Solid-phase synthesis method for preparing exenatide

[0061] Example 1

[0062] (1) Preparation of Fmoc-Rink Amide AM PEGMatrix resin

[0063] Weigh 25 grams (0.8mmol/g, 20mmol) of AM PEGMatrix resin, soak it in 500ml DCM for 30 minutes to fully swell the resin, drain it, and add Fmoc-Rink Amide linker (MW:539.58, 40mmol) 21.6g, DIEA (MW : 129.24, 80mmol) 13.5ml, TBTU (MW: 321.1, 40mmol) 12.8g, HOBT (MW: 135.1, 40mmol) 5.4g, DMF/NMP 500ml with a volume ratio of 1:1, react for 4 hours, drain it, and use DMF Wash 3 times, drain dry, then add 500ml of acetic anhydride:pyridine:DMF mixed solution with a volume ratio of 2:2:1, react for 1 hour, drain dry, wash 6 times with DMF, drain dry to obtain Fmoc-RinkAmide AM PEGMatrix Resin.

[0064] (2) Preparation of His(trt)-Gly-Glu(otbu)-{Gly-Thr(psi(me,me)pro)}-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-{Leu -Ser(psi(me,me)pro))-Lys(boc)-Gln(trt)-Met-Glu(otbu)-Glu(otbu)-Glu(otbu)-Ala-Val-Arg(pbf)-Leu -Phe-Ile-Glu(otbu)-Trp(boc)-Leu-Lys(boc)-Asn(trt)-Gly-Gly-Pro-{Ser(tbu)-Ser(psi(me,me)pro)} -Gly-Ala-Pro-Pro-Pro-Ser(tbu)-Rink Amide AM PEGMatrix resin

[0065] In the Fmoc-Rink Amide AM PEGMatrix resin obtained in step (1), add 500ml of deprotection reagent, react for 30 minutes, drain, wash 5 times with DMF, and drain; add Fmoc-Ser(tbu)-OH(MW: 383.4, 40mmol) 15.4g, HBTU (MW: 379.2, 40mmol) 15.2g, HOBT (MW: 135.1, 40mmol) 5.4g, NMM (MW: 102.1, 80mmol) 9.0ml, DMF500ml, reaction for 1 hour, ninhydrin detection The resin is colorless and transparent, drained, washed 3 times with DMF, drained to obtain Fmoc-Ser(tbu)-Rink Amide AM PEGMatrix resin; then add deprotection reagent, deprotection reaction, and then add the Fmoc protecting group Amino acid and pseudo-dipeptide fragments are repeated until the last threonine is connected, DMF is washed 3 times, then methanol and dichloromethane are alternately washed 3 times, finally methanol is added to shrink the resin, drained, and vacuum dried to obtain His(trt)-Gly-Glu(otbu)-{Gly-Thr(psi(me,me)pro)}-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-{Leu-Ser(psi (me,me)pro))-Lys(boc)-Gln(trt)-Met-Glu(otbu)-Glu(otbu)-Glu(otbu)-Ala-Val-Arg(pbf)-Leu-Phe-Ile -Glu(otbu)-Trp(boc)-Leu-Lys(boc)-Asn(trt)-Gly-Gly-Pro-{Ser(tbu)-Ser(psi(me,me)pro)}-Gly-Ala -Pro-Pro-Pro-Ser(tbu)-RinkAmide AM PEGMatrix resin total 152g;

[0066] The amounts of amino acids and pseudo-dipeptide fragments X, Y, and Z added in each step of the condensation reaction are:

[0067] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,

[0068] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,

[0069] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,

[0070] Fmoc-Ala-OH (MW: 311.3, 40mmol) 12.5g,

[0071] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,

[0072] X=Fmoc-Ser(tbu)-Ser{psi(me,me)pro}-OH(MW:510.58, 40mmol) 20.4g,

[0073] Fmoc-Pro-OH (MW: 337.4, 40mmol) 13.5g,

[0074] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,

[0075] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,

[0076] Fmoc-Asn(trt)-OH (MW: 596.7, 40mmol) 23.9g,

[0077] Fmoc-Lys(boc)-OH (MW: 468.5, 40mmol) 18.7g,

[0078] Fmoc-Leu-OH (MW: 353.4, 40mmol) 14.2g,

[0079] Fmoc-Trp(boc)-OH (MW: 526.6, 40mmol) 21.1g,

[0080] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,

[0081] Fmoc-Ile-OH (MW: 353.4, 40mmol) 14.2g,

[0082] Fmoc-Phe-OH (MW: 387.4, 40mmol) 15.5g,

[0083] Fmoc-Leu-OH (MW: 353.4, 40mmol) 14.2g,

[0084] Fmoc-Arg(pbf)-OH (MW: 648.8, 40mmol) 26.0g,

[0085] Fmoc-Val-OH (MW: 339.4, 40mmol) 13.6g,

[0086] Fmoc-Ala-OH (MW: 311.3, 40mmol) 12.5g,

[0087] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,

[0088] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,

[0089] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,

[0090] Fmoc-Met-OH (MW: 371.5, 40mmol) 14.9g,

[0091] Fmoc-Gln(trt)-OH (MW: 610.7, 40mmol) 24.5g,

[0092] Fmoc-Lys(boc)-OH (MW: 468.5, 40mmol) 18.7g,

[0093] Y=Fmoc-Leu-Ser{psi(me,me)pro}-OH(MW:480.58,40mmol) 19.2g,

[0094] Fmoc-Asp(otbu)-OH (MW: 411.5, 40mmol) 16.5g,

[0095] Fmoc-Ser(tbu)-OH (MW: 383.4, 40mmol) 15.4g,

[0096] Fmoc-Thr(tbu)-OH (MW: 397.5, 40mmol) 15.9g

[0097] Fmoc-Phe-OH (MW: 387.4, 40mmol) 15.5g,

[0098] Z=Fmoc-Gly-Thr{psi(me,me)pro}-OH(MW:438.48,40mmol)17.5g,

[0099] Fmoc-Glu(otbu)-OH (MW: 425.5, 40mmol) 17g,

[0100] Fmoc-Gly-OH (MW: 297.3, 40mmol) 11.9g,

[0101] Fmoc-His(trt)-OH (MW: 619.7, 40mmol) 24.8g,

[0102] Fmoc-Ser(tbu)-OH (MW: 383.4, 40mmol) 15.4g;

[0103] The amount of condensing agent used in each step of the condensation reaction is: HBTU (MW: 379.2, 40mmol) 15.2g, HOBT (MW: 135.1, 40mmol) 5.4g, the amount of organic base added in each step of the condensation reaction: NMM( MW: 102, 80mmol) 9.0ml.

[0104] (3) Preparation of Exenatide

[0105] Take a total of 152g of the fully protected peptide resin in step (2) and place it in a 2L round-bottomed flask, add 1.5L of cutting reagent, and the ratio is TFA: thioanisole: phenol: ethanedithiol: H 2 O=87.5:5:2.5:2.5:2.5(v/v), place in a constant temperature shaker at 25℃ and shake for 2.5 hours, filter out the resin particles with suction, collect the filtrate, then add 7.5L ether to crystallize precipitation, and centrifuge to collect Precipitate, wash with ether for 3-6 times, dry in vacuum to obtain 85g of exenatide crude product, and then separate and purify by high performance liquid chromatography and freeze-dry to obtain pure exenatide product 38g (such as figure 1 , Shown in 2), the total yield reached 45%.

[0106] Example 2

[0107] Step (1) AM PEGMatrix resin reacts with Fmoc-Rink Amide linker and DIEA for 8 hours, then add acetic anhydride: pyridine: DMF mixed solution to react for 0.5 hours, PEGMatrix resin substitution degree is 1.0mmol/g; Step (2) Fmoc -Rink Amide AM PEGMatrix resin, add deprotection reagent, react for 45 minutes; add Fmoc-Ser(tbu)-OH, NMM, reaction time is 0.5 hours; step (3) add to the peptide chain resin with full side chain protection Peptide cleavage reagent, react for 2.5 hours. The rest is the same as in Example 1.

[0108] Example 3

[0109] Step (1) AM PEGMatrix resin reacts with Fmoc-Rink Amide linker and TBTU for 2 hours, then add acetic anhydride: pyridine: DMF mixed solution to react for 2 hours, PEGMatrix resin substitution degree is 0.5mmol/g; step (2) Fmoc -Rink Amide AM PEGMatrix resin, add deprotection reagent, react for 60 minutes; add Fmoc-Ser(tbu)-OH, NMM, reaction time is 2 hours; step (3) add to the peptide chain resin with full side chain protection Cleavage reagent, react for 3 hours. The rest is the same as in Example 1.