20-(tert-Butoxy)-20-oxoicosanoic acid

The peptide backbone of is synthesized using Fluorenylmethyloxycarbonyl (Fmoc)/tert-Butyl (t-Bu) chemistry on a Symphony X peptide synthesizer (Gyros Protein Technologies. Tucson, Ariz.). The resin consists of 1% DVB cross-linked polystyrene (Fmoc-Rink-MBHA Low Loading resin, 100-200 mesh, EMD Millipore) at a substitution of 0.3-0.4 meq/g. Standard side-chain protecting groups were used. Fmoc-Lys(Mtt)-OH is used for the lysine at position 17 and Boc-Tyr(tBu)-OH) was used for the tyrosine at position 1. Fmoc groups are removed prior to each coupling step (2×7 minutes) using 20% piperidine in DMF. All standard amino acid couplings are performed for 1 hour to a primary amine and 3 hour to a secondary amine, using an equal molar ratio of Fmoc amino acid (0.3 mM), diisopropylcarbodiimide (0.9 mM) and Oxyma (0.9 mM), at a 9-fold molar excess over the theoretical peptide loading. Exceptions are couplings to Calpha-methylated amino acids, which are coupled for 3 hours. After completion of the synthesis of the peptide backbone, the resin is thoroughly washed with DCM for 6 times to remove residual DMF. The Mtt protecting group on the lysine at position 17 is selectively removed from the peptide resin using two treatments of 300 hexafluoroisopropanol (Oakwood Chemicals) in DCM (2×40-minute treatment). Subsequent attachment of the fatty acid-linker moiety is accomplished by coupling of 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic acid alpha-t-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.), mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China). 3-Fold excess of reagents (AA:PyAOP:DIPEA=1:1:1 mol/mol) are used for each coupling that is 1-hour long. After the synthesis is complete, the peptide resin is washed with DCM, and then thoroughly air-dried. The dry resin is treated with 10 mL of cleavage cocktail (trifluoroacetic acid:water:triisopropylsilane, 95:2.5:2.5 v/v) for 2 hours at room temperature. The resin is filtered off, washed twice each with 2 mL of neat TFA, and the combined filtrates are treated with 5-fold excess volume of cold diethyl ether (-20 C.) to precipitate the crude peptide. The peptide/ether suspension is then centrifuged at 3500 rpm for 2 min 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/20% Acetic acid/60% water and purified by RP-HPLC on a Luna 5 mum Phenyl-Hexyl preparative column (21*250 mm, Phenomenex) with linear gradients of 100% acetonitrile and 0.1% TFA/water buffer system (30-50% acetonitrile in 60 min). The purity of peptide is assessed using analytical RP-HPLC and pooling criteria is >95%. The main pool purity of compound 1 is found to be 98.0%. Subsequent lyophilization of the final main product pool yielded the lyophilized peptide TFA salt. The molecular weight is determined by LC-MS (obsd. M+3=1657.2; Calc M+3=1657.0).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 In a similar way as described in above and depicted below the following compound was prepared using Boc-Gly-PAM resin as starting material. TOF-MS: mass 1128.38Synthetic protocol: Wang Fmoc-Lys(Mtt) resin 0.26 mmol/g (1, 11.2 g, 2.90 mmol) was left to swell in dichloromethane (100 ml) for 45 minutes. Fmoc group was removed by treatment with 20% piperidine in N, N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 15 ml). Resin was washed with N, N-dimethylformamide (3 x 90 ml), 2-propanol (3 x 90 ml) and dichloromethane (3 x 90 ml). A solution of {2-[2-(9H-fluoren-9ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-OEG-OH, 2.23 g, 5.80 mmol), 0-(6-chlorobenzotriazol-1-yi)-N, N, N', N'-tetramethyluronium tetrafluoroborate (TCTU, 2.06 g, 5.80 mmol) and N, N-diisopropylethylamine (2.02 ml, 11.6 mmol) in N, N20 dimethylformamide (100 ml) was added to resin and the mixture was shaken for 1 hour. Resin was filtered and washed with N, N-dimethylformamide (3 x 90 ml), dichloromethane (3 x 90 ml) and N, N-dimethylformamide (3 x 90 ml). Fmoc group was removed by treatment with 20% piperidine in N, N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 ml). Resin was washed with N, N-dimethylformamide (3 x 90 25 ml), 2-propanol (3 x 90 ml) and dichloromethane (3 x 90 ml). Solution of {2-[2-(9Hfluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-OEG-OH, 2.23 g, 5.80 mmol), 0-(6-chloro-benzotriazol-1-yi)-N, N, N', N'-tetramethyluronium tetrafluoroborate (TCTU, 2.06 g, 5.80 mmol) and N, N-diisopropylethylamine (2.02 ml, 11.6 mmol) in N, N-dimethylformamide (100 ml) was added to resin and mixture was 30 shaken for 1.5 hour. Resin was filtered and washed with N, N-dimethylformamide (3 x 90ml), dichloromethane (3 x 90 ml) and N, N-dimethylformamide (3 x 90 ml). Fmoc group was removed by treatment with 20% piperidine in N, N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 ml). Resin was washed with N, N-dimethylformamide (3 x 90 ml), 2-propanol (3 x 90 ml) and dichloromethane (3 x 90 ml). Solution of (S)-2-(9H5 fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid 1-tert-butyl ester (Fmoc-LGiuOtBu, 1.85 g, 4.35 mmol), 0-(6-chloro-benzotriazol-1-yi)-N, N, N', N'-tetramethyluronium tetrafluoroborate (TCTU, 1.55 g, 4.35 mmol) and N, N-diisopropylethylamine (1.36 ml, 7.82 mmol) in N, N-dimethylformamide (100 ml) was added to resin and mixture was shaken for 1.5 hour. Resin was filtered and washed with N, N-dimethylformamide (3 x 90 10 ml), dichloromethane (3 x 90ml) and N, N-dimethylformamide (3 x 90 ml). Fmoc group was removed by treatment with 20% piperidine in N, N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 ml). Resin was washed with N, N-dimethylformamide (3 x 90 ml), 2-propanol (3 x 90 ml) and dichloromethane (3 x 90 ml). Solution of 4-[(9H-fluoren-9-ylmethoxycarbonylamino)methyl]cyclohexanecarboxylic acid 15 (Fmoc-Trx-OH, 1.65 g, 4.35 mmol), 0-(6-chloro-benzotriazol-1-yi)-N, N, N', N'tetramethyluronium tetrafluoroborate (TCTU, 1.55 g, 4.35 mmol) and N, Ndiisopropylethylamine (1.36 ml, 7.82 mmol) in N, N-dimethylformamide (100 ml) was added to resin and mixture was shaken for 2 hours. Resin was filtered and washed with N, N-dimethylformamide (3 x 90 ml), dichloromethane (3 x 90ml) and N, N20 dimethylformamide (3 x 90 ml). Fmoc group was removed by treatment with 20% piperidine in N, N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 100 ml). Resin was washed with N, N-dimethylformamide (3 x 90 ml), 2-propanol (3 x 90 ml) and dichloromethane (3 x 90 ml). Solution of The title compound was prepared according to the procedures described in Example 23, using Eicosanedioic acid (20.0 g, 58.39 mmol) was suspended in thionyl chloride (100 ml), The mixture was heated to reflux at 80C for 3 hours, and excess thionyl chloride was distilled off under reduced pressure. Add dichloromethane (200ml) to dissolve, Tert-butanol (6.14 ml, 64.26 mmol), pyridine (4.94 ml, 61.34 mmol) were added, pyridine was added within 2 hours, and the reaction was carried out at 20C for 3 hours. Evaporate the solvent under reduced pressure600 ml of a DMF/water mixed solvent pre-cooled to 0 to 4[deg.] C. was added (DMF and water were mixed in a volume ratio of 1:2), and the precipitate was collected by filtration. Dry to constant weight in vacuo and resuspend in dichloromethane (200ml) pre-chilled to 0-4C.After the filtrate was collected, the solvent was evaporated under reduced pressure, re-suspended by adding petroleum ether (400 ml), and the filtrate was evaporated to dryness under reduced pressure.Add n-heptane (80 ml) to recrystallize, collect the precipitate by filtration, and dry it under reduced pressure to constant weight.Yield 8.85 g, yield: 38%, HPLC purity: 99.4%.

Molecular Weight:398.6
XLogP3:8.8
Hydrogen Bond Donor Count:1
Hydrogen Bond Acceptor Count:4
Rotatable Bond Count:21
Exact Mass:398.33960994
Monoisotopic Mass:398.33960994
Topological Polar Surface Area:63.6
Heavy Atom Count:28
Complexity:385
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes