Genetic Engineering & Biotechnology News

MAY1 2015

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Genetic Engineering & Biotechnology News | | MAY 1, 2015 | 39 A major problem in solid-phase peptide synthesis (SPPS) is ag- gregation and poor solvation leading to incomplete depro- tection 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 hy- drophobic segments where deprotection and coupling effciencies are dramatically reduced as the peptide chain aggregates. The subsequent purifcation of the crude peptide mixture becomes a complex and time-consuming task, often requiring repeated chroma- tography runs, which leads to a reduction in yield of the tar- get peptide. 2 The application of micro- wave energy to peptide syn- thesis has made signifcant improvements in reduc- ing aggregation, increas- ing peptide purity, and shortening synthesis times. 3 More recently, the introduc- tion of High Effciency SPPS (HE-SPPS) has taken many of the advantages gained from microwave synthesis one step further. Based on chemical and instrumen- tation improvements, the HE-SPPS process has improved synthetic purities while reduc- ing 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 al- lows a 15mer peptide to be synthesized in only an hour at high purity and with mini- mum 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 proper- ties 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 Spheri Tide ® resins, which are unique compared to traditional polystyrene and PEG-based resins. They are made from poly-ε-lysine cross-linked with a defciency of homo-bifunctional carboxylic acid that pro- vides a chemical environment composed of amide bonds similar to a peptide backbone. The result is a hydrophilic, peptide-like back- bone that can reduce the tendency for intramolecular peptide aggregation. Addition- ally, the growing peptides themselves are built off the α-amine sites which, due to the highly struc- tured nature of Spheri- Tide resin, are spaced with a minimum distance between each other. This avoids clustering of link- er sites thereby allowing high purity syntheses even at loadings greater than 1 mmol/g. The fnal step in gener- ating a high-quality peptide is the cleavage step. The Accent™ Peptide Cleav- age 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 cho- sen to study. EGFRvIII and 1– 42 β−amyloid were selected based on known synthetic challenges. All peptides were frst synthe- sized using polystyrene resin, then tested with either high loading or low loading Rink amide SpheriTide resin. Results The EGFRvIII (LEEKKGNYVVTDHC) was synthesized to compare HE-SPPS and standard room temperature synthesis. Stan- dard 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, demon- strating that the unique properties of the resin can allow it to compete with other low load- ing resins in certain diffcult sequences (Table 1). The higher crude purity of the high load- ing resin could be attributed to (1) the hydro- philic nature of the resin, which prevents in- tra-chain aggregation of the growing peptide and (2) the even distribution of initiation sites. To further demonstrate the versatil- ity of HE-SPPS and SpheriTide, highly com- plex 1– 42 β-amyloid (DAEFRHDSGYEVH- HQKLVFFAEDVGSNKGAIIGLMVGGV- VIA), a notoriously diffcult sequence, was prepared. 1– 42 β-amyloid is well-known for being challenging both synthetically and ana- lytically. Using Rink amide SpheriTide (LL) (loading = 0.17 mmol/g), the sequence was synthesized in 65% crude purity by UPLC- MS (Figure 2). Conclusion 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 com- pletion, 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. n References 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, Ger- many, 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. SpheriTide ® is a registered trademark of SpheriTech CEM Peptides Michael J. Karney Life Science Product Manager APPLICATION NOTE Advertorial Rapid Synthesis of Diffcult Peptides Using a Novel Resin Keith A. Porter, E. Keller Barnhardt, Jonathan M. Collins, and Michael J. Karney Table 1. EGFRvIII Synthesis Results * # Method Resin Target Time 1 Room Temperature SPPS † 0.38 meq Rink amide MBHA PS < 10% 17h38min 2 HE-SPPS ‡ 0.38 meq Rink amide MBHA (LL) 72% 1h17min 3 0.76 meq Rink amide MBHA (HL) 49% 4 1.05 meq Rink amide SpheriTide® (HL) 67% * All synthesis performed at 0.1 mmol scale. All peptides were cleaved using typical TFA cocktail on CEM Accent Peptide Cleavage System at 38°C for 30 min followed by lyophilization and UPLC-MS analysis. † Room temperature runs used 1 hr coupling and 5+10 min deprotection steps. ‡ HE-SPPS procedure as described in Reference 4. Figure 2. UPLC-MS of 1-42 β-amyloid. Figure 1. Liberty Blue Peptide Synthesizer

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