CN106432468A - Solid-phase synthesis method for preparing exenatide - Google Patents
Solid-phase synthesis method for preparing exenatide Download PDFInfo
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Abstract
The invention discloses a method for preparing exenatide, aiming at mainly solving the technical problems in the prior art that synthesis steps are complicated, the coupling difficulty is great, post-period separation is difficult, and the like. According to the technical scheme, the method comprises the following steps: 1) mixing Fmoc-Rink Amide Linder and PEG (Polyethylene Glycol) Matrix resin, and reacting to obtain Fmoc-Rink Amide PEG Matrix resin; 2) gradually coupling amino acids with protective groups and proline pseudodipeptide segments by adopting a Fmoc solid-phase synthesis method, so as to obtain side-chain full-protection polypeptide resin; and 3) cutting the side-chain full-protection polypeptide resin, cracking polypeptides from the resin, removing side-chain protection groups so as to obtain an exenatide crude product, and carrying out high performance liquid chromatography separation and purification and freeze-drying on the crude peptide to obtain the exenatide. The process method for preparing the exenatide, provided by the invention, has the characteristics of simplicity and convenience for operation, high crude peptide purity and high comprehensive yield.
Description
Technical field
The present invention relates to Solid-phase synthesis peptides field, more particularly, to a kind of solid phase synthesis process of Exenatide.
Background technology
Exenatide (exenatide) one kind is from South America Monster (Gilka monster, Heloderma
The glucagon of a kind of polypeptide containing 39 aminoacid separated in saliva Horridum), its structure and people
(Glucagon) there is 48% homology, have 53% homology with the GLP-1 (glucagon-like-peptide-1) of people.
Research shows, as the analog of GLP-1, Exendin-4 can be with GLP-1 receptor acting, by stimulating islets of langerhans
β cell regeneration, promotes insulin secretion, the release of glucagon suppression, slows down gastric emptying rate, suppresses food intake.Its
Promoting insulin secretion is carried out according to blood sugar level, therefore can reduce hypoglycemic incidence rate, and to other pancreotropic hormones
The insensitive type ii diabetes patient of secretogogue still has blood sugar reducing function, and GLP-1 can also mitigate type ii diabetes patient's simultaneously
Body weight, is the brand-new intestinal blood sugar lowering element class medicine of a class.Intestinal blood sugar lowering element class medicine is a kind of novel therapies for type ii diabetes,
It can imitate the anti-diabetic reaction of natural gut hormone in human body or the reaction reducing concentration of glucose, these reaction bags
Include and stimulate internal generation insulin, the secretion of glucagon suppression after the meal in blood glucose rise, slow down blood and absorb nourishment
Speed and reduce food intake dose.Exenatide (Exenatide) is the type ii diabetes medicine of brand new class.Subcutaneous
Injection, daily medication 2 times, can not fill for metformin, sulfonylurea or metformin and sulfonylurea use in conjunction
The type ii diabetes patient of sub-control blood glucose.
The synthetic method of Exenatide has two methods of solid phase method and solid liquid phase set at present, and CN102174082A is open
A kind of method of solid phase synthesis Exenatide, using conventional Fmoc-Rink Amide AM Resin resin as carrier,
The solid method of Fmoc is coupled each aminoacid one by one and obtains Exenatide, be directly synthesized due to peptide sequence longer, be coupled difficulty by
One increase, easily produces residue deletions, and the peptide purification separation for the later stage causes great difficulty.CN103333237A discloses
A kind of method that solid liquid phase collection fragments synthesize Exenatide, solid liquid phase synthesis step is complicated, and amplification item controlled is many, unstable
Fixed, and the by-product producing is many, the removal of impurity will repeatedly be washed or other means purification, brings the feature of high cost, unfavorable
In industrialization.
Content of the invention
It is an object of the invention to provide a kind of high yield, low cost, reaction condition are gentle, environmental pollution is little, be conducive to
Realize the solid phase synthesis process of the Exenatide of industrialization, mainly solve synthesis step complexity, coupling hardly possible that prior art exists
Degree is big, the later stage separates the technical problems such as difficulty.
Technical solution of the present invention:A kind of solid-phase synthesis of Exenatide, comprise the steps:
(1)Fmoc-Rink Amide Linker and AM PEGMatrix reaction resin mixture are obtained Fmoc-Rink Amide
AM PEGMatrix resin;
(2)It is coupled 33 fluorenylmethyloxycarbonyl protection ammonia using Fmoc solid-phase synthesis according to the order from C-terminal to N-terminal one by one
Base acid and three class proline vacation dipeptide fragment X, Y, Z, obtain the polypeptide resin of side chain full guard, shown in formula I;
His(trt)-Gly-Glu(otbu)-X-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Y-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-Z-Gly-Ala-Pro-Pro-Pro-Ser
(tbu)-Rink Amide PEGMatrix resin
Formulas I
In formula:X= Gly-Thr(psi(me,me)pro);Y= Leu-Ser(psi(me,me)pro);Z= Ser(tbu)-Ser
(psi(me,me)pro);
(3)Addition is cut peptide reagent and the peptide chain resin of side chain full guard is cracked, and 30 nonapeptides are cleaved from resin
And remove side chain protecting group, open the five-membered ring of three false dipeptide fragment in sequence simultaneously, obtain exenatide crude product, as formula
Shown in II;Crude product is refining to obtain Exenatide through high performance liquid chromatography separation purification, lyophilizing again;
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-
Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-
Pro-Ser-NH2
Formula II.
Comprise the following steps that:
(1)Take AM PEGMatrix resin DCM to soak, make resin fully swelling, drain, add Fmoc- Rink Amide
Linker, DIEA, select TBTU/HOBT as catalyst, select volume ratio to be 1:1 DMF/NMP mixed solution as solvent,
Reaction 2-8 hour, drains, is washed with DMF 3 times, add volume ratio 2:2:1 acetic anhydride:Pyridine:The mixed solution of DMF, instead
Answer 0.5-2 hour, drain, washed with DMF 6 times, drain, obtain Fmoc-Rink Amide AM PEGMatrix resin;
The substitution value of PEGMatrix resin between 0.5mmol/g-1.0mmol/g, preferably 0.8mmol/g, Fmoc-Rink
The amount of Amide Linker is 2 times of AM PEGMatrix resin mole;The amount of TBTU/HOBT is AM PEGMatrix resin
2 times of mole, the amount of DIEA is 4 times of AM PEGMatrix resin mole;
(2)In step(1)In the Fmoc-Rink Amide AM PEGMatrix resin obtaining, add deprotecting regent, reaction
30-60 minute, drains, and is washed with DMF 5 times, drains;Add Fmoc-Ser (tbu)-OH, NMM, select HBTU/HOBT as contracting
Mixture, using DMF as solvent, reacts 0.5-2 hour, ninhydrin method detects reaction end, drains, is washed with DMF 3 times, drain,
Obtain Fmoc-Ser (tbu)-Rink Amide PEGMatrix resin;Then add deprotecting regent, deprotection reaction, then
Add and there is fluorenylmethyloxycarbonyl protected amino acid and class proline vacation dipeptide fragment, so repeatedly, until having connected last Soviet Union
Propylhomoserin.Washed with DMF 3 times, again with methanol and dichloromethane replace washing 3 times, finally plus methanol shrinkage resin, drain, vacuum
It is dried, obtain the peptide chain resin of side chain full guard, i.e. 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;
Described has fluorenylmethyloxycarbonyl protected amino acid and false dipeptide fragment, includes Fmoc-Pro-OH, Fmoc-Pro- successively
OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tbu)-Ser { psi (me, me) pro }-OH,
Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn (trt)-OH, Fmoc-Lys (boc)-OH, Fmoc-
Leu-OH, Fmoc-Trp (boc)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-
OH, Fmoc-Arg (pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (o
Tbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Met-OH, Fmoc-Gln (trt)-OH,
Fmoc-Lys (boc)-OH, Fmoc-Leu-Ser { psi (me, me) pro }-OH, Fmoc-Asp (otbu)-OH, Fmoc-Ser
(tbu)-OH, Fmoc-Thr (tbu)-OH, Fmoc-Phe-OH, Fmoc-Gly-Thr { psi (me, me) pro }-OH, Fmoc-
Glu (otbu)-OH, Fmoc-Gly-OH, Fmoc-His (trt)-OH;
The amount of Fmoc-Ser (tbu)-OH is 2 times of Fmoc-Rink Amide AM PEGMatrix resin mole,;Raise one's hat examination
Agent is the PIP/DMF mixed solution of volume ratio 20%, and the amount of each step fluorenylmethyloxycarbonyl protected amino acid is Fmoc-Rink
2 times of Amide AM PEGMatrix resin mole, the amount of condensation reagent is Fmoc-Rink Amide AM PEGMatrix
2 times of resin mole, the amount of NMM is 4 times of Fmoc-Rink Amide AM PEGMatrix resin mole, and each step is anti-
Answer detection method the used 1,2,3-indantrione monohydrate detection method of terminal;
(3)The peptide chain resin of side chain full guard adds and cuts peptide reagent, react 2-3 hour, sucking filtration filters resin particle, and receives
Collection filtrate, is subsequently adding the ice crystallizing from ether precipitation of 6 times of volumes, precipitation is collected by centrifugation, then is washed with ether 5 times, vacuum drying,
Obtain exenatide crude product.
Cut peptide reagent and select volume ratio TFA:Thioanisole:Phenol:Dithioglycol:H2O=87.5:5:2.5:2.5:2.5
Mix reagent.
In the present invention, some conventional abbreviations have following meanings
HBTU:O- benzotriazole-N, N, N, N- tetramethylurea hexafluorophosphate
TBTU:O- BTA-N, N, N', N'- tetramethylurea Tetrafluoroboric acid
HOBt:I-hydroxybenzotriazole
DIEA:N, N- diisopropylethylamine
NMM:N-methylmorpholine
Fmoc:Fluorenylmethyloxycarbonyl
Pbf:2,2,4,6,7- pentamethyl -2H- benzofuran -5 sulfonyl
Trt:Trityl
Tbu:The tert-butyl group
Otbu:Tert-butoxy
Pro:Proline
Thr:Threonine
Gly:Glycine
Ala:Alanine
Ser:Serine
Asn:Agedoite
Asp:Aspartic acid
Arg:Arginine
His:Histidine
Gln:L-Glutamine
Leu:Leucine
Ile:Isoleucine
Met:Methionine
Phe:Phenylalanine
Lys:Lysine
Glu:Glutamic acid
Val:L-Valine
Pip:Piperidines
DMF:N,N-dimethylformamide
DCM:Dichloromethane
TFA:Trifluoroacetic acid
NMP:N-Methyl pyrrolidone.
Beneficial effects of the present invention:Adopt AM PEGMatrix resin as synthesis material in the present invention, have stable
Formation of chemical bond gel network structure, all more stable in whole synthesis and cracking process, and also the PEG chain in structure is many
Reactive polypeptide provides the condition of an abundant solvation, effectively increases the coupling efficiency of polypeptide;Introduce three classes in the sequence
False dipeptide fragment X, Y, the Z of proline, effectively eliminates β-pleated sheet secondorder spatial structure, at utmost decreases residual peptide impurity
Formed, greatly improve the yield of later-period purification.
Brief description
Fig. 1 is product mass spectra figure of the present invention.
Fig. 2 is product chromatogram of the present invention.
Specific embodiment
Hereinafter with reference to example, the present invention is described in further detail, but the invention is not restricted to this instantiation.
Embodiment 1
(1)Preparation Fmoc-Rink Amide AM PEGMatrix resin
Weigh 25 grams of AM PEGMatrix resin (0.8mmol/g, 20mmol), soaked 30 minutes with 500ml DCM, make resin
Fully swelling, drain, 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, body
Amass and compare 1:1 DMF/NMP 500ml, reacts 4 hours, drains, washed with DMF 3 times, drain, add volume ratio 2:2:1 second
Anhydride:Pyridine:The mixed solution 500ml of DMF, reacts 1 hour, drains, washed with DMF 6 times, drain, and obtains Fmoc-Rink
Amide AM PEGMatrix resin.
(2)Preparation 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
In step(1)In the Fmoc-Rink Amide AM PEGMatrix resin obtaining, add 500ml deprotecting regent, instead
Answer 30 minutes, drain, washed with DMF 5 times, 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, react 1 hour, and 1,2,3-indantrione monohydrate detects resin water white transparency, drains, is washed with DMF 3 times, drain, and obtain
Fmoc-Ser (tbu)-Rink Amide AM PEGMatrix resin;Then add deprotecting regent, deprotection reaction, then
Add the aminoacid with Fmoc blocking group and false dipeptide fragment, so repeatedly, until after having connected last threonine,
DMF washs 3 times, and again with methanol and dichloromethane replace washing 3 times, finally plus methanol shrinkage resin, drains, vacuum drying, obtains
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)-Rink
The common 152g of Amide AM PEGMatrix resin;
Aminoacid and false dipeptide fragment X that each step condensation reaction is added, the amount of Y, Z is respectively:
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Ala-OH(MW:311.3,40mmol) 12.5g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
X=Fmoc-Ser(tbu)-Ser{psi(me,me)pro}-OH(MW:510.58,40mmol) 20.4g,
Fmoc-Pro-OH(MW:337.4,40mmol) 13.5g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
Fmoc-Asn(trt)-OH(MW:596.7,40mmol) 23.9g,
Fmoc-Lys(boc)-OH(MW:468.5,40mmol) 18.7g,
Fmoc-Leu-OH(MW:353.4,40mmol) 14.2g,
Fmoc-Trp(boc)-OH(MW:526.6,40mmol) 21.1g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Ile-OH(MW:353.4,40mmol) 14.2g,
Fmoc-Phe-OH(MW:387.4,40mmol) 15.5g,
Fmoc-Leu-OH(MW:353.4,40mmol) 14.2g,
Fmoc-Arg(pbf)-OH(MW:648.8,40mmol) 26.0g,
Fmoc-Val-OH(MW:339.4,40mmol) 13.6g,
Fmoc-Ala-OH(MW:311.3,40mmol) 12.5g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Met-OH(MW:371.5,40mmol) 14.9g,
Fmoc-Gln(trt)-OH(MW:610.7,40mmol) 24.5g,
Fmoc-Lys(boc)-OH(MW:468.5,40mmol) 18.7g,
Y=Fmoc-Leu-Ser{psi(me,me)pro}-OH(MW:480.58,40mmol) 19.2g,
Fmoc-Asp(otbu)-OH(MW:411.5,40mmol) 16.5g,
Fmoc-Ser(tbu)-OH(MW:383.4,40mmol) 15.4g,
Fmoc-Thr(tbu)-OH(MW:397.5,40mmol) 15.9g
Fmoc-Phe-OH(MW:387.4,40mmol) 15.5g,
Z=Fmoc-Gly-Thr{psi(me,me)pro}-OH(MW:438.48,40mmol) 17.5g,
Fmoc-Glu(otbu)-OH(MW:425.5,40mmol) 17g,
Fmoc-Gly-OH(MW:297.3,40mmol) 11.9g,
Fmoc-His(trt)-OH(MW:619.7,40mmol) 24.8g,
Fmoc-Ser(tbu)-OH(MW:383.4,40mmol) 15.4g;
The amount of the condensing agent that each step condensation reaction is adopted is:HBTU(MW:379.2,40mmol) 15.2g, HOBT (MW:
135.1,40mmol) 5.4g, the amount of the organic base that each step condensation reaction is added is:NMM(MW:102,80mmol)
9.0ml.
(3)Prepare Exenatide
Take step(2)In the common 152g of full guard peptide resin be placed in the round-bottomed flask of 2L, add 1.5L cutting reagent, proportioning is
TFA:Thioanisole:Phenol:Dithioglycol:H2O=87.5:5:2.5:2.5:2.5 (v/v), are placed in 25 DEG C of vibrations in constant-temperature table
Reaction 2.5 hours, sucking filtration filters resin particle, collects filtrate, is subsequently adding 7.5L crystallizing from ether precipitation, precipitation is collected by centrifugation,
Washed 3-6 time with ether again, vacuum drying, obtain exenatide crude product 85g, then through high performance liquid chromatography separation purification, lyophilizing
Obtain the Exenatide fine work 38g of purity afterwards(As Fig. 1, shown in 2), total recovery reaches 45%.
Embodiment 2
Step(1)AM PEGMatrix resin and 8 hours Amide linker, DIEA response time of Fmoc-Rink, add
Acetic anhydride:Pyridine:The mixed solution of DMF reacts 0.5 hour, and PEGMatrix resin substitution value is 1.0mmol/g;Step(2)
In Fmoc-Rink Amide AM PEGMatrix resin, add deprotecting regent, react 45 minutes;Add Fmoc-Ser
(tbu)-OH, NMM, the response time is 0.5 hour;Step(3)The peptide chain resin of side chain full guard adds and cuts peptide reagent, instead
Answer 2.5 hours.Remaining is with embodiment 1.
Embodiment 3
Step(1)AM PEGMatrix resin and 2 hours Amide linker, TBTU response time of Fmoc-Rink, add
Acetic anhydride:Pyridine:The mixed solution of DMF reacts 2 hours, and PEGMatrix resin substitution value is 0.5mmol/g;Step(2)
In Fmoc-Rink Amide AM PEGMatrix resin, add deprotecting regent, react 60 minutes;Add Fmoc-Ser
(tbu)-OH, NMM, the response time is 2 hours;Step(3)The peptide chain resin of side chain full guard adds and cuts peptide reagent, reaction
3 hours.Remaining is with embodiment 1.
Claims (7)
1. a kind of solid phase synthesis process preparing Exenatide is it is characterised in that comprise the steps:
(1)Fmoc-Rink Amide Linker and AM PEGMatrix reaction resin mixture are obtained Rink Amide AM
PEGMatrix resin;
(2)It is coupled 33 fluorenylmethyloxycarbonyl protection ammonia using Fmoc solid-phase synthesis according to the order from C-terminal to N-terminal one by one
Base acid and three class proline vacation dipeptide fragment X, Y, Z, obtain the polypeptide resin of side chain full guard, shown in formula I;
His(trt)-Gly-Glu(otbu)-X-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Y-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-Z-Gly-Ala-Pro-Pro-Pro-Ser
(tbu)-Rink Amide PEGMatrix resin
Formulas I
In formula:X= Gly-Thr(psi(me,me)pro);Y= Leu-Ser(psi(me,me)pro);Z= Ser(tbu)-Ser
(psi(me,me)pro);
(3)Addition is cut peptide reagent and the peptide chain resin of side chain full guard is cracked, and 30 nonapeptides are cleaved from resin
And remove side chain protecting group, open the five-membered ring of three false dipeptide fragment in sequence simultaneously, obtain exenatide crude product, as formula
Shown in II;Crude product is refining to obtain Exenatide through high performance liquid chromatography separation purification, lyophilizing again;
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-
Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-
Pro-Ser-NH2
Formula II.
2. method according to claim 1 is it is characterised in that step(1)Particular content comprises the steps
Take AM PEGMatrix resin dichloromethane to soak, make resin fully swelling, drain, add Fmoc- Rink Amide
Linker, DIPEA, select O- BTA-N, N, N', N'- tetramethylurea Tetrafluoroboric acid/1- hydroxy benzeness
And triazole is as catalyst, volume ratio is selected to be 1:1 N,N-dimethylformamide/N-Methyl pyrrolidone mixed solution is made
For solvent, react 2-8 hour, drain, washed with DMF 3 times, add volume ratio 2:2:1 acetic anhydride:
Pyridine:The mixed solution of DMF, reacts 0.5-2 hour, drains, washed with DMF 6 times,
Drain, obtain Fmoc-Rink Amide AM PEGMatrix resin.
3. method according to claim 2 is it is characterised in that the substitution value of PEGMatrix resin is in 0.5mmol/g-
Between 1.0mmol/g, the amount of Fmoc-Rink Amide Linker is 2 times of AM PEGMatrix resin mole;O- benzo
Triazole-N, the amount of N, N', N'- tetramethylurea Tetrafluoroboric acid/I-hydroxybenzotriazole is AM PEGMatrix resin mole
2 times, the amount of dichloromethane is 4 times of AM PEGMatrix resin mole.
4. method according to claim 1 is it is characterised in that step(2)Particular content comprises the steps:
In step(1)In the Fmoc-Rink Amide AM PEGMatrix resin obtaining, add deprotecting regent, react 30-
60 minutes, drain, washed with DMF 5 times, drain;Addition Fmoc-Ser (tbu)-OH, N-methylmorpholine,
Select O- benzotriazole-N, N, N, N- tetramethylurea hexafluorophosphate/I-hydroxybenzotriazole as condensing agent, with N, N- bis-
Methylformamide, as solvent, reacts 0.5-2 hour, and ninhydrin method detects reaction end, drains, uses DMF
Washing 3 times, drains, and obtains Fmoc-Ser (tbu)-Rink Amide PEGMatrix resin;Then add deprotecting regent,
Deprotection reaction, adds with fluorenylmethyloxycarbonyl protected amino acid and class proline vacation dipeptide fragment, so repeatedly, until connecting
Last threonine complete;Washed with DMF 3 times, again with methanol and dichloromethane replace washing 3 times, finally
Plus methanol shrinkage resin, drain, vacuum drying, obtain the peptide chain resin of side chain full guard, i.e. 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;
Described has fluorenylmethyloxycarbonyl protected amino acid and false dipeptide fragment, includes Fmoc-Pro-OH, Fmoc-Pro- successively
OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tbu)-Ser { psi (me, me) pro }-OH,
Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn (trt)-OH, Fmoc-Lys (boc)-OH, Fmoc-
Leu-OH, Fmoc-Trp (boc)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-
OH, Fmoc-Arg (pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (o
Tbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Glu (otbu)-OH, Fmoc-Met-OH, Fmoc-Gln (trt)-OH,
Fmoc-Lys (boc)-OH, Fmoc-Leu-Ser { psi (me, me) pro }-OH, Fmoc-Asp (otbu)-OH, Fmoc-Ser
(tbu)-OH, Fmoc-Thr (tbu)-OH, Fmoc-Phe-OH, Fmoc-Gly-Thr { psi (me, me) pro }-OH, Fmoc-
Glu (otbu)-OH, Fmoc-Gly-OH, Fmoc-His (trt)-OH.
5. method according to claim 4 is it is characterised in that the amount of Fmoc-Ser (tbu)-OH is Fmoc-Rink
2 times of Amide AM PEGMatrix resin mole,;Reagent of raising one's hat is the piperidines/N of volume ratio 20%, N- dimethyl formyl
Amine mixed solution, the amount of each step fluorenylmethyloxycarbonyl protected amino acid is Fmoc-Rink Amide AM PEGMatrix resin
2 times of mole, the amount of condensation reagent is 2 times of Fmoc-Rink Amide AM PEGMatrix resin mole, N- methyl
The amount of morpholine is 4 times of Fmoc-Rink Amide AM PEGMatrix resin mole, the detection side of each step reaction end
Method used 1,2,3-indantrione monohydrate detection method.
6. method according to claim 1 is it is characterised in that step(3)The peptide chain resin of side chain full guard adds
Cut peptide reagent reaction 2-3 hour, sucking filtration filters resin particle, and collects filtrate, the ice crystallizing from ether being subsequently adding 6 times of volumes sinks
Form sediment, precipitation is collected by centrifugation, then is washed with ether 5 times, vacuum drying, obtain exenatide crude product.
7. method according to claim 6 selects volume ratio trifluoroacetic acid it is characterised in that cutting peptide reagent:Thioanisole:
Phenol:Dithioglycol:H2O=87.5:5:2.5:2.5:2.5 mix reagent.
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