Synthesizing Unsymmetrically Branched Peptides

Table of Contents

Summary
Introduction
Materials and Methods
     Reagents
     Peptide Synthesis: LF-Chimera, (DLIWKLLSKAQEKFGKNKSR)- FKCRRWQWRNLLKGK-NH2
     Peptide Synthesis: Ub(47-76)-H2B(118-126), (AGKQLEDGRTLSDYNIQKESTLHLVLRLRG)-AVTKYTSSK-NH2
     Peptide Synthesis: Tetra-Branched Antifreeze Peptide, (TLTTTITG)4-Ac-KAAKKTAKAAKATAKEAK-NH2
     Peptide Analysis
Results
Conclusion

Summary

Microwave-enhanced SPPS increases the speed and purity of the synthesis of unsymmetrically branched peptides. It has been demonstrated that a lactoferricin-lactoferrampin antimicrobial peptide (LF Chimera)1 can be synthesized in under 5 h and with with a high 77% purity. A histone H2B-1A peptide fragment (residues 118-126)2 conjugated to a ubiquitin peptide fragment (residues 47-76) was synthesized in under 5 h with 75% purity. Synthesis of tetrabranched analog of an antifreeze peptide3 was carried out in under 5 h with 71% purity.

Introduction

Unsymmetrically branched peptides can be synthesized via SPPS by using Fmoc-Lys(ivDde)-OH (Figure 1), which includes an orthogonally protected lysine. The ivDde group is stable under conditions required for Fmoc elimination, but it is straightforwardly cleaved by hydrazinolysis.4 This enables the selective creation of an unsymmetrical branch point where a peptide of a different sequence can be coupled at the ε-amine of the lysine sidechain. Unsymmetrical branching has been used to produce peptides with a wide range of biological functions and chemical properties including compounds with antimicrobial activity1, peptides with in vitro deubiquitinase resistance2, and macromolecules with antifreeze properties3. Synthesizing branched peptides via SPPS is difficult due to the inherent closeness of the elongating peptide chains on a branched scaffold, which causes steric clashes and poor peptide coupling. Applying microwaves to the synthesis of branched peptides overcomes these steric challenges, making coupling more efficient and faster, even for complicated branched peptides with fewer deletion products (CarboMAXTM).5

Figure 1. Fmoc-Lys(ivDde)-OH

Materials and Methods

Reagents

The following Fmoc amino acids were obtained from CEM Corporation (Matthews, NC) and contain the indicated side chain protecting groups: Arg(Pbf), Asn(Trt), Asp(OtBu), Asp(OMpe), Asp(OtBu)-(Dmb)Gly-OH, Gln(Trt), Glu(OtBu), His(Boc), Lys(Boc), Ser(tBu), Tyr(tBu), Thr(tBu), and Trp(Boc). Rink Amide ProTideTM LL resin was also obtained from CEM Corporation (Matthews, NC). Boc-Phe-OH was purchased from Peptides International (Louisville, KY). Boc-Ala-OH was obtained from Alfa Aesar (Ward Hill, MA). Fmoc-Lys(ivDde)-OH was obtained from EMD Millipore (Billerica, MA). Anhydrous hydrazine, acetic anhydride (Ac2O), N,N’-Diisopropylcarbodiimide (DIC), piperidine, trifluoroacetic acid (TFA), 3,6-dioxa-1,8-octanedithiol (DODT), and triisopropylsilane (TIS) were obtained from Sigma-Aldrich (St. Louis, MO). Dichloromethane (DCM), N,N-Dimethylformamide (DMF), anhydrous diethyl ether (Et2O), acetic acid, HPLC grade water, and acetonitrile were obtained from VWR (West Chester, PA). LC-MS grade water (H2O) and LC-MS grade acetonitrile (MeCN) were obtained from Fisher Scientific (Waltham, MA).

Figure 2. LF-Chimera, Ub(47-76)-H2B(118-126), and tetra-branched antifreeze peptide

Peptide Synthesis: LF-Chimera, (DLIWKLLSKAQEKFGKNKSR)- FKCRRWQWRNLLKGK-NH2

The peptide (Figure 2) was prepared on a 0.1 mmol scale using the CEM Liberty BlueTM automated microwave peptide synthesizer on 0.526 g Rink Amide ProTide LL resin (0.19 meq/g substitution). Deprotection was performed with 20% piperidine and 0.1 M Oxyma Pure in DMF. Coupling reactions were performed in 5-fold excess of Fmoc-AA with 1.0 M DIC and 1.0 M Oxyma Pure in DMF (CarboMAXTM).5 Fmoc-Lys(ivDde) was used for K at the branched position. A solution of 5% hydrazine in DMF was used to remove ivDde. Boc-Phe-OH was used for F. Cleavage was performed using the CEM RazorTM high-throughput peptide cleavage system with 92.5:2.5:2.5:2.5 TFA/H2O/TIS/ DODT. After cleavage, the peptide was precipitated with Et2O and lyophilized overnight.

Peptide Synthesis: Ub(47-76)-H2B(118-126), (AGKQLEDGRTLSDYNIQKESTLHLVLRLRG)-AVTKYTSSK-NH2

The synthesis of the peptide in Figure 2 was completed on a 0.1 mmol scale using the CEM Liberty Blue automated microwave peptide synthesizer on 0.526 g Rink Amide ProTide LL resin (0.19 meq/g substitution). 20% piperidine and 0.1 M Oxyma Pure in DMF was used for deprotection. Coupling reactions were performed in 5-fold excess of Fmoc-AA with 1.0 M DIC and 1.0 M Oxyma Pure in DMF (CarboMAX).5 Asp(OMpe) and an Asp(OtBu)-(Dmb) Gly-OH dipeptide were used for the Asp and Asp-Gly residues respectively in order to lower aspartimide formation. To prevent epimerization, His(Boc) was used instead of His(Trt). Fmoc-Lys(ivDde) was used for K at the branched position. A solution of 5% hydrazine in DMF was used to remove ivDde. Boc-Ala-OH was used for A. Cleavage was performed using the CEM Razor high-throughput peptide cleavage system with 92.5:2.5:2.5:2.5 TFA/H2O/TIS/DODT. After cleavage, the peptide was precipitated with Et2O and lyophilized overnight.

Peptide Synthesis: Tetra-Branched Antifreeze Peptide, (TLTTTITG)4-Ac-KAAKKTAKAAKATAKEAK-NH2

The peptide (Figure 2) was prepared on a 0.1 mmol scale (resin at 0.1 mmol scale for 1st strand; resin at 0.025 mmol scale for 2nd strand) with the CEM Liberty Blue automated microwave peptide synthesizer on Rink Amide ProTide LL resin (0.526 g for the 1st strand; 0.132 g for the 2nd strand; 0.19 meq/g substitution). Deprotection was performed with 20% piperidine and 0.1 M Oxyma Pure in DMF (CarboMAX).5 Coupling reactions were performed in 5-fold excess of Fmoc-AA with 1.0 M DIC and 1.0 M Oxyma Pure in DMF. Fmoc-Lys(ivDde) was used for K at the branched position. A solution of 5% hydrazine in DMF was used to remove ivDde. Acetyl capping using 10% Ac2O in DMF was performed after deprotection of K. Cleavage was performed using the CEM Razor high-throughput peptide cleavage system with 92.5:2.5:2.5:2.5 TFA/H2O/TIS/DODT. After cleavage, the peptide was precipitated with Et2O and lyophilized overnight.

Peptide Analysis

The peptides were 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 0.05% TFA in (i) H2O and (ii) MeCN.

Results

Microwave-enhanced SPPS of LF-Chimera on the Liberty Blue automated microwave peptide synthesizer produced the target peptide in 77% purity (Figure 3). Microwave-enhanced SPPS of Ub(47-76)-H2B(118-126) on the Liberty Blue automated microwave peptide synthesizer generated the target peptide in 75% purity (Figure 4). Microwave-enhanced SPPS of tetra-branched antifreeze peptide on the Liberty Blue automated microwave peptide synthesizer generated the target peptide in 71% purity (Figure 5).

Figure 3. UPLC Chromatogram of LF-Chimera

Figure 4. UPLC Chromatogram of Ub(47-76)-H2B(118-126)

Figure 5. UPLC Chromatogram of Tetra-Branched Antifreeze Peptide

Conclusion

Microwave-enhanced SPPS enables users to synthesize unsymmetrically branched peptides quickly at with a high purity. By applying microwave energy to the synthesis of a chimeric lactoferricin-lactoferrampin peptide, the target peptide could be synthesized in under 5 h and with 77% purity. Using microwave-enhanced SPPS, a histone H2B fragment (residues 118-126) conjugated to a ubiquitin fragment (residues 47-76) was synthesized in under 5 h with 75% purity. Conventional room temperature synthesis of Ub(47-76)-H2B(118-126) requires over 53 h of manual labor time and gives a 10-20% isolated yield of target peptide.2 Conventional synthesis of a dendrimeric antifreeze peptide necessitates over 72 h of manual labor time and produces the target peptide in 40% isolated yield.3 Using microwave-enhanced SPPS, a tetra-branched antifreeze peptide was manufactured in under 5 h in 71% purity.

References

(1) Haney, E. F.; Nazmi, K.; Bolscher, J. G. M.; Vogel, H. J. Biochim. Biophys. Acta - Biomembr. 2012, 1818 (3), 762–775.

(2) Haj-Yahya, M.; Eltarteer, N.; Ohayon, S.; Shema, E.; Kotler, E.; Oren, M.; Brik, A. Angew. Chemie Int. Ed. 2012, 51 (46), 11535–11539.

(3) Vera-Bravo, R.; Scotter, A. J.; Davies, P. L.; Blanco, L. H. Rev. Colomb. Química 2012, 41 (1), 133–157.

(4) Isidro-Llobet, A.; Álvarez, M.; Albericio, F. Chem. Rev. 2009, 109 (6), 2455–2504.

(5) CEM Application Note (AP0124) - “CarboMAX - Enhanced Peptide Coupling at Elevated Temperature.”

This information has been sourced, reviewed and adapted from materials provided by CEM Corporation - Life Science.

For more information on this source, please visit CEM Corporation - Life Science.

Last updated: May 1, 2018 at 7:29 AM

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