Peptide Intermediates Difficult to Dissolve and Separate? Route Design and Workup Strategies for Tag-Assisted Liquid-Phase Peptide Synthesis (TAG-LPPS)

Introduction

The challenges in peptide synthesis often lie not only in whether peptide bonds can be formed, but also in whether the intermediates after each reaction step can dissolve, be separated, and continue to undergo subsequent transformations. For some peptides with high hydrophobicity, long sequences, or strong aggregation tendency, the reaction itself may proceed, but the workup and purification can become complicated.

Tag-Assisted Liquid-Phase Peptide Synthesis (TAG-LPPS) is a synthetic strategy developed to address these types of problems. By temporarily introducing a removable chemical tag onto the peptide chain, peptide intermediates may maintain better solubility during the reaction stage and become easier to handle during the workup stage through precipitation, filtration, washing, or phase separation. After synthesis is complete, the tag is removed to release the target peptide.

The value of this type of method lies in helping researchers address three practical issues at the same time: how to dissolve the intermediate, how to separate it after the reaction, and how to scale up the route.

1. The Challenges in Peptide Synthesis Are Not Limited to Coupling Reactions

The core reaction in peptide synthesis is the formation of an amide bond between amino acids. When protecting groups, coupling reagents, bases, and solvents are properly selected, many coupling reactions can generally proceed under small-scale conditions. However, whether a synthetic route is truly practical also depends on whether the intermediate after the reaction can be handled easily.

As the peptide chain elongates, the physicochemical properties of the intermediates may change significantly. When hydrophobic residues increase, the intermediate may show reduced solubility in common organic solvents. When there are many polar groups, extraction and separation may become difficult. When the sequence tends to aggregate, the reaction system may become turbid, precipitation may occur, or coupling may be incomplete.

These issues may further affect subsequent chain elongation and intermediate handling, mainly in the following aspects:

① Insufficient dissolution of the intermediate may lead to a heterogeneous reaction system and reduced coupling efficiency;

② When coupling is incomplete, incompletely elongated chains may continue into subsequent steps, forming deletion-sequence impurities;

③ When the target intermediate, by-products, and unreacted materials have similar properties, separation and purification become more difficult;

④ Column chromatography or preparative high-performance liquid chromatography that works at small scale may create high cost and operational pressure during scale-up.

Therefore, peptide route design should not only consider whether “the amino acid can be coupled,” but also whether “after coupling, the intermediate can dissolve, be separated, and continue to move forward.”

2. Traditional Solution-Phase Peptide Synthesis, Solid-Phase Peptide Synthesis, and TAG-LPPS All Have Their Own Application Boundaries

Traditional solution-phase peptide synthesis carries out peptide chain elongation in solution. Its advantage is that, when peptide intermediates maintain good solubility, the reaction system is usually relatively homogeneous; intermediates are convenient for sampling, characterization, and process control; and reagent amounts are relatively easy to adjust. Therefore, it has application value in certain fragment synthesis and scale-up studies. However, traditional solution-phase routes also have obvious limitations: after each coupling or deprotection step, the intermediate usually needs to be separated, purified, or at least effectively worked up. As the peptide chain elongates, the solubility, polarity, and aggregation tendency of the intermediate continue to change, and the difficulty of separation and purification often gradually increases.

Solid-Phase Peptide Synthesis (SPPS) attaches the starting amino acid to a solid-phase resin, and then completes peptide chain elongation through repeated deprotection and coupling. Its advantages are a clear operating workflow and the ability to remove excess reagents and small-molecule by-products through washing. It is widely used in research and development, small-scale preparation, and automated synthesis. However, SPPS is not suitable for all sequences or all scales. For certain long peptides, hydrophobic peptides, or sequences prone to aggregation, the solid-phase system may be limited by factors such as resin loading, solid–liquid mass transfer, incomplete coupling, excessive reagent use, solvent consumption, and scale-up cost.

TAG-LPPS is a tag-assisted strategy developed based on the concept of liquid-phase peptide synthesis. By temporarily introducing a removable chemical tag onto the peptide chain, it adjusts the solubility and separation behavior of tagged peptide intermediates in specific solvent systems, thereby providing an alternative route option for certain peptide projects that are difficult to dissolve, separate, purify, or scale up. Compared with traditional solution-phase routes, the core value of TAG-LPPS lies in retaining, as much as possible, the homogeneous features of liquid-phase reactions in suitable systems, while using the tag to convert some intermediate handling steps into operations that are more amenable to scale-up, such as precipitation, filtration, washing, or phase separation.

Traditional solution-phase peptide synthesis, SPPS, and TAG-LPPS are not simply substitutes for one another; each has its own applicable scenarios. The selection of a specific route should be based on a comprehensive assessment of the target sequence’s solubility, coupling efficiency, intermediate separation difficulty, final tag-removal conditions, purification requirements, and scale-up feasibility. The characteristics of the three synthetic approaches are compared in the table below:

Synthetic Approach	Main Features	Main Advantages	Application Limitations
Traditional solution-phase peptide synthesis	Peptide chain elongation is carried out in solution, usually without relying on solid-phase resin or temporary tags to assist separation	The reaction system is relatively homogeneous; intermediates are convenient to characterize; reagent amounts are relatively controllable	Intermediates usually need to be separated and purified after each reaction step; separation becomes more difficult as the peptide chain grows
SPPS	The starting amino acid is attached to a resin, followed by repeated deprotection and coupling	Clear operating workflow; excess reagents and small-molecule by-products can be removed by washing; convenient for R&D and automated synthesis	May be limited by resin loading, solid–liquid mass transfer, excessive reagent use, solvent consumption, and scale-up cost
TAG-LPPS	A removable chemical tag is temporarily introduced during liquid-phase synthesis to regulate intermediate solubility and separation behavior	Retains liquid-phase reaction features in suitable systems while improving intermediate separation and workup	Tag attachment, solubility improvement, separation efficiency, and final tag-removal conditions all need to be verified

3. What Is a “Tag”?

In TAG-LPPS, a “tag” is a chemical auxiliary group temporarily attached to the peptide chain. It may also be referred to as:

① Soluble tag;

② Hydrophobic tag;

③ Chemical tag;

④ Soluble support;

⑤ Anchor group.

These terms emphasize different aspects, but their core function is the same: to temporarily change the solubility and separation behavior of peptide intermediates, making the synthesis process easier to operate.

A suitable tag generally needs to meet the following requirements.

3.1 It Must Be Stably Attached to the Peptide Chain

The tag needs to remain stably attached to the peptide chain during synthesis and tolerate subsequent coupling, deprotection, washing, concentration, and other operations. Many tags are designed at the C-terminus of the peptide chain, while some strategies may use other attachment modes.

3.2 It Must Improve the Solubility of Peptide Intermediates

Through structural units such as hydrophobic chains, aromatic structures, silyl structures, or other solubility-modulating groups, the tag improves the dissolution behavior of tagged peptides in specific organic solvent systems. For example, in silylated tag-assisted peptide synthesis, the silylated tag can increase peptide solubility in organic solvents such as cyclopentyl methyl ether, allowing the reaction to proceed at higher concentration.

3.3 It Must Assist Separation After the Reaction

After the reaction is complete, separation can be achieved by using the solubility differences introduced by the tag. For example, peptide intermediates bearing a hydrophobic tag can remain soluble in the reaction solvent in certain systems. After the addition of a poor solvent, their solubility decreases and precipitation occurs, allowing the target intermediate to be recovered by filtration and washing while removing excess reagents, small-molecule by-products, and some soluble impurities.

Here, a “poor solvent” refers to a solvent in which the target tagged peptide intermediate has low solubility. The purpose of adding a poor solvent is to reduce the solubility of the target intermediate in the mixed solvent system and cause it to precipitate from solution. It should be noted that precipitation is only a separation method and does not mean purification is complete. If impurities co-precipitate with the target intermediate, washing conditions or subsequent purification still need to be further optimized.

The workup logic of different tags is not exactly the same. Hydrophobic benzyl alcohol tags often use poor solvents to reduce the solubility of tagged peptide intermediates, allowing them to precipitate and then be recovered by filtration and washing. Silylated tag (STag) systems place more emphasis on improving the solubility of tagged peptides in organic solvents such as cyclopentyl methyl ether (CPME), and on completing intermediate handling through water washing or liquid–liquid phase separation.

3.4 It Must Be Removable at the Final Stage

The tag is not part of the target peptide. After synthesis is complete, the tag must be removed under suitable conditions, while minimizing peptide chain degradation, side-chain side reactions, or the formation of difficult-to-remove by-products.

A tag in TAG-LPPS can be summarized as a temporary chemical group that can be attached, can regulate solubility, can assist separation, and can ultimately be removed.

4. What Problems Does Tag-Assisted Liquid-Phase Peptide Synthesis Mainly Help Address?

TAG-LPPS is mainly used to improve the operability of peptide intermediates in liquid-phase synthesis. Its role can be divided into three stages.

Stage	Main Problem	Role of TAG-LPPS	Key Evaluation Points
Reaction stage	The intermediate is difficult to dissolve, and the reaction system is heterogeneous	Improves or regulates intermediate solubility in specific organic solvent systems	Whether the coupling reaction proceeds more readily and whether incompletely coupled products are reduced
Workup stage	The intermediate is difficult to separate from a complex mixture	Uses precipitation, solubility differences, or phase-partitioning differences, together with filtration, washing, and other operations	Intermediate recovery, impurity removal efficiency, and operational convenience
Scale-up stage	Column chromatography or preparative high-performance liquid chromatography creates a heavy purification burden	Offers the possibility of converting some intermediate handling steps into precipitation, filtration, washing, or phase-separation operations	Solvent consumption, equipment occupancy, time cost, and batch-to-batch consistency

During the reaction stage, the role of the tag is mainly reflected in improving or regulating the dissolution state of the tagged peptide in a specific solvent system, making it easier to maintain a homogeneous reaction system. This is especially important for peptide intermediates that are highly hydrophobic or prone to aggregation, because such intermediates are more likely to show reduced solubility, precipitation, or reaction turbidity during chain elongation, thereby affecting the progress of subsequent coupling reactions.

During the workup and scale-up stages, the value of the tag is mainly reflected in the change in how intermediates are handled. Traditional liquid-phase peptide synthesis often requires separation and purification of intermediates after each reaction step, whereas TAG-LPPS offers the possibility of converting some intermediate handling steps into precipitation, filtration, washing, or phase-separation operations, thereby reducing the purification burden for intermediates.

TAG-LPPS does not mean that the final product no longer requires purification. For pharmaceutical peptides or high-purity research reagents, suitable final purification methods are still required to control purity, impurity profiles, and residues.

5. Which Peptide Projects Are Suitable for Considering TAG-LPPS?

TAG-LPPS is suitable as a route-evaluation option for projects involving difficult-to-dissolve, difficult-to-separate, or scale-up peptide preparation. The types of peptide projects suitable for considering TAG-LPPS are shown in the table below.

Project Type	Main Manifestation	Reason for Considering TAG-LPPS
Highly hydrophobic peptides	Many hydrophobic residues; intermediates may show reduced solubility during chain elongation	Tags can be explored to improve dissolution behavior in specific organic solvent systems
Routes in which intermediates are repeatedly difficult to purify and column chromatography needs to be reduced	Column chromatography is required after each coupling or deprotection step before the route can continue	Some purification operations may be converted into precipitation, filtration, or washing steps
Sequences with significant deletion-peptide impurities in SPPS	After incomplete coupling at a certain step, incompletely elongated chains continue to participate in subsequent reactions	A liquid-phase tag-assisted route may be considered to improve the possibility of intermediate monitoring and process control
Projects requiring scale-up preparation	Attention must be paid to reagent equivalents, solvent use, operation time, filtration performance, impurity removal capability, and batch-to-batch consistency	TAG-LPPS has evaluation value in such projects

TAG-LPPS is one route option, but it is not necessarily suitable for all peptides that are difficult to dissolve, difficult to separate, or require scale-up. Whether it is worth adopting depends on the main bottleneck of the current route. If the main problems are poor intermediate solubility, difficult post-reaction separation, or excessive purification pressure during scale-up, and if the tag can improve these issues, then TAG-LPPS has evaluation value.

For sequences that are prone to deletion-peptide impurities in SPPS, TAG-LPPS can be considered as an alternative route for evaluation. In SPPS, if coupling is incomplete at a certain step, incompletely elongated chains may continue into subsequent reactions and ultimately form deletion-peptide impurities with properties similar to those of the target peptide. The potential value of TAG-LPPS is that tagged intermediates in a liquid-phase system are generally more convenient for in-process monitoring by HPLC, LC-MS, and other methods, and there is a better opportunity to detect problems earlier through intermediate separation and process control.

TAG-LPPS does not necessarily eliminate deletion-peptide impurities. Whether it is ultimately suitable still needs to be verified based on factors such as the stability of tag attachment, whether coupling efficiency is improved, whether intermediate separation is effective, and whether tag removal proceeds smoothly.

6. How to Design a Tag-Assisted Liquid-Phase Peptide Synthesis Route

The key point in TAG-LPPS design is to determine whether the tag truly addresses the critical problem in the current route.

6.1 Analyze the Peptide Sequence Features

Before route design, sequence factors that may affect synthesis should be analyzed, including:

① Proportion of hydrophobic residues;

② Continuous hydrophobic segments;

③ Aggregation-prone sequences;

④ Amino acids with high steric hindrance;

⑤ Residues prone to racemization;

⑥ Sulfur-containing, indole-containing, or other sensitive structures;

⑦ Whether cyclization, disulfide bond formation, or C-terminal amidation is required.

These factors affect the tag type, attachment mode, and tag-removal conditions.

6.2 Determine Whether the Main Problem Comes from the Reaction or the Separation

1. If the main problem is low coupling efficiency, priority should be given to evaluating whether the tag can improve solubility and reaction concentration.

2. If the main problem is difficult intermediate separation, the focus should be on evaluating precipitation, filtration, washing, or phase-separation performance.

3. If the main problem is difficulty in scale-up, the focus should be on evaluating solvent safety, solid form, filtration rate, and impurity removal capability.

6.3 Determine the Tag Attachment Site

Most TAG-LPPS routes are designed from the C-terminus of the peptide chain, allowing the peptide chain to elongate from the C-terminus toward the N-terminus. If the target peptide requires C-terminal amidation, special side-chain modification, or subsequent cyclization, it should be confirmed in advance whether the tag attachment mode, tag-removal method, and subsequent transformation steps will affect the final structure.

6.4 Evaluate Precipitation and Washing Performance

Whether a tag is effective should not be judged only by reaction conversion. The workup performance must also be evaluated. Key points to confirm include:

① Whether a filterable solid forms after addition of the poor solvent;

② Whether the precipitate entrains many impurities;

③ Whether washing causes loss of the target intermediate;

④ Whether the filter cake is sticky or causes filter clogging;

⑤ Whether particle morphology remains stable after scale-up.

If precipitation recovery is low or filtration is difficult, the tag strategy may not be suitable for the route.

6.5 Verify the Final Tag-Removal Conditions

Tag removal is one of the key steps determining the success or failure of TAG-LPPS. The removal conditions should be as mild as possible and compatible with sensitive structures in the peptide chain. It is necessary to confirm:

① Whether the tag is completely removed;

② Whether peptide chain degradation occurs during removal;

③ Whether difficult-to-remove by-products are generated;

④ Whether the crude product after tag removal is suitable for subsequent purification;

⑤ Whether the target peptide structure remains intact.

Only when attachment, elongation, separation, and tag removal can all proceed smoothly can TAG-LPPS become a practical route.

7. Key Considerations When Using TAG-LPPS

7.1 Tag Structure Should Not Pursue Hydrophobicity Alone

Enhanced hydrophobicity can help precipitation or phase separation, but excessive hydrophobicity may also introduce new problems, such as limited reaction solvent selection, difficult solid filtration, increased washing losses, or difficult removal of residual impurities. The tag structure needs to balance solubility, separation ability, and ease of removal.

7.2 Precipitation Does Not Mean Purification Is Complete

Precipitation is only a separation method. Truly effective intermediate handling requires both high recovery and good impurity removal. If the target intermediate co-precipitates with impurities, the workup has not truly been simplified.

7.3 Small-Scale Results Need to Be Verified During Scale-Up

A precipitate that is easy to filter at small scale may become difficult to handle during scale-up due to fine particles, a sticky filter cake, uneven stirring, or overly rapid precipitation. Therefore, scale-up evaluation of TAG-LPPS should include filterability, washing efficiency, drying performance, and batch-to-batch consistency.

7.4 Tag Removal Must Not Affect the Target Peptide

Some peptides contain sensitive residues such as cysteine, methionine, tryptophan, asparagine, and glutamine. If the tag-removal conditions are too harsh, they may cause oxidation, deamidation, racemization, or chain degradation. Tag-removal conditions must be verified early in route development.

7.5 Final Quality Control Is Still Required

TAG-LPPS can reduce the difficulty of intermediate handling, but it cannot replace final quality control. For pharmaceutical peptides, final purity, related substances, residual solvents, residual reagents, salt form, moisture, and other quality attributes still need to be carefully controlled.

8. Representative Classification Tables of Chemicals Related to Tag-Assisted Liquid-Phase Peptide Synthesis

Table 1. Products Related to Tag and Support Construction

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Products related to soluble supports	25322-68-3	P615493	Poly(ethylene oxide)	Viscosity 65–115 cps	Can be used as a research material for polymer supports, polymer-assisted liquid-phase synthesis, and solubility regulation of peptide intermediates. When used for temporary anchoring of peptide chains, it generally needs to be combined with terminal-group functionalization and cleavable linker design.
Tag-building precursor	29654-55-5	D123605	3,5-Dihydroxybenzyl alcohol	≥98%	Can be used for constructing hydrophobic benzyl alcohol tag structures and for designing temporary anchoring groups for peptide chains.
Reagent for introducing hydrophobic chains	6938-66-5	B152631	1-Bromodocosane	≥95%	Can be used to introduce long alkyl chains, regulate tag hydrophobicity, and adjust the precipitation behavior of tagged peptide intermediates.

Table 2. Products Related to Protecting-Group Introduction, Deprotection, and Cleavage

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Reagent for Fmoc protecting-group introduction	28920-43-6	F106534	9-Fluorenylmethyl chloroformate (Fmoc-Cl)	For HPLC derivatization, ≥99% (HPLC)	Used for amino protection and amino acid derivatization, and can support protecting-group construction before peptide chain elongation.
Reagent for Fmoc protecting-group introduction	82911-69-1	F106173	9-Fluorenylmethyl N-succinimidyl carbonate	≥98%	Used for mild introduction of the Fmoc protecting group, suitable for protecting amino acid monomers and peptide fragments.
Reagent for Boc protecting-group introduction	24424-99-5	D432366	Di-tert-butyl dicarbonate solution	2 M in THF	Used to introduce the Boc protecting group, and can be involved in amino protection, fragment synthesis, and orthogonal protection design.
Reagent for Fmoc deprotection	110-89-4	P1506346	Piperidine (regulated precursor chemical)	Biotechnology grade, ≥99.5%	Used for removal of the Fmoc protecting group, suitable for deprotection operations during stepwise peptide chain elongation.
Reagent for acid deprotection and cleavage	76-05-1	T433655	Trifluoroacetic acid (TFA)	Anhydrous grade, ≥99%	Used for removal of acid-sensitive protecting groups, resin cleavage, and research on certain tag-removal systems.
Cleavage scavenger	6485-79-6	T420182	Triisopropylsilane (TIPS)	≥98.5%	Used as a cation scavenger in acid-cleavage systems to reduce side-chain side reactions and damage to sensitive residues.

Table 3. Coupling Reagents, Coupling Additives, Organic Bases, and Catalysts

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Base for coupling reactions	109-02-4	M104644	N-Methylmorpholine	For protein sequencing, ≥99.8% (GC)	Used for acid scavenging and regulation of reaction-system basicity during carboxylic acid activation and peptide-bond formation.
Base for coupling reactions	7087-68-5	N433364	N,N-Diisopropylethylamine solution	Suitable for peptide synthesis, ~2 M in 1-methyl-2-pyrrolidinone	Used for basicity regulation and formation of activated intermediates in peptide coupling reactions.
Carbodiimide coupling reagent	693-13-0	D433332	N,N′-Diisopropylcarbodiimide solution	1 M in THF	Used for condensation between carboxylic acids and amino components, suitable for peptide-bond construction in liquid-phase peptide synthesis.
Coupling additive	39968-33-7	H109328	1-Hydroxy-7-azabenzotriazole (HOAt)	≥99%	Used together with coupling reagents to promote active-ester formation and reduce the risk of racemization.
Coupling additive	2592-95-2	H684271	1-Hydroxybenzotriazole (HOBt)	≥99%	Used in carbodiimide coupling systems to assist active-ester formation and improve the selectivity of peptide-bond formation.
Acylation catalyst	1122-58-3	D109207	4-Dimethylaminopyridine	≥99%	Used in acylation and esterification-related steps, and can be involved in tag attachment, active-ester conversion, and fragment modification.
Uronium-type coupling reagent	148893-10-1	H109327	O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU)	≥99%	Used for efficient peptide-bond formation, suitable for sterically hindered amino acids and fragment condensation reactions.
Water-soluble carbodiimide coupling reagent	25952-53-8	E106172	1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride	≥98%	Used for carboxylic acid activation and amide-bond formation, and can be used for peptide-fragment coupling and aqueous/mixed-solvent systems.
Phosphonium-type coupling reagent	128625-52-5	P109336	Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate	≥98%	Used for peptide-fragment condensation and coupling of sterically hindered substrates, supporting peptide-bond construction in complex sequences.
Coupling additive	3849-21-6	E138773	Ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma Pure)	≥98%	Used in low-racemization coupling systems, and can be combined with carbodiimide or uronium-type coupling reagents for peptide-bond formation.
Uronium-type coupling reagent	1075198-30-9	C340003	COMU	≥98%	An Oxyma-derived uronium-type coupling reagent, suitable for peptide coupling and fragment condensation reactions; often used as one alternative to benzotriazole-based coupling systems.
Triazine-type coupling reagent	3945-69-5	D110326	4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride hydrate (DMTMM)	≥97%	Used for carboxylic acid activation and amide-bond formation, and can be used for peptide-fragment ligation under mild conditions.

Note: Benzotriazole-type coupling additives such as HOBt and HOAt require attention to thermal stability and safety risks during storage, transportation, and scale-up use. In process scale-up studies, an appropriate coupling system should be selected based on SDS information, regulatory requirements, and process safety assessment.

Table 4. Reaction Solvents, Precipitation Solvents, and Washing Solvents

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Alternative reaction solvent	5614-37-9	C119688	Cyclopentyl methyl ether (CPME)	Anhydrous grade, ≥99.9%, contains 50 ppm BHT inhibitor	Used in liquid-phase peptide synthesis and hydrophobic tag systems, and can be used for solubility screening of tagged peptide intermediates.
Reaction solvent	109-99-9	T120775	Tetrahydrofuran (THF)	Anhydrous grade, ≥99.9%, contains 250 ppm BHT stabilizer	Used in coupling, protecting-group introduction, and tag-construction-related reactions; suitable for anhydrous liquid-phase reaction systems.
Precipitation/washing solvent	67-56-1	M140298	Methanol	Anhydrous grade, ≥99.8%, H₂O ≤100 ppm	Used in experiments involving precipitation, washing, and solvent exchange of tagged peptide intermediates.
Polar aprotic reaction solvent	68-12-2	D119450	N,N-Dimethylformamide (DMF)	Anhydrous grade, ≥99.8%	Used to dissolve protected amino acids, coupling reagents, and peptide intermediates; suitable for peptide coupling reaction systems.
Precipitation/washing solvent	75-05-8	A433539	Acetonitrile (ACN)	Anhydrous grade, ≥99.8%	Used for tagged peptide precipitation, washing, crude-product handling, and sample preparation before reversed-phase purification.
Precipitation/washing solvent	108-20-3	I119694	Diisopropyl ether	Anhydrous grade, ≥99%, contains 100 ppm BHT stabilizer	Used for crude peptide precipitation after acid cleavage, intermediate washing in tag-assisted systems, and removal of small-molecule impurities.
Reaction/cleavage solvent	75-09-2	D116144	Dichloromethane	AR, ≥99.5%, contains 50–150 ppm isoamylene as stabilizer	Used for protecting-group operations, acid-cleavage systems, hydrophobic tag reactions, and solubility evaluation of intermediates.

Note: Traditional solvents such as DMF and DCM are widely used in peptide synthesis. However, in scale-up studies, toxicity, regulatory requirements, solvent recovery, waste-liquid treatment, and the feasibility of alternative solvents should be comprehensively evaluated.

Table 5. Fmoc-Protected Amino Acids and Side-Chain-Protected Amino Acid Monomers

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Hydrophobic amino acid monomer	35661-60-0	F100808	Fmoc-L-leucine	Moligand™, ≥98%	Used to introduce leucine residues, and can be used in constructing hydrophobic peptide segments and model studies of poorly soluble intermediates.
Polar side-chain-protected amino acid	71989-33-8	F116797	Fmoc-O-tert-butyl-L-serine	≥98%	Used to introduce serine residues; tert-butyl protection helps reduce side reactions of the side-chain hydroxyl group.
Acidic side-chain-protected amino acid	71989-14-5	F116773	Fmoc-L-aspartic acid β-tert-butyl ester	≥98%	Used to introduce aspartic acid residues; side-chain ester protection helps control the reactivity of acidic side chains.
Conformationally constrained amino acid monomer	71989-31-6	F113134	Fmoc-L-proline	≥98%	Used to introduce proline residues, and can be used in the synthesis of peptide segments containing turn structures and conformationally constrained sequences.
Aromatic hydrophobic amino acid monomer	35661-40-6	F105472	Fmoc-L-phenylalanine	≥98%	Used to introduce phenylalanine residues, and can be used in constructing aromatic hydrophobic peptide segments and aggregation-prone sequences.
Acidic side-chain-protected amino acid	71989-18-9	F100413	Fmoc-O-tert-butyl-L-glutamic acid	≥98%	Used to introduce glutamic acid residues; side-chain protection helps control the condensation selectivity of polycarboxyl-containing sequences.
Basic side-chain-protected amino acid	154445-77-9	F110975	Fmoc-Pbf-arginine	≥98%	Used to introduce arginine residues; Pbf protection can reduce side reactions of the guanidino group during peptide chain elongation.
Sulfur-containing side-chain-protected amino acid	103213-32-7	F100409	Fmoc-S-trityl-L-cysteine	≥98%	Used to introduce cysteine residues; trityl protection is suitable for sulfur-containing peptides and disulfide-bond precursor construction.
Low-steric-hindrance amino acid monomer	29022-11-5	F103019	Fmoc-glycine	≥98%	Used to introduce glycine residues, and can be used in flexible peptide segments, linker fragments, and low-steric-hindrance sequences.
Heterocyclic side-chain-protected amino acid	109425-51-6	F116780	N-Fmoc-N′-trityl-L-histidine	≥98%	Used to introduce histidine residues; trityl protection helps control the reactivity of the imidazole side chain.
Basic side-chain-protected amino acid	71989-26-9	F110978	Nα-Fmoc-Nε-Boc-L-lysine (Fmoc-Lys(Boc)-OH)	≥98%	Used to introduce lysine residues; side-chain protection is suitable for polyamine-containing sequences and subsequent selective modification studies.
Heterocyclic side-chain-protected amino acid	143824-78-6	F116800	Fmoc-L-tryptophan(Boc)-OH	≥97%	Used to introduce tryptophan residues; Boc protection helps reduce side reactions of the indole side chain during acidic treatment.

Note: The above are representative Aladdin products. For more product specifications, please search by “product name/CAS/catalog number” on the Aladdin website.

References

[1] Sharma A., Kumar A., de la Torre B. G., Albericio F. Liquid-Phase Peptide Synthesis (LPPS): A Third Wave for the Preparation of Peptides. Chemical Reviews, 2022, 122(16): 13516–13546.

[2] Okada Y., Suzuki H., Nakae T., Fujita S., Abe H., Nagano K., Yamada T., Ebata N., Kim S., Chiba K. Tag-Assisted Liquid-Phase Peptide Synthesis Using Hydrophobic Benzyl Alcohols as Supports. The Journal of Organic Chemistry, 2013, 78(2): 320–327.

[3] Yano S., Mori T., Kubota H. Silylated Tag-Assisted Peptide Synthesis: Continuous One-Pot Elongation for the Production of Difficult Peptides under Environmentally Friendly Conditions. Molecules, 2021, 26(12): 3497.

[4] Isidro-Llobet A., Kenworthy M. N., Mukherjee S., Kopach M. E., Wegner K., Gallou F., Smith A. G., Roschangar F. Sustainability Challenges in Peptide Synthesis and Purification: From R&D to Production. The Journal of Organic Chemistry, 2019, 84(8): 4615–4628.

[5] Ferrazzano L., Catani M., Cavazzini A., Martelli G., Corbisiero D., Cantelmi P., Fantoni T., Mattellone A., De Luca C., Felletti S., Cabri W., Tolomelli A. Sustainability in Peptide Chemistry: Current Synthesis and Purification Technologies and Future Challenges. Green Chemistry, 2022, 24: 975–1020.

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