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CAS No. : | 94744-50-0 | MDL No. : | MFCD00151913 |
Formula : | C19H19NO4 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | HOZZVEPRYYCBTO-UHFFFAOYSA-N |
M.W : | 325.36 | Pubchem ID : | 2756096 |
Synonyms : |
|
Num. heavy atoms : | 24 |
Num. arom. heavy atoms : | 12 |
Fraction Csp3 : | 0.26 |
Num. rotatable bonds : | 6 |
Num. H-bond acceptors : | 4.0 |
Num. H-bond donors : | 2.0 |
Molar Refractivity : | 90.02 |
TPSA : | 75.63 Ų |
GI absorption : | High |
BBB permeant : | Yes |
P-gp substrate : | Yes |
CYP1A2 inhibitor : | No |
CYP2C19 inhibitor : | No |
CYP2C9 inhibitor : | Yes |
CYP2D6 inhibitor : | No |
CYP3A4 inhibitor : | No |
Log Kp (skin permeation) : | -5.98 cm/s |
Log Po/w (iLOGP) : | 2.26 |
Log Po/w (XLOGP3) : | 3.24 |
Log Po/w (WLOGP) : | 3.39 |
Log Po/w (MLOGP) : | 2.55 |
Log Po/w (SILICOS-IT) : | 2.77 |
Consensus Log Po/w : | 2.84 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 0.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.56 |
Log S (ESOL) : | -3.87 |
Solubility : | 0.0436 mg/ml ; 0.000134 mol/l |
Class : | Soluble |
Log S (Ali) : | -4.5 |
Solubility : | 0.0103 mg/ml ; 0.0000315 mol/l |
Class : | Moderately soluble |
Log S (SILICOS-IT) : | -5.27 |
Solubility : | 0.00173 mg/ml ; 0.00000532 mol/l |
Class : | Moderately soluble |
PAINS : | 0.0 alert |
Brenk : | 0.0 alert |
Leadlikeness : | 0.0 |
Synthetic accessibility : | 3.46 |
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 |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido- norleucylaminomethyl resin), 100-200 mesh, loading of 0.23 mmol/g. The Fmoc- synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 Fmoc- Lys(ivDde)-OH and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin according to literature (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Hereafter Palm- yGlu-yGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5muMu) using an acetonitrile/water gradient (both buffers with 0,1 percent TFA). The purified peptide was analysed by LCMS (Method A). Deconvolution of the mass signals found under the peak with retention time 12.61 min revealed the peptide mass 4581 ,5 which is in line with the expected value of 4581 ,1 . Peptide Synthesizer (Protein Technologies Inc) or similar automated synthesizer using standard Fmoc chemistry and HBTU/DIPEA activation. DMF was used as the solvent. Deprotection : 20percent piperidine/DMF for 2 x 2.5 min. Washes: 7 x DMF. Coupling 2:5:10 200 mM AA / 500 mM HBTU / 2M DIPEA in DMF 2 x for 20 min. Washes: 5 x DMF. In cases where a Lys-side-chain was modified, Fmoc-L-Lys(ivDde)-OH or Fmoc-L- Lys(Mmt)-OH was used in the corresponding position. After completion of the synthesis, the ivDde group was removed according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4percent hydrazine hydrate in DMF. The Mmt group was removed by repeated treatment with 1 percent TFA in dichloromethane. The following acylations were carried out by treating the resin with the N-hydroxy succinimide esters of the desired acid or using coupling reagents like HBTU/DIPEA or HOBt/DIC. All the peptides that have been synthesized were cleaved from the resin with King's cleavage cocktail consisting of 82.5percent TFA, 5percent phenol, 5percent water, 5percent thioanisole, 2.5percent EDT The crude peptides were then precipitated in diethyl or diisopropyl ether, centrifuged, and lyophilized. Peptides were analyzed by analytical HPLC and checked by ESI mass spectrometry. Crude peptides were purified by a conventional preparative RP-HPLC purification procedure. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Example 1 Synthesis of SEQ ID NO: 7 (0337) The manual synthesis procedure as described in Methods was carried out on a desiccated Rink amide MBHA Resin (0.66 mmol/g). The Fmoc-synthesis strategy was applied with DIC/HOBt-activation. In position 14 <strong>[204777-78-6]Fmoc-Lys(ivDde)-OH</strong> and in position 1 Boc-His(Boc)-OH were used. The ivDde-group was cleaved from the peptide on resin according to a modified literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DMF. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 14.29 min revealed the peptide mass 4649.20 which is in line with the expected value of 4649.29. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Example 2 Synthesis of SEQ ID NO: 8 (0338) The manual synthesis procedure as described in Methods was carried out on a desiccated Rink amide MBHA Resin (0.66 mmol/g). The Fmoc-synthesis strategy was applied with DIC/HOBT-activation. In position 14 <strong>[204777-78-6]Fmoc-Lys(ivDde)-OH</strong> and in position 1 Boc-His(Boc)-OH were used. The ivDde-group was cleaved from the peptide on resin according to a modified literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DMF. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 14.05 min revealed the peptide mass 4634.80 which is in line with the expected value of 4635.27. |
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 |
---|---|---|
2.7 mg | [Example 25] (Synthesis method Y): Production of Ac-[D-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys (Hexyl)30]-PYY(23-36) (compound No. 279) Synthesis of Ac- [D-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys(Hexyl)30]-PYY(23-36) (0322) H-Aib-Thr(But)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Cha-Rink Amide resin (89.7 mg, 0.02 mmol) synthesized in Example 23 was weighed and placed in a reaction vessel, washed with DMF and, after swelling, treated with Fmoc-Lys(Mtt)-OH (125.0 mg, 0.2 mmol), 0.5 M HOAt/DMF solution (0.4 mL, 0.2 mmol), DIPCDI (31.8 muL, 0.2 mmol) for 15 hr to introduce Lys(Mtt) residue. The obtained resin was washed with toluene, treated with TFA-triisopropylsilane-trifluoroethanol-toluene (1:5:47:47) for 10 min, and an operation to remove the reaction solution was repeated until the solution was no longer colored. The resin was washed with toluene, neutralized by washing with 5% DIEA-toluene solution, and washed with DMF. The obtained resin was suspended in DMF, 1-hexanal (2.5 muL, 0.04 mmol) was added in the presence of acetic acid (50 muL) and the mixture was stirred for 15 min. NaBH3CN (6.2 mg, 0.1 mmol) was added, and the mixture was further stirred for 1.5 hr. The resin was washed with DMF, and treated with Boc2O (24.4 mg, 0.2 mmol), DIEA (34.8 muL, 0.2 mmol) in DMF at room temperature for 3 hr. The progress of the reaction was confirmed by Kaiser test, and Asn(Trt), Aib, Cha, Pya(4), Arg(Pbf), D-Pro, Ser(But) were successively introduced by manual solid phase synthesis process including repeats of removal of Fmoc by 20% piperidine/DMF treatment and condensation by a treatment with Fmoc-amino acid (0.1 mmol), 0.5 M HOAt/DMF (0.2 mL, 0.1 mmol), DIPCDI (16 muL, 0.1 mmol). Then, after removal of Fmoc, the resin was treated with Ac2O (9.4 muL), DIEA (17.4 muL) in DMF for 30 min for acetylation, and the obtained resin was washed with MeOH and dried. (0323) The obtained resin (103.9 mg) was treated with TFA: thioanisole: m-cresol: HO: EDT: TIS (80:5:5:5:2.5:2.5) (1 mL) for 90 min, an operation to add diethyl ether to the reaction solution, precipitate a white powder by centrifugation, and remove diethyl ether by decantation was repeated twice. The residue was dissolved in aqueous acetic acid solution, passed through a disc filter with a pore diameter 0.45 mum to remove fine granules, and purified by preparative HPLC using Daisopak-SP100-5-ODS-P 2×25 cm, and Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, flow rate 8 mL/min, A/B: 73/27-63/37 linear density gradient elution (60 min) was performed. The eluted object product was fractionated in test tubes, and each fraction was analyzed by HPLC to specify fractions containing only the object product. They were combined and freeze-dried to give 2.7 mg of a white powder. MALDI-TOF-MS analysis, (M+H)1891.9 (Calculated 1892.2) HPLC elution time: 9.6 min elution condition (HPLC mode d): column: Merck Chromolith Performance RP-18e(4.6×100 mm I.D.) eluent: using Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, A/B: 80/20 - 30/70 linear concentration gradient elution (25 min) flow rat: 1.0 mL/min |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
4.1 mg | The obtained resin (89.7 mg, 0.02 mmol) was weighed and placed in a reaction vessel, washed with DMF and, after swelling, treated with Fmoc-Lys(Mtt)-OH (125.0 mg, 0.2 mmol), 0.5 M HOAt/DMF solution (0.4 mL, 0.2 mmol), DIPCDI (31.8 muL, 0.2 mmol) for 15 hr to introduce Lys(Mtt) residue. The obtained resin was washed with toluene, treated with TFA-triisopropylsilane-trifluoroethanol-toluene (1:5:47:47) for 10 min, and an operation to remove the reaction solution was repeated until the solution was no longer colored. The resin was washed with toluene, neutralized by washing with 5% DIEA-toluene solution, washed again with toluene. To the obtained resin was added N,N-bis-Boc-1-guanylpyrazole (31.0 mg, 0.1 mmol), DIEA (17.4 muL, 0.1 mmol), in toluene:TFE (3:1), and treated at room temperature overnight. The progress of the reaction was confirmed by Kaiser test, and Asn(Trt), Aib, Cha, Pya(4), Arg(Pbf), D-Pro, Ser(Bu) were successively introduced by manual solid phase synthesis process including repeats of removal of Fmoc by 20% piperidine/DMF treatment and condensation by a treatment with Fmoc-amino acid (0.1 mmol), 0.5 M HOAt/DMF (0.2 mL, 0.1 mmol), DIPCDI (16 muL, 0.1 mmol). Then, after removal of Fmoc, the resin was treated with AcO (9.4 muL), DIEA (17.4 muL) in DMF for 30 min for acetylation, and the obtained resin was washed with MeOH and dried. The obtained resin (87.1 mg) was treated with TFA: thioanisole: m-cresol: HO: EDT: TIS (80:5:5:5:2.5:2.5) (1 mL) for 90 min, an operation to add diethyl ether to the reaction solution, precipitate a white powder by centrifugation, and remove ether by decantation was repeated twice. The residue was dissolved in aqueous acetic acid solution, passed through a disc filter with a pore diameter 0.45 mum to remove fine granules, and purified by preparative HPLC using Daisopak-SP100-5-ODS-P 2×25 cm, and Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, flow rate 8 mL/min, A/B: 78/22-68/32 linear concentration gradient elution (60 min) was performed. The eluted object product was fractionated in test tubes, and each fraction was analyzed by HPLC to specify fractions containing only the object product. They were combined and freeze-dried to give 4.1 mg of a white powder. MALDI-TOF-MS analysis, (M+H)1850.3 (Calculated 1850.1) HPLC elution time: 7.4 min elution condition (HPLC mode d): column: Merck Chromolith Performance RP-18e(4.6×100 mm I.D.) eluent: using Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, A/B: 80/20 - 30/70 linear concentration gradient elution (25 min) flow rate: 1.0 mL/min |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
5.1 mg | [Example 24] (Synthesis method X): Production of Ac-[D-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys(Ac)30]-PYY(23-36) (compound No. 278) Synthesis of Ac-[D-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys(Ac)30]-PYY (23-36) (0321) H-Aib-Thr (But) -Arg (Pbf) -Gln(Trt)-Arg(Pbf)-Cha-Rink Amide resin (89.7 mg, 0.02 mmol) synthesized in Example 23 was weighed and placed in a reaction vessel, washed with DMF and, after swelling, treated with Fmoc-Lys(Mtt)-OH (125.0 mg, 0.2 mmol), 0.5 M HOAt/DMF solution (0.4 mL, 0.2 mmol), DIPCDI (31.8 muL, 0.2 mmol) for 15 hr to introduce Lys(Mtt) residue. The obtained resin was washed with toluene, treated with TFA-triisopropylsilane-trifluoroethanol-toluene (1:5:47:47) for 10 min, and an operation to remove the reaction solution was repeated until the solution was no longer colored. The resin was washed with toluene, neutralized by washing with 5% DIEA-toluene solution, washed with DMF, and the obtained resin was treated overnight with AcO (9.4 muL), DIEA (17.4 muL) in DMF at room temperature. The progress of the reaction was confirmed by Kaiser test, and Asn(Trt), Aib, Cha, Pya(4), Arg(Pbf), D-Pro, Ser(Bu) were successively introduced by manual solid phase synthesis process including repeats of removal of Fmoc by 20% piperidine/DMF treatment and condensation by a treatment with Fmoc-amino acid (0.1 mmol), 0.5 M HOAt/DMF (0.2 mL, 0.1 mmol), DIPCDI (16 muL, 0.1 mmol). Then, after removal of Fmoc, the resin was treated with AcO (9.4 muL), DIEA (17.4 muL) in DMF for 30 min for acetylation, and the obtained resin was washed with MeOH and dried. The obtained resin (102.8 mg) was treated with TFA: thioanisole: m-cresol: HO: EDT: TIS (80:5:5:5:2.5:2.5) (1 mL) for 90 min, an operation to add diethyl ether to the reaction solution, precipitate a white powder by centrifugation and remove ether by decantation was repeated twice. The residue was dissolved in aqueous acetic acid solution, passed through a disc filter with a pore diameter 0.45 mum to remove fine granules, and purified by preparative HPLC using Daisopak-SP100-5-ODS-P 2×25 cm, and Solution A: 0.1% TFA-water, Solution B:. 0.1% TFA-containing acetonitrile, flow rate 8 mL/min, A/B: 77/23-67/33 linear concentration gradient elution (60 min) was performed. The eluted object product was fractionated in test tubes, and each fraction was analyzed by HPLC to specify fractions containing only the object product. They were combined and freeze-dried to give 5.1 mg of a white powder. MALDI-TOF-MS analysis, (M+H)1850.2 (Calculated 1850.1) HPLC elution time: 8.0 min elution condition (HPLC mode d): column: Merck Chromolith Performance RP-18e(4.6×100 mm I.D.) eluent: using Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, A/B: 80/20 - 30/70 linear concentration gradient elution (25 min) flow rate: 1.0 mL/min |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
515 mg | Synthesis of tetrahydro-2H-pyran-4-ylcarbamoyl-[D-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]-PYY(23-36) (0317) H-Asn(Trt)-Lys(Boc)-Aib-Thr(But)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Cha-Sieber Amide resin (0.289 mmol/g, 1.73 g, 0.5 mmol) obtained by condensing amino acids in the same manner as in Example 12 and using Sieber Amide resin as a starting material, ABI433A peptide synthesizer DCC/HOBt 0.25 mmol protocol was swollen with DMF. Then, the resin was treated with Fmoc-Aib-OH (651 mg, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol) for 1.5 hr. Fmoc group was removed with 20% piperidine in DMF, and the resin was treated with Fmoc-Cha-OH (787 mg, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol) for 1.5 hr. The resin was washed, and further treated overnight with Fmoc-Cha-OH (787 mg, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol). The resin was washed, and subjected to a capping treatment with decanoic anhydride (737 muL, 2. 0 mmol), DIEA (348 muL, 2.0 mmol) in DMF for 30 min. Fmoc group was removed with 20% piperidine in DMF, and the resin was treated with Fmoc-Pya(4)-OH (767 mg, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIEA (348 muL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol) for 2.5 hr. The resin was washed with DMF, Fmoc group was removed with 20% piperidine in DMF, and the resin was treated with Fmoc-Arg(Pbf)-OH (1.30 g, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol) for 2 hr. The resin was washed, and subjected to a capping treatment with decanoic anhydride (737 muL, 2.0 mmol), DIEA (348 muL, 2.0 mmol) in DMF for 30 min. Fmoc group was removed with 20% piperidine in DMF, and the resin was treated with Fmoc-D-Pro-OH (674 mg, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol) for 12 hr. The resin was washed, and further treated overnight with Fmoc-D-Pro-OH (674 mg, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol). The resin was washed, and subjected to a capping treatment with decanoic anhydride (737 muL, 2.0 mmol), DIEA (348 muL, 2.0 mmol) in DMF for 30 min. The resin was washed with DMF, Fmoc group was removed with 20% piperidine in DMF, and the resin was treated with Fmoc-Ser(But)-OH (767 mg, 2.0 mmol), HOAt in DMF (0.5 M, 4.0 mL, 2.0 mmol), DIPCDI (318 muL, 2.0 mmol) for 2 hr. The resin was washed successively with DMF, MeOH, and dried under reduced pressure. The total amount of the obtained H-Ser(But)-D-Pro-Arg(Pbf)-Pya(4)-Cha-Aib-Asn(Trt)-Lys(Boc)-Aib-Thr(But)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Cha-Sieber Amide resin was swollen again with DMF, and treated with CDI (405 mg, 2.5 mmol), DIEA (436 muL, 2.5 mmol) in DMF for 2 hr. The resin was washed with DMF, and treated overnight with 4-aminotetrahydropyrane (404 mg, 4.0 mmol) in DMF. The resin was washed successively with DMF, MeOH, dried under reduced pressure and the total amount of the obtained resin was suspended in TFA: thioanisole: m-cresol: H2O: EDT: TIS (80:5:5:5:2.5:2.5) (15 mL), and the mixture was stirred at room temperature for 4 hr. The reaction solution was added to stirring diethyl ether under ice-cooling while removing the resin by a filter to obtain precipitation, and an operation to remove the supernatant after centrifugation was repeated 3 times. The residue was extracted with 50% aqueous acetic acid solution, passed through a disc filter with a pore diameter 0.45 mum to remove fine granules, and purified in 9 portions by HPLC. The HPLC conditions were YMC Pack R&D-ODS-5-B S-5 120A column (30x250 mm), Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, flow rate 15 mL/min, A/B: 77/23-67/33 linear concentration gradient elution (60 min). Each fraction was analyzed by HPLC to specify fractions containing only the object product. The fractions with low purity obtained by the first purification were concentrated, and subjected to HPLC separation in 2 portions under the same conditions. All the fractions containing only the object product were combined and freeze-dried to give 685 mg of a white powder. The obtained purified sample (685 mg) was dissolved in CHCN/HO (15/30 mL), and AG 1x8 AcO resin (7.54 mL, 9.05 mmol equivalents) was added. The solution was stood for 1 hr while occasionally stirring with hand, passed through a disc filter with a pore diameter 0.45 mum to remove fine granules, concentrated in an evaporator to reduce the liquid amount to about 5 mL, and the solution was freeze-dried to give 515 mg of a white powder. MALDI-TOF-MS analysis, (M+H)1893.2 (Calculated 1893.1) HPLC elution time: 7.5 min elution condition (HPLC mode g): column: SHISEIDO CAPCELL PAK C18 MGII(4.6×100 mm) eluent: using Solution A: 0.1% TFA-water, Solution B: 0.1% TFA-containing acetonitrile, A/B: 80/20 - 30/70 linear concentration gradient elution (25 min) flow rate: 1.0 mL/min |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The solid phase peptide syntheses were performed on a Prelude Peptide Synthesizer (Protein Technologies Inc) using standard Fmoc chemistry and HBTU/DIPEA activation. DMF was used as the solvent. Deprotection: 20% piperidine/DMF for 2×2.5 min. Washes: 7×DMF. Coupling 2:5:10 200 mM AA/500 mM HBTU/2M DIPEA in DMF 2× for 20 min. Washes: 5×DMF. In cases where a Lys-side-chain was modified, Fmoc-L-Lys(ivDde)-OH was used in the corresponding position. After completion of the synthesis, the ivDde group was removed according to a literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). The following acylations were carried out by treating the resin with the N-hydroxy succinimide esters of the desired acid or using coupling reagents like HBTU/DIPEA or HOBt/DIC. (0270) All the peptides that had been synthesized were cleaved from the resin with King's cleavage cocktail consisting of 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% EDT. The crude peptides were then precipitated in diethyl or diisopropyl ether, centrifuged, and lyophilized. Peptides were analyzed by analytical HPLC and checked by ESI mass spectrometry. Crude peptides were purified by a conventional preparative HPLC purification procedure. Example 2 The solid phase synthesis was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.34 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire, Prep C18) using an acetonitrile/water gradient (both buffers with 0.1% TFA. Finally, the molecular mass of the purified peptide was confirmed by LC-MS. M.W. (calculated)=4259.6 g/mol; M.W. (found)=4259.0 g/mol. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Example 1: Synthesis of SEQ ID NO: 12 (0213) The solid phase synthesis was carried out on Novabiochem Rink-Amide resin (4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.34 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 1 Boc-Tyr(tBu)-OH and in position 14 <strong>[204777-78-6]Fmoc-Lys(ivDde)-OH</strong> were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DMF. Hereafter Palm-Glu(gammaOSu)-OtBu was coupled to the liberated amino-group. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire, Prep C18) using an acetonitrile/water gradient (both buffers with 0.05% TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 13.25 min revealed the peptide mass 4490.46 which is in line with the expected value of 4491.12. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Example 3: Synthesis of SEQ ID NO: 14 (0215) The solid phase synthesis was carried out on Rink-resin with a loading of 0.38 mmol/g, 75-150 mum from the company Agilent Technologies. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 1 Boc-Tyr(tBu)-OH and in position 14 <strong>[204777-78-6]Fmoc-Lys(ivDde)-OH</strong> were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin according to literature (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Hereafter Palm-Glu(gammaOSu)-OtBu was coupled to the liberated amino-group employing the coupling reagents HBTU/DIPEA followed by Fmoc-deprotection with 20% piperidine in DMF. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5muM) using an acetonitrile/water gradient (both buffers with 0,1% TFA). The purified peptide was analysed by LCMS (Method C). Deconvolution of the mass signals found under the peak with retention time 27.09 min revealed the peptide mass 4503.6 which is in line with the expected value of 4503.18. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Example 2: Synthesis of SEQ ID NO: 31 (0214) The solid phase synthesis was carried out on Novabiochem Rink-Amide resin (4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.34 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 1 Boc-Tyr(tBu)-OH and in position 14 <strong>[204777-78-6]Fmoc-Lys(ivDde)-OH</strong> were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DMF. Hereafter Stea-Glu(gammaOSu)-OtBu was coupled to the liberated amino-group. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire, Prep C18) using an acetonitrile/water gradient (both buffers with 0.05% TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 12.78 min revealed the peptide mass 4603.59 which is in line with the expected value of 4604.24. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: All peptides were synthesized in the same manner following thesynthesis procedure for 4-imidazolecarbonyl-[D-Hyp24,Iva25,Pya(4)26,Cha27,36,cMeLeu28,Lys30,Aib31]PYY(23-36) (31). Using a commerciallyavailable Sieber amide resin (391 mg, 0.25 mmol) as astarting material and the ABI 433A peptide synthesizer (DCC/HOBt0.25-mmol protocol), amino acids were successively condensed togive H-Asn(Trt)-Lys(Boc)-Aib-Thr(tBu)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Cha-NH-Sieber Amide Resin (903 mg, 0.289 mmol/g). A 34.6-mg(0.01 mmol) aliquot of the obtained resin was weighed, washedwith DMF, and after swelling, treated with Fmoc-cMeLeu-OH(18.4 mg, 0.05 mmol), DIPCDI (8.0 lL, 0.05 mmol), and 0.5 MHOAt/DMF (0.1 mL, 0.05 mmol) in DMF for 90 min to introducecMeLeu residue on position 28. The resin was treated with 20%piperidine/DMF to remove N-terminal Fmoc group, then Cha wasintroduced on position 27 in the same manner. Pya(4), Iva,D-Hyp, Ser(tBu) and N-terminal 1-trityl-1H-imidazole-4-carboxylicacid were introduced by repeating the same steps. The resin waswashed with DMF, methanol, and dried to give 1-trityl-1Himidazole-4-carbonyl-Ser(tBu)-D-Hyp-Iva-Pya(4)-Cha-cMeLeu-Asn(Trt)-Lys(Boc)-Aib-Thr(tBu)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Cha-Sieberamide resin (43.0 mg, 0.01 mmol). Trifluoroacetic acid (TFA):thioanisole:m-cresol:H2O:1,2-ethanedithiol:triisopropylsilane (80:5:5:5:2.5:2.5) (0.4 mL) was added to the entire amount of the obtainedresin, then the mixture was stirred at ambient temperature for90 min, and diethyl ether was added to the reaction solution to allowprecipitation of a white powder. Diethyl ether was removed bydecantation after centrifugation of the suspension, and the procedurewas repeated to remove acid and the scavenger. The residuewas extracted with an aqueous acetic acid solution and purified bypreparative HPLC using a Daisopak-SP100-5-ODS-P column(250 20mmi.d.) to give 5.1 mgof a white powder. Mass spectrum:MALDI-TOF (a-cyano-4-hydroxycinnaminic acid, monoisotopic) [M+H]+ 1861.25 (calcd. 1861.09). Elution time on RP-HPLC: 6.43 min.Elution conditions: a Phenomenex Kinetex XB-C18 column(1.7 mm, 100 2.1 mm i.d.), linear density-gradient elution witheluents A/B = 95/5-45/55 (10 min) using 0.1% TFA in water as eluentA and 0.1% TFA-containing acetonitrile as eluent B; flow rate:0.5 mL/min. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 350 mg of the beads (1.0 x 106 beads) were split equally to distribute into15 reaction vessels and the beads in each reaction vessel (23 mg, 6.6 x 104 beads) was treated with 6 equivalent ofFmoc-aa-OH (36 mumol), 6 equivalent of Oxyma (36 mumol, 5.1 mg), and 6 equivalent of DIC (36 mumol, 5.7 muL) in 182muL of DMF subjected to coupling of one of the following 15 amino acids; Fmoc-D-Ala-OH, Fmoc-D-Asp-OH,Fmoc-D-Glu-OH, Fmoc-D-Phe-OH, Fmoc-D-His(Trt)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH,Fmoc-D-Asn(Trt)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Ser(OtBu)-OH, Fmoc-D-Thr(OtBu)-OH, Fmoc-D-Val-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. The coupling reaction was carried out at RT for2-4 h with gentle shaking. The beads were washed with DMF three times. The beads were pooled and mixed. Fmocgroup was deprotected by exposing resin to 20% piperidine in DMF. First resin was treated with 20% piperidine in DMFfor 3 min then with fresh 20% piperidine in DMF for 12 min. The beads were washed with DMF three times. Thisprocedure of split, coupling, mix, and Fmoc deprotection was repeated 12 times to synthesize 12-mer random peptides.For X3, X7, X11 residues, beads were split into 7 reaction vessels and the following seven amino acids, instead of theabovementioned 15 remino acids, were used for coupling; Fmoc-D-Ala-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Val-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Tyr(OtBu)-OH, and Fmoc-Aib-OH. After completion of the 12thresidue, beads were mixed and stored at -20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The peptide component of Compound 1 is synthesized by automated solid-phase synthesis using Fluorenylmethyloxycarbonyl (Fmoc)/tert-Butyl (t-Bu) chemistry on a Symphony 12-channel multiplex peptide synthesizer (Protein Technologies, Inc. Tucson, Ariz.). The synthesis resin consists of 1% DVB cross-linked polystyrene (Fmoc-Rink-MBHA Low Loading resin, 100-200 mesh, EMD Millipore, Temecula, Calif.) at a substitution 0.3-0.4 meq/g. Standard side-chain protecting groups are as follows: tert-butyloxycarbonyl (Boc) for Trp and Lys; tert-butyl ester (OtBu) for Asp and Glu; tBu for Ser, Thr and Tyr; and triphenylmethyl (Trt) for Gln; N-alpha-Fmoc-N--4-methyltrityl-L-lysine (Fmoc-Lys(Mtt)-OH) was used for the lysine at position 20 of SEQ ID NO: 3 and Nalpha,N(im)-di-Boc-L-histidine (Boc-His(Boc)-OH) was used for the histidine at position 1. Fmoc groups were removed prior to each coupling step (2×7 minutes) using 20% piperidine in dimethylformamide (DMF). All standard amino acid couplings are performed for 1 hour, using an equal molar ratio of Fmoc amino acid (EMD Millipore, Temecula, Calif.), diisopropylcarbodiimide (DIC)(Sigma-Aldrich, St. Louis, Mo.) and Oxyma (Oxyma Pure, Iris Biotech, Marktredwitz, Germany), at a 9-fold molar excess over the theoretical peptide loading and at a final concentration of 0.18 M in DMF. Two exceptions are the glutamine residue at position 3 of SEQ ID NO: 5, which is double-coupled (2×1 hour), and the histidine residue at position 1 of SEQ ID NO: 5, which was coupled at a 6-fold molar excess using 1-Hydroxy-7-azabenzotriazole (HOAt) instead of Oxyma for 18 hours. After completion of the synthesis of the linear peptide, the resin was transferred to a disposable fritted 25 mL polypropylene syringe (Torviq, Niles, Mich.) equipped with a polytetrafluoroethylene (PTFE) stopcock (Biotage, Charlotte, N.C.) and the 4-Methyltrityl (Mtt) protecting group on the lysine at position 20 of SEQ ID NO: 5 was selectively removed from the peptide resin using three treatments with 20% hexafluoroisopropanol (Oakwood Chemicals, West Columbia, S.C.) in DCM (2×10 minutes and 1×45 minutes) to expose the free epsilon amine of the lysine at position 20 and make it available for further reaction. Subsequent attachment of the fatty acid-linker moiety is accomplished by performing two succeeding couplings of [2-(2-(Fmoc-amino)ethoxy)ethoxy]acetic acid (Fmoc-AEEA-OH) (ChemPep, Inc. Wellington, Fla.; 3-fold excess of amino acid (AA):1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU): N,N-diisopropylethylamine (DIPEA) [1:1:5 mol/mol] for a 3 hour coupling time), followed by coupling of Fmoc-glutamic acid alpha-t-butyl ester (Fmoc-Glu-OtBu)(Ark Pharm, Inc. Libertyville Ill., 3-fold excess of AA:HATU:DIPEA [1:1:5 mol/mol] for a 3 hour coupling time). In each case, the Fmoc moiety is removed as described above. Finally, mono-OtBu-octadecanedioic acid (WuXi AppTec, Shanghai, China) is coupled to the resin over 18 hours using a 3-fold excess of acid:HATU:DIPEA (1:1:5 mol/mol). After the synthesis is complete, the peptide resin is washed with dichloromethane (DCM), diethyl ether and thoroughly air dried by applying vacuum suction to the syringe for 5 minutes. The dry resin is treated with a cleavage cocktail (trifluoroacetic acid (TFA): anisole: water: triisopropylsilane, 88:5:5:2 v/v) for 2 hours at room temperature to release the peptide from the solid support and remove all side-chain protecting groups. The resin is filtered off, washed twice with neat TFA, and the combined filtrates are treated with cold diethyl ether to precipitate the crude peptide. The peptide/ether suspension is then centrifuged at 4000 rpm to form a solid pellet, the supernatant is decanted, and the solid pellet is triturated with ether two additional times and dried in vacuo. The crude peptide is solubilized in 20% acetonitrile/water and purified by RP-HPLC on a C8 preparative column (Luna 21×250 mm, Phenomenex, Torrance, Calif.) with linear gradients of acetonitrile and water using three different buffer systems: 1) 0.1 M ammonium acetate in water, pH 5.0; 2) 0.1% TFA in water; and 3) 5% acetic acid in water. Subsequent lyophilization of the final main product pool yields the lyophilized peptide acetate salt. In a synthesis performed essentially as described above, the purity of Compound 1 is assessed using analytical RP-HPLC and found to be >97%. The molecular weight is determined by analytical electrospray MS. The molecular weight of Compound 1 is calculated to be 4535.0 Daltons while the observed deconvoluted averaged molecular weight was determined to be 4535.0 Daltons and the following ions were observed: 1512.3 (M+3H), 1134.3 (M+4H), 908 (M+5H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. In an analogous way, the other peptides listed in Table 3 were synthesized and characterized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0374) Deconvolution of the mass signals found under the peak with retention time 9.935 min revealed the peptide mass 4853.73 which is in line with the expected value of 4853.67. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. In an analogous way, the other peptides listed in Table 3 were synthesized and characterized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. In an analogous way, the other peptides listed in Table 3 were synthesized and characterized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 ODB 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 9.824 min revealed the peptide mass 4839.67 which is in line with the expected value of 4839.67. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2?,4?-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 <strong>[159857-60-0]Fmoc-Lys(Mmt)-OH</strong> and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 OBD 5 mum 50×150 mm) using an acetonitrile/water gradient (both buffers with 0.1percent TFA). The purified peptide was analysed by LCMS (Method B). (0376) Deconvolution of the mass signals found under the peak with retention time 9.828 min revealed the peptide mass 4894.63 which is in line with the expected value of 4894.64. In an analogous way, the other peptides listed in Table 3 were synthesized and characterized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
53.2 mg | H-Pro-Ser(tBu)-Ser(tB u)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Arg(Pbf)-Sieber amide resin (0.394 mmol/g, 127 mg) prepared in Reference Example 3 was added to a reaction tube, which was then set in a peptide synthesizer. Amino acids were sequentially condensed according to the protocol using 20% piperidine/NMP [50C, 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDI/Oxyma [50C, 15 minutesj to condense the Fmoc-amino acids. After elongation, the resin was washed with MeOH and dried under reduced pressure to thereby obtain the protected peptide resin of interest BocMe-Tyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Iva-Ile-Ser(tBu)-Asp(OtB u)-Tyr(tB u)-S er(tBu)-Ile-Ala-Leu-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-As n(Trt)-Trp(Boc)-Leu-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tB u)-Gly-Ala-ProPro-Pro-Ser(tBu)-Arg(Pbf)-Sieber amide resin. To the total amount of the obtained resin, 4 mL of TFA:m-cresol:thioanisole :ethandithiol:H20 :triisopropylsilane (80:5:5:5:2.5:2.5) was added and the resulting mixture was stuffed for 1.5 hours. The operation wherein diethyl ether was added to the reaction solution to obtain a precipitate and after centrifugation the supernatant was removed was repeated twice and thereby washed the precipitate. The residue was extracted with a 50% acetic acid aqueous solution and after removal of the resin by filtration, the purification was carried out by preparative HPLC using a daisopak SP-100-5-ODS-P 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: 62/3 8 to 52/48, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 81 mg of a white powder.The total amount of the obtained powder was dissolved in a small amount of an acetonitrile aqueous solution. An ion exchange resin (AG 1 X8 resin (acetate form), 1.2 meq/mL, 78 1iL) was added to the solution, which was then allowed to stand for 1 hour while occasionally shaken. After removal of the resin by filtration, the filtrate was freeze-dried to thereby obtain 53.2 mg of a white powder.Mass spectrometry result: (M+H)÷ 4345.1 (calculated 4344.3)HPLC elution time: 7.7 minutesElution conditions:Column Merck Chromolith Performance RP-18e (4.6 x 100 mm I.D.)Eluents: Using solution A: 0.1% TFA-water, solution B: 0.1% TFA-containing acetonitrile, A/B: 95/5 to 35/65. Linear concentration gradient elution (10 minutes).Flow rate: 3.0 mL/minutes |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
62.8 mg | H-Pro-Ser(tB u)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tB u)-Arg(Pbf)-Sieber amide resin (0.394 mmol/g, 127 mg) prepared in Reference Example 3 was added to a reaction tube, which was then set in a peptide synthesizer. Amino acids were sequentially condensed according to the protocol using 20% piperidine/NMP [50C, 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDI/Oxyma [50C, 15 minutesj to condense the Fmoc-amino acids. After elongation, the resin was washed with MeOH and dried under reduced pressure to thereby obtain the protected peptide resin of interest BocMe-Tyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Iva-Ile-Ser(tBu)-Asp(OtB u)-Tyr(tB u)-Ser( tB u)-Ile-Aib-Leu-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn( Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Arg(Pbf)-Sieber amide resin. To the total amount of the obtained resin, 4 mL of TFA:m-cresol:thioanisole :ethandithiol:H20 :triisopropylsilane (80:5:5:5:2.5:2.5) was added and the resulting mixture was stuffed for 1.5 hours. The operation wherein diethyl ether was added to the reaction solution to obtain a precipitate and after centrifugation the supernatant was removed was repeated twice and thereby washed the precipitate. The residue was extracted with a 50% acetic acid aqueous solution and after removal of the resin by filtration, the purification was carried out by preparative HPLC using a daisopak SP-100-5-ODS-P 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: 61/39 to 51/49, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 71.1 mg of a white powder.The total amount of the obtained powder was dissolved in a small amount of an acetonitrile aqueous solution. An ion exchange resin (AG 1 X8 resin (acetate form), 1.2 meq/mL, 68 1iL) was added to the solution, which was then allowed to stand for 1 hour while occasionally shaken. After removal of the resin by filtration, the filtrate was freeze-dried to thereby obtain 62.8 mg of a white powder.Mass spectrometry result: (M+H) 4345.0 (calculated 4344.3)HPLC elution time: 7.6 minutesElution conditions:Column Merck Chromolith Performance RP-18e (4.6 x 100 mm I.D.)Eluents: Using solution A: 0.1% TFA-water, solution B: 0.1% TFA-containing acetonitrile, A/B: 95/5 to 35/65. Linear concentration gradient elution (10 minutes). Flow rate: 3.0 mL/minutes |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
170 mg | Sieber amide resin (0.71 mmol/g, 352 mg) was added to a reaction tube, which was then set in a peptide synthesizer. Amino acids were sequentially condensed according to the protocol using 20% piperidine/NMP [50C, 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDI/Oxyma [50C, 15 minutesj to condense the Fmoc-amino acids. After elongation, the resin was washed with MeOH and dried under reduced pressure to thereby obtain the protected peptide resin of interest BocMe-Tyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Phe-Ile-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser (tBu)-Ile-Ala-Leu-Asp(OtB u)-Arg(Pbf)-Aib-His(Trt)-Gln(Trt)-Aib-Asn(Trt)-Phe-ValAsn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-Gly-Ala-Pr o-Pro-Pro-Ser(tBu)-Arg(Pbf)-Sieber amide resin. To the total amount of the obtained resin, 4 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 at room temperature. The operation wherein diethyl ether was added to the reaction solution to obtain a precipitate and after centrifugation the supernatant was removed was repeated twice and thereby washed the precipitate. The residue was extracted with a 50% acetic acid aqueous solution and after removal of the resin by filtration, the purification was carried out by preparative HPLC using a daisopak SP-100-5-ODS-P 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: 69/31 to 59/41, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 190.8 mg of a white powder.The total amount of the obtained powder was dissolved in a small amount of an acetonitrile aqueous solution. An ion exchange resin (AG 1 X8 resin (acetate form), 1.2 meq/mL, 179 1iL) was added to the solution, which was then allowed to stand for 1 hour while occasionally shaken. After removal of the resin by filtration, the filtrate was freeze-dried to thereby obtain 170.0 mg of a white powder.Mass spectrometry result: (M+H) 4443.3 (calculated 4444.3)HPLC elution time: 6.5 minutesElution conditions:Column Merck Chromolith Perfomiance RP-18e (4.6 x 100 mm I.D.)Eluents: Using solution A: 0.1% TFA-water, solution B: 0.1% TFA-containing acetonitrile, A/B: 95/5 to 35/65. Linear concentration gradient elution (10 minutes).Flow rate: 3.0 mL/minutes |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70.1 mg | H-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tB u)-Sieber amide resin (SEQ ID NO: 481) (260.4 mg, 0.05 mmol) synthesized in Reference Example A was weighed into a reaction tube, which was then set in a peptide synthesizer. Amino acids were sequentially extended according to the protocol using 20% piperidine/NMP [reacted at 50C for 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDI/Oxyma [reacted at 50C for 15 minutesj to condense the Fmoc-amino acids. In this case, the condensation reaction of Boc-MeTyr(tBu) at position 1, Thr(tBu) at position 5, Ile at position 12, Arg(Pbf) at position 16, Gln(Trt)position 19, and Trp(Boc) at position 25 was carried out at 50C for 30 minutes. The operation wherein the obtained BocMeTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tB u)-Iva-Ile-Ser(tB u)-Asp(OtBu)-Tyr(tBu)-Ser(tu)-Ile-Aib-Lys(ivDde)-Asp(OtB u)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pr o-Pro-Pro-Ser(tBu)-Sieber amide resin was suspended in a 2% hydrazine/NMP solution, the resulting suspension was stirred at 50C for 10 minutes, and then the solution was removed by filtration was repeated 8 times to deprotect the ivDde group of Lys at position 14. Subsequently, Fmoc-Gly-Gly-Gly-OH was introduced using the peptide synthesizer and then Gly, Gly, and eicosanedioic acid were sequentially introduced. In this case, 20% piperidine/NMP was used [reacted at 50C for S minutesjdeprotect the Fmoc group, and the condensation reaction was carried out using the double coupling method in which after all the reactions at 50C for 15 minutes, the solution was removed by filtration, and the same condensation reaction was repeated. After completion of solid-phase synthesis, the resin was washed with MeOH, and dried under reduced pressure to thereby obtain 421.5 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(tu)-Ile-Aib-Lys(Eda-GGGGG-)-Asp(OtB u)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-G ly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin. To the total amount of the obtained resin, 4.6 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 Phenomenex Kinetex 5 im XB-C18 (250 x 30.0 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 15 mL/ minute from A/B: 40/60 to 50/50, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 70.1 mg of a white powder. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
109.2 mg | H-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg (Pbf)-Pro-Ser(tB u)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tB u)-Sieber amide resin (SEQ ID NO: 481) (520.0 mg, 0.1 mmol) synthesized in Reference Example A was weighed into a reaction tube, which was then set in a peptide synthesizer. Amino acids were sequentially extended according to the protocol using 20% piperidine/NMP [reacted at 50C for 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDJIOxyma [reacted at 50C for 15 minutesj to condense the Fmoc-aminoacids. In this case, the condensation reaction of Boc-MeTyr(tBu) at position 1, Thr(tBu) at position 5, lie at position 12, Gln(Trt) at position 19, and Trp(Boc) at position 25 was carried out at 50C for 30 minutes. The operation wherein the obtained Boc-MeTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tB u)-Iva-Ile-Ser(tB u)-Asp(OtBu)-Tyr(tBu)Ser(tB u)-Ile-Aib-Leu-Asp(OtBu)-Arg(Pbf)-Lys(ivDde)-Ala-Gln(Trt)-Aib-Asn(Trt)-Ph e-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tB u)-GlyAla-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin was suspended in a 2% hydrazine/NMP solution, the resulting suspension was stirred at 50C for 10 minutes, and then the solution was removed by filtration was repeated 8 times to deprotect the ivDde group of Lys at position 17. Subsequently, Fmoc-Gly-Gly-Gly-OH was introduced using the peptide synthesizer and then Gly, Gly, and octadecanedioic acid were sequentially introduced. In this case, 20% piperidine/NMP was used [reacted at 50C for 5 minutesj to deprotect the Fmoc group, and the condensation reaction was carried out using the double coupling method in which after all the reactions at 50C for 15 minutes, the solution was removed by filtration, and the same condensation reaction was repeated. After completion of solid-phase synthesis, the resin was washed with MeOH, and dried under reduced pressure to thereby obtain 817.0 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(Oda-GGGGG-)-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-G ly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin. To the total amount of the obtained resin, 8 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 Phenomenex Kinetex S im XB-C18 (250 x 30.0 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 15 mL/ minute from A/B: 40/60 to 50/50, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 109.2 mg of a white powder. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
3.4 mg | Fmoc-Ser(tBu)-Alko resin (0.68 meq/g, 147.1 mg, 0.1 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% piperidine/NMP [reacted at room temperature for 15 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDJIOxyma [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-Lys(ivDde)-Asp(OtB u)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pr o-Pro-Pro-Ser(tBu)-Alko resin was suspended in a 2% hydrazine/NMP solution, the resulting suspension was stuffed at 50C for 10 minutes, and then the solution was removed by filtration was repeated 8 times 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 664.0 mg of the protected peptide resin of interest.; Boc-MeTyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tB u)-Iva-Ile-Ser(tB u)-Asp(OtBu)-Tyr(tBu)Ser(tB u)-Ile-Aib-Lys-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-A sn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-Gly-Ala-ProPro-Pro-Ser(tBu)-Alko resin (SEQ ID NO: 483) (166.0 mg, 0.025 mmol) synthesized in Reference Example C was weighed into a reaction tube, which was then set in a peptide synthesizer. 20% piperidine/NMP [reacted at 50C for 5 minutesj was used to deprotect the Fmoc group, subsequently Fmoc-Gly-Gly-Gly-OH was introduced using the peptide synthesizer, and then Gly, Gly, and octadecanedioic acid were sequentially introduced. In this case, 20% piperidine/NMP was used [reacted at 50C for 5 minutesj to deprotect the Fmoc group, and 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 solid-phase synthesis, the resin was washed with MeOH, and dried under reduced pressure to thereby obtain 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-Lys(Oda-GGGGG-)-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-G ly-Ala-Pro-Pro-Pro-Ser(tBu)-Alko resin. Subsequently, to the total amount of the obtained resin, 2.5 mL of TFA:m-cresol:thioanisole :ethandithiol:H2 O: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 50% 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 30 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 15 mL/minute from A/B: 40/60 to 50/50, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 3.4 mg of a white powder.Mass spectrometry result: (M+H)+ 4784.51 (calculated 4785.49) HPLC elution time: 5.88 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 |
---|---|---|
2.9 mg | Sieber amide resin (0.71 meq/g, 352 mg, 0.25 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% piperidine/NMP [reacted at room temperature for 15 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDJIOxyma [reacted at room temperature for 150 minutesj to condense the Fmoc-amino acids.The operation wherein the obtained BocMe-Tyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Iva-Ile-Ser(tBu)-Asp(OtB u)-Tyr(tB u)-Ser( tB u)-Ile-Aib-Lys(ivDde)-Asp(OtBu)-Arg(Pbf)-Aib-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-Pro-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 was repeated twice 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 1.87 g of BocMe-Tyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Iva-Ile-Ser(tBu)-Asp(OtB u)-Tyr(tB u)-Ser( tB u)-Ile-Aib-Lys-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn( Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin.37.3 mg (0.005 mmol) of the obtained resin was weighed into a reaction tube, which was then set in a peptide synthesizer. Subsequently, Fmoc-Gly-Gly-OH and FmocGly-Gly-Gly-OH was introduced using the peptide synthesizer, according to the protcol using 20% piperidine/NMP [reacted at 50C for 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDJIOxyma [50C, 15 minutesj to condense the Fmoc-amino acids, and then BocMe-Tyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Iva-Ile-Ser(tBu)-Asp(OtB u)-Tyr(tB u)-Ser( tB u)-Ile-Aib-Lys(H-Gly-Gly-Gly-Gly-Gly-)-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-A ib-Asn(Trt)-Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tB u)Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber amide resin was obtained. In this case, the condensation reaction was carried out using the double coupling method in which after all the reactions at 50C for 15 minutes, the solution was removed by filtration, and the same condensation reaction was repeated. To the obtained resine, 6.4 mg of adipic anhydride, 6.4 mg of DIPEA, and NMP (0.1 ml) were added, and then the solution was stirred at room temperature for 2 hours. After removal of the reaction solution by filtration, the resin was washed with MeOH, and dried under reduced pressure to thereby obtain 36.9 mg of the protected peptide resin of interest, BocMe-Tyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Iva-Ile-Ser(tBu)-Asp(OtB u)-Tyr(tB u)-Ser( tB u)-Ile-Aib-Lys(Hda-GGGGG-)-Asp(tBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Pro-Ser(tBu)-Ser(tBu)-Gl y-Ala-Pro-Pro-Pro-Ser(tB u)-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 HPLCusing Phenomenex Kinetex 5iim XB-C18 (250x20.0 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: 66/34 to 56/44, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 2.9 mg of a white powder.Mass spectrometry result: (M+H)+ 46 16.573 (calculated 4616.32) HPLC elution time: 4.09 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 |
---|---|---|
Sieber amide resin (0.71 meqg, 140.8 mg, 0.1 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. In this case, the condensation reaction of Boc-MeTyr(tBu) at position 1, Thr(tBu) at position 5, Ile at position 12, Arg(Pbf) at position 16, Gln(Trt) at position 19, and Trp(Boc) at position 25 was caffied out at 50C for 30 minutes. 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-Lys-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-Val-Asn(T rt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Sieber amide resin was suspended in a 2% hydrazineNMP solution, the resulting suspension was stirred at 50C for 10 minutes, and then the solution was removed by filtration was repeated 8 times to deprotect the ivDde group of Lys at position 14. The obtained resin was washed with MeOH, and dried under reduced pressure to thereby obtain 762.1 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-Lys-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)rt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Sieber amide resin.; -Phe-Val-Asn(TBoc-MeTyr(tBu)-Aib-Glu(OtB u)-Gly-Thr(tBu)-Iva-Ile-Ser(tBu)-Asp(OtB u)-Tyr(tB)-Ser(tBu)-Ile-Aib-Lys-Asp(OtBu)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)-Phe-ValAsn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Sieber amide resin (SEQ ID NO:482) (38.1 mg, 0.005 mmol) synthesized in Reference Example B was weighed into areaction tube, which was then set in a peptide synthesizer. Gly, Gly, FmocGly-Gly-Gly-OH, and octadecanedioic acid were introduced according to the protocol using 20% piperidine/NMP [reacted at 50C for 5 minutesj to deprotect the Fmoc group and 5 equivalents of Fmoc-amino acids/DIPCDI/Oxyma [reacted at 50C for 15 minutesj to condense the Fmoc-amino acids. In this case, 20% piperidine/NMP was used [reacted at 50C for 5 minutesj to deprotect the Fmoc group, and the condensation reaction was carried out using the double coupling method 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 solid-phase synthesis, the resin was washed with MeOH, and dried under reduced pressure to thereby obtain 45.3 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-Lys(Oda-GGGGG-)Asp(OtB u)-Arg(Pbf)-Aib-Ala-Gln(Trt)-Aib-Asn(Trt)Phe-Val-Asn(Trt)-Trp(Boc)-Iva-Leu-Ala-Gln(Trt)-Arg(Pbf)-Sieber amide resin. To the total amount of the obtained resin, 0.5 mL of TFA:m-cresol:thioanisole:ethandithiol:H2 O: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 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 Phenomenex Kinetex 5 im XB-C18 (250 x 20.0 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: 59/41 to 49/51, and fractions containing the product of interest were collected and freeze-dried to thereby obtain 3.7 mg of a white powder. Mass spectrometry result: (M+H)+ 4006.32 (calculated 4007.14) HPLC elution time: 5.97 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 |
---|---|---|
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 |
---|---|---|
2.A Loading of Fmoc-Lys(Ac)-OH on Rink Amide Resin (0433) In a 100 ml reactor equipped with a sintered glass at the bottom, 6 g of Novabiochem or ChemImpex Rink amide AM resin (Low Loading 0.47 mmol/g) was swelled in 40 ml of DMF. The solvent was drained and 30 ml of 20% piperidine in DMF solution were added. After 15 min shaking, the solvent was drained. This was repeated twice to ensure complete Fmoc protecting group removal. The resin was washed with 5×30 ml DMF. (0434) In a separate flask a solution containing Fmoc-Lys(Ac)-OH (3.5 g, 8 mmol, 3 eq.) HOBT.H2O (1.3 g 8.5 mmol) in 30 ml DMF was prepared. Diisopropylcarbodiimide (DIC) (1 g, 8.5 mmol) was added to this solution and after 5 min the resulting mixture was added to the resin. The suspension was shaken on a stirring plate for 4 h or until completion of the reaction as judged by Kaiser Test (Ninhidrin test) on an aliquot part of the resin. (0435) The solvent was then drained and the resin washed 3 times with 30 ml DMF. Fmoc-Lys(Ac)-NH2 loaded resin was used immediately for subsequent steps or stored wet at 4 C. until needed. (0436) 2.D. Synthesis of Peptide Having the SEQ ID NO: 6 A batch of resin obtained in 2.A. corresponding to 0.1 mmol of Fmoc-Lys(Ac)-NH2 was placed in the reactor of a CEM Liberty Blue microwave peptide synthesizer. Peptide synthesis was performed by using DIC 0.5M/Oxyma 1M in DMF. (0468) All amino acids were introduced with double couplings at 90 C. as above, with the exception of amino-isobutyric acid at position 21 and serine at position 29 for which a triple coupling at 90 for 2 minutes was performed. <strong>[204777-78-6]Fmoc-Lys(ivDde)-OH</strong> was used at position 25. (0469) At the end of peptide assembly, the resin was transferred to a 20 ml polypropylene syringe and peptide was acetylated at N-terminus with of acetic anhydride (95 muL, 1 mmol) and DIPEA (174 muL, 1 mmol) in DMF (10 mL) for 20 minutes, repeating the cycle twice. (0470) Then, ivDde protecting group on lysine 25 side chain was removed by stirring with 10 mL of a solution of hydrazine 5% w/v in DMF for 20 min as many times as necessary until no starting material could be detected after cleavage of an aliquot part of resin and UPLC/MS analysis. When deprotection was judged complete, resin was washed with DMF (5×10 ml). (0471) Two TTDS spacer units were introduced by single coupling by performing twice the following procedure: To the resin a solution of Fmoc-TTDS-OH (163 mg, 0.3 mmol) HOAt (42 mg, 0.3 mmol) and DIC (77 muL, 0.5 mmol) 7 mL of DMF was added. The syringe was agitated on an orbital table for 18 h. The reaction was monitored by Kaiser Test. When needed, a double coupling was performed. The resin was washed with DMF (2×10 mL). Then to the resin, 10 mL of 20% v/v of piperidine in DMF was added. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated a second time and the resin was washed with DMF (2×10 mL) and dichloromethane (3×10 mL). (0472) The three gamma-glutamic acids spacers were introduced by performing a double coupling of each Fmoc-Glu-OtBu. Thus the following procedure was applied three times: (0473) To the resin a solution (4S)-5-tert-butoxy-4-(9H-fluoren-9-yl methoxy carbonylamino)-5-oxo-pentanoic acid (Fmoc-Glu-OtBu) (127 mg, 0.3 mmol), HOAt (42 mg, 0.3 mmol) and DIC (77 muL, 0.5 mmol) in 7 mL of DMF. The syringe was agitated on an orbital table for 18 h. The reaction was monitored by Kaiser Test. The resin was washed with DMF (2×10 mL). Then to the resin, 10 mL of 20% v/v of piperidine in DMF was added. The syringe was agitated on an orbital table for 20 min. This deprotection procedure was repeated a second time and the resin was washed with DMF (3×10 mL) and dichloromethane (3×30 mL). Finally, the peptide was acylated with Stearoyl chloride (62 mg, 0.2 mmol) and DIPEA (54 muL, 0.3 mmol) in 5 ml DCM for 2.5 h. The resin was washed with DMF (2×30 mL) and dichloromethane (3×30mL) and dried under vacuum. (0475) The cleavage of the peptide from the resin was performed using a solution phenol (0.5 g), water (0.5 mL) and TIPS (0.2 mL) in TFA (QSP 10 mL) for 2.5 hours at room temperature. The resin was filtered off, and washed with 2×4 mL TFA. The combined filtrates were transferred to a 100 mL round bottom flask and partially concentrated under vacuum at T<30 C. and the peptide was precipitated by the addition of 50 mL ice-cold MTBE and centrifuged at 3600 rpm for 30 minutes. (0476) The centrifuged pellet was then washed with ice-cold diethyl ether and centrifuged. This process was repeated three times. 240 g of crude peptide were obtained. Purification was performed using purification system B and fractions containing pure desired peptide were lyophilized. The peptide as trifluoroacetate salt was obtained as a white solid. (0477) m=38 mg (8%) (0478) UPLC/MS: (0479) RT: 5.40 min. (Analytical condition A), purity 97% (UV) (0480) Observed mass m/z (ion type): 1376.1 (M+3H); 1032.0 (M+4H); 826.0 (M+5H). |
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). |
[ 1311992-97-8 ]
2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-allylpent-4-enoic acid
Similarity: 0.91
[ 1231709-22-0 ]
(R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-methylbutanoic acid
Similarity: 0.91
[ 288617-71-0 ]
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpent-4-enoic acid
Similarity: 0.91
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