Automated Synthesis of Peptoids and Peptoid-Peptide Hybrids

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Peptoids are polymers of various N-substituted glycines. Though similar in structure to peptides (Figure 1), peptoids are resistant to proteolytic degradation, attributed to the complete substitution of their amide bonds. The increased stability of peptoids in vivo makes them an attractive peptidomimetic target for drug discovery and development.1,2

 Comparison of peptide and peptoid structure

Figure 1.  Comparison of peptide and peptoid structure


Peptoids and peptoid-peptide hybrids are typically synthesized through a "sub-monomer" process, which consists of two steps: (1) acylation with bromoacetic acid and N,N’diisopropylcarbodiimide (DIC) and (2) nucleophilic displacement with a monosubstituted amine (Figure 2).1,3


Typical synthesis of peptoids

Figure 2.  Typical synthesis of peptoids


Because many structurally diverse monosubstituted amines are commercially available, peptoids with a wide variety of side chains can be readily synthesized.2,3 However, conventional synthesis of peptoids can take up to three hours per residue.1 Microwave irradiation has been shown to significantly reduce this time, making production of peptoid libraries and peptoid-peptide hybrids much more viable.1–3


Materials and Methods


All amino acids were obtained from CEM Corporation (Matthews, NC) and contained the following side chain protecting groups: Glu(OtBu) and Lys(Boc). Oxyma Pure and Rink Amide ProTideTM LL resin were obtained from CEM Corporation (Matthews, NC). Bromoacetic acid, N,N-diisopropylcarbodiimide (DIC), benzylamine, β-alanine t-butyl ester hydrochloride, piperidine, trifluoroacetic acid (TFA), 3,6-dioxa-1,8-octanedithiol (DODT), triisopropylsilane (TIS), and acetic acid were obtained from Sigma-Aldrich (St. Louis, MO). N-Boc-1,4-diaminobutane and isobutylamine were obtained from Alfa Aesar (Ward Hill, MA).  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).


Peptoid-Peptide Hybrid Synthesis: Pro-Glu-(NLeu)-(NPhe)-Gly-(NLys)-NH2

 The peptoid-peptide hybrid (Figure 3) 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). For peptoid residues, deprotection was performed with piperidine in DMF, acylation was performed with bromoacetic acid and DIC, and nucleophilic displacement was performed with monosubstituted amine in DMF. For peptide residues, deprotection was performed with piperidine in DMF, and coupling reactions were performed with a 5-fold excess of Fmoc-AA-OH, DIC in DMF and Oxyma Pure in DMF. 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.



Target Peptoid-Peptide Hybrid: Pro-Glu-(NLeu)-(NPhe)-Gly-(NLys)- NH2

Figure 3.  Target Peptoid-Peptide Hybrid: Pro-Glu-(NLeu)-(NPhe)-Gly-(NLys)-NH2



Peptoid-Peptide Hybrid Analysis

The peptoid-peptide hybrid was analyzed on a Waters Acquity UPLC system with PDA detector equipped with an Acquity UPLC BEH C8 column (1.7 mm and 2.1 x 100 mm). The UPLC system was connected to a Waters 3100 Single Quad MS for structural determination. Peak analysis was achieved on Waters MassLynx software. Separations were performed with a gradient elution of 0.1% TFA in (i) H2O and (ii) MeCN.



Microwave-enhanced SPPS of Pro-Glu-(NLeu)-(NPhe)-Gly-(NLys)-NH2 on the Liberty Blue automated microwave peptide synthesizer produced the target peptide in 81% purity (Figure 4).


UPLC Chromatogram of Pro-Glu-(NLeu)-(NPhe)-Gly-(NLys)-NH2

Figure 4.  UPLC Chromatogram of Pro-Glu-(NLeu)-(NPhe)-Gly-(NLys)-NH2



The CEM Liberty Blue automated microwave peptide synthesizer allows quick and efficient access to peptides, peptoids, and peptoid-peptide hybrids. Microwave-enhanced SPPS produced peptoid-peptide hybrid, Pro-Glu-(NLeu)-(NPhe)-Gly-(NLys)-NH2, in 81% purity.



(1) Olivos, H. J.; Alluri, P. G.; Reddy, M. M.; Salony, D.; Kodadek, T. Org. Lett. 2002, 4, 4057–4059.
(2) Unciti-Broceta, A.; Diezmann, F.; Ou-Yang, C. Y.; Fara, M. A.; Bradley, M. Bioorg. Med. Chem. 2009, 17, 959–966.
(3) Gorske, B. C.; Jewell, S. A.; Guerard, E. J.; Blackwell, H. E. Org. Lett. 2005, 7, 1521–1524.


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