Peptide Knowledge Center

A complete guideline for solid-phase peptide synthesis

solid-phase peptide synthesis

Solid-phase organic synthesis began in the 1960s and is a new branch of organic synthetic chemistry. For half a century, solid-phase organic synthesis has expanded from the initial peptide synthesis to various organic synthesis of small molecules and complex natural products.


In particular, the special advantages of solid-phase peptide synthesis reactions have led to combinatorial chemistry and Instrument automated synthesis technology unprecedented attention from scientists to this subject.


This guideline is divided into 6 chapters, focusing on the principle, basic concepts, application scope and corresponding synthesis examples of solid-phase peptide synthesis, which are expanded in the following order:

1. Basic principles and characteristics of solid-phase organic synthesis

2. The basis for the implementation of solid-phase peptide synthesis: carrier and linker

3. A complete guide for solid-phase peptide synthesis

4. Monitoring methods in solid-phase organic synthesis reactions

5. Synthesis of various organic small molecules, heterocyclic molecules and natural products.

6. Non-classical solid phase organic synthesis

1. Basic principles and characteristics of solid-phase organic synthesis

1.1 Development history of solid phase synthesis

The ancient Greece philosopher Aristotle believed that there would be no chemical reaction without a solution. Later generations were influenced by this idea, and the chemical reaction carried out in homogeneous solution occupied a dominant position in modern chemistry history for more than 200 years


With the deepening of research, people have discovered that many important chemical phenomena that exist in nature, such as the oxidation of metals, the formation of fossil fuels, the absorption and digestion of food, the combination of sperm and eggs, and the division of cells, are all occurs in a heterogeneous medium.


In 1963, American chemist Professor Merrifield published the first article on the synthesis of tetrapeptide compounds (Leu-Ala-Gly-Val) using solid phase resin as a carrier.This marked the beginning of solid-phase organic chemical synthesis.Due to the unique advantages of solid-phase peptide synthesis, a large number of peptides, hormones and proteins with important biological activities were synthesized in just a few years, which greatly promoted the in-depth development of life science research. For this reason, Professor Merrifield was awarded Nobel Chemistry Prize in 1984 .


From the history of chemical development, the current solid-phase organic synthesis is developed on the combination of traditional organic chemistry and polymer chemistry:

The early solid-phase organic synthesis was based on the synthesis of peptide molecules. The reactions involved were mainly to form amide bonds, and the carrier type and linker structure were relatively simple. With the development of solid-phase organic synthesis chemistry, the synthesis of non-oligomeric organic small molecules has gradually become the main content in the field of solid-phase synthesis, which in turn has increased the diversity of solid-phase synthesis reaction types, solid-phase peptide reagents, and carrier linkers for big development.


The current solid-phase organic synthesis chemistry is still in the stage of rapid development. Compared with traditional organic synthesis, solid-phase organic synthesis still has huge room for development. Statistics show that in the rankings related to organic chemistry in the world today, the top 15 journals almost every issue have papers related to solid-phase organic synthesis, huge treasures are waiting to be mined.


1.2 Principles and characteristics of solid-phase organic synthesis

The progress of solid-phase organic synthesis is not a complete reaction without any solvent as a medium. During the reaction, the substrate connected to the solid-phase carrier is in an appropriate solvent, and then chemically reacts with the added foreign molecules and reagent molecules at the junction of the solid-liquid phase. After the reaction, only to remove the excess reagents and by-products in the solvent to obtain the product which attached to the solvent-insoluble carrier. Finally, after the appropriate reaction cuts the linker bond, the target product is released from the carrier. This is the basic principle of solid-phase organic synthesis:

Since simple filtration-rinsing-refiltration can remove all soluble impurities and obtain products attached to the carrier, the treatment of intermediate products after solid-phase synthesis reaction completely avoids the recrystallization, distillation, column chromatography and other complex purification operations in traditional liquid-phase synthesis. This is one of the most important features of solid phase peptide synthesis.


Since filtration can separate the product and all other soluble impurities, a large excess of soluble reagents than the substrate can be used in the reaction to promote the reaction conversion rate to approach 100%, which is another characteristic of solid-phase organic synthesis.


The pseudo-dilution effect is the third characteristic of solid-phase organic synthesis. In the classic liquid phase synthesis, in order to ensure that the product larger than the seven-membered ring is generated in the molecule, the reaction substrate is often fully diluted by the solvent to avoid the intramolecular bonding effect. 


Another function of the pseudo-dilution effect is to ensure that only a single regional group reaction occurs in molecules containing polycyclic groups.The reason why the solid-phase carrier has the characteristics of pseudo-dilution effect is because the substrate molecules participating in the chemical reaction are fixed at various positions of the carrier through the linker bond. These molecules cannot move freely like liquid phase reactions, and there is no chance for them to approach each other or collide with each other. Therefore, there is no possibility of binding between molecules of the same kind, just like they are in an infinitely diluted solution.


1.22:Pseudo dilution effect: avoid intermolecular reactions

2.What you may need to know about solid carrier and linker

2.1 A solid phase carrier which cannot be dissolved but swelled in various solvents is the most basic structural requirement for solid phase organic synthesis. Only with the carrier can the intermediate product become a solid phase component through the linker, the separation and purification can be achieved through simple filtration.The solid phase carrier and the reaction substrate are connected as a whole through a structure called linker.


Insoluble carriers must have the following properties:

• Chemical stability. Except for the initial interaction with the linker structure, the solid carrier is neither involved nor affected in the subsequent synthesis reaction

• Has good swelling properties in solvents.

• Has mechanical strength: good anti-grinding and anti-extrusion functions. Because once the carrier becomes powder, it is difficult to perform rapid filtration operations


The solid-phase peptide synthesis established by Professor Merrifield in 1963 uses polystyrene and 1%-2% styrene-based resin (bio-beads) as a solid-phase carrier.To this day, this resin (poly-styrene resin) is still the most widely used and most important carrier in solid-phase organic synthesis. In addition, polyphenylene amide-based carriers were also a common carrier in the 1970s. It is characterized by solubility in organic solvents and water, but poor chemical stability.


Later developed a PS-PEG resin combination with polystyrene and polyethylene glycol (brand name Tenta-gel) began to play a more important role. It has the advantages of water solubility and chemical stability, but also expensive. The following are a few commonly used solid phase resins:



Performance and application



Good strength, suitable for various organic solvents, low price


Polyphenylene amide

Suitable for water and organic solvents, poor stability



Various polar and non-polar solvents ,expensive



Suitable for parallel multiple synthesis, no swelling

Paper scrap


For parallel multiple synthesis, small synthesis volume

2.2 You need to know about linker

The relationship between carrier and linker

The linker is the fragment between the carrier and the target compound. Its chemical activity is closely related to the reaction type of the assembled target compound, the form of the substrate, and the final cleavage reaction type.


The principle of choosing a suitable linker:

There are many types of linkers, how to choose a linker suitable for a certain synthetic peptide is the first step to success.There are two types of mature and classic matching methods in solid-phase peptide synthesis: 1. Boc-AA(Bzl)-OH/strong acid hydrolysis resin (Merrifield, PAM, MBHA). 2.Fmoc-AA(tBu)-OH/Weak acid hydrolysis resin (Wang, Rink, Trt)


In addition to the above two types of classic matching methods, many hybrid methods that break the classics are being widely used. Of course, there is still a cooperative relationship between the linker used in these methods and the subsequent synthesis reaction. The following synthesis strategy considerations should be paid attention to by synthesis designers:


• Match the reaction conditions and product structure formation

• Ester bond linker priority principle

• Removal of all protecting groups by solid phase

• Economic principles

3.A complete guideline to solid-phase peptide synthesis

The traditional liquid phase peptide synthesis method has a history over one hundred years. The solid-phase peptide synthesis method (SPPS) created by Merrifield in 1963 is much younger. The unique advantages of the SPPS method, replacing each step of separation, recrystallization, column purification and other operations of the classic method with simple diafiltration operations, greatly improving the peptide synthesis efficiency.


Due to the wide application of the solid phase peptide synthesis method, scientists from various countries have successfully completed the synthesis of a large number of complex active peptides, which has facilitated the development of life sciences. Therefore, SPPS inventor Merrifield won the 1984 Nobel Prize in Chemistry. With the continuous maturity and development of SPPS, it has also promoted the emergence of automated peptide synthesizers and combinatorial chemistry.


In this chapter we will talk about the following aspects:

•Basic principles of solid-phase peptide synthesis


3.1 Principles of solid-phase peptide synthesis

The mainstream way of solid-phase synthesis of peptides is the C to N assembly sequence. The first step of the synthesis is to link the carboxy-terminal amino acids to the functionalized carrier, and then proceed to the assembly (Coupling cycle reaction) of each amino acid in sequence. Since the amino acid, which is the material of the synthetic peptide, has two active groups:the amino group and the carboxyl group, the amino group must be temporarily protected during the condensation reaction. When the condensation is complete, use specific conditions to remove the temporary protective group on the amino group. Therefore, each condensation reaction includes two reactions, condensation and deprotection.


In the condensation cycle, the conditions are not always same. According to the difference between the carboxyl component and the amino component, the condensation reaction maybe difficult or easy, and the speed is different. For example, Val which has a large steric hindrance, Arg which is too solvated, and Pro which has a secondary amino group are all amino acids which are difficult to connect.


In this special case, it may be necessary to increase the input of carboxyl components, high-activity condensing agent (HBTU, TBTU instead of DCC), extend the reaction time, appropriately increase the temperature, and use microwave assistance. In addition to the structure of the amino acids involved in the reaction, the length of the assembled peptide chain and the degree of hydrophobicity of the peptide chain also have an important influence on the condensation reaction.


3.2 Combination of carboxyl terminal and carrier

This refers to the reaction between the carboxyl end of the amino acid and the carrier. Due to the different structure of the linker on the carrier, an ester or amide linker can be formed after connection. After the peptide chain is assembled and cleaved, the amide linker can provide peptide amides, while the ester linker can provide peptide carboxylic acids, peptide alcohols, peptide aldehydes and peptide amides in various forms due to different cleavage conditions.


3.2.1 Connection to amino resin

MBHA, PAL, Knorr, Rink-NH2 resins all contain amino groups that react with carboxylic acids in the linker. The reaction of the first amino acid at the carboxyl terminal is the same as the coupling reaction: using DCC as a condensing agent, first mix the N-protected amino acid with DCC and HOBt in a solvent (DMF, THF, DCM) to generate an active ester of amino acid. After the activation reaction is completed, the by-product precipitate is removed by filtration, and the carboxyl activation component and the amino resin are coupled to react.


There are two points to note in this process: 1. The amino groups of most amino acid resins are not sold in a free state. Some amino groups are in the form of hydrochloride, and some have temporary protective groups (Fmoc,Boc). Therefore, the neutralization or removal of the temporary protective group must be carried out when connecting with the first amino acid at the C-terminal. 2.The amount of the amino acid activation component must be much higher than that of the amino resin component. The general molar ratio is (2-5) : 1, in order to make the connection reaction close to 100%. The use of excess substrate is common to all solid-phase organic synthesis.


3.2.2 Connection to hydroxyl resin

Wang, PAM, HMPA, and Sasrin resins all contain a glycol group in the linker structure. It is different from the coupling reaction of the first amino acid at the C terminal. In most cases, the symmetric anhydride of the N-protected amino acid and the catalyst DMAP are reacted with the hydroxyl resin.


3.2.3Connection to chloromethyl resin

The condensation of the carboxyl group of N-protected amino acid with the benzyl chloride on these type of resin is actually an ester-forming reaction with the hydrogen chloride removed. Therefore, no condensation reagent with dehydration function is needed, only a suitable alkaline reagent is needed.


3.2.4 Connection to bromoacetyl resin

The reaction conditions are similar to those of chloromethyl resin, except that the conditions are milder because the bromine atom at the alpha position of the carbonyl group is more active than benzyl chloride. When many alkaline conditions and ethyl acetate are used as the solvent, the reaction can be completed in 5-6 hours at room temperature.


3.2.5 Connection to trityl resin

Trityl resin contains a triple benzyl structure, so the reaction activity is very high. Generally, protected amino acids and four times the amount of DIEA and Trt resin are mixed in dichloromethane. The reaction is complete after 30-120 minutes at room temperature. Based on the gentle condensation and cleavage reaction of Trt linker, it is an ideal way to conduct peptide fragment condensation reaction on this resin.


3.2.6 Determination of the first amino acid content on the carrier

The degree of connection between the first amino acid at the c-terminal end of the target peptide and the carrier ,it will affect the degree of subsequent peptide assembly. Therefore, the degree of connection between the amino acid and the carrier must be quantitatively determined. The determination methods are as follows:


Resin test

Weigh the actual increase in resin and compare it with the theoretical increase to calculate the yield in this step:


Yield %= Actual increase /Theoretical increase *100%

This method is slightly less accurate, and the resin should be thoroughly dried before weighing  to reduce errors.


Fmoc assay

When the first amino acid connected to the carrier is protected by fmoc, the strong absorption of Fmoc at a wavelength of 290nm can be used for quantitative determination.


Other methods include halogen analysis, nitrogen analysis, amino acid content analysis, and free chlorine content determination. The appropriate method should be selected according to the actual situation.


3.3 Condensation reagent

There are many condensation methods to form amide bonds, but the most suitable for solid-phase peptide synthesis are the active ester method and the symmetric anhydride method.No matter what method is used to condense peptides, the most important thing is to choose a suitable condensation reagent. Among many condensation reagents for peptide synthesis, carbodiimide-type DCC is the earliest and most widely used classic reagent. It still a common condensing agent in solid phase peptide synthesis now.


The carboxyl group reacts with DCC to form an active intermediate I, which has high activity and can directly react with the amino group to form a peptide bond product. If there is an excess of carboxyl components, I can also generate symmetrical anhydrides II, and then react with amino groups to produce peptides.If HOBt is added to the condensation reagent, I can also form OBt type active ester III, and amino groups can also generate peptides. In most cases, liquid phase synthesis is based on the route from active intermediate I to final product.


In the solid-phase reaction, the amino component is attached to the solid-phase carrier, and the chance of colliding with the active intermediate I is not as much as the liquid phase. Therefore, the two approaches of I -symmetric anhydride - product and I - active ester - product are main mechanism of solid phase peptide synthesis.


The main by-product in the condensation reaction of DCC is N-acylurea by I (active intermediate) rearrangement . Its formation will consume a part of the active intermediates, so it will affect the yield of the product.


Using DCC alone as a condensing agent is often accompanied by some side reactions, and the use of composite reagents is an effective way to solve these side reactions. Commonly used compound reagents are N-hydroxy compound additives with DCC and other substances, such as HOSu, HBNb, HOBt, HOAt, etc., which can effectively reduce the degree of racemization and increase the yield of peptides.


With the further development of peptide chemistry, the synthesis of many heterocyclic peptides with complex structures and unnatural amino acids with large steric hindrance requires more efficient and functionally specific condensing agents. Many new condensing agents are constantly being developed and used in the synthesis of different types of peptides. From the structural point of view, peptide condensation reagents are divided into carbodiimide type, BOP type, etc. The following is a brief introduction


3.3.1 Carbodiimide condensation reagent 

In addition to DCC, DIC and EDC are also more commonly used carbodiimide type condensing agents. DIC has a short application history, but the DIU produced by its reaction is soluble in many organic solvents. This feature has great advantages compared to the insoluble DCU produced by the DCC reaction.


EDC is the only water-soluble carbodiimide type condensing agent, and some literatures call it WSC (water-soluble carbodiimide). The double solubility of EDC in organic solvents and water makes its application range wider than DCC and DIC.


3.3.2 BOP type condensation reagent 

The BOP-type condensing agent makes a major change in peptide condensing reagents. Structurally, the condensation mode of carbodiimide and water to form urea is completely eliminated. And the speed of the condensing agent's connecting peptide far exceeds that of DCC, and the risk of racemization is significantly reduced.


BOP condensing agent must have the participation of tertiary amine in the reaction (ex DIEA). In the application, it was found that the by-product HMPA produced by the BOP reaction was carcinogenic, and then many condensing agents of the same type appeared to replace BOP, and their reactivity was better than BOP. For example BOP, PyBOP, HBTU, TBTU, HBPyU, HBPipU, BOI.


3.3.3 AOP type condensation reagent 

The difference between the basic structure of this type of condensing agent and the BOP type is that the pyridine ring replaces the original benzene, and the condensation activity of AOP is stronger than that of BOP. It has been widely used in the synthesis of peptide libraries. This type of condensing agent is characterized by fast speed and low racemization rate.


Such condensing agents include PyAOP, HATU, HAPyU, etc.


3.4.4 Phosphate type condensation reagent 

Representative compounds of this type of condensing agent include DPPA, DEPBT, FDPP, etc. They are relatively mild condensing agents and have been successfully used in solid-phase peptide synthesis.


DPPA was used in the synthesis of solid-phase peptides in 1980. It has the smallest racemization reaction compared to other condensation methods. Therefore, the use of DPPA as a condensing agent can make Ser, Thr, His, Trp and other amino acid side chains need not be protected.It should be noted that it is necessary to control the temperature below 5 degrees Celsius. Otherwise it will generate highly active by-products。


The other two phosphate condensing agents FDPP and DEPBT are also relatively mild peptide condensing agents, which can be used to synthesize peptides with minimal protection (Ser, Thr, Tyr side chains are not protected).


3.4 Protecting group

The protection and deprotection of active groups occupies a very important position in organic synthesis. There are many new types of protecting groups that are constantly emerging. The following is an introduction according to the classification of protected chemical groups.


3.4.1 Amino protecting group

3.4.2 Carboxyl protecting group

3.4.3 Hydroxyl protecting group

3.4.4 Guanidine Protecting Group

3.4.5 Imidazole protecting group

3.4.6 Sulfhydryl protecting group

3.4.7 Indole protecting group

3.4.8 Amide protecting group


3.5 Peptide connection

3.5.1 The choice of amino acid protection

Before peptide synthesis, it is first necessary to determine which protection method to be used. Because different protection methods require different linker / resins and different cleavage conditions. The protection mode of solid phase peptide synthesis is simpler than that of liquid phase synthesis. The groups used to protect the alpha amino group are mainly Boc, Moz, and Fmoc in solid phase synthesis.


The groups used for side chain protection are matched with different amino protecting groups. The side chain protecting groups used in solid-phase peptide synthesis with Boc and Fmoc are as follows:

Using of side chain protecting groups in solid phase peptide synthesis:

Active side chain group




































In addition, with the increasing marketization of various raw materials, reagents, and instruments, peptide synthesis using Fmoc chemistry has gradually become the primary choice:

Comparison of Boc and Fmoc peptide synthesis:

Related conditions



Conditions for removing protecting groups

30%-50% of TFA /DCM

20% Piperidine /DMF

Type of solid carrier

Merrifield ,Pam,MBHA


Cleavage reagent

Strong acid

Weak acid

Automated synthesis



Raw material average price ratio


Environmental impac

More acid emissions


3.5.2 Peptide assembly

In 1963, Merrifield's first solid-phase peptide synthesis was to synthesize Leu-Ala-Gly-Val tetrapeptide on a chloromethyl resin by condensing one amino acid residue at a time.In the early stage after that, because the various technologies are not yet complete, most of the products are oligopeptides with less than 10 amino acids, and they are completed by gradual condensation. Since the solid-phase reaction cannot fully carry out purification operations such as recrystallization, fractionation, or column chromatography, the missing peptides or other by-products that may exist in each condensation step can only accumulate until the end of the reaction.


Therefore, the purity of the final product is very poor, especially the solid phase synthesis of large peptide molecules, this problem is more serious.The specific situation is as follows

Length (AA)

Cumulative conversion rate

Cumulative yield of by-products

Final actual yield

















































It can be seen from the table that the yield of short peptides with more than a dozen amino acids synthesized by the stepwise synthesis method is satisfactory. After more than 20AA peptides, the content of by-products increases surprisingly. What's more troublesome is that the by-products of peptide synthesis are mainly oligopeptides with various amino acid deletions. Their differences in polarity, solubility, molecular weight, etc. are small, making separation and purification difficult.


In order to overcome this shortcoming, in addition to using efficient new condensing agents and improving reaction conditions (such as time, temperature, solvent), it is a very effective method to use fragment condensation instead of stepwise synthesis. Its method is that instead of connecting one amino acid at a time on the solid-phase carrier, it connects with a peptide fragment each time. This peptide can contain 2-10 amino acids in length.


The connected fragments must meet the following requirements: 1.N-terminal and side chain reactive groups must be protected. 2.Try to use GLY and PRO as the C-terminal amino acid fragments with less racemization risk. 3.It is easily soluble in organic solutions (such as DMF, NMP). 4.Single pure product.


The advantage of fragment condensation is that the overall yield and purity of the product are significantly improved. Even if the by-products need to be removed after the target long peptide is assembled, the corresponding purification operation is much easier than the stepwise method. The structure difference between these by-products and the target product is large (not an amino acid, but a fragment difference). It can be separated by solvent precipitation method or sephadcx method.


3.5.3 Problems encountered in the peptide condensation reaction


(1)Difficult amino acids

Since the substrates and reagents of the solid-phase synthesis reaction are in a heterogeneous medium, the collision opportunities of various molecules are not as many as the homogeneous liquid phase reaction, so the reaction rate is slow or not complete are common. In this regard, the following methods can be used to solve the problem of incomplete condensation.


When encountering difficult amino acids, it is necessary to further increase the amount of carboxyl component and condensation reagent (5 to 10 times).In peptide synthesis, 20 amino acids have different activities. Even if it is the same amino acid, it will still have different activities because of different protective groups or whether the amino acid is involved in the reaction as an amino component or a carboxyl component.To use this method, you need to know enough about the amino acid activity of the substrate to be condensed.


Repeat the condensation. Two to three condensation methods can be used to completely react the amino acid components. It should be noted that the repeated condensation requires the use of a different condensation reagent or solvent medium from the first condensation. This change will improve the solvation state of the resin and peptide chain, exposing free amino acid groups that are not condensed for the first time.


Block the reaction. If three repeated condensations still show a positive reaction of ninhydrin, it indicates that a small amount of non-condensed amino components are difficult to acylate. In this case, only the acetic anhydride/pyridine acylation method can be used to acetylate the remaining amino groups.


For the difficult amino acid situation, some people has conducted a special study. This is the difficulty sequence of the Fmoc-amino acid as the carboxyl component in the condensation reaction.





Condensation activity


Condensation activity


Condensation activity































We can see that no matter what the protection method of Arg is, it is typical of difficult amino acids. According to the activity of various amino acids in the above table, the corresponding reaction conditions should be prepared in the early stage of peptide design.


(2) Condensation method selection

There are fewer ways to condense peptides in the solid phase than in the liquid phase, and basically use carbodiimide-type condensing agents, and various BOP condensing agents can also be used. The BOP-type condensation reagent has high activity and low degree of racemization. When used in solid-phase peptide synthesis, it is not necessary to consider the carboxyl component too much, and the ratio of 2:1 to the amino component is sufficient.


(3) Difficult sequence

Many solid-phase peptide synthesis examples show that in some cases where the condensation is not complete, even if improved reaction conditions are used, such as changing the condensation reagents, prolonging the reaction time, and repeating the condensation many times, the effect is not good. These incomplete condensations are often not related to the types of amino acids, but are closely related to the length of the peptide chain that has grown.


A large number of studies have found that certain peptide chains are within 6-16 amino acids in length, and it is difficult to completely condense each step. The peptides in this interval are called “ difficult sequences ”. The reason for this is that the peptide chain is prone to intra- or intermolecular hydrogen bonding within a certain length. In addition, there is a phenomenon of molecular aggregation between the peptide chain and the resin carrier in solid-phase peptide synthesis. Therefore, the influence of “ difficult sequences” in solid-phase peptide synthesis is much greater than that in liquid phase.


A large number of hydrogen bonds around the peptide chain lead to tight aggregation of the peptide chain, so the solubility is greatly reduced. Later studies found that the peptide molecules that undergo molecular aggregation have a secondary structure dominated by beta folding. Beta transfer and folding make the amino or carboxyl end of the peptide molecule buried deep in the secondary structure, making it difficult for condensation reactions with other molecules.


For the customized beta structure of difficult sequence, the following measures can be taken in peptide synthesis:


1. Reasonable fragment condensation: The biggest hazard of the beta structure is that the reaction components cannot be fully dissolved. Containing Pro amino acids in the middle of the peptide chain can effectively destroy the formation of the beta structure and facilitate the condensation reaction of the fragment peptide.


2.N temporary substitution method

According to the principle that Pro can terminate the Beta folding structure, a substituent with a certain volume can be introduced into the N atom of the amide bond in the target peptide molecule without Pro to make it a secondary amine amino acid (similar to Pro). After the synthesis is completed , The linker is cleaved and the substituent is removed at the same time to restore the structure of the target peptide. Na Dmb is the first substituent introduced into the N atom of the amide bond to prevent beta folding.


3.High chaotropic sequence additives

It was the first to use the interference effect of urea molecules on hydrogen bonds to destroy the Beta structure of the peptide chain. It was later discovered that the salt composed of bulky anions and cations (NA,K )is an ideal additive to destroy the beta structure.


3.6 Release of peptide product

After the peptide is assembled, the linker must be cleaved, and the solid-phase carrier must be excised before the target peptide can be obtained. The release method of peptide synthesis is simple,mainly including acid hydrolysis, saponification, ammonia hydrolysis, and transesterification.



The most important and most widely used cleavage reaction in solid-phase peptide synthesis is accomplished in various acidic environments. This cleavage method is suitable for most types of carriers. At the same time, the side chain protecting group and carrier are removed at the same time, and the active peptide product close to the natural structure can be obtained.


Resins on different peptide chains require different acid reagents, such as Trt type resins require <20% TFA. Wang resin requires 80-95% TFA, Merrifield resin requires low concentration of HF or TFMSA, PAM and MBHA resin requires high concentration of HF or TFMSA to complete the cleavage.


Most side chain protecting groups can be removed under different acidic conditions, but there are several protecting groups used in orthogonal strategies, which must be removed before or after acid hydrolysis.

Side chain situation

Cleavage method

Side chain situation

Cleavage method

Cys (Acm)












50%Piperidine /DMF


20% Piperidine /DMF


0.1% Piperidine /H2O-DMF/DMS/TFA

Cleavage of Fmoc resin

Using Fmoc amino acids as substrates, weak acid-sensitive linker carriers, such as wang resin, HMPA resin, and Rink resin, can be cleaved by TFA. Different peptides have different amino acid groups and side chain protecting groups, so the choice of cleavage reagent is also different:


When the peptide chain contains Arg and Met groups, the acid hydrolysis reagent generally uses 8.4% PhOH-EDT (3:1), 4.3% Sulfanisole,83.0 %TFA and 4.3% H2O;

95% TFA and 5% H2O can be used when the peptide chain does not contain Arg, Met, Trt,and Trp protecting groups.

When peptide chain contains Trp and Trt can use 2.5%EDT,2.5%H2O and 95.0% TFA.


Use of scavengers

There are many types of scavengers, with different structures and activities. Therefore, a reasonable choice of scavengers is very important to improve the cleavage and yield of peptides. Commonly used scavengers are anisole, m-cresol, DMS, EDT, PhOH, p-cresol, etc., but the scavenger that needs attention is H2O.Water must not be used as a scavenger in strong acid cleavage reactions, which will cause the amide bond to break. Thioanisole is an excellent scavenger in various acidolysis reactions.