CN116813693B - Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis - Google Patents

Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis Download PDF

Info

Publication number
CN116813693B
CN116813693B CN202311093705.8A CN202311093705A CN116813693B CN 116813693 B CN116813693 B CN 116813693B CN 202311093705 A CN202311093705 A CN 202311093705A CN 116813693 B CN116813693 B CN 116813693B
Authority
CN
China
Prior art keywords
polypeptide
arg
linker
pbf
boc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202311093705.8A
Other languages
Chinese (zh)
Other versions
CN116813693A (en
Inventor
向双春
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Jinding Biological Co ltd
Original Assignee
Suzhou Jinding Biological Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Jinding Biological Co ltd filed Critical Suzhou Jinding Biological Co ltd
Priority to CN202311093705.8A priority Critical patent/CN116813693B/en
Publication of CN116813693A publication Critical patent/CN116813693A/en
Application granted granted Critical
Publication of CN116813693B publication Critical patent/CN116813693B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a tag compound for polypeptide synthesis and application thereof in polypeptide synthesis, and relates to the technical field of organic synthesis. The tag compound introduces amino acid containing longer side chains on the 2, 4-disubstituted carbon-based tags, reduces the degree of ester-based aminolysis in the deprotection process, and increases the synthesis yield of the polypeptide. In addition, the invention introduces the C-terminal amide linker to prepare the polypeptide with the C-terminal amide, thereby expanding the application range of the 2, 4-disubstituted carbon-based label in the prior art.

Description

Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis
Technical Field
The invention relates to the technical field of organic synthesis, in particular to a tag compound for polypeptide synthesis and application thereof in polypeptide synthesis.
Background
Polypeptide compounds are mostly prepared by conventional solid-phase polypeptide synthesis (solid-phase-phase peptide synthesis, SPPS). However, solid-phase polypeptide synthesis methods suffer from several drawbacks, including: the purity of the final product is lower, the consumption of raw materials is overlarge, and more three wastes are generated. The Liquid phase polypeptide synthesis method (LPPS) meets the requirements of green chemistry and large-scale production, and the current Liquid phase synthesis method adds a soluble C-terminal Tag (Tag) to solidify the growing polypeptide chain. However, because of the great lipophilicity of the labels, the polypeptide-label adducts formed are soluble in organic solvents and are significantly different from reagents and byproducts, and the polypeptide-label adduct intermediates can be obtained by simple precipitation, filtration or extraction, thereby achieving the purpose of removing excess reagents and byproducts by elution similar to solid phase synthesis. Representative C-terminal tags are shown below:
LPPS can be produced on a kilogram or ton scale, can be automated, and has low energy input in the production process; the labor force is less; low energy and material consumption cost. The presence of long fatty chains can significantly increase the hydrophobicity of the polypeptide-tag, which is not greatly affected by the growth of the polypeptide chain, and therefore, long fatty chain based tags have also been used in LPPS. These carbon-based tags can be precipitated in a polar solvent such as acetonitrile or methanol, providing a basis for the separation and extraction of intermediates in amino acid condensation and Fmoc deprotection. Methods for producing polypeptides using C-terminal tags are disclosed in patent US20180215782, CN107406480B, US20140107319A 1.
The representative C-terminal tags described above have unique properties. More than 80% trifluoroacetic acid is required for releasing the polypeptide from the carrier, and in the preparation of the polypeptide, the 3, 5-disubstituted carbon-based label is not subjected to color reaction by acid treatment. The 3, 5-disubstituted carbon-based label is suitable for an acid-resistant Boc method polypeptide synthesis strategy. The polypeptide-carrier formed by the 2, 4-disubstituted carbon-based label is sensitive to acidic conditions, and only 2% TFA can release the polypeptide from the carrier, so that the polypeptide-carrier is suitable for preparing polypeptide fragments containing protecting groups. And the 2, 4-disubstituted carbon-based label shows deeper red when meeting acid, and the reaction is conveniently tracked in the process of preparing the polypeptide. The carbon-based tag is only suitable for the preparation of middle-short peptides because of the reduced properties of long-chain alkanes, the enhanced properties of polypeptides, and the difficulty of subsequent operations with the growth of peptide chains attached to the carbon-based tag.
However, in the process of preparing the protected polypeptide fragment by actually applying the 2, 4-disubstituted carbon-based label, the condition that the carrier is removed during the coupling and Fmoc removal of the polypeptide is found, so that the final weight gain of the polypeptide-carrier is small, and particularly, in the Fmoc removal step of the dipeptide, some dipeptides are separated more from the synthetic carrier. In order to overcome the above-mentioned defects of 2, 4-disubstituted carbon-based labels and utilize the characteristic of convenient central control, the method is effectively applied to the preparation of polypeptides, and the improvement of the polypeptide synthesis vector is very necessary.
Disclosure of Invention
The invention aims to provide a tag compound for polypeptide synthesis and application thereof in polypeptide synthesis, and the tag compound increases the yield of polypeptide synthesis, can regulate the solubility of a subsequent polypeptide-carrier, and is convenient for polypeptide coupling or purification.
Abbreviations used in the present invention and meanings corresponding to English are shown in the following table:
DIEA n, N-diisopropylethylamine
DMF N, N-dimethylformamide
NMP N-methylpyrrolidone
Fmoc 9-fluorenylmethoxycarbonyl
DBU 1, 8-diazabicyclo undec-7-ene
DIC N, N' -diisopropylcarbodiimide
HOBt 1-hydroxybenzotriazoles
HATU 2- (7-Oxybenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
HBTU Benzo (E) benzo (Etriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate
TBTU O- (IH-benzotriazol-1-yl) -N, N, N ', N' -tetramethylisourea boron tetrafluoride
COMU (2-oximino-cyanoacetic acid ethyl ester) -N, N-dimethyl-morpholinyl urea hexafluorophosphate
Trt Triphenylmethyl radical
Aloc (2-propenoxy) carbonyl group
OtBu Tert-butyl ester group
tBu Tert-butyl group
Boc Boc-group
Cbz Benzyloxycarbonyl group
Z Benzyloxycarbonyl group
Oxyma pure 2-Oxime cyanoacetic acid ethyl ester
Pbf Azodicarboxylic acid diethyl ester
HMPA Linker 4-hydroxymethyl-3-methoxyphenoxyacetic acid
Rink amide 4- [ (2, 4-Dimethoxyphenyl) (Fmoc-amino) methyl]Phenoxyacetamide
Fmoc-Rink amide Linker 4- [ (2, 4-Dimethoxyphenyl) (FMOC-amino) methyl]Phenoxyacetic acid
In order to achieve the above object, the present invention has the following technical scheme:
in one aspect, the present invention provides a tag compound for polypeptide synthesis, wherein the chemical structural formula of the tag compound is as follows:
wherein R is a linear alkane of 10 to 30C atoms or an alkane containing a branched chain with a main chain length of 14 to 40C atoms;
-O-A-L is-O-AA 1 -AA 2 -L or-O-AA 1 -AA 2 -AA 3 -L；
AA 1 Selected from Lys (Boc), lys (Z), lys (Aloc) or Nle;
AA 2 selected from Arg (pbf), lys (Boc), lys (Z), glu (OtBu), asp (OtBu), trp (Boc), gln (Trt), ser (tBu), thr (tBu), leu, val, ala, met, ile, pro, gly, tyr (tBu), asn (Trt) or Cys (Trt);
AA 3 selected from Arg (pbf), lys (Boc), lys (Z), glu (OtBu), asp (OtBu), trp (Boc), gln (Trt), ser (tBu), thr (tBu), leu, val, ala, met, ile, pro, gly, tyr (tBu), asn (Trt) or Cys (Trt).
In one aspect of the inventionIn some embodiments, the AA 1 And AA (alpha) 2 The materials in (a) can be repeatedly selected.
Preferably, L in the general formula is a Linker synthesized by polypeptide, and the Linker is selected from one or more of HMPA Linker and Rink amide Linker;
the HMPA Linker structure is as follows:
the structure of Rink amide Linker is as follows:
。
preferably, in the general formula of the tag compound described above, R is a linear alkane having 14 to 30C atoms or an alkane having a branched chain and a main chain length of 14 to 30C atoms.
Preferably, the present invention provides a method for preparing the above-mentioned tag compound, the method comprising the steps of:
(1) Introducing Fmoc-amino acid 1 into a tag compound to obtain Fmoc-amino acid-2, 4 disubstituted tag compound;
(2) Fmoc is removed by using organic alkali to obtain an H-amino acid 1-2,4 disubstituted label compound;
(3) Condensing Fmoc-amino acid 2 with an H-amino acid 1-tag compound to obtain Fmoc-amino acid 2-amino acid 1-2,4 disubstituted tag compounds;
(4) Fmoc is removed by using organic alkali to obtain an H-amino acid 2-amino acid 1-2,4 disubstituted label compound;
(5) Introducing a polypeptide synthesis linker to obtain a synthon-amino acid 2-amino acid 1-2,4 disubstituted label compound;
(6) Repeating (4) and (5) to obtain the synthon-amino acid 3-amino acid 2-amino acid 1-2,4 disubstituted label compound.
Further preferably, taking a as an example of dipeptide a, when the Linker is HMPA Linker, the reaction scheme of the tag compound is as follows:
。
further preferably, the preparation method comprises the following steps:
(1) 2,4 disubstituted carbon based tags and Fmoc-AA 1 Obtaining 2, 4-disubstituted carbon-based label-Fmoc-AA through PCB condensation 1 A lipid;
(2) 2,4 disubstituted carbon based label-Fmoc-AA 1 Fmo group removal of fat by piperidine to obtain 2, 4-disubstituted carbon-based label-AA 1 -NH 2 ；
(3) Fmoc-AA using polypeptide condensing reagent 2 With 2, 4-disubstituted carbon label-AA 1 -NH 2 Coupling to obtain 2, 4-disubstituted carbon label-AA 1 -AA 2 -Fmoc；
(4) 2,4 disubstituted carbon based label-AA 1 -AA 2 Fmoc protecting group is removed by piperidine to obtain 2, 4-disubstituted carbon-based label-AA 1 -AA 2 -NH 2 ；
(5) Coupling of Linker HMPA Linker to 2,4 disubstituted carbon based label-AA with polypeptide condensing agent 1 -AA 2 -NH 2 Obtaining 2, 4-disubstituted carbon-based label-AA 1 -AA 2 -NH-HMPA Linker 8。
Still further preferably, the 2, 4-disubstituted carbon based label of step (1) is attached to Fmoc-AA 1 The molar ratio of (2) is 1:2.
still further preferably, the 2, 4-disubstituted carbon label of step (2) -Fmoc-AA 1 The molar ratio of lipid to piperidine was 1:15.
still more preferably, the concentration of piperidine is 10%.
Still further preferably, fmoc-AA in step (3) 2 With 2, 4-disubstituted carbon label-AA 1 -NH 2 The molar ratio of (2) is 1.5:1.
still further preferably, the 2, 4-disubstituted carbon-based label-AA in step (3) 1 -NH 2 The mass volume ratio of the polypeptide condensation reagent is 1:1.5.
still further preferably, the 2, 4-disubstituted carbon-based label-AA in step (4) 1 -AA 2 -Fmoc to piperidine molar ratio of 1:15.
still more preferably, the piperidine concentration is 10%.
Still further preferably, the Linker HMPA Linker in step (5) is used with the 2, 4-disubstituted carbon based tag-AA 1 -AA 2 -NH 2 The molar ratio of (2) is 1.5:1.
still further preferably, the 2, 4-disubstituted carbon-based label-AA in step (5) 1 -AA 2 -NH 2 The molar ratio of the polypeptide condensing agent to the polypeptide condensing agent is 1:1.5.
still further preferably, the polypeptide condensing agent in step (3) and step (5) is selected from one or more of DIC, HOBt, 6-chloro-1-hydroxy-benzotriazol (6-Cl-HOBt), 1-hydroxy-7-azabenzotriazol (HOAt), N' -Dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), carbonyldiimidazole (CDI), benzotriazol-1-yl-oxy-tripyrrolidinyl (PyBOP), tripyrrolidinylphosphonium bromide hexafluorophosphate (PyBroP), 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4-one (debt), TBTU, HATU and HBTU.
Preferably, taking a as an example of dipeptide a, when the linker is Rink amide Linker, the reaction scheme of the tag compound is as follows:
。
further preferably, the method for preparing the tag compound is as follows: after introduction of linker Rink amide Linker 9 according to step (5) above, intermediate 10 was freed of Fmoc protecting group with piperidine solution to give 2, 4-disubstituted carbon label-AA 1 -AA 2 -NH-Rink amide Linker 11。
In the structural formulae of Compound 8 and Compound 11, the group-OA 1 A-AA 2 the-NH-of the-NH-is intended to represent AA 2 Specific connection form of amino group and linker HMPA, compoundThe label compounds represented by 8 and 11 correspond to the general formula of the label compounds described above.
Still more preferably, the piperidine concentration is 5-15%.
Still more preferably, the piperidine concentration is 10%.
Still further preferably, the molar ratio of intermediate 10 to piperidine is 1: (10-20).
Still more preferably, the molar ratio of intermediate 10 to piperidine is 1:15.
in yet another aspect, the present invention provides the use of the above-described tag compounds in polypeptide synthesis.
Preferably, the use of the compounds of the invention for polypeptide synthesis comprises the steps of:
(1) Introducing a first protected amino acid onto the tag compound;
(2) And obtaining a polypeptide crude product through coupling, deprotection and acidolysis.
It is further preferred that the tag compounds described in step (1) include, but are not limited to, 2,4 disubstituted carbon based tags-AA 1-AA2-NH-HMPA Linker 8, 2,4 disubstituted carbon based tags-AA 1-AA2-NH-Rink amide Linker 11.
Further preferably, the coupling, deprotection and acidolysis described in step (2) are conventional methods in the art.
The beneficial effects of the invention are as follows:
(1) According to the invention, amino acids (Lys and Nle) containing longer side chains are introduced on the 2, 4-disubstituted carbon-based label, so that the ester group at the root of the 2, 4-disubstituted carbon-based label is shielded, the degree of ester group aminolysis in the deprotection process is greatly reduced, the polypeptide is not easy to fall off from the carrier, and the polypeptide synthesis yield is increased.
(2) The number and combination of amino acids between the 2, 4-disubstituted carbon-based tag and the linker can be selected to adjust the solubility of the subsequent polypeptide-carrier, thereby facilitating the coupling or purification of the polypeptide.
(3) The C-terminal amide linker is introduced to prepare the polypeptide with the C-terminal amide, so that the application range of the 2, 4-disubstituted carbon-based label in the prior art is enlarged.
(4) The 2, 4-disubstituted carbon-based tag-A-L polypeptide synthesis carrier improves the polypeptide synthesis performance of the 2, 4-disubstituted carbon-based tag.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA in example 1.
FIG. 2 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker in example 2.
FIG. 3 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Arg (pbf) -HMPA of example 3.
FIG. 4 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Leu-HMPA in example 3.
FIG. 5 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -HMPA in example 3.
FIG. 6 is a nuclear magnetic resonance spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -Rink Amide Linker in example 3.
FIG. 7 is a high performance liquid chromatogram of crude Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH in example 4.
FIG. 8 is a mass spectrum of crude Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH in example 4.
FIG. 9 shows the reaction sequence of Ac-Ser-Val-Val-Val-Arg-Thr-NH according to example 5 2 High performance liquid chromatography.
FIG. 10 shows the structure of Ac-Ser-Val-Val-Val-Arg-Thr-NH according to example 5 2 Mass spectrum of crude product.
FIG. 11 is a high performance liquid chromatogram of the crude H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-OH in example 6.
FIG. 12 is a mass spectrum of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-OH crude product in example 6.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.
Example 1 1-methoxy-2, 4-twenty-eight alkoxy benzene-Lys (Boc) -Arg (pbf) -HMPALink
1.1Fmoc-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene Synthesis
Fmoc-Lys (Boc) -OH 93.7g (0.2 mol), DIC 25.2g (0.2 mol), HOBt 27g (0.2 mol) and 2, 4-octacosanol 64.5g (0.1 mol) were mixed in 700mL of methylene chloride and reacted at room temperature for 16 hours. The solvent was dried under reduced pressure and the resulting paste was stirred with 1000mL of acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried once again with acetonitrile, filtered and dried to obtain 101g of purified Fmoc-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in 88.6% yield.
Synthesis of 1.2H-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
Fmoc-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 100g (0.9 mol) was dissolved in 1000mL of dichloromethane, DBU 136.6g (0.9 mol) and 80mL of piperidine were added sequentially, the reaction was stirred at room temperature for 20 min and Fmoc was removed completely. Cooling the reaction liquid to 5-10 ℃, regulating the pH of the reaction liquid to 7.0 by phosphoric acid, drying the solvent by spin-drying under reduced pressure, and stirring the obtained paste by acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, and then the solid obtained by filtration was slurried with water 2 times and then with acetonitrile 2 times, and H-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 754g was obtained after filtration and air drying in 94% yield.
1.3 Fmoc-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene Synthesis
Fmoc-Arg (pbf) -OH 818.6g (1.26 mol), DIC 162.6g (1.26 mol), oxyma pure 179g (1.26 mol) and H-Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 750g (0.84 mol) were mixed in 8000mL of dichloromethane and stirred at room temperature for 40 minutes. The solvent was dried under reduced pressure and the resulting paste was stirred with acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried twice with acetonitrile to obtain 1151g of Fmoc-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in a yield of 90%.
1.4 Synthesis of H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
1151g of Fmoc-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 914g was synthesized according to the feed ratio and the procedure of step 1.2 of example 1 to yield 914g of H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in 94% yield.
1.5 Synthesis of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA Linker
140.3g (0.77 mol) of p-hydroxymethylphenoxyacetic acid (MW: 182.17), 99.3g (0.77 mol) of DIC, 104g (0.77 mol) of HOBt and 910g (0.7 mol) of H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene were mixed in 8000mL of methylene chloride and stirred at room temperature for 70 minutes. The solvent was dried under reduced pressure and the resulting paste was stirred with 5000mL of acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried twice with acetonitrile to obtain 974g of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA, the nuclear magnetic pattern of which is shown in FIG. 1, and the yield was 95%. The structural formula is as follows:
。
example 2 1-methoxy-2, 4-twenty-eight-alkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker
2.1 Fmoc-Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene synthesis
Fmoc-Rink Amide Linker 701.5.5 g (0.13 mol), H-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxy obtained in example 1.4 (130 g (0.1 mol), HBTU 49.3g (0.13 mol), HOBt 17.6g (0.13 mol) and DIEA 53mL (0.3 mol) were mixed in 7500mL of dichloromethane and stirred at room temperature for 40 minutes. The solvent was dried under reduced pressure and the resulting paste was stirred with 5000mL of acetonitrile to form a solid. The solid was stirred with acetonitrile for 10 minutes, then filtered, and the obtained solid was slurried twice with acetonitrile to obtain 229g of Fmoc-Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene in a yield of 95%.
2.2 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker
220g (0.12 mol) of Fmoc-Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 1100mL of tetrahydrofuran, 21g of DBU and 100mL of piperidine were added sequentially, and the reaction was stirred at room temperature for 20 minutes, and Fmoc was removed completely. Cooling the reaction liquid to 5-10 ℃, regulating the pH value of the reaction liquid to 7.0 by phosphoric acid, decompressing and spin-drying the solvent, and stirring the obtained paste by acetonitrile to form a solid. The solid is stirred with 1200mL of acetonitrile for 10 minutes, the solid obtained by filtration is pulped once again by acetonitrile, and 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide Linker g is obtained after filtration and air drying, the nuclear magnetic spectrum is shown in figure 2, and the yield is 87%. The structural formula is as follows:
example 3
Following the dosing ratios and operating methods of example 1 and example 2, the following compounds were prepared:
1-methoxy-2, 4-twenty-eight alkoxy benzene-Nle-Arg (pbf) -HMPA, its nuclear magnetic spectrum is shown in figure 3.
The nuclear magnetic spectrum of the 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Leu-HMPA is shown in figure 4.
The nuclear magnetic spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -HMPA is shown in figure 5.
The nuclear magnetic spectrum of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -Rink Amide Linker is shown in FIG. 6.
EXAMPLE 4 Synthesis of Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH
4.1 Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPa-Arg (pbf) -Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-twenty-eight-alkoxybenzene;
starting material 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Arg (pbf) -HMPA 186g (0.1 mol) was synthesized using the procedure of steps 1.1-1.4 of example 1 to yield 194.1g with a total yield of 78.03%.
4.2 Synthesis of Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH
150g of Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPa-Arg (pbf) -Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 1800mL of a polypeptide cleavage solution (80% TFA:5% anisole: 5% phenol: 5% water: 3% EDT:2% TIS), and the solution was stirred at room temperature for 3 hours, and 3000mL of cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 41.1g of crude polypeptide is obtained after vacuum drying at room temperature, and the purity is 92.7%. The high performance liquid chromatogram is shown in FIG. 7, and the mass spectrum is shown in FIG. 8.
Comparative example 4-1
1) Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -1-methoxy-2, 4-twenty-eight-alkoxy benzene
54.5g (0.1 mol) of starting material 2, 4-octaalkoxybenzyl alcohol were synthesized using the procedure of steps 1.1 to 1.4 of example 1 to give 131.1g, with a total yield of 41%.
2) 210g of Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -1-methoxy-2, 4-twenty-eight-alkoxy benzene was dissolved in 2600mL of polypeptide cleavage liquid (80% TFA:5% anisole: 5% phenol: 5% water: 3% edt:2% tis), stirring at room temperature for 3 hours, and adding 5000mL of cold diethyl ether into the reaction solution to precipitate polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 79.5g of crude polypeptide is obtained after vacuum drying at room temperature, and the purity is 92.3%.
Comparative example 4-2
1) Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPA-Leu-Nle-1-methoxy-2, 4-octacosalkoxybenzene
104g (0.1 mol) of 1-methoxy-2, 4-octacosalkoxybenzene-Nle-Leu-HMPA was synthesized using the procedure of steps 1.1-1.4 of example 1 to give 247.9. 247.9 g in a total yield of 69.14%.
2) Synthesis of Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Tyr-Ser-Trp-Tyr-OH
170g of Ac-Tyr (tBu) -Arg (pbf) -Ser (tBu) -Arg (pbf) -Lys (Boc) -Tyr (tBu) -Tyr (tBu) -Ser (tBu) -Trp (Boc) -Tyr (tBu) -HMPA-Leu-Nle-1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 2000ml of a polypeptide cleavage solution (80% TFA:5% anisole: 5% phenol: 5% water: 3% EDT:2% TIS), stirred at room temperature for 3 hours, 4000ml of cold diethyl ether was added to the reaction solution, and a polypeptide solid was precipitated. After repeated centrifugation and methyl tertiary ether washing, 57.1g of crude polypeptide with the purity of 92.1% is obtained after vacuum pumping at room temperature.
The above comparative examples 4-1 and 4-2 were each compared with example 4.
The data for example 4 above and comparative examples 4-1 and 4-2 are summarized in Table 1.
Table 1.
As can be seen from the above table, both comparative example 4-1 and example 4 differ little in product purity, but the unmodified 1-methoxy-2, 4-octacosalkoxybenzene polypeptide synthesis vector is significantly lower in yield than the Lys (Boc) -Arg (pbf) -Arg (pbf) modified vector. The comparison test shows that Lys (Boc) with longer side chain can shade ester group at root of 2, 4-disubstituted carbon-based label, greatly reduce the degree of degradation of ester group by amine during deprotection process and improve product yield.
The yield in comparative example 4-2 was 69.14%, which indicates that the cleavage of the ester group was largely suppressed by blocking the ester group at the root of the label with Nle.
Example 5 Ac-Ser-Val-Val-Val-Arg-Thr-NH 2 Synthesis
5.1 Ac-Ser (tBu) -Val-Val-Val-Arg (pbf) -Thr (tBu) -Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene Synthesis
Starting material 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -Rink Amide 133g (0.1 mol) was synthesized by the method of example 1, steps 1.2-1.4 to give Ac-Ser (tBu) -Val-Val-Val-Arg (pbf) -Thr (tBu) -Rink Amide-Arg (pbf) -Nle-1-methoxy-2, 4-octacosalkoxybenzene 192.7. 192.7 g in 81.2% yield.
5.2 Ac-Ser-Val-Val-Val-Arg-Thr-NH 2 Synthesis
197g Ac-Ser (tBu) -Val-Val-Val-Arg (pbf) -Thr (tBu) -Rink Amide-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 2100mL polypeptide cleavage solution (80% TFA:5% anisole: 5% phenol: 5% water: 5% TIS), stirred at room temperature for 2.5 hours, and 4000mL cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 46.5g of crude polypeptide is obtained after vacuum drying at room temperature, and the purity is 95.1%. The high performance liquid chromatogram is shown in FIG. 9, and the mass spectrum is shown in FIG. 10.
EXAMPLE 6 Synthesis of H-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
6.1 H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
144.6g (0.1 mol) of 1-methoxy-2, 4-octacosalkoxybenzene-Lys (Boc) -Arg (pbf) -HMPA (prepared by the method of example 1, steps 1.1-1.4) was synthesized to yield 278.6g, 82.1% yield.
6.2 Synthesis of H-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene 490g was dissolved in 6000mL of the polypeptide cleavage solution (80% TFA:5% anisole sulfide: 5% phenol: 5% water: 3% EDT:2% TIS), stirred for 2 hours, and 10 liters of cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 143.8g of crude polypeptide is obtained after vacuum pumping at room temperature, and the purity is 91.2%. The high performance liquid chromatogram is shown in FIG. 11, and the mass spectrum is shown in FIG. 12.
6.3 Performance test of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene
100mg of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene were dissolved in 0.5mL of each of dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydrofuran: DMF was 1:3 to obtain a clear and transparent solution. The polypeptide carrier can be continuously coupled with Fmoc-Lys (Boc) -OH, fmoc-Ala-OH and other protective amino acids by adopting DIC/HOBt, HBTU/HOBT and COMU condensing agents to form longer polypeptides.
Comparative example 6-1
1) H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-octacosalkoxybenzene
Starting material 2, 4-octacosalkoxybenzyl alcohol 64.5g (0.1 mol) the method of step 1.1-1.4 of example 1 was used, and the product was found to be poorly soluble in dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, DMF in the penultimate step, i.e. Fmoc-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-octacosalkoxybenzene. After the completion of the treatment, 99.45g of a product was obtained, and the yield was 51%.
2) Synthesis of H-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
80g H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-octacosalkoxybenzene was dissolved in 1000mL of a solution of the polypeptide in cleavage liquid (80% TFA:5% anisole sulfide: 5% phenol: 5% water: 3% EDT:2% TIS), stirred for 2 hours, and 1.2L of cold diethyl ether was added to the reaction solution to precipitate a polypeptide solid. After repeated centrifugation and methyl tertiary ether washing, 31.7g of crude polypeptide is obtained after vacuum pumping at room temperature, and the purity is 90.9%.
3) Performance test of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-1-methoxy-2, 4-twenty-eight-alkoxybenzene
100mg of H-Glu (tBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-HMPa-Arg (pbf) -Lys (Boc) -1-methoxy-2, 4-octacosalkoxybenzene was dissolved in each of 2mL of dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydrofuran/DMF (1:3) and not completely dissolved. The polypeptide carrier and Fmoc-Lys (Boc) -OH, fmoc-Ala-OH and other protective amino acids cannot react by adopting DIC/HOBt, HBTU/HOBT and COMU condensing agents.
The data for example 6 and comparative example 6-1 are summarized in Table 2.
Table 2.
As can be seen from the above table, the unmodified 1-methoxy-2, 4-octacosalkoxybenzene polypeptide synthesized vector was not only significantly lower in yield than the modified vector, but the coupling reaction was terminated when the performance of the whole polypeptide-vector reached a certain stage. From this, it can be seen that the solubility of the subsequent polypeptide-carrier can be adjusted by selecting the number and combination of amino acids between the 2, 4-disubstituted carbon based tag and the linker, facilitating the coupling or purification of the polypeptide.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (8)

1. A tag compound for polypeptide synthesis, said tag compound having the chemical structural formula:
wherein R is a linear alkane of 18 to 22C atoms;
-O-A-L is-O-AA 1 -AA 2 -L or-O-AA 1 -AA 2 -AA 3 -L；
AA 1 -AA 2 Is Lys (Boc) -Arg (pbf) or Nle-Leu;
AA 1 -AA 2 -AA 3 lys (Boc) -Arg (pbf);
l in the general formula is a Linker synthesized by polypeptide, wherein the Linker is selected from one or more of HMPA Linker and Rink amide Linker;
the HMPA Linker structure is as follows:
the structure of Rink amide Linker is as follows:
。
2. a process for producing A labeled compound according to claim 1, wherein when-O-A-L is-O-AA 1 -AA 2 When the Linker is HMPA Linker, the reaction scheme of the tag compound is as follows:
。
3. the method according to claim 2, wherein Fmoc-AA is condensed with a polypeptide condensing reagent in step (3) 2 With 2, 4-disubstituted carbon label-AA 1 -NH 2 Coupling to obtain 2, 4-disubstituted carbon label-AA 1 -AA 2 -Fmoc; the polypeptide condensation reagent is selected from one or more of DIC, HOBt, 6-chloro-1-hydroxy-benzotriazole, 1-hydroxy-7-azabenzotriazole, N' -dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, carbonyldiimidazole, benzotriazol-1-yl-oxy-tripyrrolidinyl hexafluorophosphate, tripyrrolidinylphosphonium bromide hexafluorophosphate, 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4-one, TBTU, HATU and HBTU.
4. The method for producing a labeled compound according to claim 1, wherein when the linker is Rink amide Linker, the reaction scheme of the labeled compound is as follows:
。
5. the process of claim 4, wherein intermediate 10 is prepared by removing Fmoc protecting groups from a piperidine solution to provide 2, 4-disubstituted carbon label-AA 1 -AA 2 -NH-Rink amide Linker 11; the mass concentration of the piperidine is 5-15%.
6. The process of claim 5, wherein the molar ratio of intermediate 10 to piperidine is 1: (10-20).
7. Use of a tag compound according to claim 1 or a tag compound according to any one of claims 2 to 6 in polypeptide synthesis.
8. The use according to claim 7, wherein the use of a tag compound for polypeptide synthesis comprises the steps of:
(1) Introducing a first protected amino acid onto the tag compound;
(2) And obtaining a polypeptide crude product through coupling, deprotection and acidolysis.
CN202311093705.8A 2023-08-29 2023-08-29 Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis Active CN116813693B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311093705.8A CN116813693B (en) 2023-08-29 2023-08-29 Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311093705.8A CN116813693B (en) 2023-08-29 2023-08-29 Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis

Publications (2)

Publication Number Publication Date
CN116813693A CN116813693A (en) 2023-09-29
CN116813693B true CN116813693B (en) 2023-12-01

Family

ID=88114888

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311093705.8A Active CN116813693B (en) 2023-08-29 2023-08-29 Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis

Country Status (1)

Country Link
CN (1) CN116813693B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107406480A (en) * 2015-03-04 2017-11-28 Jitsubo株式会社 Peptide symthesis method
CN113929763A (en) * 2021-11-22 2022-01-14 申联生物医药（上海）股份有限公司 Method for preparing semenotide by using soluble label as carrier

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1451207B1 (en) * 2001-11-05 2009-08-26 Irm Llc Labeling reagent and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107406480A (en) * 2015-03-04 2017-11-28 Jitsubo株式会社 Peptide symthesis method
CN113929763A (en) * 2021-11-22 2022-01-14 申联生物医药（上海）股份有限公司 Method for preparing semenotide by using soluble label as carrier

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A Pot-economical Liquid-phase Peptide Nucleic Acid Synthesis Enabled by a Soluble Tag-assisted Method;Keisuke Ogami 等;Chem. Lett.;第138-140页 *
Tag-Assisted Liquid-Phase Peptide Synthesis Using Hydrophobic Benzyl Alcohols as Supports;Yohei Okada 等;The Journal of Organic Chemistry;第320-327页 *

Also Published As

Publication number Publication date
CN116813693A (en) 2023-09-29

Similar Documents

Publication Publication Date Title
CN106749613B (en) Synthetic method of somaglutide
JP6713983B2 (en) Peptide synthesis method
US20100137559A1 (en) Process for production of cyclic peptides
EP2334314A1 (en) Process for the preparation of pramlintide
US8377891B2 (en) Process for synthesis of cyclic octapeptide
KR20100036326A (en) Process for the production of pramlintide
CN108218957B (en) Method for preparing AMG416 by combining solid phase and liquid phase
EP2566883A1 (en) Novel process for the preparation of leuprolide and its pharmaceutically acceptable salts thereof
CN113121673B (en) Method for preparing elcatonin by solid-liquid combination method
CN116813693B (en) Label compound for polypeptide synthesis and application of label compound in polypeptide synthesis
CN110878119A (en) Solid-phase synthesis method of abamectin
XUE et al. Synthesis of S‐alkyl and C‐terminal analogs of the Saccharomyces cerevisiae a‐factor: Influence of temperature on the stability of Fmoc and OFm groups toward HF
JPWO2010016551A1 (en) Method for removing dibenzofulvene
CN109354608A (en) A method of the synthesis alarelin based on Fmoc dipeptides
JP2022527041A (en) An improved way to make precanatides
CN112175046A (en) Method for synthesizing triptorelin by polypeptide solid-liquid combination
CN112279891A (en) Improved solid-phase synthesis method of triptorelin
CN113135988B (en) Preparation method of thymosin beta 4
CN113651875B (en) Oligopeptide-34 synthesis method
CN110041407B (en) Method for synthesizing dessertraline acetate based on Fmoc dipeptide
US20140296144A1 (en) Process for the preparation of octreotide acetate
CN111793125B (en) Preparation method of pure solid-phase synthesized salmon calcitonin
CN117683114A (en) Preparation method of thymalfasin
Akai et al. The dipicolylamino group as an efficient leaving group for amide bond formation via hexafluoroisopropyl ester
WO2021255639A1 (en) Synthesis of a guanylate cyclase agonist by fragments based approach

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant