From Analytical Instrument to Synthetic Platform: A New Strategy for Reconstructing Automated Flow Solid-Phase Peptide Synthesis with Standard Analytical HPLC

1. Why Automated Flow Peptide Synthesis Driven by Standard Analytical HPLC Deserves Attention

Peptide synthesis is not a new problem. Since Merrifield established solid-phase peptide synthesis, resin supports, protecting-group strategies, coupling reagents, and heating methods have continued to evolve, with the same fundamental goals: improving chain-extension efficiency, reducing side reactions, and obtaining target sequences more reliably. Over the past decade or so, automated flow peptide synthesis has further demonstrated that peptide-chain assembly can be carried out with shorter cycle times and a higher degree of automation: representative work in 2017 compressed the total synthesis time per amino acid residue to about 40 seconds, and in 2020 the chain length accessible by automated fast-flow chemistry synthesis was extended to 164 aa.

However, this direction has long faced a practical limitation: many methods rely on specially designed or custom-built flow synthesis equipment. Even if a laboratory understands the underlying principles, it may still lack the instrumentation needed to carry out the experiments directly. In 2025, the Winssinger group reported a ChemRxiv preprint, Peptides on Tap: Automated Flow Synthesis with Standard HPLC, proposing a more accessible approach: directly using an analytical HPLC equipped with an autosampler, stable solvent delivery, temperature control, and online UV detection modules to perform automated flow solid-phase peptide synthesis inside a short guard column packed with resin.

This work achieved the synthesis of 30 aa GLP-1 and 20 aa bivalirudin on the milligram scale, indicating that the concept is not merely an instrument modification exercise, but already has practical synthetic capability. Because this work is currently available as a preprint (a publicly released manuscript that has not yet undergone formal peer review), this method is better understood as a newly proposed approach that has completed key experimental validation, but still requires further peer review and external verification.

2. Basic Configuration and Key Design Features of Automated Flow Solid-Phase Peptide Synthesis Using Standard Analytical HPLC

Romanens, P.; Barluenga, S.; Dockerill, M.; Winssinger, N. Peptides on Tap: Automated Flow Synthesis with Standard HPLC. ChemRxiv, 2025, Version 1. DOI: 10.26434/chemrxiv-2025-6v687.

In essence, this work still belongs to automated flow solid-phase peptide synthesis rather than liquid-phase peptide synthesis. The authors packed about 10 mg of resin into a 20 × 2 mm short guard column, and then used an analytical HPLC system equipped with an autosampler, high-pressure solvent delivery, column-temperature control, and online UV/DAD detection to pass activated Fmoc amino acid solutions, deprotection reagent, and washing solvents through the resin bed in a preset sequence, thereby completing the coupling, deprotection, and washing cycles. The peptide chain is actually borne by the resin, so the reaction still occurs on the solid phase. The key advance in this work is therefore not a change in the reaction nature of peptide synthesis itself, but the reorganization of a common laboratory analytical HPLC into an automated platform capable of executing solid-phase peptide synthesis cycles.

Specific roles of the standard analytical HPLC components in this method

HPLC component	Role in this method	Significance for the experiment
Autosampler	Injects activated amino acid solutions, deprotection reagent, and washing solvents	Makes each step a programmable and reproducible quantitative operation
Solvent delivery system	Controls flow rate and residence time	Determines the contact efficiency between reagents and the resin bed
Column oven	Provides controlled heating conditions	Accelerates coupling and deprotection
Short guard column packed with resin	Serves as the actual reaction zone	Keeps the overall process within the framework of solid-phase peptide synthesis
DAD/UV detector	Records absorption signals associated with Fmoc deprotection online	Gives the synthesis process real-time observability

The reason this method can work may be summarized in three key points:

1. The operating conditions are programmable and reproducibly controllable.

The system operates at 80 °C; the activated amino acid solution passes through the resin bed at a flow rate of 0.1 mL/min, corresponding to about 1 minute of exposure time; deprotection is carried out with 20% piperidine/DMF. This means that coupling, deprotection, and washing are all converted into configurable injection sequences, flow rates, temperatures, and residence times, rather than relying on step-by-step manual operation.

2. Online detection removes dependence on after-the-fact judgment.

The authors used DAD to monitor Fmoc deprotection-related signals at 360 nm, thereby turning questions such as “Has deprotection been completed?” and “Is the cycle stable?” into real-time data. This is especially important for condition screening, method optimization, and process control.

3. The choice of coupling system balances efficiency and process controllability.

The authors employed T-BEC/Oxyma rather than simply following the common HATU-centered route. Oxyma is widely regarded as a lower-risk alternative to benzotriazole-based additives; studies on the thermal stability of peptide coupling reagents, together with subsequent work on TBEC/Oxyma, also provide support for the use of this combination.

3. Performance of This Method in Synthetic Efficiency, Stereochemistry, and Validation with Representative Peptides

1. Basic efficiency

The authors first optimized conditions using pentaphenylalanine as a model peptide. According to LC-MS evaluation, the crude purity exceeded 93%, and the completion of each individual step exceeded 99%. This indicates that the method already achieves a high level of completion in the fundamental coupling and deprotection cycles.

On this basis, the authors further synthesized a 20-mer sequence containing all 20 natural amino acids to observe how different residues behaved under this workflow. This shows that the method is not limited to a few simple residues, but has reasonably good compatibility with many common side-chain types. Even so, this remains a coverage-style validation and cannot yet be regarded as the establishment of a fully systematic scope of applicability.

2. Stereochemistry

The authors specifically examined epimerization. For residues such as proline, serine, and cysteine, no obvious epimerization was observed. For the more sensitive histidine residue, when a freshly prepared activated solution of Fmoc-His(Trt)-OH was used within a few minutes, the epimerization level was about 3.6%; under the control condition using HATU/iPr2EtN at room temperature with 5 minutes of preactivation, the value was about 4.8%. If the activated histidine solution was left standing for 4 hours before use, epimerization increased markedly to above 20%; if Fmoc-His(Bom)-OH was used instead, it could be further reduced to below 1%.

These results indicate that the method does not inherently eliminate the risk of epimerization. However, under the model residues and optimized conditions examined by the authors, stereochemical integrity can be maintained at an acceptable level. For sensitive residues such as histidine, the freshness of the activated solution and the choice of protecting group remain highly important.

3. Representative sequences

The authors did not stop validation at the short-peptide level, but instead selected two representative sequences of real practical significance: GLP-1 and bivalirudin. GLP-1 is 30 aa and could be completed within 4 hours under these conditions; bivalirudin is a 20-mer and required 2 hours and 40 minutes.

In terms of results, the crude GLP-1 product was dominated by the target peptide, although a small amount of a truncated byproduct lacking the terminal histidine was still present; for bivalirudin, no detectable truncation products were observed. According to the reported data, under the small-scale condition using 10 mg of resin, the crude amount of GLP-1 obtained was 4.98 mg, compared with a theoretical value of 9.56 mg, corresponding to about 52.1%. This scale is not a route for large-scale production, but it is already sufficient for many research-scale sample preparations, methodological validation studies, and subsequent biological testing.

4. Functional validation

The authors did not stop at LC-MS data or crude-product purity, but further compared the thrombin inhibitory activity of crude bivalirudin with that of a purified reference sample prepared by conventional SPPS. According to the reported results, the two showed comparable activity, and the crude bivalirudin had a Ki of 1.8 nM. This indicates that the crude product obtained from this method is not merely “analytically similar,” but has already reached a practically usable level in terms of function.

Key experimental results of this method and their implications

Evidence	Original result	What it demonstrates
Model peptide optimization	Crude purity of pentaphenylalanine >93%; single-step completion >99%	The basic coupling and deprotection cycles have a high degree of completion
20-mer coverage test	A sequence containing all 20 natural amino acids was synthesized	The method is not restricted to only a few simple residues
Histidine epimerization assessment	Freshly activated His(Trt): about 3.6%; HATU/iPr2EtN control: about 4.8%; aged activated solution: >20%; His(Bom): <1%	Sensitive residues require careful management, but stereochemical control is practically achievable
GLP-1 synthesis	30 aa, completed in 4 hours; 4.98 mg crude product versus a theoretical value of 9.56 mg	The method has entered the medium-length peptide range and already provides research-scale output capability
Bivalirudin synthesis	20-mer, completed in 2 hours 40 minutes	The method is applicable to representative peptide sequences of real practical significance
Bivalirudin activity	The crude product showed thrombin inhibitory activity comparable to the reference sample, Ki = 1.8 nM	The crude-product quality is already sufficient to support functional validation

4. Practical Significance of Automated Flow Solid-Phase Peptide Synthesis Using Standard Analytical HPLC

1. It lowers the entry barrier to automated flow peptide synthesis.

By transferring automated flow peptide synthesis from a workflow often tied to custom-built equipment to the more commonly available analytical HPLC platform found in laboratories, this approach improves method accessibility.

2. It integrates synthetic execution and process monitoring within a single instrument.

This method uses the built-in online UV/DAD detection of the HPLC system to record the Fmoc deprotection process in real time, so that the coupling–deprotection cycle no longer depends entirely on offline judgment. This has practical value for both condition optimization and process control.

3. It is better suited to research-scale, small-scale peptide preparation and validation.

The current results show that this approach can accomplish the synthesis of medium-length to longer peptides on the milligram scale and support subsequent activity validation. It is therefore better suited to methodological studies, sequence validation, and functional testing, rather than direct scale-up production.

5. What Types of Research Tasks Are Better Suited to Automated Flow Solid-Phase Peptide Synthesis Using Standard Analytical HPLC

Research task	Suitability	Reason
Rapidly obtaining milligram-scale medium-length to longer peptides for research validation, biochemical testing, or some cell-based experiments	Relatively high	The reported study already provides research-scale synthesis results for GLP-1 and bivalirudin, showing that this method can deliver milligram-scale samples and support subsequent functional validation
Comparing different coupling conditions, protecting groups, or sequence behaviors	High	Online UV/DAD monitoring is helpful for assessing the deprotection process and the impact of condition changes on the synthesis cycle
Using an existing analytical HPLC to carry out small-scale automated peptide synthesis	High	One of the central meanings of this work is precisely the transfer of a workflow previously dependent on specialized equipment onto a common analytical HPLC platform
Industrial scale-up or routine large-batch peptide supply	Low	This work demonstrates analytical-scale, milligram-level output and is not yet sufficient to support conclusions about scale-up production scenarios
A general solution for all difficult sequences	Low	The existing results show broad residue compatibility, but GLP-1 still produced a small amount of terminal histidine truncation byproduct, indicating that complex sequence problems have not been fully solved

6. Product Selection Guide for Standard HPLC-Driven Automated Flow Peptide Synthesis (Tables 1–5)

Current research or experimental need	Recommended table to consult first	Why this table should be consulted first	Typical points of attention
To build or optimize the resin reaction zone used for automated flow solid-phase peptide synthesis, such as selecting a column-packed resin, considering resin loading mode, or determining the preswelling and main reaction solvents	Table 1	This type of work must first address “where the reaction takes place, how the resin is handled, and which solvent system is used”; resin type, resin swelling state, and the main reaction solvent directly affect column-packing feasibility, mass transfer, and subsequent coupling performance	Rink Amide-AM resin, 2-chlorotrityl chloride resin, DMF, NMP, dichloromethane, 1,2-dichloroethane
To optimize the chain-extension cycle itself, such as coupling efficiency, deprotection rate, cleavage conditions, or side-reaction control	Table 2	The core of this type of task lies in reagent-system selection, including deprotection bases, coupling reagents, additives, and cleavage/post-treatment reagents; Table 2 usually needs to be consulted first before deciding how monomers and sequences should be matched	Piperidine, DIPEA, DIC, DCC, T-BEC, Oxyma, HOBt, HOAt, HBTU, HATU, PyBOP, TFA, TIPS
The target peptide sequence has already been determined, and the next step is to directly select Fmoc-protected amino acid monomers suitable for instrument use	Table 3	The focus at this stage is “which monomers can directly enter the Fmoc solid-phase peptide synthesis workflow,” especially key residues bearing side-chain protection; Table 3 is the most suitable for direct material selection for instrument operation	Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-His(3-Bom)-OH, Fmoc-Lys(Boc)-OH
To start from the composition of the target sequence and first understand which amino acid residues are present and which positions may be more sensitive, before deciding which Fmoc monomers to purchase	Table 4	Table 4 is not a direct monomer list for instrument loading, but helps readers first understand sequence composition, residue properties, and possible synthetic difficulties at the natural amino acid level, and then match them with the monomers in Table 3	Properties and potential synthetic challenges of residues such as His, Cys, Arg, Lys, Asp, Ser, Thr, Trp, Val, Ile, and Pro
To evaluate whether a peptide sequence may belong to a “more difficult to synthesize” category, such as having greater steric hindrance, many heterocyclic residues, or multiple sulfur- or hydroxyl-containing sites	Consult Table 4 first, then Table 3	Table 4 is used first to determine which residues make up the sequence, and Table 3 is then used to see whether the corresponding protected monomers require special attention; this way of reading is better suited to early-stage route evaluation	β-branched residues (Val, Ile), His, Cys, Ser, Thr, Arg, Lys, etc.
To compare different coupling systems and screen condensation conditions better suited to regular and difficult sites	Table 2	The selection of the coupling system determines the activation mode, reaction rate, and side-reaction risk; this step mainly focuses on the pairing of coupling reagents and additives rather than first looking at the amino acids themselves	Systems such as DIC/Oxyma, T-BEC/Oxyma, HATU/DIPEA, HBTU/DIPEA, and PyBOP
To focus on residues such as histidine, cysteine, lysine, and arginine, which are more likely to raise protecting-group or side-reaction issues	Consult Table 3 first, then Table 2	First identify the appropriate protected monomer, then return to the selection of coupling and deprotection systems; these issues usually cannot be addressed by looking only at the monomer or only at the coupling reagent	Fmoc-His(Trt)-OH, Fmoc-His(3-Bom)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH
To perform crude peptide testing after synthesis, UHPLC/LC-MS analysis, sample redissolution, or purity evaluation	Table 5	This type of work mainly takes place after synthesis and focuses on analytical and purification mobile phases rather than the earlier column-packing or coupling steps	Acetonitrile solution, water
To use the synthesized peptide for cell experiments, culture-system evaluation, or subsequent biological studies	Table 5	At this stage, the concern is no longer “how to make the peptide,” but “how to move the prepared sample into the downstream evaluation system”; the culture-related materials in Table 5 are more suitable for this stage	L-glutamic acid solution, L-glutamine solution, L-asparagine
To sort out a complete experimental workflow from scratch: resin packing → coupling/deprotection → monomer preparation → post-synthesis analysis	Read in the order of “Table 1 → Table 2 → Table 3 → Table 5”; insert Table 4 when sequence properties need to be considered	This order best matches the actual experimental workflow: first determine the solid-phase system and solvent, then define the cycle reagents, next prepare the monomers, and finally move into analysis and downstream application; Table 4 is more suitable for insertion when evaluating sequence characteristics and anticipating synthesis difficulties	Suitable for establishing a new route, reproducing a method, optimizing conditions, and understanding the overall product system as a whole

Table 1. Solid Supports, Materials Related to Resin Column Packing, and Main Reaction / Pretreatment Solvents

Category	CAS No.	Aladdin Catalog No.	Name	Specification or Purity	Product Features and Applications
Solid support / acid-labile loading resin	42074-68-0	C432714	2-Chlorotrityl chloride, polymer-bound	200–400 mesh, loading range: 1.0–1.5 mmol/g Cl loading, 1% cross-linked	A commonly used acid-labile loading resin suitable for initial amino acid loading and solid-phase chain extension in peptide synthesis; cleavage conditions are relatively mild, and it is often used for the preparation of C-terminal carboxylic acid peptides or resin-bound intermediates for further transformation.
Solid support / amide-type peptide resin	183599-10-2	R118279	Rink Amide AM resin	0.3–0.8 mmol/g, 100–200 mesh, 1% DVB	A commonly used amide-type solid-phase peptide synthesis resin that usually affords C-terminal amidated peptides after cleavage; suitable for packing into small reaction columns or guard columns for automated flow solid-phase synthesis.
Resin preswelling / washing solvent	75-09-2	D433565	Dichloromethane	Anhydrous grade, ≥99.8%, containing 40–150 ppm amylene as stabilizer	Commonly used for preswelling, washing, and solvent exchange of solid-phase resins, helping improve resin swelling state and mass-transfer performance; also often used for resin treatment before column packing.
Resin preswelling / washing solvent	107-06-2	D119458	1,2-Dichloroethane	Anhydrous grade, ≥99.8%, H2O ≤0.005%	Can be used for preswelling, washing, or solvent switching in some resin systems, and has practical value when adjustment of resin-bed state and improvement of the solubility of some components are needed.
Main reaction solvent for automated flow solid-phase peptide synthesis	68-12-2	D119450	N,N-Dimethylformamide (DMF)	Anhydrous grade, ≥99.8%	One of the most commonly used main reaction solvents in Fmoc solid-phase peptide synthesis, suitable for amino acid activation, coupling, deprotection, and washing steps, and serves as a fundamental solvent throughout peptide chain extension.
Alternative polar reaction solvent	872-50-4	M119668	1-Methyl-2-pyrrolidinone (NMP)	Anhydrous grade, ≥99.5%	A commonly used highly polar aprotic solvent that can serve as an alternative or supplement to DMF to improve the solubility and reaction performance of certain protected amino acids, coupling reagents, or resin systems.

Table 2. Reagents Related to Coupling, Deprotection, Cleavage, and Post-Treatment

Category	CAS No.	Aladdin Catalog No.	Name	Specification or Purity	Product Features and Applications
Key base for Fmoc deprotection	110-89-4	P1506346	Piperidine solution	Biotechnology grade, ≥99.5%	One of the most commonly used bases for deprotection in the Fmoc strategy, typically used together with DMF to remove the Fmoc protecting group; a fundamental reagent in each chain-extension cycle.
Common organic base for coupling activation	7087-68-5	D109322	N,N-Diisopropylethylamine	Distilled grade, ≥99.5%	A commonly used organic base in peptide coupling, which can be used together with coupling reagents such as HATU, HBTU, and PyBOP for carboxylic acid activation and condensation reactions; also often used for neutralization and condition adjustment.
Classical carbodiimide coupling reagent	693-13-0	N420184	N,N'-Diisopropylcarbodiimide	≥98.5%	A commonly used carbodiimide coupling reagent, usually employed together with Oxyma, HOBt, or HOAt for amino acid coupling and peptide-bond formation; a common basic reagent in solid-phase peptide synthesis.
Traditional / reference carbodiimide coupling reagent	538-75-0	D106074	N,N′-Dicyclohexylcarbodiimide	≥99%	A classical carboxylic acid activation reagent that can be used for amidation, esterification, and peptide coupling; in peptide synthesis, it is often used as a representative reagent of traditional carbodiimide systems. Suitable as a traditional reference system.
Carbodiimide coupling reagent / key reagent in the main line of this article	1433-27-8	T340901	1-tert-Butyl-3-ethylcarbodiimide	≥98%	A carbodiimide reagent suitable for peptide coupling, commonly used together with Oxyma for amino acid activation and peptide-bond formation; suitable for coupling reactions under automated and flow conditions.
Coupling additive / key reagent in the main line of this article	3849-21-6	E138773	Ethyl (hydroxyimino)cyanoacetate	≥98%	Also known as Oxyma, commonly used as a peptide coupling additive; when combined with carbodiimide reagents, it can improve coupling efficiency and help reduce certain side reactions.
Coupling additive / traditional promoter	123333-53-9	H106176	1-Hydroxybenzotriazole Monohydrate	≥97%	Also known as HOBt·H2O, a classical coupling promoter commonly used together with carbodiimide reagents such as DIC and DCC to improve carboxylic acid activation efficiency and coupling performance.
Coupling additive / high-activity promoter	39968-33-7	H109328	1-Hydroxy-7-azabenzotriazole	≥99%	A high-activity coupling additive, commonly used together with carbodiimides or uronium/phosphonium coupling reagents, helping improve the efficiency of difficult couplings.
Common uronium-type coupling reagent	94790-37-1	H106174	HBTU	≥99%	A commonly used uronium-type coupling reagent that can be combined with DIPEA for amino acid activation and peptide-bond formation; suitable for routine Fmoc solid-phase peptide synthesis.
High-efficiency uronium-type coupling reagent	148893-10-1	H109327	HATU	≥99%	A highly efficient uronium-type coupling reagent suitable for peptide-bond formation at difficult coupling sites, sterically hindered residues, or under rapid-activation conditions.
Phosphonium-type coupling reagent	128625-52-5	P109336	1H-Benzotriazol-1-yloxytripyrrolidinophosphonium Hexafluorophosphate	≥98%	Also known as PyBOP, a commonly used phosphonium-type coupling reagent suitable for carboxylic acid activation and amide-bond formation in peptide synthesis.
Acidic reagent for peptide cleavage / purification	76-05-1	T103293	Trifluoroacetic acid	For protein sequencing, ≥99%	One of the most commonly used strong acids in peptide synthesis; it can be used to cleave the peptide chain from acid-labile resin while simultaneously removing most side-chain protecting groups; it is also commonly used as an additive in reversed-phase HPLC purification mobile phases.
Scavenger for peptide cleavage	6485-79-6	T420182	Triisopropylsilane (TIPS)	≥98.5%	A common scavenger in TFA cleavage systems that can quench reactive intermediates formed during the reaction and reduce side reactions involving sensitive residues.

Table 3. Key Fmoc Monomers That Can Be Directly Used in Automated Flow Fmoc Solid-Phase Peptide Synthesis

Category	CAS No.	Aladdin Catalog No.	Name	Specification or Purity	Product Features and Applications
Key Fmoc-protected amino acid monomer (low-epimerization choice for histidine)	84891-19-0	F332700	Fmoc-His(3-Bom)-OH	≥99%	A protected Fmoc monomer of histidine suitable for solid-phase peptide synthesis of His residues; the Bom protecting strategy helps improve certain aspects of histidine coupling and epimerization control.
Directly instrument-compatible Fmoc monomer (hydroxyl-containing residue)	71989-33-8	F116797	Fmoc-Ser(tBu)-OH	≥98%	A protected Fmoc monomer of serine suitable for chain extension at Ser residues; tBu protection prevents the side-chain hydroxyl group from participating in side reactions.
Directly instrument-compatible Fmoc monomer (conformation-sensitive residue)	71989-31-6	F113134	Fmoc-Pro-OH	≥98%	An Fmoc monomer of proline that can be used directly in solid-phase peptide synthesis; suitable for introducing Pro residues and for investigating the effect of sequence conformation on coupling performance.
Directly instrument-compatible Fmoc monomer (aromatic hydrophobic residue)	35661-40-6	F105472	Fmoc-Phe-OH	≥98%	An Fmoc monomer of phenylalanine, one of the commonly used basic monomers, suitable for constructing hydrophobic or aromatic peptide sequences.
Directly instrument-compatible Fmoc monomer (protected arginine residue)	154445-77-9	F110975	Fmoc-Arg(Pbf)-OH	≥98%	A protected Fmoc monomer of arginine; Pbf protection reduces side reactions involving the guanidino group and is suitable for the stable introduction of Arg residues and subsequent global deprotection.
Directly instrument-compatible Fmoc monomer (protected cysteine residue)	103213-32-7	F100409	Fmoc-Cys(Trt)-OH	≥98%	A protected Fmoc monomer of cysteine; Trt protection helps reduce thiol oxidation and side reactions, making it suitable for the assembly of sulfur-containing peptide segments.
Directly instrument-compatible Fmoc monomer (protected histidine residue)	109425-51-6	F116780	Fmoc-His(Trt)-OH	≥98%	A commonly used protected Fmoc monomer of histidine, suitable for solid-phase chain extension at His residues; Trt protection improves chemical stability during assembly.
Directly instrument-compatible Fmoc monomer (protected lysine residue)	71989-26-9	F110978	Fmoc-Lys(Boc)-OH	≥98%	A protected Fmoc monomer of lysine; Boc protection of the ε-amino group prevents side-chain participation in side reactions and is suitable for the stable introduction of Lys residues.

Table 4. Natural Amino Acid Reference Materials Corresponding to Target Peptide Sequences

Category	CAS No.	Aladdin Catalog No.	Name	Specification or Purity	Product Features and Applications
Natural amino acid reference corresponding to target peptide sequences (simplest residue)	56-40-6	G432934	Glycine	UltraBio™, molecular biology grade, ultrapure grade, ≥99%(NT)	Can be used for studies of peptide-sequence composition, amino acid properties, and analytical reference; if used for solid-phase peptide synthesis, the corresponding Fmoc-protected derivative is generally selected.
Natural amino acid reference corresponding to target peptide sequences (neutral aliphatic residue)	56-41-7	L432936	L-Alanine	UltraBio™, ultrapure grade, ≥99.5%(NT)	Can serve as a basic reference material for Ala residues in sequence analysis, methodological studies, or control experiments; actual chain extension usually uses Fmoc-Ala-OH.
Natural amino acid reference corresponding to target peptide sequences (β-branched hydrophobic residue)	72-18-4	L755656	L-Valine	UltraBio™, ≥99.5%(NT)	Can be used for composition analysis and property reference of Val residues; in solid-phase peptide synthesis, the corresponding Fmoc-protected monomer is generally used.
Natural amino acid reference corresponding to target peptide sequences (hydrophobic aliphatic residue)	61-90-5	L433129	L-Leucine	UltraBio™, ultrapure grade, ≥99.5%(NT)	Can be used for composition analysis of Leu residues and as a reference for sequence design; Fmoc-Leu-OH is usually selected for actual solid-phase assembly.
Natural amino acid reference corresponding to target peptide sequences (β-branched hydrophobic residue)	73-32-5	L755616	L-Isoleucine	UltraBio™, ≥99.5%(NT)	Can serve as a basic reference material for Ile residues in amino acid composition analysis and steric-property evaluation; actual synthesis usually employs Fmoc-Ile-OH.
Natural amino acid reference corresponding to target peptide sequences (conformation-sensitive residue)	147-85-3	L476193	L-Proline	UltraBio™, ≥99.5%	Can be used for structural and conformational studies of Pro residues; in solid-phase peptide synthesis it is commonly used in the form of Fmoc-Pro-OH for chain extension.
Natural amino acid reference corresponding to target peptide sequences (sulfur-containing hydrophobic residue)	63-68-3	M101130	L-Methionine	Moligand™, ≥99%	Can serve as a composition reference material for Met residues in sequence analysis and method development; actual synthesis usually employs Fmoc-Met-OH.
Natural amino acid reference corresponding to target peptide sequences (aromatic hydrophobic residue)	63-91-2	P110424	L-Phenylalanine	Moligand™, ≥99%	Can be used for analysis of aromatic hydrophobic residues and model-peptide design; in actual solid-phase assembly, Fmoc-Phe-OH is generally used.
Natural amino acid reference corresponding to target peptide sequences (phenolic aromatic residue)	60-18-4	L755704	L-Tyrosine	UltraBio™, Moligand™, ≥99%	Can be used as a reference for the composition and structure of Tyr residues; in solid-phase peptide synthesis, side-chain-protected Fmoc-Tyr(tBu)-OH is usually employed.
Natural amino acid reference corresponding to target peptide sequences (aromatic heterocyclic residue)	73-22-3	L755707	L-Tryptophan	UltraBio™, ≥99.5%(NT)	Can be used for composition analysis and methodological reference of Trp residues; in actual synthesis, protected Fmoc-Trp(Boc)-OH is usually used.
Natural amino acid reference corresponding to target peptide sequences (hydroxyl-containing polar residue)	56-45-1	S432938	(S)-(+)-Serine	Moligand™, suitable for synthesis	Can serve as a basic reference material for Ser residues in sequence analysis and synthetic design; in solid-phase peptide synthesis, Fmoc-Ser(tBu)-OH is usually selected.
Natural amino acid reference corresponding to target peptide sequences (hydroxyl-containing polar residue)	72-19-5	L433393	L-Threonine	UltraBio™, ultrapure grade, ≥99.5%(NT)	Can be used for composition analysis of Thr residues and studies of polar residues; in actual solid-phase synthesis, protected Fmoc-Thr(tBu)-OH is usually used.
Natural amino acid reference corresponding to target peptide sequences (acidic residue)	56-84-8	L476204	L-Aspartic acid	UltraBio™, ultrapure grade, ≥99.5%(T)	Can serve as a basic reference material for Asp residues in sequence-composition studies and methodological control work; in actual solid-phase synthesis, side-chain-protected Fmoc-Asp(OtBu)-OH is usually used.
Natural amino acid reference corresponding to target peptide sequences (heterocyclic / epimerization-sensitive residue)	71-00-1	L755708	L-Histidine	UltraBio™, ≥99.5%(NT)	Can be used for composition analysis and methodological studies of His residues; in solid-phase peptide synthesis, protected Fmoc-His(Trt)-OH or other corresponding derivatives are generally used.
Natural amino acid reference corresponding to target peptide sequences (basic amino residue)	56-87-1	L598932	L-Lysine	Moligand™, ≥98%, Metal <500 ppm	Can be used for composition analysis and structural reference of Lys residues; in actual solid-phase peptide synthesis, Fmoc-Lys(Boc)-OH is usually employed.
Natural amino acid reference corresponding to target peptide sequences (basic guanidino residue)	74-79-3	A137768	L-Arginine	Moligand™, ≥99%(HPLC)	Can be used for composition analysis of Arg residues and studies of amino acid properties; in actual solid-phase synthesis, side-chain-protected Fmoc-Arg(Pbf)-OH is usually employed.
Natural amino acid reference corresponding to target peptide sequences (sulfur-containing sensitive residue)	52-90-4	L755721	L-Cysteine	UltraBio™, ≥98.5%(RT)	Can be used for basic studies and control analysis of Cys residues; in actual solid-phase peptide synthesis, protected Fmoc-Cys(Trt)-OH is usually selected.

Table 5. Mobile Phases for Peptide Analysis/Purification and Materials Related to Downstream Biological Evaluation

Category	CAS No.	Aladdin Catalog No.	Name	Specification or Purity	Product Features and Applications
Reagent for peptide analysis / purification mobile phase	75-05-8	A433526	Acetonitrile solution	MS grade, UltraPureChrom™, UHPLC grade, contains 0.1% (v/v) formic acid	Suitable for UHPLC and LC-MS analysis; can be used as a reversed-phase mobile phase compatible with peptide detection and mass spectrometry, and is also commonly used for crude-peptide purity evaluation and sample characterization.
Reagent for peptide analysis / purification mobile phase	7732-18-5	W433885	Water	MS grade, UltraPureChrom™, UHPLC grade	Suitable for preparing aqueous mobile phases for peptide analysis and purification, and can also be used for sample redissolution, dilution, and pretreatment before analysis.
Amino acid supplement related to downstream biological evaluation / cell culture	56-86-0	L1509602	L-Glutamic Acid Solution (0.2 mol/L, sterile)	Sterile-filtered, BioReagent, for cell culture, 0.2 mol/L	Suitable for supplementation of cell-culture systems, medium preparation, or experiments related to downstream biological evaluation of peptide samples.
Amino acid supplement related to downstream biological evaluation / cell culture	56-85-9	L1509609	L-Glutamine Solution (0.1 mol/L, Sterile)	Sterile-filtered, BioReagent, for cell culture, 0.1 mol/L	Commonly used for supplementation of cell-culture systems and preparation of culture conditions for functional experiments, and is suitable for downstream biological studies of peptide samples.
Amino acid supplement related to downstream biological evaluation / cell culture	70-47-3	L478521	L-Asparagine	Moligand™, BioReagent, for cell culture, for insect cell culture	Suitable for nutritional supplementation and culture-system preparation in cell culture, insect cell culture, and related biological experiments.

Note: The above are representative Aladdin products. For more product specifications, please refer to the product list at the end of the article or search by “product name / CAS / catalog number” on the Aladdin official website.

For more related articles, see below:

A Complete Guide to Choosing Resins for SPPS (Solid-Phase Peptide Synthesis): Fmoc/Boc Routes, C-Terminal Acid/Amide, and a Key-Parameter Navigator

HPLC-Grade Solvents for High-Performance Liquid Chromatography: From Concept to Practice

References

[1] Merrifield, R. B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Journal of the American Chemical Society, 1963, 85(14), 2149–2154. DOI: 10.1021/ja00897a025.

[2] Mijalis, A. J.; Thomas, D. A.; Simon, M. D.; Adamo, A.; Beaumont, R.; Jensen, K. F.; Pentelute, B. L. A fully automated flow-based approach for accelerated peptide synthesis. Nature Chemical Biology, 2017, 13(5), 464–466. DOI: 10.1038/nchembio.2318.

[3] Hartrampf, N.; Saebi, A.; Poskus, M.; Gates, Z. P.; Callahan, A. J.; Cowfer, A. E.; Hanna, S.; Antilla, S.; Schissel, C. K.; Quartararo, A. J.; Ye, X.; Mijalis, A. J.; Simon, M. D.; Loas, A.; Liu, S.; Jessen, C.; Nielsen, T. E.; Pentelute, B. L. Synthesis of proteins by automated flow chemistry. Science, 2020, 368(6494), 980–987. DOI: 10.1126/science.abb2491.

[4] Schiefelbein, K.; Hartrampf, N. Flow-based Methods in Chemical Peptide and Protein Synthesis. CHIMIA, 2021, 75(6), 480. DOI: 10.2533/chimia.2021.480. PMID: 34233808.

[5] Romanens, P.; Barluenga, S.; Dockerill, M.; Winssinger, N. Peptides on Tap: Automated Flow Synthesis with Standard HPLC. ChemRxiv, 2025. DOI: 10.26434/chemrxiv-2025-6v687.

[6] Sperry, J. B.; Minteer, C. J.; Tao, J. Y.; Johnson, R.; Duzguner, R.; Hawksworth, M.; Oke, S.; Richardson, P. F.; Barnhart, R.; Bill, D. R.; Giusto, R. A.; Weaver, J. D. Thermal Stability Assessment of Peptide Coupling Reagents Commonly Used in Pharmaceutical Manufacturing. Organic Process Research & Development, 2018, 22(10), 1262–1275. DOI: 10.1021/acs.oprd.8b00193.

[7] Subirós-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F. Oxyma: an efficient additive for peptide synthesis to replace the benzotriazole-based HOBt and HOAt with a lower risk of explosion. Chemistry – A European Journal, 2009, 15(37), 9394–9403. DOI: 10.1002/chem.200900614.

[8] Pawlas, J.; Choi, J. H.; von Bargen, C.; Maibom-Thomsen, S.; Rasmussen, J. H.; Ludemann-Hombourger, O. Elevating 1-tert-Butyl-3-ethylcarbodiimide (TBEC) as a Reagent for Sustainable Peptide Synthesis: Quality Assessment and Minimizing Racemization, Precipitation, and Radical-Induced Side Reactions by TBEC/Oxyma Couplings in an Environmentally Sensible Solvent. Organic Process Research & Development, 2023, 27(7), 1348–1364. DOI: 10.1021/acs.oprd.3c00120.