Clickable Polyprolines from Azido-proline N-Carboxyanhydride

Abstract

Polyproline is a material of great interest in biomedicine due to its helical scaffold of structural importance in collagen and mucins and its ability to gel and to change conformations in response to temperature. Appending of function-modulating chemical groups to such a material is desirable to diversify potential applications. Here, we describe the synthesis of high-molecular-weight homo, block, and statistical polymers of azide-functionalized proline. The azide groups served as moieties for highly efficient click-grafting, as stabilizers of the polyproline PPII helix, and as modulators of thermoresponsiveness. Saccharides and ethylene glycol were utilized to explore small-molecule grafting, and glutamate polymers were utilized to form polyelectrolyte bottlebrush architectures. Secondary structure effects of both the azide and click modifications, as well as lower critical solution temperature behavior, were characterized. The polyazidoprolines and click products were well tolerated by live human cells and are expected to find use in diverse biomedical applications.

Figure 1

(A) Extended PPII helix with 3 residues per turn and 9.3 Å helical pitch vs compact PPI helix with 3.3 residues per turn and 6.3 Å helical pitch; (B) trans vs cisl-Pro peptide bonds where PPII structures are predominantly trans and PPI are predominantly cis; (C) ring pucker conformations of 4-substituted l-Pro where 4R stereochemistry results in substituents at R′ (i.e., Hyp or Azp, R′ = OH or N₃; R″ = H).

Scheme 1. (A) Synthesis of Azp, Conversion to Azp NCA Monomer, and Two ROP Routes to polyAzp; DIAD = Diisopropyl Azodicarboxylate, PPh₃ = Triphenylphosphine, MeOH = Methanol, DPPA = Diphenylphosphoryl Azide, DEAD = Diethyl Azodicarboxylate, TEA = Triethylamine; (B) Preparation of Statistical Copolypeptides by Pre-mixing of NCAs Followed by Treatment with Ni Initiator (Copolymers with BnGlu Are Not Pictured); (C) Preparation of Block Copolypeptides by Sequential Addition of NCAs after Treatment with Co Initiator; (D) Structures of Initiators Used in This Study; (E) Cartoon Representation of Clickable PPro

Figure 2

(A) ATR–FTIR data for conversion of Azp NCA to PAzp with intact azide group using Ni initiator in DMF at ambient temperature. (B) Polymerization data for growth of PAzp chains from a BnGlu₇₃ macroinitiator indicating controlled polymerization; M_n is for the PAzp segment, R² = 0.9872.

Figure 3

CD spectra at 20 °C of (A) PHyp and statistical copolymers of Hyp and Azp in water; (B) PGlu and statistical copolymers of Glu and Azp in water; and (C) PPro and statistical copolymers of Pro and Azp in 95% n-propanol 5% water.

Figure 4

(A) Copper-catalyzed cycloadditions of helical Azp-polypeptides and alkyne-functionalized EG, Gal, Glc, and polyelectrolyte brush precursor PBnGlu. (B) ATR–FTIR absorbance spectra, normalized to the amide peak at ∼1650 cm^–1, for Azp₃₅ before and after the click reaction with Gal-Alk. (C) SEC/MALS/RI analysis of Azp₁₅-s-AcOP₄₅ before and after click reaction with Gal-Alk (* denotes the solvent signal).

Figure 5

Aqueous CD spectra at 20 °C of (A) Gal clicked homo- and co-Tzp polymers; (B) Tzp₁₅-s-Hyp₄₅ backbones before and after grafting of various sugars and monomethyl EG; (C) Glu₇₀ and Tzp₁₅-s-Hyp₄₅ backbones before click grafting, and polyelectrolyte bottlebrushes.

Figure 6

Cell viability and proliferation assay in HEK293 cells after treatment with various concentrations of Hyp₇₅, Azp₁₅-s-Hyp₄₅, and GalTzp₁₅-s-Hyp₄₅. Media and Triton are negative and positive controls, respectively. Data were collected in triplicate and are represented as mean ± SEM, and * represents statistical significance.