Article pubs.acs.org/Macromolecules
Block-Sequence-Specific Polypeptides from α‑Amino Acid N‑Carboxyanhydrides: Synthesis and Influence on Polypeptide Properties Cristina Lavilla,*,† Mark Byrne,‡ and Andreas Heise*,†,‡,§ †
Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Den Dolech 2, 5612AZ Eindhoven, The Netherlands ‡ School of Chemical Sciences, Dublin City University, Dublin 9, Ireland § Department of Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland S Supporting Information *
ABSTRACT: Sequential addition of benzyl-L-glutamate and tert-butyl-Lglutamate N-carboxyanhydrides (NCAs) under optimized reaction conditions was used to synthesize block-sequence-defined polypeptides. Alternating octablock, tetrablock, and diblock as well as statistical polypeptides were obtained with comparable total compositions and total number of units. All of them were able to adopt helical secondary structures. Selective deprotection of the tert-butyl side chain yielded polypeptides with alternating benzyl-L-glutamate and L-glutamic acid block sequences available for further selective modification of individual block sequences. This was exploited for the conjugation with PEG side chains selectively on the glutamic acid block sequences. A detailed investigation revealed significant differences in properties as a function of the block-sequenced composition. Polypeptides with shorter alternating block sequences showed better water solubility. Dynamic light scattering revealed the presence of individual polypeptide chains in water in the case of the octablock polypeptide, while increasing aggregation was observed with increasing block length as well as for the statistical polypeptide. Moreover, the octablock polypeptide displayed a defined cloud point at 60 °C, while no defined transition could be observed with any of the other polypeptide block sequences. The results demonstrate the dependence of polypeptide properties on their block-sequenced composition and open opportunities for a polymerization approach complementary to the stepwise synthesis of peptidomimetics.
1. INTRODUCTION Nature produces peptides that are optimized for individual biochemical tasks. Their exact folding and properties arise from precisely controlled amino acid sequences and compositions, which let them perform a multitude of functions such as enzymatic catalysis, recognition events, and regulating processes.1,2 Pioneered by Merrifield, short synthetic peptide sequences (83% at 25 °C, the diblock and statistical copolypeptides only reach a helicity of around 73% (Table 2). Considering that 3.6 amino acid units form an α-helical turn, the block sequence length in [(BG)5-(PEGG)5]4 is approximately in the range of 1.5 helical turns. As amino acids at both ends of the polypeptide cannot participate in the helix, a helicity of >83% suggests the formation of a perfect α-helix, while changes in the polypeptide block sequence reduce the helical content. As in the DLS results, the statistical copolypeptide is more similar to the diblock copolypeptide. The results highlight that the shorter the PEGylated block sequences for a polypeptide with alternatingly equal-length block sequence distribution, the better the water solubility and the helix stabilization. A statistical copolymerization may not necessarily provide the same result unless full randomness may be assured. Finally, the thermoresponsiveness of the PEGylated polypeptides in water was investigated by measuring the transmittance at a wavelength of 500 nm, from 5 to 90 °C (concentration 5 mg/mL). No change was detected for the already turbid dispersions of (BG)20-b-(PEGG)20 and (BG)20st-(PEGG)20 (Figure 3). Conversely, when heating the clear solution of [(BG)5-(PEGG)5]4 (initial transmittance of 83%), a cloud point was detected at 60 °C, reaching a final transmittance of 4%. This transition was reversible upon cooling. [(BG)10-(PEGG)10]2, with an initial transmittance of 84%, did not show such a visible cloud point, but it decreased D
DOI: 10.1021/acs.macromol.6b00498 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This project has received funding from the European Union’s Seventh Framework Program for research, technological development and demonstration (Marie Curie Intra-European Fellowship for Career Development FP7-PEOPLE-2013-IEF625116 awarded to Cristina Lavilla). Mark Byrne gratefully acknowledges funding from Science Foundation Ireland (SFI) (07/IN1/B1792).
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Figure 3. Transmittance at 500 nm vs temperature upon heating (straight lines) and cooling (dashed lines), for [(BG)5-(PEGG)5]4, [(BG)10-(PEGG)10]2, (BG)20-b-(PEGG)20, and (BG)20-st-(PEGG)20 in H2O (5 mg/mL).
its transmittance to 58% when heating to 90 °C, and recovered the initial value of 84% when cooling again to 5 °C. These results evidenced important differences in the thermal behavior of the two multiblock copolypeptides, i.e., [(BG)5-(PEGG)5]4 and [(BG)10-(PEGG)10]2, despite having the same overall chemical composition and molecular weights. Therefore, the positioning of the thermoresponsive PEG units along the backbone is a factor of utmost importance in the resulting thermal properties of the α-helical polypeptides.
3. CONCLUSIONS The synthesis of well-defined, block-sequence-specific synthetic polyglutamates was demonstrated using two glutamic acid derivatives with different protecting groups. The synthesis yielded polypeptides with different block-sequenced composition while keeping the molecular weights and total monomer composition constant. All polypeptides were able to adopt secondary structures irrespective of their primary structure. The selective deprotection of one type of repeating unit followed by PEGylation yielded polypeptides with PEG side-chains in defined positions along the polypeptide chain. It was observed that the solution properties such as water solubility, helicity, and thermoresponsiveness were ultimately dependent on the block-sequenced structure of the polypeptides. The results reported here open possibilities to further design simplistic peptidomimetics, controlling folding and environmental interaction properties in a polymerization approach complementary to the stepwise synthesis of peptidomimetics.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b00498. Additional NMR, FTIR, CD spectra, DSC, and TGA data as well as tables and experimental procedures (PDF)
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DOI: 10.1021/acs.macromol.6b00498 Macromolecules XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.macromol.6b00498 Macromolecules XXXX, XXX, XXX−XXX
