroacetic acid to yield the title compound (Moiety D-OSu). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The fatty acid side chain was prepared using solid phase synthesis using 2-chlorotrityl chloride resin as schematically represented in FIG. 4. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine and the free amino group was then activated using p-nitrophenylchloroformate in THF and DIPEA followed by reaction with 1,3-diaminopropane in THF: DMAc in presence of DIPEA using HOBt to form NH2-(CH2)3-NH-C(O)-{(2-(2-amino-ethoxy)-ethoxy}-acetic acid-2-Cl-Trt-Resin. The free amino group was then coupled to Fmoc-Glu-OtBu using HOBt and DIPC, which yielded 2-[2-[2-[3-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]propylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The resultant 2-[2-[2-[3-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]propylcarbamoylamino]ethoxy]-ethoxy]acetic acid-2-Cl-Trt-Resin was selectively deblocked using piperidine and then coupled with octadecanedioic acid mono tert butyl ester to give 2-[2-[2-[3-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-propyl carbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1) to obtain 2-[2-[2-[3-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-propylcarbamoylamino]ethoxy]ethoxy]acetic acid (LCMS=m/z: 801.41 (M+H+)). The resultant was then reacted with HOSu in presence of dicyclohexyl carbodiimide (DCC) to yield succinimide protected intermediate, which was de-protected with trifluoroacetic acid to yield the title compound (Moiety E-OSu). |
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. |
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. |
Precautionary Statements-General | |
Code | Phrase |
P101 | If medical advice is needed,have product container or label at hand. |
P102 | Keep out of reach of children. |
P103 | Read label before use |
Prevention | |
Code | Phrase |
P201 | Obtain special instructions before use. |
P202 | Do not handle until all safety precautions have been read and understood. |
P210 | Keep away from heat/sparks/open flames/hot surfaces. - No smoking. |
P211 | Do not spray on an open flame or other ignition source. |
P220 | Keep/Store away from clothing/combustible materials. |
P221 | Take any precaution to avoid mixing with combustibles |
P222 | Do not allow contact with air. |
P223 | Keep away from any possible contact with water, because of violent reaction and possible flash fire. |
P230 | Keep wetted |
P231 | Handle under inert gas. |
P232 | Protect from moisture. |
P233 | Keep container tightly closed. |
P234 | Keep only in original container. |
P235 | Keep cool |
P240 | Ground/bond container and receiving equipment. |
P241 | Use explosion-proof electrical/ventilating/lighting/equipment. |
P242 | Use only non-sparking tools. |
P243 | Take precautionary measures against static discharge. |
P244 | Keep reduction valves free from grease and oil. |
P250 | Do not subject to grinding/shock/friction. |
P251 | Pressurized container: Do not pierce or burn, even after use. |
P260 | Do not breathe dust/fume/gas/mist/vapours/spray. |
P261 | Avoid breathing dust/fume/gas/mist/vapours/spray. |
P262 | Do not get in eyes, on skin, or on clothing. |
P263 | Avoid contact during pregnancy/while nursing. |
P264 | Wash hands thoroughly after handling. |
P265 | Wash skin thouroughly after handling. |
P270 | Do not eat, drink or smoke when using this product. |
P271 | Use only outdoors or in a well-ventilated area. |
P272 | Contaminated work clothing should not be allowed out of the workplace. |
P273 | Avoid release to the environment. |
P280 | Wear protective gloves/protective clothing/eye protection/face protection. |
P281 | Use personal protective equipment as required. |
P282 | Wear cold insulating gloves/face shield/eye protection. |
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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