solid phase synthesis

Polymer carrier and linker for solid phase synthesis of polypeptide

1.The solid-phase synthesis of polypeptides with polymer carriers requires solid-phase carriers and connecting molecules between the solid and reactants. The correct choice of carriers and connecting molecules determines the success of solid-phase synthesis.Most of the carriers used for solid-phase peptide synthesis are polystyrene, divinylbenzene and styrene copolymer derivatives, such as < strong > chlorine resin, PAM resin, Wang's resin and amino resin < / strong >.The swelling state of the carrier resin has an obvious influence on the free diffusion of the condensation reagent and the carboxyl group component, the aggregation of peptide chains and other factors related to the condensation reaction.In order to make the carrier have better swelling property, and have large network space enough to accommodate the growing long peptide chain, and facilitate the reactants to enter the interior of the carrier, generally 1% - 2% cross-linked polystyrene bead resin or microporous resin is used.
2.The solid-phase synthesis of peptides by linkers used to bond polymers with different linkers, which are bifunctional compounds containing chloromethyl, mercaptomethyl, acyl chloride, p-benzoyl, arylsulfonyl chloride, allyl, succinyl, o-nitrobenzylalcohol and diphenylchlorosilane.An ideal linker must be very stable in the whole synthesis process, and can be cut down quantitatively after synthesis without destroying the target molecules.According to the type of C-terminal structure of the peptide linked to the resin, the appropriate linker should be selected to generate corresponding derivatives such as carboxylic acid, amide or amino alcohol.
Detection of peptide synthesized by solid phase
Even the coupling technology with high efficiency can not guarantee the acylation reaction to be carried out 100%.Moreover, the efficiency of the coupling reaction is greatly reduced when there are three-dimensional obstacles or β 2-slice sequences.There are always polypeptide chains with missing or truncated order on the polymer carrier. When they are released, they also enter into the product, which brings great difficulties to the separation.Therefore, the condensation rate of each amino acid should reach 99.9% in the solid phase synthesis of peptide, especially for the longer peptide, otherwise the product will be very impure.Therefore, it is very important to monitor the process of each step.
1 The qualitative color reaction ninhydrin method (Kaiser method) is to determine the amino groups on the resin quickly by Ninhydrin color reaction, so as to determine whether the acylation reaction is complete.The sensitivity of ninhydrin method for the determination of amino group in polystyrene resin can reach 5 μ mol / g.This sensitivity has been able to detect whether the condensation reaction has been carried out more than 99%.The color of ninhydrin is different because of different amino acid residues and sequences.Aspartic acid (ASP), asparagine (ASN) can produce very weak blue or light brown.The reaction of chromogenic reagent 2,4,62 trinitrobenzene sulfonic acid with amino group on the resin showed orange red, with a sensitivity of 5 μ mol / g resin.
Kaiser reagents include:
A,Ethanol solution of 6% ninhydrin
B,Ethanol solution of 80% phenol
C,Pyridine solution of 2% 0.001mkcn
The pyridine in the preparation needs to be treated with ninhydrin and then re evaporated before use.In the detection process, take a small amount of resin, add 2-3 drops of a, B and C respectively, and heat at 100 ℃ for 1-2min. If the solution has blue color, or the resin appears blue and reddish brown, it indicates that there is free amino group, otherwise, it indicates that the connection is complete.There are other methods to detect free amino group: trinitrobenzene sulfonic acid method, picric acid method, bromine Finland method, etc.
2 This method can be used to determine the amount of residual amino group on the resin after peptide grafting and the total amino group after removing the protective group. It can be used to quantitatively detect whether the condensation reaction and the removal of the protective group are complete.If not, it can be handled repeatedly in time.2% salicylaldehyde + 6% pyridine alcohol solution reacted with the amino group on the resin (60 ℃, 30-35 minutes). After washing, the salicylaldehyde was replaced by 5% benzylamine alcohol solution (60 ℃, 30 minutes). After diluting with benzylamine alcohol solution, the light absorption value of 315 nm was read and the amount of ammonia group was calculated (Ε = 4.36 × 103).The relative deviation of this method is less than 5% for samples with - NH2 above 0.15 μ mol / Mg resin.
3 In the middle of peptide synthesis, a small amount of peptide resin (3-10mg) was taken for cracking, ether precipitated, dissolved in appropriate solvent and analyzed directly by HPLC.The N-terminal protecting group of peptide is retained or removed as needed.When the synthesized peptide is a short polar peptide, the protective group can be retained, otherwise, the retention time in HPLC is too short, which is not conducive to analysis.Although this method is troublesome, it takes a short time and has a high accuracy.
Reaction solvent
Dimethylformamide (DMF) and dichloromethane (DCM) are commonly used solvents in peptide solid-phase synthesis, especially DMF is widely used in many reaction systems due to its high solubility for reactants and products.Although DMF has better solubility, its boiling point is higher and it needs to be decompressed to be evaporated.Moreover, the byproduct n-acylurea is easy to form in high dielectric constant solvents (DMF, CH3CN, DMSO, H2O, etc.), but not in low dielectric constant solvents (CH2Cl2, CCl4, C6H6, etc.).In nonpolar solvents, N-protected amino acids can react with DCC to form symmetric anhydrides.Therefore, as long as the reactants can be dissolved, the solvent with low dielectric constant should be selected as far as possible.In the solid-phase synthesis, DCM is mostly used as the solvent, which has two advantages: low racemization rate and slow formation of n-acylurea.When the carboxyl component is difficult to dissolve, several drops of DMF can be added to help dissolve.
Condensation reagents mainly include:Carbodiimide type, onium salt type(Uronium)
Carbodiimide type
Mainly including: DCC, DIC, edc.hcl, etc.DCC is used in the reaction, because the DCU generated in the reaction has little solubility in DMF and produces white precipitate, so it is generally not used in the solid-phase synthesis, but because of its low price, it can be removed by filtration in the liquid-phase synthesis, which is still widely used.Because of its water solubility, edc.hcl is widely used in the connection of peptides and proteins, and it is also quite successful.However, one of the biggest disadvantages of this type of condensation reagent is that if it is used alone, there will be more side reactions. However, the research shows that the side reactions can be controlled in a very low range if HOBt, hoat and other reagents are added in the activation process.
Because of its high reaction activity and high speed, onium salt type condensation reagent is widely used now, mainly including HBTU, tbtu, Hatu, pybop, etc.During the use of the reagent, it is necessary to add organic bases, such as diisopropyl ethylamine (diea) and N-Methylmorpholine (NMM). Only after the reagent is added can amino acids be activated.  

Solid phase synthesis process

1、Synthesis method with hydroxy resin as carrier (Wang resin as an example: Fig. 1)
 Wang resin, PAM resin, HMPA resin and sasrin resin all contain benzyl alcohol group in linker structure.In most cases, the prepared symmetrical anhydrides of N-protected amino acids and DMAP (p-dimethylaminopyridine) are reacted with hydroxy resin.In this bonding reaction.DMAP is necessary, but it also brings some troubles.For example, when some Fmoc amino acids with higher steric hindrance and lower reactivity need longer reaction time, and the purity of DMAP is not ideal.Some Fmoc groups will be removed and some dipeptides will be formed.The more common risk is that DMAP often racemizes Cys and his as the first amino acid at the C-terminal when they react with hydroxy resin.Therefore, the solution is to use CL TRT resin to bond with these two amino acids, which can also generate ester linker.Another solution is to use MSNT reagent [FRA i388, Ren 1998], which can effectively avoid the racemization risk of Cys, his and a variety of hydroxy resins.

Figure 1

2.Synthesis method with triphenylmethyl resin as carrier (Fig. 2)
Triphenylmethyl (TRT) resin has triphenylbenzyl structure, so the reaction activity is very high.In general, the reaction of 30-12 min at room temperature is complete when equal molar amount of protective amino acid and 4 times amount of diea and TRT resin are mixed in dichloromethane.For the unresponsive sites, methanol can be used to block the sites.Based on the mild bonding and cracking characteristics of TRT linker, it is an ideal way to condense skin segments on this resin and prepare skin segments with full protection.

Solid phase synthesis of Glu Trp by Fmoc
Figure 2

3.Synthesis method with amino resin as carrier
Mbha resin, pal resin, Knorr resin and rink-nh2 resin all contain amino groups which can react with carboxylic acid in linker.The reaction conditions of C-terminal first amino acid binding with it are exactly the same as the operation in the peptide binding cycle.If DCC is used as condensation agent, N-protected amino acids, DCC and HOBt are usually mixed in solvents (DMF, THF, DCM, etc.) to form HOBt active ester of amino acids, which is placed for 3-5h. After the activation reaction is basically complete, DCU precipitate is filtered out, and the solution of carboxyl activated component is mixed with amino resin for bonding reaction.There are two points worth noting in this step: ① most amino resins are not sold in free state.Some amino acids are in the form of hydrochloride, some have temporary protective groups (such as Fmoc, BOC, etc.), so they must be neutralized or removed before binding with the first amino acid at the C-terminal
Treatment; ② the amount of amino acid activation component should be much higher than that of amino resin component, the general molar ratio is (2-5): 1, the purpose is to make the bonding reaction close to 100%, because the amino resin that has not been acylated can not be removed from the product.The use of large excess components is common to all solid-phase organic synthesis.
     In addition to DCC, other common condensation agents are DIC, EDC, EEDQ, HBTU, etc.

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