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CAS No. : | 73731-37-0 | MDL No. : | MFCD00077072 |
Formula : | C19H19NO5 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | OYULCCKKLJPNPU-DIFFPNOSSA-N |
M.W : | 341.36 | Pubchem ID : | 6992530 |
Synonyms : |
|
Num. heavy atoms : | 25 |
Num. arom. heavy atoms : | 12 |
Fraction Csp3 : | 0.26 |
Num. rotatable bonds : | 7 |
Num. H-bond acceptors : | 5.0 |
Num. H-bond donors : | 3.0 |
Molar Refractivity : | 91.14 |
TPSA : | 95.86 Ų |
GI absorption : | High |
BBB permeant : | No |
P-gp substrate : | Yes |
CYP1A2 inhibitor : | Yes |
CYP2C19 inhibitor : | No |
CYP2C9 inhibitor : | No |
CYP2D6 inhibitor : | No |
CYP3A4 inhibitor : | No |
Log Kp (skin permeation) : | -6.65 cm/s |
Log Po/w (iLOGP) : | 2.05 |
Log Po/w (XLOGP3) : | 2.44 |
Log Po/w (WLOGP) : | 2.36 |
Log Po/w (MLOGP) : | 1.74 |
Log Po/w (SILICOS-IT) : | 2.15 |
Consensus Log Po/w : | 2.15 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 0.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.56 |
Log S (ESOL) : | -3.39 |
Solubility : | 0.14 mg/ml ; 0.00041 mol/l |
Class : | Soluble |
Log S (Ali) : | -4.1 |
Solubility : | 0.0274 mg/ml ; 0.0000801 mol/l |
Class : | Moderately soluble |
Log S (SILICOS-IT) : | -4.35 |
Solubility : | 0.0152 mg/ml ; 0.0000446 mol/l |
Class : | Moderately soluble |
PAINS : | 0.0 alert |
Brenk : | 0.0 alert |
Leadlikeness : | 0.0 |
Synthetic accessibility : | 3.94 |
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 |
---|---|---|
91% | Stage #1: With caesium carbonate In methanol for 0.25 h; Inert atmosphere Stage #2: at 20℃; Inert atmosphere |
At r.t. a solution of FmocONSu (3.37 g, 10.0 mmol) in 25 mL of Dioxane12 is added over 1h to a solution of L-threonine (1.19 g, 10.0 mmol) and Na2CO3 (1.06 g, 10.0 mmol) in 10 mL Dioxane and 25 mL water. The resulting suspension is stirred overnight and becomes clear. The solvent is evaporated in vacuo and the product is precipitated with 1M HClaq. at pH 4, filtered, washed with water and dissolved in EtOAc. The organic phase is dried (Na2SO4) and evaporated to yield the product quantitatively as a white solid which does not need any further purification. Under argon atmosphere Fmoc-threonine (2.67 g, 7.80 mmol) is dissolved in 32 mL dry MeOH. Then Cs2CO3 (1.40 g, 4.30 mmol) is added.11 After 15 min the solvent is evaporated and the resulting solid is three times suspended in CH2Cl2 and evaporated to remove all the MeOH. The Cs-salt is then suspended in 36 mL of dry DMF under argon atmosphere and stirred with benzylbromide (1.02 mL, 1.47 g, 8.60 mmol) over night at r.t. After evaporation of the solvent in vacuo, the residue is partitioned between water and CH2Cl2. The water phase is subsequently extracted two times with EtOAc. The combined organic phases are dried (Na2SO4) and evaporated. The crude product is purified either by recrystallisation from EtOAc or by silica column chromatography (cyclohexane/EtOAc 4 : 1) to get Fmoc-threoninebenzylester (3.07 g, 7.10 mmol, 91percent, Rf = 0.17 (cyclohexane/EtOAc 3 : 1)) as a white solid. The Fmoc-threoninebenzylester (2.16 g, 5.00 mmol) is dissolved in 48 mL dry CH2Cl2 under argon atmosphere. The solution is cooled to 0°C and NEt3 (770 μL, 560 mg, 5.50 mmol) and Triisopropylsilyltriflat (1.42 mL, 1.61 g, 5.25 mmol) is added subsequently.12 The mixture is allowed to warm to r.t. and is stirred 1h. Then 30 mL of dilute K2CO3- solution is added. The phases are separated and the water phase is extracted two times with CH2Cl2. The combined organic phases are dried (Na2SO4) and evaporated. The crude product is purified by silica column chromatography (cyclohexane/EtOAc 10 : 1) to get Fmoc(OTIPS)benzylester (2.47 g, 4.20 mmol, 84percent, Rf = 0.61 (cyclohexane/EtOAc 3 : 1)) as a colourless oil. Fmoc(OTIPS)benzylester (0.87 g, 1.48 mmol) is dissolved in 7.4 mL dry CH2Cl2 under argon atmosphere. The solution is cooled to 0°C and 7.4 mL of 40percent (v/v) Piperidine solution in dry CH2Cl2 are added dropwise.13 After 15 min of stirring at 0°C, the mixture is concentrated in vacuo at 25°C to an oil and put immediately onto the silica column (cyclohexane/EtOAc 5 : 1 + 0.5percent NEt3, elution of product with 1 : 1 + 0.5percent NEt3). Yield of the title compound 17d is 0.44 g (1.20 mmol, 81percent) as a colourless oil. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
100% | Stage #1: With sodium carbonate In 1,4-dioxane; water at 20℃; Inert atmosphere Stage #2: With hydrogenchloride In water |
At r.t. a solution of FmocONSu (3.37 g, 10.0 mmol) in 25 mL of Dioxane12 is added over 1h to a solution of L-threonine (1.19 g, 10.0 mmol) and Na2CO3 (1.06 g, 10.0 mmol) in 10 mL Dioxane and 25 mL water. The resulting suspension is stirred overnight and becomes clear. The solvent is evaporated in vacuo and the product is precipitated with 1M HClaq. at pH 4, filtered, washed with water and dissolved in EtOAc. The organic phase is dried (Na2SO4) and evaporated to yield the product quantitatively as a white solid which does not need any further purification. Under argon atmosphere Fmoc-threonine (2.67 g, 7.80 mmol) is dissolved in 32 mL dry MeOH. Then Cs2CO3 (1.40 g, 4.30 mmol) is added.11 After 15 min the solvent is evaporated and the resulting solid is three times suspended in CH2Cl2 and evaporated to remove all the MeOH. The Cs-salt is then suspended in 36 mL of dry DMF under argon atmosphere and stirred with benzylbromide (1.02 mL, 1.47 g, 8.60 mmol) over night at r.t. After evaporation of the solvent in vacuo, the residue is partitioned between water and CH2Cl2. The water phase is subsequently extracted two times with EtOAc. The combined organic phases are dried (Na2SO4) and evaporated. The crude product is purified either by recrystallisation from EtOAc or by silica column chromatography (cyclohexane/EtOAc 4 : 1) to get Fmoc-threoninebenzylester (3.07 g, 7.10 mmol, 91percent, Rf = 0.17 (cyclohexane/EtOAc 3 : 1)) as a white solid. The Fmoc-threoninebenzylester (2.16 g, 5.00 mmol) is dissolved in 48 mL dry CH2Cl2 under argon atmosphere. The solution is cooled to 0°C and NEt3 (770 μL, 560 mg, 5.50 mmol) and Triisopropylsilyltriflat (1.42 mL, 1.61 g, 5.25 mmol) is added subsequently.12 The mixture is allowed to warm to r.t. and is stirred 1h. Then 30 mL of dilute K2CO3- solution is added. The phases are separated and the water phase is extracted two times with CH2Cl2. The combined organic phases are dried (Na2SO4) and evaporated. The crude product is purified by silica column chromatography (cyclohexane/EtOAc 10 : 1) to get Fmoc(OTIPS)benzylester (2.47 g, 4.20 mmol, 84percent, Rf = 0.61 (cyclohexane/EtOAc 3 : 1)) as a colourless oil. Fmoc(OTIPS)benzylester (0.87 g, 1.48 mmol) is dissolved in 7.4 mL dry CH2Cl2 under argon atmosphere. The solution is cooled to 0°C and 7.4 mL of 40percent (v/v) Piperidine solution in dry CH2Cl2 are added dropwise.13 After 15 min of stirring at 0°C, the mixture is concentrated in vacuo at 25°C to an oil and put immediately onto the silica column (cyclohexane/EtOAc 5 : 1 + 0.5percent NEt3, elution of product with 1 : 1 + 0.5percent NEt3). Yield of the title compound 17d is 0.44 g (1.20 mmol, 81percent) as a colourless oil. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
80% | With potassium carbonate In acetonitrile at 20℃; for 2 h; | General procedure: To a solution of H-Phe-OH (100 mg, 60.5 mmol) in 50 percent MeCN (6.1 mL)were added Fmoc-OPhth (233 mg, 60.5 mmol) and K2CO3 (167 mg, 121 mmol) and stirred at room temperature. After 2 h of stirring saturated sodium bicarbonate solution and H2O were added and the resulting solution was washed with diethyl ether. The aqueous phase is acidified to pH 1 with 1M HCl and extracted with diethyl ether. The organic phase was washed with 1 M HCl, H2O, brine, dried over MgSO4. The filtrate was evaporatedevaporated under reduced pressure to give yellow solid as crude product. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
67% | Stage #1: With phosphorus trichloride In tetrahydrofuran at -5 - 5℃; for 0.0833333 h; Inert atmosphere Stage #2: With 2,6-dimethylpyridine In tetrahydrofuran at -5 - 5℃; Inert atmosphere |
General Procedure for Amino Acid PhosphorylationIn the following description, a "volume" equivalent is one mL of liquid per gram of the limiting reagent (e.g., the dry amino acid).Amino acid dryingHydrated forms of amino-group-protected amino acids (hydrated specified as >0.5 wtpercent water) are dried prior to use. The amino-group-protected amino acid (1.0 equivalents of dry compound) is charged to a flask. Dry THF (4 volumes, EMD OmniSolv HPLC grade, >99.99percent) is added and the total volume is marked on the outside of the flask. The mixture is heated in a 70°C bath and THF (about 2 volumes) is removed via distillation at 700 mbar. The solution is then diluted to the original, marked volume with dry THF. This distillation and dilution cycle is repeated two additional times. The final water content of the amino acid solution should be <0.20 wtpercent by Karl-Fischer (KF) titration.Step One: Dichlorophosphite preparationTo a flask flushed with nitrogen, THF (8 volumes) is charged. The solvent is cooled to 0°C and PC13 (1.30 equivalents, Aldrich, Reagent Plus grade, 99percent) is charged to the flask at a rate that maintains the solution at 0-5 °C. When the addition is complete and the solution is below 2.5 °C, a suitable base such as pyridine (Aldrich, anhydrous, 99+percent) (1 equivalent) may optionally be added at a rate that keeps the reaction temperature at - 5°C to 5°C. This optional step is especially useful when the dichlorophosphite is prepared from tert-butanol. With the reaction temperature below 2.5°C, a suitable alcohol such as tert-butanol or benzyl alcohol (BnOH; Aldrich, ACS grade, 99+percent) (1.50 equivalents) is added at a rate that keeps the reaction at -5 to 5°C. The solution is then allowed to stir for 5 minutes at 0-5°C and the consumption of PC13 is confirmed by 31P NMR (in CDCI3). A suitable base such as 2,6-lutidine (Aldrich, >99percent) (3.00 equivalents; 2.00 equivalents if a base was added previously) is then added to the flask at a rate that keeps the reaction at -5 to 5 °C. This forms a thick slurry.Step Two: Phosphite ester intermediate preparationA suitable base such as 2,6-lutidine (1.0 equivalents) is added to the dried amino- group-protected amino acid (APG-A.A.) solution. This solution is then added to the mixture from step one at a rate that keeps the reaction at -5 to 5°C. Additional THF (1 volume) is used to rinse the flask containing the APG-A.A. solution into the reaction. HPLC assays (Method 1) are taken at 10 min intervals starting 5 minutes after the addition to confirm reaction completion (no change in the ratio of APG-A.A. to intermediate phosphite ester at 225/210 nm).Step Three: Hydrolysis to phosphite esterAfter three consistent HPLC traces are observed, H20 (3.6 volumes) is added to the flask at a rate that does not allow the reaction to go above 10°C. During the addition, a two-phase solution is formed.Step Four: Oxidation to phosphateA suitable oxidant is then added to the two-phase mixture. When the oxidant isNaBr03/NaBr, solid NaBr (2.32 equivalents, Aldrich, ACS grade, 99+percent) is added to the two-phase mixture in the reactor at 0°C; an aqueous solution (20 wtpercent in water) of NaBr03 (0.48 equivalents, Riedel-de-Haen, puriss grade, 98percent> NaBr03 in DI water) is then added at a rate that keeps the reaction at 0-5°C. After the oxidant addition is complete the cooling is removed to allow the reaction to warm to ambient temperature, being careful not to allow the reaction to exotherm above 30°C. HPLC assays (Method 1) are taken to confirm reaction completion (phosphite ester intermediate <1.5 Apercent at 225/210 nm). When reaction completion is observed an aqueous solution (10 wtpercent> in water) of Na2S205 (1 volume, Sigma- Aldrich, ACS grade, 97+percent> Na2S205 in DI water) is added to the flask in one portion.Example 2. Fmoc (BnO) Phosphothreonine.67percent yield. 1H NMR (400 MHz, DMSO): δ 7.89 (d, 2H), 7.78 (d, 2H), 7.71 (d, 1H), 7.38 (m, 9H), 4.92 (d, 2H), 4.81 (m, 1H), 4.26 (m, 4H), 1.30 (d, 3H); 13C NMR (400 MHz, DMSO): δ 170.95 (s, 1C), 156.51 (s, 1C), 143.74 (d, 2C), 140.65 (d, 2C), 136.96 (d, 1C), 128.29 (s, 2C), 127.89 (s, 1C), 127.61 (s, 2C), 127.50 (s, 2C), 127.03 (s, 2C), 125.40 (d, 2C), 120.04 (s, 2C), 73.10 (d, 1C), 67.35 (d, 1C), 66.05 (s, 1C), 58.59 (d, 1C), 46.59 (s, 1C), 18.17 (d, 1C); 31P NMR (400 MHz, DMSO): δ -2.27; HRMS: [MH+] = 512.14700 (Calc =512.14688); Specific Rotation: +5.99° |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Solid phase peptide synthesis was performed on a CEM Liberty Peptide Synthesizer using standard Fmoc chemistry. TentaGel S Ram resin (1 g; 0.25 mmol/g) was swelled in NMP (10 ml) prior to use and transferred between tube and reaction vessel using DCM and NMP. Coupling (0148) An Fmoc-amino acid in NMP/DMF/DCM (1:1:1; 0.2 M; 5 ml) was added to the resin in a CEM Discover microwave unit together with HATU/DMF or COMU/DMF (0.5 M; 2 ml) and DIPEA/NMP (2.0 M; 1 ml). The coupling mixture was heated to 75° C. for 5 min while nitrogen was bubbled through the mixture. The resin was then washed with NMP (4×10 ml). Deprotection (0149) Piperidine/DMF (20percent; 10 ml) was added to the resin for initial deprotection and the mixture was heated by microwaves (30 sec; 40° C.). The reaction vessel was drained and a second portion of piperidine/NMP (20percent; 10 ml) was added and heated (75° C.; 3 min.) again. The resin was then washed with DMF (6×10 ml). Side Chain Acylation (0150) Fmoc-Lys(ivDde)-OH or alternatively another amino acid with an orthogonal side chain protective group was introduced at the position of the acylation. The N-terminal of the peptide backbone was then Boc-protected using Boc2O or alternatively by using a Boc-protected amino acid in the last coupling. While the peptide was still attached to the resin, the orthogonal side chain protective group was selectively cleaved using freshly prepared hydrazine hydrate (2-4percent) in NMP for 2×15 min. The unprotected lysine side chain was first coupled with Fmoc-Glu-OtBu or another spacer amino acid, which was deprotected with piperidine and acylated with a lipophilic moiety using the peptide coupling methodology as described above. Alternatively, the acylation moiety was introduced as a premade building block e.g. Fmoc-Lys(hexadecanoyl-gamma-Glu)-OH where gamm-Glu is the coupling of Glutamic acid through the side-chain. Abbreviations employed are as follows: COMU: 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexaflourophosphate ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl DCM: dichloromethane DMF: N,N-dimethylformamide (0151) DIPEA: diisopropylethylamine EtOH: ethanol Et2O: diethyl ether HATU: N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide MeCN: acetonitrile NMP: N-methylpyrrolidone (0152) TFA: trifluoroacetic acid TIS: triisopropylsilane Cleavage (0153) The resin was washed with EtOH (3×10 ml) and Et2O (3×10 ml) and dried to constant weight at room temperature (r.t.). The crude peptide was cleaved from the resin by treatment with TFA/TIS/water (95/2.5/2.5; 40 ml, 2 h; r.t.). Most of the TFA was removed at reduced pressure and the crude peptide was precipitated and washed three times with diethylether and dried to constant weight at room temperature. HPLC Purification of the Crude Peptide (0154) The crude peptide was purified to greater than 90percent by preparative reverse phase HPLC using a PerSeptive Biosystems VISION Workstation equipped with a C-18 column (5 cm; 10 mum) and a fraction collector and run at 35 ml/min with a gradient of buffer A (0.1percent TFA, aq.) and buffer B (0.1percent TFA, 90percent MeCN, aq.). Fractions were analyzed by analytical HPLC and MS and relevant fractions were pooled and lyophilized. The final product was characterized by HPLC and MS. (0155) The synthesized compounds are shown in Table 1 and Table 2 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20% 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20percent 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20percent 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20% 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: tGLP-1 and its analogues 2?13 were all synthesized using general solid-phase peptide synthesis of N-Fmoc/tBu chemistry. 63Fmoc Rink Amide-MBHA resin (0.1 mmol) was added to a 25 ml peptide synthetic vessel and swollen with DMF for 40 min. After deprotected by 25percent piperidine in DMF, a solution of Fmoc-AA-OH (0.4 mmol), HATU (0.4 mmol), HoAt (0.4 mmol) and DIPEA (0.8 mmol) in DMF was added to the vessel. After reacted for 1 h, the resin was washed three times with DMF and three times with CH2Cl2, then qualitative ninhydrin testing was performed to monitor whether some free amino groups still existed on the resin ornot. If not, the resin was washed three times with DMF again and repeated the procedures of deprotection and coupling. Forthe coupling of some unnatural amino acids, NMM instead of DIPEA and NMP instead of DMF were used. Besides, the reaction time was prolonged to 4 h. Following the final deprotection of N-terminus, the target peptide was cleaved from resin with Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 2 h atroom temperature. After filtration, the residue solution was concentrated, precipitated with cold diethyl ether and centrifuged for three times. The residue was dissolved in water and purified by Waters 2545 preparative RP-HPLC system. Sephadex G-25 was used for the further purification to remove some short peptide impurities. The molecular mass of the target peptide was confirmed by MALDI-TOF. The purity of peptide was tested with analytical RP-HPLC, and the conditions were as follows: a linear gradient of 20percent mobile phase A and 80percent mobile phase B to 80percent mobile phase A and 20percent mobile phase B (A: acetonitrile containing 0.1percent TFA; B: H2O containing 0.1percent TFA) in 30 min, at a flow rate of 1 mL/minute with UV detection at 214 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1B(1) Coupling of the Trityl-Linker to the Solid Support (0337) 200 g of Aminomethyl-polystyrene resin (crosslinked with 1% divinyl benzene, loading of aminomethyl groups 1 mmol/g) (supplier: Senn Chemicals AG, Dielsdorf/Switzerland) were stirred alternately with several portions of dimethylformamide (1600 mL) and isopropanol (1600 mL) After two final washes with dimethlyformamide, the resin was treated with a previously prepared solution of 4-hydroxy-diphenylmethyl-benzoic acid (91.3 g 300 mmol), 1-hydroxy-benzotriazole monohydrate (45.9 g, 300 mmol) and diisopropylcarbodiimide (75.7 g, 600 mmol) in dimethylformamide (1600 mL). The reaction mixture was stirred for 1.5 h and a Ninhydrin test was performed. The test still showed free amino groups and thus diisopropylcarbodiimide (7.6 g, 60 mmol) was added and the reaction stirred over night. A further ninhydrin test in the morning was negative and the reaction mixture was filtered off. The resin was washed with dimethylformamide and isopropanol alternatingly. The resin was dried in vacuo and yielded 257 g of dry linker-resin. The material was used for the next synthesis step without further analysis. 1B(2) Coupling of Fmoc-Leu-OH Preparation of Fmoc-Leu-Linker-Resin (0338) Linker-Resin (190 g, 147.8 mmol) was swollen by stirring in toluene (1400 mL). The solvent was filtered off and replaced by a solution of toluene (1400 mL) and acetyl chloride (53 mL, 1478 mmol). This mixture was stirred for 2 h, filtered off, replaced by an identical mixture which was stirred for another 2 h before filtering off. The chlorinated resin was washed twice with toluene and three times with dichloromethane. (0339) In a round bottom flask, a solution of Fmoc-Leu-OH (104.8 g, 296 mmol) and of N-Methylmorpholine (49 mL, 444 mmol) in dichloromethane (600 mL) was prepared. This solution was added to the resin and stirred over night. In the morning, the solution was filtered of and the resin was washed with dichloromethane and isopropanol alternatingly. The resin 0was dried in vacuo and yielded 234.7 g of dry Fmoc-Leu-linker-Resin. The loading with Fmoc-groups was determined at 0.787 mmol/g what led to a yield of 185 mmol (125% of theory). Amino acid analysis at an external contractor confirmed 1B(3) Coupling of Fmoc-Thr-OH Preparation of Fmoc-Thr-Leu-Linker-Resin. Fmoc-Leu-Linker-Resin (140 g, 109 mmol) was swollen by stirring in two successive portions of dimethylformamide (1100 mL) for 30 min each. (0341) Fmoc protecting group was cleaved by two subsequent washings of 20% piperidine in dimethylformamide for 5 min and 15 min respectively. The resin was washed by several alternating washes with dimethylfomamide and isopropanol. Phenolphtalein and water were added to a sample of the final wash solution. The absence of pink colour confirmed successful removal of piperidine. (0342) The resin was washed with tetrahydrofurane (1200 mL) three times to prepare for the following coupling step. (0343) In a round bottomed flask a solution of Fmoc-Thr-OH (112.1 g, 328 mmol), hydroxybenzotriazole monohydrate (51.25 g, 334 mmol) and diisopropylcarbodiimide (51 mL, 655 mmol) in tetrahydrofuran (600 mL) was prepared (0344) The solution was added to the resin and the pH checked immediately (pH=6.5). The reaction mixture was stirred for 1.5 h until a ninhydrin test showed complete reaction. The solution was filtered off and the resin was washed with dimethylformamide and isopropanol alternatingly. A small sample of the resin was dried and sent for amino acid analysis (0.13% D-Leu, 1B(4) Coupling of Fmoc-Gln(Trt)-OH Preparation of Fmoc-Gln(Trt)-Thr-Leu-Linker-Resin (0345) The Fmoc-Thr-Leu-Linker-Resin from the previous step was swollen by stirring in two subsequent portions of dimethylformamide (1100 mL) for 30 min each. (0346) Fmoc protecting group was cleaved by two subsequent washings with 20% piperidine in dimethylformamide for 5 min and 15 min respectively. The resin was washed by several alternating washes with dimethylfomamide and isopropanol. Phenolphtalein and water were added to a sample of the final wash solution. The absence of pink colour proved successful removal of piperidine. (0347) The resin was washed with dimethylformamide (1100 mL) three times to prepare for the following coupling step. (0348) In a round bottomed flask a solution of Fmoc-Gln(Trt)-OH (138.6 g, 226 mmol), HATU (86.2 g, 226 mmol) and Ethyldiisopropylamine (58.4 g, 452 mmol) in dimethylformamide (400 mL) was prepared.. The solution was added to the resin and the pH checked immediately (pH=10). The reaction mixture was stirred for 3 h until a ninhydrin test showed complete reaction. The solution was filtered of and the resin was washed with dimethylformamide and isopropanol alternatingly. (0350) The resin was dried in vacuo and yielded 170 g of dry Fmoc-Gln(Trt)-Thr-Leu-Linker-Resin. The loading with Fmoc-groups was determined at 0.60 mmol/g indicating a yield of 102 mmol (94% of theory over the last two steps). Amino ... |
Yield | Reaction Conditions | Operation in experiment |
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Equipment: (0492) Peptide-synthesizer equipped with a 250 ml glass-reactor with frit and manifold for automatic solvent delivery, shaking and sucking off reagents. 2A(1) Synthesis of Fmoc-Thr-Leu-Trt-Tentagel-S (0493) Fmoc-Leu-Trt-Tentagel-S-Resin (18.7 g loading 0.37 mmol/g (supplied by Rapp Polymere GmBH, Tuebingen/Germany)) were swollen in DMF by shaking for 30 min. (0494) The Fmoc protecting group was cleaved by two subsequent treatments with 20% piperidine in DMF for 5 min and 15 min respectively. After resin washing by several alternating washes with DMF and isopropanol the complete removal of bases was checked with the absence of a pink color after Phenolphthalein and water addition to the last washing step. (0495) 4.7 g of Fmoc-Thr-OH, 5.26 g of HATU and 1.8 g of DIPEA were dissolved in 50 ml of DMF. After 5 min of stirring additional 1.8 g of DI PEA were added. After checking the pH (>11) the mixture was added to the deprotected resin and shaken for 2 h. The performed Kaiser test was OK and the resin was washed by several alternating washes with DMF and isopropanol. After drying the resin weight was 19.01 g. A small sample was cleaved and checked by HPLC. A single main peak showed a successful conversion. 2A(2) Synthesis of Fmoc-Gln(Trt)-Thr-Leu-Trt-Tentagel-S (0496) 19.01 g Fmoc-Thr-Leu-Trt-Tentagel-S-Resin (6.6 mmol) were pre-swollen in DMF and the Fmoc protecting group was cleaved by two subsequent treatments of 20% piperidine in DMF for 5 min and 15 min respectively. After resin washing by several alternating washes with DMF and isopropanol the complete removal of bases was checked with the absence of a pink color after Phenolphthalein and water addition to the last washing step. (0497) 8.07 g of Fmoc-Gln(Trt)-OH, 5.01 g of HATU and 3.4 g of DIPEA were dissolved in 50 ml of DMF. After checking the pH (>11) the mixture was added to the deprotected resin and shaken for 1.5 h. The performed Kaiser test was OK and the resin was washed by several alternating washes with DMF and isopropanol. The resulting resin was directly used in the following step below, only a small sample was cleaved and checked by HPLC. A single main peak showed successful conversion. 2A(3) Synthesis of Ac-Gln(Trt)-Thr-Leu-Trt-Tentagel-S (0498) The Fmoc-Gln(Trt)-Thr-Leu-Trt-Tentagel-S resin from above was re-swollen in DMF by shaking in DMF for 10 min. The Fmoc protecting group was cleaved by two subsequent treatments with 20% piperidine in DMF for 5 min and 15 min respectively. The resin was washed by several alternating washes with DMF and isopropanol. Phenolphthalein and water were added to a sample of the final wash solution. The absence of pink color proved successful removal of piperidine. (0499) 0.774 g of acetic-acid, 6.707 g of PyBOP and 3.33 g of DIPEA were dissolved in 50 ml of DMF. After checking the pH (>11) the mixture was added to the deprotected resin and shaken for 2.5 h. The performed Kaiser test was OK and the resin was washed by several alternating washes with DMF and isopropanol. The resulting resin was directly used in the following step below, only a small sample was cleaved and checked be HPLC. A single main peak showed successful conversion. 2A(4) Synthesis of Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S (Previously Named: Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S)(Side-Chain Esterification) 2A(4) Synthesis of Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S (Previously Named: Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S)(Side-Chain Esterification) (0500) The Ac-Gln(Trt)-Thr-Leu-Trt-Tentagel-S resin from above was re-swollen in DMF by shaking in DMF for 10 min. (0501) 8.7 g of Fmoc-Val-OH and 8.3 g of DIPEA were dissolved in 25 ml of DCM. In parallel 2.76 g of MSNT were dissolved in another 25 ml of DCM. Both solutions were combined and after 3 min pre-activation put to the peptide resin and shaken for 2 h. The resin was washed by several alternating washes with DMF and isopropanol. The resulting resin was directly used in the following step below, only a small sample was cleaved and checked be HPLC. A single main peak showed successful conversion. 2A(5) Synthesis of Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Fmoc)-Leu-Trt-Tentagel-S (Previously Named: Ac-Gln(Trt)-Thr(Val-N-me-Tyr(tBu)-Fmoc)-Leu-Trt-Tentagel-S) (0502) The Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S resin from above was re-swollen in DMF by shaking in DMF for 10 min. (0503) The Fmoc protecting group was cleaved by two subsequent treatments with 20% piperidine in DMF for 5 min and 15 min respectively. The resin was washed by several alternating washes with DMF and isopropanol. Phenolphthalein and water were added to a sample of the final wash solution. The absence of pink color proved successful removal of piperidine. (0504) 6.1 g of Fmoc-N-Me-Tyr(tBu)-OH, 4.8 g of HATU and 3.3 g of DIPEA were dissolved in 50 ml of DMF. After checking the pH (>11) the mixture was added to the deprotected resin and shaken for 2.5 h. The performed Kaiser test was OK an... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 100 mg 2-chlorotrityl resin (0.5 mmol/g) was placed in a 6 mL polypropylene syringe with a polyethylene filter in the bottom. It was swelled with DCM for 1 h. Then it was washed by DMF(3 2 mL) and DCM (3 2 mL). The first building block was added by using <strong>[118904-37-3]<strong>[118904-37-3]Fmoc-D-allo-Ile</strong>-OH</strong> (35.3 mg, 0.1 mmol) and DIEA(34.8 mL, 0.2 mmol) in 2 mL DCM and shaken for 1 h. Then 80 mL MeOH was added and shaken for another 20 min. Then the resin was washed by DCM (3 2 mL) and DMF (3 2 mL). The following amino acids were coupled through the general Fmoc-SPPS strategy. Then the resin was treated by a mixture of 3 mL DCM/AcOH/TFE(v/v/v = 8:1:1) for 1.5 h to obtain the crude linear peptide 3(36.7 mg, 0.037 mmol). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The peptide chain was elongated on CTC resin commencing with Fmoc-Dab(Boc)-O-CTCResin.20% piperidine in DMF (for de-blocking) was added with mixing for 30 minutes. The reaction was drained and washed with DMF (x5). Construction of the desired peptide sequence wascontinued using HATU (1.9-2.85 eq) and DIPEA (4-6.0 eq) in DMF (10 mL) followed by 20% piperidine in DMF for each amino acid to afford H2N-Ahx-Ahx-[L-octylGly]-Dab(Boc)- Thr(OH)-Dab(Boc)-Dab(Dde)-Dab(Boc)-[D-Phe]-Leu-Dab(Boc)-O-CTC-resin. The peptide was then treated with a mixture of DCM and DIPEA (4 eq) with Cbz-Cl (2 eq).At this point the resin was treated with 3% hydrazine hydrate in DMF to effect Ddedeprotection. The resin was washed with DMF (x5) and the peptide was further elongated as above with the required remaining amino acids. The peptide was treated with 1 %TFA/DCM (2 x 50 mL) for 2 minutes and adjusted to pH=7 with DIPEA and diluted with DCM. TBTU (2 eq) and HOBt (2 eq) were added followed by DIPEA (2 eq), and the mixture was stirred for 1 hour to effect cyclisation. The reaction was washed with 5% aqueous HCI and concentratedin vacuo to afford Cbz-Abx-Abx-[L-octylGly]- Dab(Boc)-Thr(OH)- Dab(Boc)Dab*Dab(Boc) [D- Phe]-Leu-Dab(Boc)- Dab(Boc)Thr(OH)*.The crude peptide was treated with 4:1 DMF:MeOH (50 mL) and Pd(OH)2/C (50%, 2 g) was added under nitrogen. The suspension was purged with hydrogen several times and stirred under 15 psi hydrogen for 4 hours at 35 00. The crude peptide was purified usingpreparative HPLC as described above using a gradient of between 30-70% MeCN in water(with 0.075% TFA) over 60 minutes to afford the title compound.HPLC Rt = 9.54 minutes, ES MS mlz 980 [M+2H]/2 and 930 [M-Boc+2H]/2; theoretical mass:1958 |
Tags: 73731-37-0 synthesis path| 73731-37-0 SDS| 73731-37-0 COA| 73731-37-0 purity| 73731-37-0 application| 73731-37-0 NMR| 73731-37-0 COA| 73731-37-0 structure
A987330[ 1236189-72-2 ]
(2S,3R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxybutanoic acid-15N
Reason: Stable Isotope
[ 940301-35-9 ]
(2S,3R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxy-4-methylpentanoic acid
Similarity: 0.96
[ 1217603-41-2 ]
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxy-3-methylbutanoic acid
Similarity: 0.95
[ 884880-39-1 ]
(R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxy-3-methylbutanoic acid
Similarity: 0.95
[ 158257-40-0 ]
(3S,4S)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxy-6-methylheptanoic acid
Similarity: 0.94
[ 131545-63-6 ]
(2R,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(benzyloxy)butanoic acid
Similarity: 0.92
Precautionary Statements-General | |
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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 |
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