May 1, 2015 (Vol. 35, No. 9)

Keith A. Porter CEM Peptides
E. Keller Barnhardt CEM Peptides
Jonathan M. Collins CEM Peptides
Michael J. Karney Life Science Product Manager CEM Peptides

A major problem in solid-phase peptide synthesis (SPPS) is aggregation and poor solvation leading to incomplete deprotection and/or coupling steps, resulting in low crude purity peptides.1 This phenomenon is aggravated in the case of longer peptides and those which contain hydrophobic segments where deprotection and coupling efficiencies are dramatically reduced as the peptide chain aggregates. The subsequent purification of the crude peptide mixture becomes a complex and time-consuming task, often requiring repeated chromatography runs, which leads to a reduction in yield of the target peptide.2

The application of microwave energy to peptide synthesis has made significant improvements in reducing aggregation, increasing peptide purity, and shortening synthesis times.3 More recently, the introduction of High Efficiency SPPS (HE-SPPS) has taken many of the advantages gained from microwave synthesis one step further. Based on chemical and instrumentation improvements, the HE-SPPS process has improved synthetic purities while reducing cycle times down to 4 minutes with up to 90% reduction in waste.4 The Liberty Blue™ Peptide Synthesizer (Figure 1) was designed to automate the HE-SPPS process and allows a 15mer peptide to be synthesized in only an hour at high purity and with minimum waste.

The properties of a resin are well known to have a major impact on the quality of a peptide made by SPPS. Therefore, we searched for improvements to resin properties to build on the synthetic improvements realized with the Liberty Blue automated microwave peptide synthesizer using HE-SPPS methodology. In our investigation we focused on Spheri­Tide® resins, which are unique compared to traditional polystyrene and PEG-based resins. They are made from poly-ε-lysine cross-linked with a deficiency of homo-bifunctional carboxylic acid that provides a chemical environment composed of amide bonds similar to a peptide backbone. The result is a hydrophilic, peptide-like backbone that can reduce the tendency for intramolecular peptide aggregation. Additionally, the growing peptides themselves are built off the α-amine sites which, due to the highly structured nature of Spheri­Tide resin, are spaced with a minimum distance between each other. This avoids clustering of linker sites thereby allowing high purity syntheses even at loadings greater than 1 mmol/g.

Figure 1. Liberty Blue Peptide Synthesizer

The final step in generating a high-quality peptide is the cleavage step. The Accent™ Peptide Cleavage System can perform microcleavage to check progress during a complicated synthesis as well as full cleavage upon synthesis completion. Unlike traditional cleavage techniques, the Accent can complete microcleavage in as little as 2 minutes and full-scale cleavage in 30 minutes or less.

In order to test the SpheriTide resin and further explore the HE-SPPS methodology of the Liberty Blue, two peptides were chosen to study. EGFRvIII and 1–42β−amyloid were selected based on known synthetic challenges. All peptides were first synthesized using polystyrene resin, then tested with either high loading or low loading Rink amide SpheriTide resin.


The EGFRvIII (LEEKKGNYVVTDHC) was synthesized to compare HE-SPPS and standard room temperature synthesis. Standard room temperature conditions with low loading Rink amide MBHA PS (Loading = 0.38 mmol/g) produced only a trace amount of product. This is consistent with published reports where multiple deprotections were required after each coupling (up to 8 in some cases) and >12 hour couplings were used to obtain crude purities of 40–70%.5 The same resin under HE-SPPS conditions produced product with 72% crude purity in little over an hour total time.

Advantageously, the higher loading Rink amide SpheriTide resin (loading = 1.05 mmol/g) achieved the same result, demonstrating that the unique properties of the resin can allow it to compete with other low loading resins in certain difficult sequences (Table 1). The higher crude purity of the high loading resin could be attributed to (1) the hydrophilic nature of the resin, which prevents intra-chain aggregation of the growing peptide and (2) the even distribution of initiation sites.

To further demonstrate the versatility of HE-SPPS and SpheriTide, highly complex 1–42β-amyloid (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), a notoriously difficult sequence, was prepared. 1–42β-amyloid is well-known for being challenging both synthetically and analytically. Using Rink amide SpheriTide (LL) (loading = 0.17 mmol/g), the sequence was synthesized in 65% crude purity by UPLC-MS (Figure 2).

Figure 2. UPLC-MS of 1-42ß-amyloid.


SpheriTide resin, when coupled with HE-SPPS techniques on the Liberty Blue Peptide Synthesizer, results in high-quality peptides in a short timeframe. Upon sequence completion, the peptide can easily be cleaved and high yields retained using the Accent. Even challenging sequences can be prepared with high crude purities in high yields.

CEM Peptides

Michael J. Karney
Life Science Product Manager

1. Rovero, P. In Solid Phase Synthesis. A Practical Guide; Kates, S. A.; Albericio, F., Eds.; Marcel Dekker: New York, 2000; Chapters 1-6.
2. Quibell, M.; Johnson, T. In Fmoc Solid Phase Peptide Synthesis. A Practical Approach; W. C. Chan, P. D. White, (Eds.); Oxford University Press: New York, 2000; Chapters 1-2.
3. References include: (a)Yu, H. M.; Chen, S. T.; Wang, K. T.; J. Org. Chem., 1992, 57, 4781. (b) Erdélyi, M.; Gogoll, A.; Synthesis, 2002, 11, 1592. (c) Vanier, G. S. In Microwave Heating as a Tool for Sustainable Chemistry; Leadbeater, N. E., Ed.; CRC Press: Boca Raton, FL, 2010; Chapter 9, p 231. (d) Palasek, S. A.; Cox, Z. J.; Collins, J. M.; J. Pept. Sci., 2007, 13, 143. (e) Collins, J. M. Microwaves in Organic Synthesis, 3rd ed.; Hoz, A., Loupy, A., Eds.; Wiley-VCH: Weinheim, Germany, 2013; Vol. 2, Chapter 20, p 897.
4. Collins, J.M.; Porter, K.A.; Singh, S.K.; Vanier, G.S.; Org. Lett., 2014, 16, 940.
5. Finneman, J.I.; Pozzo, M. J.; J. Pep. Sci. 2012, 8, 511.

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