CN103374058B - Solid-phase synthesis process of angiotensinamide as well as intermediate and application thereof - Google Patents
Solid-phase synthesis process of angiotensinamide as well as intermediate and application thereof Download PDFInfo
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Abstract
The invention discloses a process for producing angiotensinamide with a chemical structure of H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH by adopting solid-phase synthesis technology and an intermediate adopted in the method. The method comprises the following steps of: (a) preparing protected angiotensinamide with a chemical structure of R1-Asn-Arg(R2)-Val-Tyr(R3)-Val-His(R4)-Pro-Phe-R5, wherein R1 is an amino protecting group, R2, R3 and R4 are side chain protecting groups, and R5 is carboxyl resin; and (b) under the action of a cutting reagent, deprotecting and separating the protected angiotensinamide obtained in the step (a) from the resin to generate angiotensinamide with the chemical structure of H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH. The method for synthesizing angiotensinamide is environment-friendly and efficient, has low requirements for equipment, and can be applied to large-scale industrial production.
Description
Technical field
The invention belongs to medicine synthesising process field.More specifically, the present invention relates to a kind of process for solid phase synthesis of vasopressin and intermediate thereof and application.
Background technology
Vasopressin is for increasing Cardura, CAS registration number 53-73-6, English name angiotensinamide or [Asn
1, Val
5]-angiotensin II, chemical structure is H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH (2003 editions national drug standards WS-10001-(HD-1448)-2003).Hypotension etc. caused when vasopressin is used for wound or postoperative shock and general anesthesia or lumbar anesthesia, can prevent or treat peri-operation period ypotension, as first aid using medicine treatment shock stage ypotension, also can be used for treatment angiotensin converting enzyme inhibitor over administration and conventional treatment invalid time.
When cardiovascular homeostasis is on the hazard, neurohormone mechanism-comprise sympathetic nervous system, vasopressing system and renin-angiotensin system to be activated, causes vasoconstriction to keep blood pressure and critical organ blood flow.Under ypotension (comprise dehydration and hemorrhage) state, catecholamine, Angiotensin and vassopressin play a crucial role regulating in arteriotony.Vasopressin medicine is the Angiotensin II analogs of chemosynthesis, and both pharmacological actions are similar.The direct excited arteriole vascular smooth muscle of vasopressin energy, makes arteriole brute force shrink, thus rapid raising blood pressure.Vasopressin mainly acts on skin, internal organ and renal blood vessels to increase Peripheral resistance, to skeletal muscle and cerebrovascular contraction less, faint to the excitation of cardiac muscle, without significant vein shrinking effect.Vasopressin also can promote adrenocortical secretion steroid hormone aldosterone, and aldosterone acts on uriniferous tubules, plays and protects sodium, water conservation, the effect of row's potassium, thus cause hypervolemia, elevation of blood pressure; Aldosterone also can act on hypothalamus, and hypothalamus secreting hormone impels hypophysis to discharge antidiuretic hormone (vassopressin), promotes that uriniferous tubules is to the heavily absorption of moisture.N holds Asn to remove formation seven peptide angiotonin III by vasopressin after aminopeptidase effect, and the contracting blood vessel function of Angiotensin II I is more weak, but promotes that the effect of Aldosterone Secretion is better than vasopressin.Vasopressin also can induce adrenal medulla and sympathetic nerve terminal to discharge catecholamine in a large number, catecholamine by adrenergic receptor vasoconstriction, strengthen myocardial contraction and raising blood pressure.Vasopressin causes heart rate decrease but rare arrhythmia sometimes; Coronary artery also can be caused to shrink and temporary infringement myocardial function, but continue medication and can improve ventricular function.
The chemosynthesis of polypeptide mainly contains liquid phase synthesis and solid phase synthesis two kinds of methods at present.The advantage of liquid phase synthesis is that the synthesis often walking intermediate product directly can carry out process control, and has an opportunity to be able to purifying; Shortcoming is complex process, takies more man-hour and manpower, needs more equipment and place.Process for solid phase synthesis is simple, takies man-hour with manpower is less, and material shifts less and saves equipment and place; Its shortcoming be often walk intermediate product cannot purifying, each step reaction conditions must be optimized and make transformation efficiency close to 100% and avoid the generation of side reaction as far as possible.A kind of method of liquid phase synthesis vasopressin is described in patent US2978444, this operational path has used gathering synthesis strategy, there is higher efficiency, but following two problems makes the more difficult acquisition high purity product of this route: in peptide fragment condensation reaction, peptide fragment C terminal amino acid, the particularly easy racemization of His; Reusability saponification reaction in this route, in the strong alkaline aqueous solution easy deacylated tRNA amine of Asn side chain.The method use hydrogenation in addition, need specific installation and working conditions, also will use metal catalyst costly and easily cause residual heavy metal problem in product.What is more important, this patented method cannot realize continuous synthesis, needs to take more production unit (as reactor etc.) and manpower, man-hour.Document Biochemistry1965, Vol4:2394 report a kind of linear synthesis strategy, and owing to employing solid phase synthesis technique, technique is relatively simple, can ensure higher yield.But this route employs chloromethyl resin, finally need to use Hydrogen bromide to be disintegrated down from resin by peptide chain, and side chain protected group nitro and benzyl are difficult to dissociate completely in hydrogenation.
One of key issue of polypeptide drugs synthesis technique is control to protected amino acid racemization (Chinese medicine mix 2010, Vol 19:102).Patent US6015881 provides a kind of current solution to suppressing the racemization of protected amino acid in condensation reaction.The program thinks that preactivated at low temperatures amino acid is very important to suppression racemization.Amino acid is pre-activate in such a way: be cooled to 0-5 DEG C after Fmoc protected amino acid, HOBt, DIPEA at room temperature dissolve, and is then joined in above-mentioned solution by HBTU and is stirred to dissolving.HBTU is finally reinforced is because activation and racemization all can not occur when it does not exist.The program needs a set of independently reactor A for batching, temperature control and activates relay amino acid in production implementation process, is transferred to by activates relay amino acid solution in condensation reaction still B, and equipment cleaning work subsequently.What is more important; although the OBt ester of protected amino acid has good stability, if period of storage is slightly long in reactor A, because racemization path can not be moved towards with amino condensation; so need strict control (J.Org.Chem.1997, Vol62:4307) to the pre-activate time.So pre-activate scheme adds the complexity of synthesis technique.
In sum, also lack now a kind of method of efficient synthesis vasopressin, therefore still in the urgent need to developing the production technique of new vasopressin.
Summary of the invention
Therefore; the technical problem to be solved in the present invention overcomes existing vasopressin solid phase synthesis process exactly and adopts chloromethyl resin; finally need to use Hydrogen bromide to be disintegrated down from resin by peptide chain; side chain protected group nitro and benzyl are difficult to the deficiency of dissociating completely in hydrogenation; a kind of preparation method of new vasopressin is provided; the method does not need to use Hydrogen bromide to be dissociated from resin by peptide chain, and side chain protected group nitro and benzyl dissociate completely.The present invention also provides the important intermediate and application thereof that relate in above-mentioned preparation method.In addition; the present invention is also to suppressing the racemization of protected amino acid in condensation reaction to provide a kind of current solution; in the program, batching and the activation of protected amino acid and condensation reaction are all carried out continuously in same reactor, carry out condensation reaction without the need to being transferred in condensation reactor by protected amino acid low-temperature activation in activator.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
One of technical scheme of the present invention is: a kind of preparation method of vasopressin, is characterized in that, comprises following steps:
(a) preparation protection vasopressin R
1-Asn-Arg (R
2)-Val-Tyr (R
3)-Val-His (R
4)-Pro-Phe-R
5, wherein, R
1for amino protecting group, R
2, R
3, R
4for Side chain protective group, R
5for carboxy resin;
(b) under the effect of cutting reagent, the protection vasopressin deprotection of step (a) gained and and resin isolation, generate vasopressin H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH.
Preferably, the cutting reagent described in step (b) is conventional weak acid reagent TFA.Preferred, also containing TIS and water in described weak acid reagent.Most preferred, described weak acid reagent is TFA: TIS: H
2o volume ratio is the mixture of 95: 2.5: 2.5.The consumption of cutting reagent is also conventional, preferably 100g peptide resin/L cutting reagent according to appointment.
Preferably, R
1, R
2, R
3, R
4, R
5be respectively Boc, Pbf, tBu, Trt, Wang resin.Belong to acid labile blocking group.
Protection vasopressin of the present invention is under TFA effect, and can efficiently being dissociated by a step chemical reaction, to obtain C end be the peptide section of free carboxy, and deprotection simultaneously, generate vasopressin.
Preferably, step (a) comprises the following steps:
(1) adopt solid-phase synthesis to be fixed on carboxy resin by the phenylalanine (blocking group-Phe-OH) of amido protecting, then amido protecting group is removed, obtain the resin (H-Phe-carboxylic acid resin) connecting phenylalanine;
(2) proline(Pro) (blocking group-Pro-OH) of amido protecting is added, condensation reaction is carried out under the existence of condensing agent, then amido protecting group is removed, obtain the resin (H-Pro-Phe-carboxylic acid resin) connecting proline(Pro), phenylalanine;
(3) Histidine (blocking group-His (Trt)-OH) of amino-protected side chain protection is added, condensation reaction is carried out under the existence of condensing agent, then amido protecting group is removed, obtain the resin (H-His (Trt)-Pro-Phe-carboxylic acid resin) connecting side chain protection group propylhomoserin, proline(Pro), phenylalanine;
(4) α-amino-isovaleric acid (blocking group-Val-OH) of amido protecting is added, condensation reaction is carried out under the existence of condensing agent, then amido protecting group is removed, obtain the resin (H-Val-His (Trt)-Pro-Phe-carboxylic acid resin) connecting α-amino-isovaleric acid, side chain protected Histidine, proline(Pro), phenylalanine;
(5) tyrosine (blocking group-Tyr (tBu)-OH) of amino-protected side chain protection is added, condensation reaction is carried out under the existence of condensing agent, then amido protecting group is removed, obtain the resin (H-Tyr (tBu)-Val-His (Trt)-Pro-Phe-carboxylic acid resin) connecting side chain protected tyrosine, α-amino-isovaleric acid, side chain protected Histidine, proline(Pro), phenylalanine;
(6) α-amino-isovaleric acid (blocking group-Val-OH) of amido protecting is added, condensation reaction is carried out under the existence of condensing agent, then amido protecting group is removed, obtain the resin (H-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-carboxylic acid resin) connecting α-amino-isovaleric acid, side chain protected tyrosine, α-amino-isovaleric acid, side chain protected Histidine, proline(Pro), phenylalanine;
(7) arginine (blocking group-Arg (Pbf)-OH) of amino-protected side chain protection is added, condensation reaction is carried out under the existence of condensing agent, then amido protecting group is removed, obtain the resin (H-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-carboxylic acid resin) connecting side chain protected arginine, α-amino-isovaleric acid, side chain protected tyrosine, α-amino-isovaleric acid, side chain protected Histidine, proline(Pro), phenylalanine;
(8) the amino l-asparagine (Boc-Asn-OH) protected by Boc is added; condensation reaction is carried out under the existence of condensing agent; obtain connecting amino protected by Boc l-asparagine, side chain protected arginine, α-amino-isovaleric acid, side chain protected tyrosine, α-amino-isovaleric acid, side chain protected Histidine, proline(Pro), phenylalanine resin, namely protect vasopressin R
1-Asn-Arg (R
2)-Val-Tyr (R
3)-Val-His (R
4)-Pro-Phe-R
5, wherein, R
1for amino protecting group Boc, R
2, R
3, R
4for Side chain protective group, R
5for carboxy resin (Boc-Asn-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-carboxylic acid resin).
Preferably, the amido protecting group described in step (1)-any one of (7) is Fmoc.
Preferably, the Side chain protective group of the Histidine of the amino-protected side chain protection described in step (3) is (Trt); The Side chain protective group of the tyrosine of the amino-protected side chain protection described in step (5) is (tBu); The arginic Side chain protective group of the amino-protected side chain protection described in step (7) is (Pbf).
Preferably; phenylalanine (Fmoc-Phe-OH) method be fixed on carboxy resin protected by Fmoc by amino described in step (1) can be ordinary method; also can be following preferred method; in the solvent that volatile organic solvent and non-volatile organic solvent form; under the effect of condensing agent; under the startup helping condensing agent; the amino phenylalanine protected by Fmoc and carry out condensation reaction through the carboxy resin of volatile organic solvent swelling treatment, obtains connecting the resin that Fmoc protects phenylalanine.
Preferred, step (a) comprises the following steps:
(1) volatile organic solvent and non-volatile organic solvent composition solvent in, under condensing agent effect, under the startup helping condensing agent, the phenylalanine of amino Fmoc protection and carry out condensation reaction through the carboxy resin of volatile organic solvent swelling treatment, obtain connecting the resin that Fmoc protects phenylalanine, then amido protecting group Fmoc is removed, obtain the resin connecting phenylalanine;
(2) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the proline(Pro) that the resin of the connection phenylalanine of step (1) gained and amino Fmoc protect carries out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting proline(Pro), phenylalanine;
(3) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the connection proline(Pro) of step (2) gained, the resin of phenylalanine and amino Fmoc protect the Histidine of side chain Trt protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(4) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the α-amino-isovaleric acid that the resin of the connection side chain Trt protection group propylhomoserin of step (3) gained, proline(Pro), phenylalanine and amino Fmoc protect carries out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(5) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the resin of the connection α-amino-isovaleric acid of step (4) gained, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine and amino Fmoc protect the tyrosine of side chain tBu protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain connecting the resin that side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(6) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the α-amino-isovaleric acid that the connection side chain tBu of step (5) gained protects the resin of tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine and amino Fmoc to protect carries out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(7) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the connection α-amino-isovaleric acid of step (6) gained, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin of phenylalanine and amino Fmoc protect the arginine of side chain Pbf protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain connecting side chain Pbf and protect arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin of phenylalanine,
(8) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the connection side chain Pbf of step (7) gained protects arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the l-asparagine that the resin of phenylalanine and amino Boc protect carries out condensation reaction, obtain the l-asparagine connecting amino Boc protection, side chain Pbf protects arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin of phenylalanine, namely vasopressin R is protected
1-Asn-Arg (R
2)-Val-Tyr (R
3)-Val-His (R
4)-Pro-Phe-R
5, wherein, R
1for amino protecting group Boc, R
2, R
3, R
4for Side chain protective group Pbf, tBu, Trt, R
5for carboxy resin.
Preferred, the condensation system of the condensation reaction described in step (1)-(8) comprise following several in any one, wherein the 1st row AA represents amino acid, and last list is shown and helped condensing agent, middle column represents condensing agent, is separated by between row and row by "/":
(1)AA/HOBt/DIC
(2)AA/HOBt/DCC
(3) AA/TBTU/HOBT/DIPEA or NMM
(4) AA/HATU/HOBT/DIPEA or NMM
(5) AA/HCTU/HOBT/DIPEA or NMM
(6) AA/PyBop/HOBT/DIPEA or NMM
(7) AA/Bop/HOBT/DIPEA or NMM
(8) AA/HATU/HOAT/DIPEA or NMM
(9) AA/HATU/HOBT/DIPEA or NMM
(10) AA/HCTU/HOBt/DIPEA or NMM
(11)AA/CDI/HOBt
(12)AA/CDI/HOAT。
Wherein, DCC character is similar with DIC, and partial amino-acid coupling effect is slightly better than DIC, but easily lumps, and operation inconvenience, in condensation course, intermediate not easily washs and loses no time, therefore the preferred DIC of the present invention.BOP, PyBOP, HATU, HBTU, HCTU are all salt condensing agents, and the effect of HATU, HCTU is better than HBTU, and condensation efficiency is high, but its price.Organic bases NMM is weak compared with DIPEA alkalescence, other character basic simlarity, according to circumstances interchangeable.
Most preferred, the condensing agent described in step (1) is DIC and HOBt, helps condensing agent to be DMAP; Condensing agent described in step (2)-(7) is HBTU and HOBT, and help condensing agent to be organic bases, described organic bases is DIPEA, NMM or collidine; Condensing agent described in step (8) is HOBT, helps condensing agent to be DIC.
Preferred further, condensing agent described in step (7) is HBTU and HOBT, condensing agent is helped to be organic bases, and step (7) also comprises second time condensation reaction, comprise the following steps, in the solvent that volatile organic solvent and non-volatile organic solvent form, under the existence of condensing agent HOBT, under the startup of DIC, the resin of the connection peptides of gained and amino Fmoc protect the arginine (Fmoc-Arg (Pbf)-OH) of side chain Pbf protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain connecting side chain Pbf and protect arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin (H-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-carboxylic acid resin) of phenylalanine.
Preferably, all chemical reactions that the present invention synthesizes vasopressin complete continuously successively in same reactor, comprise washing, drain.When arbitrary protected amino acid condensation, transfer in condensation reactor without the need to low-temperature activation in activator.Can see the schematic diagram of Fig. 6.
Preferably; when carrying out the condensation reaction in step (1)-(8); protected amino acid, condensing agent are first dissolved in the reaction mixture containing peptide resin; containing the solvent (preferred DMF and DCM) that volatile organic solvent and non-volatile organic solvent form in this mixture; then utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted; and utilize the volatilization of volatile organic solvent heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, then add condensation reaction and start agent startup condensation reaction.The present invention connect at every turn protected amino acid all have employed nitrogen be blown into make DCM volatilize falling temperature technique, pass in reaction mixture nitrogen make volatile solvent volatilize temperature of reaction system can be kept at 15-20 DEG C.Before adding organic bases DIPEA or DIC startup reaction, all need to utilize DCM to volatilize be cooled to 15-20 DEG C, temperature of reaction system is at 15-20 DEG C, after adding organic bases startup condensation reaction, make condensation reaction system temperature be stabilized in 22-28 DEG C by attemperator always.Therefore, preferably, the temperature that step (1)-(8) start condensation reaction after condensation reaction is 22-28 DEG C, is more preferably 25 DEG C.And the reaction times of condensation reaction is as routine, be generally till monitoring reaches reaction end.
Preferred, described non-volatile organic solvent be selected from DMF and DMSO one or both.
Preferred, described volatile organic solvent is selected from DCM and Et
2one or both in O.
Preferably, the amount ratio of described non-volatile organic solvent and volatile organic solvent is 3: 1-3.5: 1 (volume ratio).
Preferred, described organic bases is selected from DIPEA, NMM (N-methylmorpholine) or collidine (2，4，6-trimethylpyridine).
Preferred, the volume containing the sample of the resin of the connection phenylalanine of step (1) gained is 0.7-0.8mmol/g.
In method of the present invention; step before or after condensation reaction is all conventional steps; as deprotection reaction, polypeptide is cut down from resin to the washing of reaction product peptide resin, finally and slough the steps such as protecting group, certainly also comprise the step such as swelling to resin.
The reaction removing amido protecting group Fmoc described in the present invention is this area routine techniques, and such as adding piperidines can remove under mild conditions.Preferably, in DMF solvent, peptide resin and piperidines are carried out deprotection reaction.Preferably 25 DEG C are reacted 20 minutes, carry out two secondary responses.
In solid phase synthesis of the present invention, after the reaction of each step completes, the separation and purification for reaction product is all adopt the technology in conventional solid synthesis, will connect peptide resin washing, drain.Washing adopts the washing agent in conventional solid synthesis, generally comprises reaction solvent or methyl alcohol etc.
Two of technical scheme of the present invention is: a kind of protection vasopressin, its be connect amino protected by Boc l-asparagine, side chain protected arginine, α-amino-isovaleric acid, side chain protected tyrosine, α-amino-isovaleric acid, side chain protected Histidine, proline(Pro), phenylalanine carboxy resin (i.e. R
1-Asn-Arg (R
2)-Val-Tyr (R
3)-Val-His (R
4)-Pro-Phe-R
5, wherein, R
1for amino protecting group Boc, R
2, R
3, R
4for Side chain protective group, R
5for carboxy resin).
Three of technical scheme of the present invention is: described protection vasopressin is preparing the application in vasopressin polypeptide drugs.
The present invention provides a kind of current solution to suppressing the racemization of protected amino acid in condensation reaction; in the program, batching and the activation of protected amino acid and condensation reaction are all carried out continuously in same reactor, carry out condensation reaction without the need to being transferred in condensation reactor by protected amino acid low-temperature activation in activator.The program is as follows: a kind of single step reaction does not need pre-activate that the solid-phase synthesis of the racemization of protected amino acid in condensation reaction but can be suppressed to prepare the method for polypeptide, comprise the following steps: by protected amino acid, condensing agent is first dissolved in the reaction mixture containing peptide resin, containing the solvent (preferred DMF and DCM) that volatile organic solvent and non-volatile organic solvent form in this mixture, then utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of volatile organic solvent heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, then add condensation reaction and start agent startup condensation reaction.The temperature starting condensation reaction after condensation reaction is preferably 22-28 DEG C, more preferably 25 DEG C.
A preferred embodiment of the present invention is: a kind of preparation method of vasopressin; comprise the following steps: in DMF and DCM solvent; with HBTU/HOBt or DIC/HOBt for condensing agent; be that condensation reaction starts agent with DIPEA; Fmoc or Boc protected amino acid is connected on Wang resin; then deaminize acid N holds blocking group, iterative cycles, and the protected amino acid connected one by one is followed successively by:
(1)Fmoc-Phe-OH，
(2)Fmoc-Pro-OH，
(3)Fmoc-His(Trt)-OH，
(4)Fmoc-Val-OH，
(5)Fmoc-Tyr(tBu)-OH，
(6)Fmoc-Val-OH，
(7)Fmoc-Arg(Pbf)-OH，
(8)Boc-Asn-OH，
Thus obtain protection octapeptide vasopressin resin, then use cutting reagent TFA: TIS: H
2o (volume ratio 95: 2.5: 2.5) process obtains vasopressin.
The raw material that the present invention is used or reagent except special instruction, all commercially.
Compared to prior art, beneficial effect of the present invention is as follows:
The method of synthesis vasopressin provided by the invention does not need to use Hydrogen bromide, environmental protection, efficient, low for equipment requirements, can be applicable to large-scale industrial and produces.And product purity is high, productive rate is high, and purity can reach more than 99.5%.In the present invention, preferably, respond just can be carried out in simple reaction vessel, can avoid the loss produced because of manual operations and material repetitive displacement.Condensation reaction, without the need to low temperature pre-activate outside reactor, saves production unit, manpower and man-hour.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, characteristic sum beneficial effect of the present invention is described.
Fig. 1: vasopressin building-up process figure of the present invention.
Fig. 2: HBTU condensation Boc-Asn-OH gained vasopressin crude product HPLC schemes.
Fig. 3: DIC/HOBt condensation Boc-Asn-OH gained vasopressin crude product HPLC schemes.
Fig. 4: the vasopressin acetate sterling HPLC collection of illustrative plates that test obtains.
Fig. 5: the vasopressin acetate mass spectrum that test obtains.
Fig. 6: online low temperature pre-activate schematic diagram.
Embodiment
In the present invention, the resin of term " carboxy resin " to be the reactive group be directly connected with (first) amino acid in resin be carboxyl.As Wang resin.Carboxy resin, adds suitable condensing agent usually, and ester is amino acid whose fixing to complete altogether to make protected amino acid and resin formation.
The present invention is condensation amino acid on Wang resin, and all chemical reactions all complete continuously in a reactor, simplifies the low temperature pre-activate step used in conventional solid synthesis.This alternating temperature to reaction system controls to propose particular requirement.While raising combined coefficient, also in purification of intermediate, be subject to a lot of restriction.So there is higher requirement the efficiency aspect of continuous synthesis to chemical reaction, particularly condensation reaction.Special feature of the present invention is the optimum reaction condition optimizing each condensation step, makes efficiently, synthesizes high yield, the high purity product that also can obtain industrial application value rapidly, continuously.This obtains more concrete embodiment by following in some.
The first, the invention provides a kind of can the synthetic route (Fig. 1) of mass-producing High-efficient Production vasopressin.Use Wang resin linearly to synthesize protection vasopressin, without the need to process such as HBr or HF, efficiently can dissociate under TFA effect and obtain the peptide section that C end is free carboxy; Protect Arg side chain, Trt to protect His side chain, tBu to protect Tyr side chain with Pbf, guaranteeing efficiently dissociates when TFA process obtains vasopressin.Final step condensation reaction raw material uses Boc-Asn-OH, and non-common raw material Fmoc-Asn (Trt)-OH, can reduce by a step like this and take off Fmoc reaction.What is more important; if there is bibliographical information peptide chain N to hold Asn side chain Trt protection, this blocking group, to trifluoroacetic acid quite stable, needs long time treatment could remove (Pept.Res.1992 completely; Vol5:145), other side reaction can be caused like this.DIC and HBTU is the conventional condensing agent in Peptide systhesis industry.HOBt is conventional condensation additive, and the OBt Acibenzolar that protected amino acid and HOBt are formed under condensing agent exists has good amidate action performance and stability concurrently.Generally speaking, the peptide bond condensation speed of HBTU is very fast, correspondingly also can reduce side reaction odds, and protected amino acid 2 doubly feeds intake, within excessive 1 times can condensation complete.
The second, the present invention effectively avoids using Asn side chain dehydration side reaction.If Asn unprotected side chain is protected, with easily causing side chain dehydration side reaction during DCC or BOP condensation, use DCC/HOBt can avoid this side reaction (J.Org.Chem.1980, Vol45:55; Int.J.Pept.ProteinRes.1989, Vol34:287).When with HBTU condensation Fmoc-Asn-OH, there is the impurity that molecular weight is [M-18] in vasopressin product, supposition may dewater (Fig. 2) for Asn amide side chain.After using DCC/HOBt instead, restrained effectively the generation of this by product.But consider in suitability for industrialized production, the product of DCC is difficult to process, attempts replacing DCC/HOBt with DIC/HOBt, also can suppress the generation of [M-18] by product, relative area ratio can be controlled below 2% (Fig. 3).
3rd, present invention, avoiding the preactivated troublesome operation of protected amino acid.HBTU is very efficient condensation reagent, needs organic bases if DIPEA is to start condensation reaction.Reaction system adds in DIPEA process has a large amount of heat energy to discharge, and there will be temperature and to rise sharply effect.This exothermic effect is often associated, as Fmoc-His (Trt)-OH with the racemization side reaction in the condensation process of some amino acid.The experience of some Peptide systhesis shows, if by Fmoc-His (Trt)-OH and HBTU, DIPEA in a pre-activate reactor low-temperature mixed to suppress exothermic effect, then the solution through pre-activate process is joined in amino acid condensation reactor, significantly can suppress the racemization (patent US6015881) of His.But in actual mechanical process, this preactivated time is difficult to grasp, if amino acid is not had and amino contact reacts by activating, also can increase the probability of racemization side reaction.Therefore, best solution is in amino acid condensation reactor, realize instant alternating temperature operation, makes the temperature in condensation whole process remain at fixed value.The present invention by the condensation temp stability contorting of reactor at 22-28 DEG C, even the condensation of Fmoc-His (Trt)-OH is also without the need to pre-activate.Protected amino acid, HBTU and HOBT are first dissolved in the reaction mixture containing peptide resin, and the solvent of this mixture contains DMF and DCM.Then utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM to absorb heat whole reactive system is cooled to 15-20 DEG C rapidly.Then drip DIPEA, after this ensure that protected amino acid activation, be not subject to the impact of high ambient temperature, and once amino acid can be had an opportunity after being activated and amino condensation.This method is simple to operate, and products obtained therefrom quality is suitable with pre-activate scheme.
4th, the present invention is high volume containing the sample continuous synthesis protection vasopressin on Wang resin.Wang resin is a kind of good continuous synthesis upholder, but increasing along with volume containing the sample, spatially can cause condensation reaction difficulty, cause condensation incomplete.Obviously, consider from industrial application angle, high volume containing the sample effectively can reduce resin Material Cost ratio shared in synthesis, reduces the volume of reactor, reduce solvent load.Fmoc-Arg (the Pbf)-OH needing condensation bulky in the synthesis of protection vasopressin; after the volume containing the sample of Fmoc-Phe-Wang resin is higher than 0.7mmol/g; Fmoc-Arg (Pbf)-OH condensation often can be caused to be difficult to completely; and increase feed ratio can not solve this problem, could obtain negative findings after with acetic anhydride end socket in ninhydrin reaction.The present invention is by the secondary condensation of Fmoc-Arg (Pbf)-OH; solve high volume containing the sample and the incomplete contradiction of condensation reaction; achieve high volume containing the sample (Fmoc-Phe-Wang resin, 0.7-0.8mmol/g) continuous synthesis protection vasopressin on Wang resin.
Further describe technical characteristic of the present invention below, these technical characteristics make the present invention be different from general method, can guarantee the efficient suitability for industrialized production of vasopressin.
The preparation of 1.Fmoc-Phe-Wang resin:
Resin pre-treatment: Wang resin DMF is washed once, then uses the swelling 30min of DCM, drain.To mix with Fmoc-Phe-OH, HOBt, DIC and DMF, DCM through pretreated Wang resin, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, after slow dropping DMAP, control temperature is at 22-28 DEG C of reaction 16-20 hour, drain, DMF washes twice, and adds Pyridine/Ac
2o/DCM solution, reaction 2-3 hour, with DMF, MeOH cross washing twice, drains and obtains Fmoc-Phe-Wang resin.The 6-8 that DMF volume number (mL) is Wang resin quality number (g) doubly.The total mole number of active group on Wang resin is defined as n, then the mole number of Fmoc-Phe-OH, HOBt, DIC is 3n; The mole number of DMAP is 0.2n; Pyridine and Ac
2the mole number of O is 10n.
The preparation of 2.Fmoc-Pro-Phe-Wang resin:
In the Fmoc-Phe-Wang resin of step 1, add 20%Pip/DMF solution and take off Fmoc, 22-28 DEG C of reaction twice, first time 10min, second time 20min, drains, with DMF, MeOH, DCM cross washing, drains.Add Fmoc-Pro-OH, HBTU, HOBt and DMF, DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, after dripping DIPEA, control temperature is little negative up to triketohydrindene hydrate detection at 22-28 DEG C of reaction 2-3, drain, MeOH, DMF cross washing, drains; Obtain Fmoc-Pro-Phe-Wang resin.The volume number (mL) of 20%Pip/DMF, DMF, DCM and MeOH is respectively the 6-8 of Fmoc-Phe-Wang resin quality number (g) doubly; The mole number of Fmoc-Pro-OH, HOBt and HBTU is respectively 2 times of Fmoc-Phe-Wang resin mole number; The mole number of DIPEA is 3 times of Fmoc-Phe-Wang resin mole number.
The preparation of 3.Fmoc-His (Trt)-Pro-Phe-Wang resin:
In the Fmoc-Pro-Phe-Wang resin of step 2, add 20%Pip/DMF solution and take off Fmoc, 22-28 DEG C of reaction twice, first time 10min, second time 20min, drains, with DMF, MEOH, DCM cross washing, drains.Add Fmoc-His (Trt)-OH, HBTU, HOBt and DMF, DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, after dripping DIPEA, control temperature is little negative up to tetrachlorobenzoquinone detection at 22-28 DEG C of reaction 2-3, drain, MeOH, DMF cross washing, drains; Obtain Fmoc-His (Trt)-Pro-Phe-Wang resin.The volume number (mL) of 20%Pip/DMF, DMF, DCM and MeOH is respectively the 6-8 of Fmoc-Phe-Wang resin quality number (g) doubly; The mole number of Fmoc-His (Trt)-OH, HOBt and HBTU is respectively 2 times of Fmoc-Phe-Wang resin mole number; The mole number of DIPEA is 3 times of Fmoc-Phe-Wang resin mole number.
The preparation of 4.Fmoc-Val-His (Trt)-Pro-Phe-Wang resin:
In Fmoc-His (the Trt)-Pro-Phe-Wang resin of step 3, add 20%Pip/DMF solution and take off Fmoc, 22-28 DEG C of reaction twice, first time 10min, second time 20min, drains, with DMF, MEOH, DCM cross washing, drains.Add Fmoc-Val-OH, HBTU, HOBt and DMF, DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, after dripping DIPEA, control temperature is little negative up to triketohydrindene hydrate detection at 22-28 DEG C of reaction 2-3, drain, MeOH, DMF cross washing, drain and obtain Fmoc-Val-His (Trt)-Pro-Phe-Wang resin.The volume number (mL) of 20%Pip/DMF, DMF, DCM and MeOH is respectively the 6-8 of Fmoc-Phe-Wang resin quality number (g) doubly; The mole number of Fmoc-Val-OH, HOBt and HBTU is respectively 2 times of Fmoc-Phe-Wang resin mole number; The mole number of DIPEA is 3 times of Fmoc-Phe-Wang resin mole number.
The preparation of 5.Fmoc-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin:
In Fmoc-Val-His (the Trt)-Pro-Phe-Wang resin of step 4, add 20%Pip/DMF solution and take off Fmoc, 22-28 DEG C of reaction twice, first time 10min, second time 20min, drains, washes intersection and wash, drain with DMF, MEOH, DCM.Add Fmoc-Tyr (tBu)-OH, HBTU, HOBt and DMF, DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, after dripping DIPEA, control temperature is little negative up to triketohydrindene hydrate detection at 22-28 DEG C of reaction 2-3, drain, MeOH, DMF cross washing, drain and obtain Fmoc-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin.The volume number (mL) of 20%Pip/DMF, DMF, DCM and MeOH is respectively the 6-8 of Fmoc-Phe-Wang resin quality number (g) doubly; The mole number of Fmoc-Tyr (tBu)-OH, HOBt and HBTU is respectively 2 times of Fmoc-Phe-Wang resin mole number; The mole number of DIPEA is 3 times of Fmoc-Phe-Wang resin mole number.
The preparation of 6.Fmoc-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin:
In Fmoc-Tyr (tBu)-Val-His (the Trt)-Pro-Phe-Wang resin of step 5, add 20%Pip/DMF solution and take off Fmoc, 22-28 DEG C of reaction twice, first time 10min, second time 20min, drains, with DMF, MEOH, DCM cross washing, drains.Add Fmoc-Val-OH, HBTU, HOBt and DMF, DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, after dripping DIPEA, control temperature is little negative up to triketohydrindene hydrate detection at 22-28 DEG C of reaction 2-3, drain, MeOH, DMF cross washing, drain and obtain Fmoc-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin.The volume number (mL) of 20%Pip/DMF, DMF, DCM and MeOH is respectively the 6-8 of Fmoc-Phe-Wang resin quality number (g) doubly; The mole number of Fmoc-Val-OH, HOBt and HBTU is respectively 2 times of Fmoc-Phe-Wang resin mole number; The mole number of DIPEA is 3 times of Fmoc-Phe-Wang resin mole number.
The preparation of 7.Fmoc-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin:
In Fmoc-Val-Tyr (tBu)-Val-His (the Trt)-Pro-Phe-Wang resin of step 6, add 20%Pip/DMF solution and take off Fmoc, 22-28 DEG C of reaction twice, first time 10min, second time 20min, drains, with DMF, MEOH, DCM cross washing, drains.Add Fmoc-Arg (Pbf)-OH, HBTU, HOBt and DMF, DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to 15-20 DEG C rapidly, after dripping DIPEA, control temperature is at 22-28 DEG C of reaction 2-3 hour.If triketohydrindene hydrate detects negative, drain, with MeOH, DMF cross washing, obtain Fmoc-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin, the mole number of Fmoc-Arg (Pbf)-OH, HOBt and HBTU is respectively 2 times of Fmoc-Phe-Wang resin mole number; The mole number of DIPEA is 3 times of Fmoc-Phe-Wang resin mole number.If triketohydrindene hydrate detects non-feminine gender, DMF washes twice, drain, add Fmoc-Arg (Pbf)-OH, HOBt and DMF, DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM to absorb heat the 15-20 DEG C that to be lowered the temperature rapidly by whole reactive system, after dripping DIC, control temperature is little negative up to triketohydrindene hydrate detection at 22-28 DEG C of reaction 2-3, drain, with MeOH, DMF cross washing, obtain Fmoc-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin, the mole number of Fmoc-Arg (Pbf)-OH is 1 times of Fmoc-Phe-Wang resin mole number, the mole number of HOBt and DIC is respectively 2 times of Fmoc-Phe-Wang resin mole number.The volume number (mL) of 20%Pip/DMF, DMF, DCM and MeOH is respectively the 6-8 of Fmoc-Phe-Wang resin quality number (g) doubly;
The preparation of 8.Boc-Asn-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin:
In Fmoc-Arg (Pbf)-Val-Tyr (tBu)-Val-His (the Trt)-Pro-Phe-Wang resin of step 7, add 20%Pip/DMF solution and take off Fmoc, 22-28 DEG C of reaction twice, first time 10min, second time 20min, drains, with DMF, MeOH, DCM cross washing, drains.Add Boc-Asn-OH, HOBt and DMF, DCM; utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted; and utilize the volatilization of DCM to absorb heat the 15-20 DEG C that to be lowered the temperature rapidly by whole reactive system; after dripping DIC; control temperature is little negative up to triketohydrindene hydrate detection at 22-28 DEG C of reaction 2-3, drains, with DMF; MeOH cross washing, obtains protecting group vasopressin.The volume number (mL) of 20%Pip/DMF, DMF, DCM and MeOH is respectively the 6-8 of Fmoc-Phe-Wang resin quality number (g) doubly; The mole number of Boc-Asn-OH is 2 times of Fmoc-Phe-Wang resin mole number; The mole number of DIC, HOBt is 4 times of Fmoc-Phe-Wang resin mole number.
9. the preparation of vasopressin
Protection vasopressin is joined and cuts peptide reagent TFA: TIS: H
2in O, reaction 2-3 hour, suction filtration, filtrate is concentrated by underpressure distillation, and add ice-cold methyl tertiary butyl ether precipitation, vasopressin crude product is collected in centrifugal settling.Thick peptide obtains white sterling through RPLC purifying.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.
The preparation of embodiment 1:Fmoc-Phe-Wang resin
Weigh Wang resin 50g (100-200,1.23mmol/g), load core reactor, wash once with 500mL DMF, drain, then make resin fully swelling with 500mL DCM, drain.
Add Fmoc-Phe-OH (MW:387.4,3 times of Wang resin mole number) 71.5g, HOBt (MW:135.13,3 times of Wang resin mole number) 24.9g, DIC (MW:126.2,3 times of Wang resin mole number) 28.9mL, 300mL DMF, 100mL DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM to absorb heat whole reactive system to be cooled to rapidly 16 DEG C, slowly to add DMAP (MW:122,0.2 times of Wang resin mole number) 1.5g, stirs mixture 18 hours at 26 DEG C.Vacuum is drained, and DMF washes twice, and vacuum is drained.Add Pyridine (10 times of MW:79.1, Wang resin mole number) 49mL, Ac
2o (10 times of MW:102.09, Wang resin mole number) 58mL, 500mL DCM, stirred 3 hours at 27 DEG C by mixture, vacuum is drained, and with DMF, MeOH cross washing twice, vacuum is drained to constant weight.The substitution degree recording Fmoc-Phe-Wang resin is 0.80mmol/g (Fmoc-Phe-Wang resin gross weight 75g, total mole number 60mmol).
The preparation of embodiment 2:Fmoc-Pro-Phe-Wang resin
Add 500mL 20%Pi p/DMF solution, stir 10 minutes at 25 DEG C, vacuum is drained, then adds 500mL 20%Pi p/DMF solution, stir 20 minutes at 25 DEG C, vacuum is drained, and wash once with DMF, MeOH washes twice, once, once, vacuum is drained in DCM washing in DMF washing.Add Fmoc-Pro-OH (MW:337.4, 2 times of Fmoc-Phe-Wang resin mole number) 40.5g, HOBt (MW:135.13, 2 times of Fmoc-Phe-Wang resin mole number) 16.2g, HBTU (MW:379.25, 2 times of Fmoc-Phe-Wang resin mole number) 45.5g, 350mL DMF, 100mL DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 15 DEG C, drip DIPEA (MW:129.24, 2 times of Fmoc-Phe-Wang resin mole number) 31.4mL, mixture is stirred 2.5 hours at 25 DEG C, tetrachlorobenzoquinone detects and is negative, vacuum is drained, MeOH washing once, DMF washes twice, vacuum is drained.
The preparation of embodiment 3:Fmoc-Hi s (Trt)-Pro-Phe-Wang resin
Add 500mL 20%Pip/DMF solution, stir 10 minutes at 25 DEG C, vacuum is drained, then adds 500mL 20%Pip/DMF solution, stir 20 minutes at 25 DEG C, vacuum is drained, and wash once with DMF, MeOH washes twice, once, once, vacuum is drained in DCM washing in DMF washing.Add Fmoc-His (Trt)-OH (MW:619.7, 2 times of Fmoc-Phe-Wang resin mole number) 74.4g, HOBt (MW:135.13, 2 times of Fmoc-Phe-Wang resin mole number) 16.2g, HBTU (MW:379.25, 2 times of Fmoc-Phe-Wang resin mole number) 45.5g, 350mL DMF, 100mLDCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 18 DEG C, drip DIPEA (MW:129.24, 2 times of Fmoc-Phe-Wang resin mole number) 31.4mL, mixture is stirred 2 hours at 23 DEG C, triketohydrindene hydrate detects and is negative, vacuum is drained, MeOH washing once, DMF washes twice, vacuum is drained.
The preparation of embodiment 4:Fmoc-Val-His (Trt)-Pro-Phe-Wang resin
Add 500mL 20%Pip/DMF solution, stir 10 minutes at 25 DEG C, vacuum is drained, then adds 500mL 20%Pip/DMF solution, stir 20 minutes at 25 DEG C, vacuum is drained, and wash once with DMF, MeOH washes twice, once, once, vacuum is drained in DCM washing in DMF washing.Add Fmoc-Val-OH (MW:339.4, 2 times of Fmoc-Phe-Wang resin mole number) 40.7g, HOBt (MW:135.13, 2 times of Fmoc-Phe-Wang resin mole number) 16.2g, HBTU (MW:379.25, 2 times of Fmoc-Phe-Wang resin mole number) 45.5g, 350mL DMF, 100mL DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 20 DEG C, drip DIPEA (MW:129.24, 2 times of Fmoc-Phe-Wang resin mole number) 31.4mL, mixture is stirred 2 hours at 25 DEG C, triketohydrindene hydrate detects and is negative, vacuum is drained, MeOH washing once, DMF washes twice, vacuum is drained.
The preparation of embodiment 5:Fmoc-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin
Add 500mL 20%Pip/DMF solution, stir 10 minutes at 25 DEG C, vacuum is drained, then adds 500mL 20%Pip/DMF solution, stir 20 minutes at 25 DEG C, vacuum is drained, and wash once with DMF, MeOH washes twice, once, once, vacuum is drained in DCM washing in DMF washing.Add Fmoc-Tyr (tBu)-OH (MW:459.5, 2 times of Fmoc-Phe-Wang resin mole number) 55.1g, HOBt (MW:135.13, 2 times of Fmoc-Phe-Wang resin mole number) 16.2g, HBTU (MW:379.25, 2 times of Fmoc-Phe-Wang resin mole number) 45.5g, 350mL DMF, 100mLDCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 18 DEG C, drip DIPEA (MW:129.24, 2 times of Fmoc-Phe-Wang resin mole number) 31.4mL, mixture is stirred 2.5 hours at 26 DEG C, triketohydrindene hydrate detects and is negative, vacuum is drained, MeOH washing once, DMF washes twice, vacuum is drained.
The preparation of embodiment 6:Fmoc-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin
Add 500mL 20%Pip/DMF solution, stir 10 minutes at 25 DEG C, vacuum is drained, then adds 500mL 20%Pip/DMF solution, stir 20 minutes at 25 DEG C, vacuum is drained, and wash once with DMF, MeOH washes twice, once, once, vacuum is drained in DCM washing in DMF washing.Add Fmoc-Val-OH (MW:339.4, 2 times of Fmoc-Phe-Wang resin mole number) 40.7g, HOBt (MW:135.13, 2 times of Fmoc-Phe-Wang resin mole number) 16.2g, HBTU (MW:379.25, 2 times of Fmoc-Phe-Wang resin mole number) 45.5g, 350mL DMF, 100mL DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 15 DEG C, drip DIPEA (MW:129.24, 2 times of Fmoc-Phe-Wang resin mole number) 31.4mL, mixture is stirred 2.5 hours at 28 DEG C, triketohydrindene hydrate detects and is negative, vacuum is drained, MeOH washing once, DMF washes twice, vacuum is drained.
Embodiment 7:
The preparation of Fmoc-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin
Add 500mL 20%Pip/DMF solution, stir 10 minutes at 25 DEG C, vacuum is drained, then adds 500mL 20%Pip/DMF solution, stir 20 minutes at 25 DEG C, vacuum is drained, and wash once with DMF, MeOH washes twice, once, once, vacuum is drained in DCM washing in DMF washing.Add Fmoc-Arg (Pbf)-OH (MW:648.8, 2 times of Fmoc-Phe-Wang resin mole number) 77.9g, HOBt (MW:135.13, 2 times of Fmoc-Phe-Wang resin mole number) 16.2g, HBTU (MW:379.25, 2 times of Fmoc-Phe-Wang resin mole number) 45.5g, 350mL DMF, 100mL DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 15 DEG C, drip DIPEA (MW:129.24, 2 times of Fmoc-Phe-Wang resin mole number) 31.4mL, mixture is stirred 3 hours at 25 DEG C, triketohydrindene hydrate detects in non-feminine gender, vacuum is drained, DMF washes twice, vacuum is drained.Add Fmoc-Arg (Pbf)-OH (MW:648.8, 1 times of Fmoc-Phe-Wang resin mole number) 39.0g, HOBt (MW:135.13, 2 times of Fmoc-Phe-Wang resin mole number) 16.2g, 350mL DMF, 100mL DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 18 DEG C, drip DIC (MW:126.2, 2 times of Fmoc-Phe-Wang resin mole number) 18.8mL, mixture is stirred 2.5 hours at 27 DEG C, triketohydrindene hydrate detects and is negative, MeOH washing once, DMF washes twice, vacuum is drained.
Embodiment 8:
The preparation of Boc-Asn-Arg (Pbf)-Val-Tyr (tBu)-Val-His (Trt)-Pro-Phe-Wang resin
Add 500mL 20%Pip/DMF solution, stir 10 minutes at 25 DEG C, vacuum is drained, then adds 500mL 20%Pip/DMF solution, stir 20 minutes at 25 DEG C, vacuum is drained, and wash once with DMF, MeOH washes twice, once, once, vacuum is drained in DCM washing in DMF washing.Add Boc-Asn-OH (MW:232.1, 2 times of Fmoc-Phe-Wang resin mole number) 27.8g, HOBt (MW:135.13, 4 times of Fmoc-Phe-Wang resin mole number) 32.5g, 350mL DMF, 100mL DCM, utilize that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilize the volatilization of DCM heat absorption that whole reactive system is cooled to rapidly 19 DEG C, drip DIC (MW:126.2, 4 times of Fmoc-Phe-Wang resin mole number) 37.5mL, mixture is stirred 3 hours at 25 DEG C, triketohydrindene hydrate detects and is negative, vacuum is drained, MeOH washing once, DMF washes twice, MeOH washs three times, vacuum is drained to constant weight, obtain protection vasotonia resin 157.4g.
The preparation of embodiment 9:H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH
By TFA: TIS: H
2peptide solution 1.6L is cut in O=95: 2.5: 2.5 (volume ratio) preparation, after freezing, when stirring, joined by the protection vasopressin resin of 157.4g and cutting in peptide solution, and 26 DEG C are stirred 2.5 hours.Filter, filtrate is concentrated by underpressure distillation, and methylate tertbutyl ether precipitates, and 70.5g vasopressin (MW:1030.5) crude product is collected in centrifugal settling, and synthesis yield is 114%.
Embodiment 10:H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH's is refining
70.5g vasopressin crude product 5%ACN/ water (V/V) is dissolved, be made into 10g/L, utilize preparative high performance liquid phase at 0.1%TFA/ water (V/V) and 0.1%TFA/ACN (V/V) gradient elution, the component that fraction collection needs also detects, qualified component is gone up preparative column desalination again, with water and ACN gradient elution, HAc is added to obtaining in elutriant, concentrate through vacuum rotary steam and remove ACN, obtain H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH xHAc white solid finally by lyophilize, HPLC purity assay is 99.5% (see Fig. 4).Mass Spectrometric Identification structure correct (see Fig. 5).Instrument: Waters, US QTOF Premier mass spectrograph, polarity: just, capillary voltage: 3.0kV, sampling spiroid: 45V, collision energy: 4EV ion source temperature: 100 DEG C, desolvation temperature: 350 DEG C, desolvation gas: 600l/hr, sweep limit: M/z is 1002000, sweep time: 0.3 second, the time of scanning room: 0.02 second.
Discuss
The significant obstacle using Wang resins synthesis polypeptide is diketopiperazine problem; namely after condensation two amino acid; when with organic bases deaminize protecting group Fmoc, two peptide recirculations easily occur and forms diketopiperazine and come off from resin, synthetic yield reduces.In the present invention, the detailed examination yield of synthetic product, does not find obvious yield decline phenomenon, infers thus without serious diketopiperazine problem.
The method technique is simple, process control, can stablize preparation high purity vasopressin, be very suitable for the High-efficient Production of automatization.The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
The title of raw material used and production firm:
Raw material production manufacturer
Wang resin Tianjin Nankai Hecheng S&T Co., Ltd.
The biochemical company limited of protected amino acid Shanghai gill
The definite title of shorteningss all in the present invention is as follows:
DMF:N, N '-dimethyl methane amide
DCM: methylene dichloride
Fmoc:9-fluorenylmethyloxycarbonyl
Boc: tertbutyloxycarbonyl
HOBt:1-hydroxy benzo-triazole
DIC:N, N-DIC
DMAP:4-Dimethylamino pyridine
Pyridine (PipPy): piperidines pyridine
Piperidine: piperidines
Ac
2o: diacetyl oxide
MeOH: methyl alcohol
HBTU: benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate
DIPEA:N, N-diisopropylethylamine
Trt: trityl
TBu: the tertiary butyl
PbfPbf:2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl
TFA: trifluoroacetic acid
TIS: tri isopropyl silane
DCC: carbodicyclo hexylimide
BOP: benzotriazole-1-base oxygen base three (dimethylamino) phosphorus hexafluorophosphate
ACN: acetonitrile
HAc: acetic acid
Claims (5)
1. a preparation method for vasopressin, is characterized in that, comprises following steps:
(a) preparation protection vasopressin R1-Asn-Arg (R2)-Val-Tyr (R3)-Val-His (R4)-Pro-Phe-R5, wherein, R1 is amino protecting group, and R2, R3, R4 are Side chain protective group, and R5 is carboxy resin;
(b) under the effect of cutting reagent, the protection vasopressin deprotection of step (a) gained and and resin isolation, generate vasopressin H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-OH;
All chemical reactions of described step (a) preparation protection vasopressin complete continuously successively in same reactor; when arbitrary protected amino acid condensation; transfer in condensation reactor without the need to low-temperature activation in activator; when carrying out condensation reaction; protected amino acid, condensing agent are first dissolved in the reaction mixture containing peptide resin, containing volatile organic solvent DCM or Et in this mixture
2the solvent that O and non-volatile organic solvent DMF or DMSO form, then utilizes that nitrogen gas stream and peptide resin are full and uniform to be contacted, and utilizes the volatilization of volatile organic solvent to absorb heat whole reactive system to be cooled to 15-20 DEG C rapidly, then to start condensation reaction; The temperature starting condensation reaction after condensation reaction is 22-28 DEG C, and the reaction times is till monitoring reaches reaction end;
Described protection vasopressin R1, R2, R3, R4, R5 are respectively Boc, Pbf, tBu, Trt, Wang resin;
The cutting reagent of described step (b) is TFA ︰ TIS ︰ H
2o volume ratio is the mixture of 95 ︰ 2.5 ︰ 2.5.
2. preparation method as claimed in claim 1, it is characterized in that, step (a) comprises the following steps:
(1) volatile organic solvent and non-volatile organic solvent composition solvent in, under condensing agent effect, under the startup helping condensing agent, the phenylalanine of amino Fmoc protection and carry out condensation reaction through the carboxy resin of volatile organic solvent swelling treatment, obtain connecting the resin that Fmoc protects phenylalanine, then amido protecting group Fmoc is removed, obtain the resin connecting phenylalanine;
(2) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the proline(Pro) that the resin of the connection phenylalanine of step (1) gained and amino Fmoc protect carries out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting proline(Pro), phenylalanine;
(3) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the connection proline(Pro) of step (2) gained, the resin of phenylalanine and amino Fmoc protect the Histidine of side chain Trt protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(4) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the α-amino-isovaleric acid that the resin of the connection side chain Trt protection group propylhomoserin of step (3) gained, proline(Pro), phenylalanine and amino Fmoc protect carries out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(5) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the resin of the connection α-amino-isovaleric acid of step (4) gained, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine and amino Fmoc protect the tyrosine of side chain tBu protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain connecting the resin that side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(6) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the α-amino-isovaleric acid that the connection side chain tBu of step (5) gained protects the resin of tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine and amino Fmoc to protect carries out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain the resin connecting α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), phenylalanine;
(7) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the connection α-amino-isovaleric acid of step (6) gained, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin of phenylalanine and amino Fmoc protect the arginine of side chain Pbf protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain connecting side chain Pbf and protect arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin of phenylalanine,
(8) volatile organic solvent and non-volatile organic solvent composition solvent in, under the existence of condensing agent, under the startup helping condensing agent, the connection side chain Pbf of step (7) gained protects arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the l-asparagine that the resin of phenylalanine and amino Boc protect carries out condensation reaction, obtain the l-asparagine connecting amino Boc protection, side chain Pbf protects arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin of phenylalanine, namely vasopressin R is protected
1-Asn-Arg (R
2)-Val-Tyr (R
3)-Val-His (R
4)-Pro-Phe-R
5, wherein, R
1for amino protecting group Boc, R
2, R
3, R
4for Side chain protective group Pbf, tBu, Trt, R
5for carboxy resin.
3. preparation method as claimed in claim 2, it is characterized in that, the condensation system of the condensation reaction described in step (1)-(8) comprise following several in any one, wherein the 1st row AA represents amino acid, last list is shown and is helped condensing agent, middle column represents condensing agent, is separated by between row and row by "/":
(1)AA/HOBt/DIC
(2)AA/HOBt/DCC
(3) AA/TBTU/HOBT/DIPEA or NMM
(4) AA/HATU/HOBT/DIPEA or NMM
(5) AA/HCTU/HOBT/DIPEA or NMM
(6) AA/PyBop/HOBT/DIPEA or NMM
(7) AA/Bop/HOBT/DIPEA or NMM
(8) AA/HATU/HOAT/DIPEA or NMM
(9) AA/HATU/HOBT/DIPEA or NMM
(10) AA/HCTU/HOBt/DIPEA or NMM
(11)AA/CDI/HOBt
(12)AA/CDI/HOAT。
4. preparation method as claimed in claim 3, it is characterized in that, the condensing agent described in step (1) is DIC and HOBt, helps condensing agent to be DMAP; Condensing agent described in step (2)-(7) is HBTU and HOBT, and help condensing agent to be organic bases, described organic bases is DIPEA, NMM or collidine; Condensing agent described in step (8) is HOBT, helps condensing agent to be DIC.
5. preparation method as claimed in claim 4, it is characterized in that, step (7) also comprises second time condensation reaction, comprise the following steps, in the solvent that volatile organic solvent and non-volatile organic solvent form, under the existence of condensing agent HOBT, under the startup helping condensing agent DIC, the resin of the connection peptides of gained and amino Fmoc protect the arginine of side chain Pbf protection to carry out condensation reaction, then carry out amido protecting group Fmoc and remove reaction, obtain connecting side chain Pbf and protect arginine, α-amino-isovaleric acid, side chain tBu protects tyrosine, α-amino-isovaleric acid, side chain Trt protection group propylhomoserin, proline(Pro), the resin of phenylalanine.
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Non-Patent Citations (2)
| Title |
|---|
| Synthesis of a New Class of Druglike Angiotensin II C-Terminal Mimics with Affinity for the AT2 Receptor;Jennie Georgsson et al.;《J. Med. Chem.》;20070315;第50卷(第7期);全文 * |
| 固相合成血管紧张素II及其反相高效液相色谱纯化与分析;浦迎秋等;《中南药学》;20071008;第5卷(第4期);第309页左栏第1段、图1，第309页左栏最后一段至右栏第3段 * |
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Address after: 200240 Shanghai city Minhang District Jianchuan Road No. 1317 Patentee after: Add medicine to the first biochemical pharmaceutcal corporation, Ltd in Shanghai Address before: 200240 Shanghai city Minhang District Jianchuan Road No. 1317 Patentee before: Shanghai No.1 Biochemical & Pharmaceutical Co., Ltd. |