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Microwave Assisted SPPS of Hindered, Non-Standard Amino Acids

October 31, 2017
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Introduction

Hindered, non-standard amino acids such as α-aminoisobutyric acid (Aib) and N-methyl alanine ((N-Me)-A) (Figure 1) can be found in many biologically relevant compounds. 1-3  The synthesis of peptides including Aib or N-methylated amino acids has proved challenging, however; the steric hindrance introduced by the second methyl group, whether on the α-carbon or the amide nitrogen, makes coupling these amino acid derivatives difficult in conventional SPPS.

 

      Sterically-hindered, non-standard amino acids

Figure 1.  Sterically-hindered, non-standard amino acids.

 

Through the use of microwave-enhanced SPPS, though, difficulties associated with hindered, non-standard amino acids have been minimized. The employment of microwave energy in SPPS drives conventionally-difficult couplings of bulky amino acids, like Aib and N-methyl alanine, quickly and efficiently to completion.4

 

Materials and Methods

Reagents

N-α-Fmoc-α-aminoisobutyric acid was obtained from AnaSpec (Freemont, CA). Fmoc-N-Me-Ala-OH was obtained from Peptides International (Louisville, KY). All other amino acids were obtained from CEM Corporation (Matthews, NC) and contained the following side chain protecting groups: Asn(Trt), Asp(OMpe), Gln(Trt), Glu(OtBu), Lys(Boc), Ser(OtBu), and Tyr(tBu). Oxyma Pure and Rink Amide ProTideTM LL resin were obtained from CEM Corporation (Matthews, NC). N,N-Diisopropylcarbodiimide (DIC) was obtained from CreoSalus (Louisville, KY). Fmoc-Gly-Wang Resin LL was obtained from NovaBiochem (St. Louis, MO). Piperidine was obtained from Alfa Aesar (Ward Hill, MA). Trifluoroacetic acid (TFA), 3,6-dioxa-1,8-octanedithiol (DODT), triisopropylsilane (TIS), and acetic acid were obtained from Sigma-Aldrich (St. Louis, MO). Dichloromethane (DCM), N,N-dimethylformamide (DMF), and anhydrous diethyl ether (Et2O) were obtained from VWR (West Chester, PA). HPLC-grade water (H2O), and HPLC-grade acetonitrile (MeCN) were obtained from Fisher Scientific (Waltham, MA).

 

Peptide Synthesis: GEQKLGAibAibAibASEESLG-NH2

The peptide was prepared at 0.1 mmol scale using the CEM Liberty Blue automated microwave peptide synthesizer on Rink Amide ProTide LL resin (0.18 meq/g substitution).  Deprotection was performed with piperidine and Oxyma Pure in DMF.  Coupling reactions were performed with DIC in DMF, Oxyma Pure in DMF, and a 5-fold excess of Fmoc-AA-OH. Cleavage was performed using the CEM Razor high-throughput peptide cleavage system with TFA/H2O/TIS/ DODT. Following cleavage, the peptide was precipitated in Et2O and lyophilized overnight.

 

Peptide Synthesis: VQAibAibIDYING-OH

 The peptide was prepared at 0.1 mmol scale using the CEM Liberty Blue automated microwave peptide synthesizer on Fmoc-Gly-Wang LL Resin (0.33 meq/g substitution). Deprotection was performed with piperidine and Oxyma Pure in DMF. Coupling reactions were performed with DIC in DMF, Oxyma Pure in DMF, and a 5-fold excess of Fmoc-AA-OH. Cleavage was performed using the CEM Razor high-throughput peptide cleavage system with TFA/H2O/TIS/ DODT. Following cleavage, the peptide was precipitated in Et2O and lyophilized overnight.

 

Peptide Synthesis: VQ(N-Me-A)(N-Me-A)IDYING-OH

The peptide was prepared at 0.1 mmol scale using the CEM Liberty Blue automated microwave peptide synthesizer on Fmoc- Gly-Wang LL Resin (0.19 meq/g substitution). Deprotection was performed with piperidine and Oxyma Pure in DMF.  Coupling reactions were performed with DIC in DMF, Oxyma Pure in DMF, and a 5-fold excess of Fmoc-AA-OH. Cleavage was performed using the CEM Razor high-throughput peptide cleavage system with TFA/H2O/TIS/ DODT. Following cleavage, the peptide was precipitated in Et2O and lyophilized overnight.

 

Results

Microwave-enhanced SPPS of GEQKLGAibAibAibASEEDLG-NH2 on the Liberty Blue automated microwave peptide synthesizer produced the target peptide in 89% purity (Figure 2).

 

UPLC chromatogram of GEQKLGAibAibAibSEEDLH-NH2 

Figure 2.  UPLC chromatogram of GEQKLGAibAibAibASEEDLG-NH2

 

Microwave-enhanced SPPS of VQAibAibIDYING-OH on the Liberty Blue automated microwave peptide synthesizer produced the target peptide in 95% purity (Figure 3).

 

UPLC chromatogram

Figure 3.  UPLC chromatogram of VQAibAibIDYING-OH

 

Microwave-enhanced SPPS of VQ(N-Me-A)(N-Me-A)IDYING-OH on the Liberty Blue automated microwave peptide synthesizer produced the target peptide in 86% purity (Figure 4).

 

UPLC chromatogram

Figure 4.  UPLC chromatogram of VQ(N-Me-A)(N-Me-A)IDYING-OH

 

Conclusion

Microwave-enhanced SPPS enables conventionally-difficult couplings of bulky amino acids, like Aib and N-Me-A, to occur quickly and efficiently. Though conventional synthesis produces GEQKLGAibAibAibASEEDLG-NH2 in 40 h and < 10% purity, microwave-enhanced SPPS produces the target peptide in under 3 h and in 89% purity. Additionally, syntheses of acyl carrier protein derivatives VQAibAibIDYING-OH and VQ(N-Me-A)(N-Me-A)IDYING-OH are completed in under 2 h and in 95% and 86% purities respectively. Microwave-enhanced SPPS has proven an effective tool in minimizing the difficulties associated with hindered, non-standard amino acids in SPPS.

 

References

(1) Mueller, P.; Rudin, D. O. Nature. 1968, 217, 713–719.

(2) Rebuffat, S.; Goulard, C.; Hlimi, S.; Bodo, B. J. Pept. Sci. 2000, 6, 519–533.

(3) Ahmed, G.; Elger, W.; Meece, F.; Nair, H. B.; Schneider, B.; Wyrwa, R.; Nickisch, K. Bioorg. Med. Chem. 2017, 25, 5569–5575.

(4) Collins, J. M. Microwave-Enhanced Synthesis of Peptides, Proteins, and Peptidomimetics. In Microwaves in Organic Synthesis, Third Edition; de la Hoz, A.; Loupy, A.; Wiley-VCH: Weinheim, 2012; 897–959.

 

Want to learn more? Contact CEM at peptide.support@cem.com for method programming details!

 

 

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