Polymerization of Protecting-Group-Free Peptides via ROMP

Abstract

A study was conducted to survey the tolerance of ring-opening metathesis polymerization (ROMP) with respect to amino acid (a.a) identity of pentapeptide-modified norbornene-based monomers. A library of norbornyl-pentapeptides were prepared with the general structure, norbornyl-GX₂PLX₅, where residue 'X' was changed at each of the two positions (2 or 5) alternately to consist of the natural amino acids F, A, V, R, S, K, N, T, M, Q, H, W, C, Y, E, Q, and D. Each peptide monomer, free of protecting groups, was mixed in turn under a standard set of polymerization conditions with the ROMP initiator (IMesH₂)C₅H₅N)₂(Cl)₂Ru=CHPh. Two sets of polymerization reactions were performed, one with Monomer:Initiator (M:I) ratio of 20:1, and another with M:I of 200:1. For the nucleophilic amino acids cysteine and lysine, polymerization reactions were quantitatively compared to those of their protected analogues. Furthermore, we describe polymerization of macromonomers containing up to 30 a.a. to test for tolerance of ROMP to peptide molecular weight. These reactions were studied via SEC-MALS and NMR. Finally, with knowledge of sequence scope in hand, we prepared a set of enzyme-substrate containing brush polymers and studied them with respect to their bioactivity.

Figure 1

The template Norbornene-Gly-X₂-Pro-Ile-X₅ peptide, where X₂ and X₅ were systematically substituted for different amino acids and polymerized using initiator, I.

Figure 2

Representative examples of polymer characterization using NMR and SEC for a successful and failed polymerization (a and b respectively), together with an example of a higher degree of polymerization (c). (a) ROMP of 19 (Norb-GFPLR) at M:I = 20:1 reached completion within 3 hours as indicated by NMR and confirmed by SEC-MALS. (b) ROMP of 13 (Norb-GCPLI) at M:I = 20:1 reached 16% conversion at 24 hours as indicated by NMR with polymeric products barely detectable by SEC-MALS. (c) ROMP of 26 (Norb- GFPLW) at M:I = 200:1 reached 99% conversion at 24 hours as indicated by NMR (see ESI) with polymeric products detectable and confirmed by SEC-MALS. Standard SEC conditions given in ESI.

Figure 3

Rate of polymerization of monomers containing lysine/glycine repeats. (a) Percent conversion as determined by integration of the olefin peak via ¹H NMR. (b) log plots of the polymerization of each monomer. The following slopes (k_obs) were determined by linear least-squares fitting of the plots: of 3.4 hr⁻¹,1.4 hr⁻¹, 0.74 hr⁻¹, and 0.14 hr⁻¹ for 35, 33, 34,and 32 respectively.

Figure 4

Higher molecular weight peptide monomers 36 (a 15-mer) and 37 (a 30-mer).

Figure 5

Enzymatic studies of trypsin cleavage of monomers 38 and 39 and their corresponding polymers.