Solid-Phase Peptide Synthesis – More Efficient with Sonication
Solid Phase Peptide Synthesis (SPPS) is the common method for peptide synthesis. Ultrasonication is a reliable tool to intensify solid phase peptide synthesis resulting in higher yields, improved purity, no racemization and significantly accelerated reaction speed. Hielscher Ultrasonics offers various ultrasonic solutions for peptide synthesis, cleavage, and dissolving.
Ultrasonic Solid-Phase Peptide Synthesis
Ultrasonication is already widely applied as intensifying method in organic synthesis and is well known for its advantages such as drastically reduced reaction times, higher yields, less by-products, the initiation of pathways, which could not be achieved in other ways, and/or better selectivity. Great benefits can be also obtained, when sonication is coupled into peptide synthesis reactions. Research results have demonstrated that ultrasonically assisted peptide synthesis achieves optimized yield of peptides with high purity, without racemization within a short reaction time.
- High peptide yields
- Significantly faster synthesis
- Higher peptide purity
- No racemization
- Parallel synthesis of various peptides
- Scalable to any volume
Ultrasonic cuphorn for the uniform sonication of several reactor vessels for improved peptide synthesis.
Graphic demonstrating the Merrifield solid phase peptide synthesis. Ultrasonication is used to promote and enhance the synthesis reaction as well as for cleavage of the synthesized peptides from the resin.
Graphic: ©Conejos-Sanchez et al., 2014)
| Study | Peptide(s) Studied | Reaction Time (Classical vs US-SPPS) | Crude Purity (Classical vs US-SPPS) | Racemization and Side Reactions | Key Takeaways |
|---|---|---|---|---|---|
| Wołczański et al. (2019) | Met-enkephalin, UB33-43-OCam, TAT48-60-NH2, B2m58-69-NH2, ACP65-74-NH2, β-endorphin | 600–1920 min → 48–284 min | 24.2–71.3% → 60.6–89.9% | None observed for Cys/His, even at 70°C | ~7–12x faster synthesis; major purity improvements, especially for aggregation-prone sequences like TAT. |
| Silva et al. (2021) | Pep1 (12-mer), Pep2 (9-mer), Pep3 (25-mer) | >3515/365 min → ~250/85 min (Pep1/Pep2); Pep3: 347 min (US only) | 73–84% → 72–82% (Pep1/Pep2) | Not tested; slight purity drop in Pep2 noted | 14x time reduction for Pep1; crude yields significantly increased. |
| Merlino et al. (2019) | Model pentapeptides, peptides up to 44-mer, “difficult sequences” (Aβ 1-42, ACP 65-74) | 60 min → 30 min (model) | 84% → 93% (model); significantly higher than thermal heating (~45°C) | No significant increase for Cys/His enantiomers | Highly effective for difficult/aggregation-prone sequences; allows reduced reagent excess while maintaining high purity. |
Solid Phase Peptide Synthesis Improved with Ultrasound
Solid Phase Peptide Synthesis (SPPS) is a chemical reaction that allows the assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous support. However, the traditional solid-phase peptide synthesis is a relatively inefficient and slow process. Therefore, ultrasonic intensification of peptide synthesis is a highly regarded tool for a more efficacious and rapid synthesis of peptides.
Silva et al. (2021) compared “classical” fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound (US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH2) and the novel peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2).
US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the “classical” method. Interestingly, ultrasound-assisted SPPS yielded Pep1 in higher purity (82%) than the “classical” SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted the research team to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained within less than 6 hours (347 min) in moderate purity (approx. 49%).
Faster peptide synthesis via solid phase peptide synthesis using ultrasonic agitation.
(Study and analysis: Wołczański et al., 2019)
Merlino et al. (2019) also conducted a comprehensive study of the ultrasonic effects on Fmoc-based solid-phase peptide synthesis, which allowed for the synthesis of different biologically active peptides (up to 44-mer), with a remarkable savings of material and reaction time. They demonstrated that ultrasonication did not exacerbate the main side reactions and improved the synthesis of peptides endowed with “difficult sequences”, placing the ultrasonically-promoted solid-phase peptide synthesis (US-SPPS) among the current high-efficient peptide synthetic strategies.
The availability of high-performance systems for the ultrasonic (sonical) synthesis of peptides allows for significantly improved synthesis rates and an increase of the purity of raw products. (cf. Wołczański et al., 2019)
The investigation of racemization. Comparison of significant 1H NMR spectra of models peptides synthesized manually using the classical approach at room temperature vs ultrasonic method at elevated temperature. Chemical shifts of His and Cys α-protons and methylene group of Acm (left panels), ɣ-methyl protons of Val (right panels) show that sonication at 70°C does not cause racemization.
(Study and analysis: Wołczański et al., 2019)
Ultrasonic Cleavage of Peptides
After solid-phase peptide synthesis (SPPS), synthesised peptides must be cleaved from the polymeric resins. This step is also known as deprotection. When common shaking and ultrasonication for peptide cleavage from resin are compared, the shaking method requires approx. 1 hour, whilst ultrasonic cleavage can be accomplished in 15 to 20 min. The ultrasonic peptide cleavage can be applied to the cleavage of protected amino acids and peptides linked to polystyrene resins through benzylic ester bonds.
Ultrasonic cleavage of peptides from polystyrene resin gives high yields of peptides in high purity, without racemization, within a rapid procedure.
(study and graphic: ©Anuradha and Ravindranath, 1995)
Ultrasonically agitated reactor for improved and accelerated peptide synthesis. The picture shows the ultrasonicator UP200St in a stirred glass reactor.
High-Performance Ultrasonicators for Peptide Synthesis
Hielscher Ultrasonics offers various ultrasonic solution for direct and indirect sonication. Powerful and precisely controllable ultrasonic processors supply exactly the right amount of ultrasound energy to the reaction vessel. Whether you use syringes, tubes, multi-well plates, or glass reactors as synthesis vessel, Hielscher Ultrasonics offers the most suitable ultrasonicator for your peptide application.
- customized peptides
- large-scale peptide production
- peptide libraries
Many peptide syntheses are performed in syringes (e.g., fritted syringe reactors). Hielscher’s ultrasonic syringe agitator sonicates the peptide solution coupling the ultrasound waves through the syringe wall into the liquid. The ultrasonic syringe agitator is one of the most popular ultrasonic solutions for the ultrasonically-assisted synthesis of peptides.
The ultrasonic cuphorn is a suitable tool to sonicate up to 5 reactor vessels, whilst the VialTweeter can hold up to ten reaction tubes plus additionally five larger vessel via clamp-on accessory.
For other reactor types such as the Merrifield or Kamysz solid-phase reactor and other polypropylene or borosilicate vessels and reactors, Hielscher offers sonicators for indirect and direct sonication with customized clamp-on mounting or flange.
Of course, larger stirred glass reactors, e.g. for solution-phase synthesis, can be easily equipped with ultrasonic probes (a.k.a. sonotrodes or ultrasonic horns) of any size.
- various ultrasonicator types
- direct and indirect sonication
- precise intensity control
- precise temperature control
- continuous or pulsed ultrasound
- smart features, programmable devices
- available for any volume
- linear scalability
For solid phase peptide synthesis in multi-well/microtiter plates, the UIP400MTP is the ideal device. Ultrasonic cavitation is indirectly coupled uniformly into the numerous sample wells for superior mass transfer and synthesis reaction. Watch the video below to see the microplate sonicator UIP400MTP in action!
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Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers for mixing applications, dispersion, emulsification and extraction on lab, pilot and industrial scale.
Facts Worth Knowing about Ultrasonic Peptide Synthesis
What are Peptides?
Peptides are compounds where multiple amino acids are linked via amide bonds, so-called peptide bonds. When bound in complex structures – typically consisting of 50 or more amino acids -, these large peptide structures are termed proteins. Peptides are an essential building block of life and fulfil numerous functions in the body.
How does Peptide Synthesis Work?
In organic chemistry, molecular biology, and life science, peptide synthesis is the process of producing peptides. Peptides are chemically synthesized via condensation reaction of the carboxyl group of one amino acid to the amino group of another amino acid. Protecting groups (also protective groups) strategies are usually used in order to avoid undesired side reactions with the various amino acid side chains.
Chemical (in-vitro) peptide synthesis most often starts by coupling the carboxyl group of the incoming amino acid (C-terminus) to the N-terminus of the growing peptide chain. In contrary to this C-to-N synthesis, natural protein biosynthesis of long peptides in living organisms occurs in the opposite direction. This means that in biosynthesis, the N-terminus of the incoming amino acid is linked to the C-terminus of the protein chain (N-to-C).
Most research and development protocols for peptide synthesis are based on solid-phase methods, whilst solution-phase synthesis methods can be found in large-scale industrial production of peptides.
What is the Current State of the Art in Peptide Synthesis?
SUS-SPPS (Sustainable Ultrasound-assisted SPPS) has been demonstrated to reduce the process to just two steps per cycle (combined coupling/capping/deprotection + single wash), significantly reducing solvent consumption and time. (cf. Mottola et al., 2025)
Ultrasonic-assisted TAPS is an emerging combination with demonstrated success in total syntheses (e.g., lipobactin) and is being explored by CDMOs for API manufacturing.
Discover the advantages of ultrasonically-assisted TAGGING peptide synthesis!
Continuous processing is being explored as a further enhancement – silylated tag-assisted continuous one-pot synthesis platforms have been described.
The field is moving toward green chemistry principles, with ultrasound and TAPS both contributing to reduced solvent use, lower reagent loading, and improved sustainability.
Literature / References
- Merlino, F., Tomassi, S., Yousif, A. M., Messere, A., Marinelli, L., Grieco, P., Novellino, E., Cosconati, S., Di Maro, S. (2019): Boosting Fmoc Solid-Phase Peptide Synthesis by Ultrasonication. Organic Letters, 21(16), 2019. 6378–6382.
- Andrew M. Bray; Liana M. Lagniton; Robert M. Valerio; N.Joe Maeji (1994): Sonication-assisted cleavage of hydrophobic peptides. Application in multipin peptide synthesis. Tetrahedron Letters 35(48), 1994. 9079–9082.
- Silva, R., Franco Machado, J., Gonçalves, K., Lucas, F. M., Batista, S., Melo, R., Morais, T. S., & Correia, J. (2021): Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6. Molecules (Basel, Switzerland), 26(23), 7349.
- Conejos-Sanchez, Inmaculada; Duro Castaño, Aroa; Vicent, María (2014): Peptide-Based Polymer Therapeutics. Polymers. 6. 515-551.
- Raheem, Shvan J; Schmidt, Benjamin W; Solomon, Viswas Raja; Salih, Akam K; Price, Eric W (2020): Ultrasonic-Assisted Solid-Phase Peptide Synthesis of DOTA-TATE and DOTA-linker-TATE Derivatives as a Simple and Low-Cost Method for the Facile Synthesis of Chelator-Peptide Conjugates. ACS Bioconjugate Chemistry, 2020.
- Salvatore Mottola, Alessandra Del Bene, Vincenzo Mazzarella, Roberto Cutolo, Ida Boccino, Francesco Merlino, Sandro Cosconati, Salvatore Di Maro, Anna Messere (2015): Sustainable Ultrasound-Assisted Solid-Phase peptide synthesis (SUS-SPPS): Less Waste, more efficiency. Ultrasonics Sonochemistry, Volume 114, 2025.
- M.V. Anuradha, B. Ravindranath (1995): Ultrasound in peptide synthesis. 4: Rapid cleavage of polymer-bound protected peptides by alkali and alkanolamines. Tetrahedron Volume 51, Issue 19, 1995. 5675-5680.
- Wołczański, G., Płóciennik, H., Lisowski, M., Stefanowicz, P. (2019): The faster peptide synthesis on the solid phase using ultrasonic agitation. Tetrahedron Letters, 2019.
Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers from lab to industrial size.