Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Feb 24;9(8):nwac033.
doi: 10.1093/nsr/nwac033. eCollection 2022 Aug.

Water-assisted and protein-initiated fast and controlled ring-opening polymerization of proline N-carboxyanhydride

Yali Hu  1   2 , Zi-You Tian  1 , Wei Xiong  1 , Dedao Wang  3 , Ruichi Zhao  1 , Yan Xie  3 , Yu-Qin Song  3 , Jun Zhu  3 , Hua Lu  1
Affiliations

Water-assisted and protein-initiated fast and controlled ring-opening polymerization of proline N-carboxyanhydride

Yali Hu et al. Natl Sci Rev. .

Abstract

The production of polypeptides via the ring-opening polymerization (ROP) of N-carboxyanhydride (NCA) is usually conducted under stringent anhydrous conditions. The ROP of proline NCA (ProNCA) for the synthesis of poly-L-proline (PLP) is particularly challenging due to the premature product precipitation as polyproline type I helices, leading to slow reactions for up to one week, poor control of the molar mass and laborious workup. Here, we report the unexpected water-assisted controlled ROP of ProNCA, which affords well-defined PLP as polyproline II helices in 2-5 minutes and almost-quantitative yields. Experimental and theoretical studies together suggest the as-yet-unreported role of water in facilitating proton shift, which significantly lowers the energy barrier of the chain propagation. The scope of initiators can be expanded from hydrophobic amines to encompass hydrophilic amines and thiol-bearing nucleophiles, including complex biomacromolecules such as proteins. Protein-mediated ROP of ProNCA conveniently affords various protein-PLP conjugates via a grafting-from approach. PLP modification not only preserves the biological activities of the native proteins, but also enhances their resistance to extreme conditions. Moreover, PLP modification extends the elimination half-life of asparaginase (ASNase) 18-fold and mitigates the immunogenicity of wt ASNase >250-fold (ASNase is a first-line anticancer drug for lymphoma treatment). This work provides a simple solution to a long-standing problem in PLP synthesis, and offers valuable guidance for the development of water-resistant ROP of other proline-like NCAs. The facile access to PLP can greatly boost the application potential of PLP-based functional materials for engineering industry enzymes and therapeutic proteins.

Keywords: N-carboxyanhydride; PPII helix; proline; protein-polymer conjugates; ring-opening polymerization.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Comparison of the ROP of ProNCA in (a) pure ACN and (b) mixed ACN/H2O. (c) Conversions of ProNCA over time and (d) the zoomed-in period of the first 160 s. (e) SEC of the PLP produced in ACN and mixed ACN/H2O. (f) Photographs of the reactions in ACN (left) and mixed ACN/H2O (right). (g) Snapshots of the ROP of ProNCA in mixed ACN/H2O showing visible bubbles. [ProNCA]0/[I] = 100/1, [ProNCA]0 = 100 mg/mL, 10oC for the ROP in ACN/H2O and r.t. for the one in dry ACN.
Figure 2.
Figure 2.
Controlled ROP of ProNCA in mixed ACN/H2O mediated by benzyl amine. (a) Conversion of ProNCA over time and (b) plots of first-order kinetics of the ROP at different [ProNCA]0/[I]0 ratios in ACN-d3/D2O (1/1); [ProNCA]0 = 100 mg/mL. (c, d) Plots of Mn and Đ of PLP, (c) as a function of [ProNCA]0/[I]0 ratio and (d) conversion of ProNCA ([ProNCA]0/[I]0 = 100/1). (e) SEC traces showing the chain extension of PLP from a 20-mer to a 50-mer. (f) MALDI-TOF mass spectrum of PLP 25-mer. (g) CD spectra of PLP, PDP and PDLP showing typical left-handed and right-handed PPII helices, and no secondary conformation, respectively.
Figure 3.
Figure 3.
DFT calculations of model chain propagation reactions reveal plausible pathways for the ROP of ProNCA in dry ACN (black) and mixed ACN/H2O (red).
Figure 4.
Figure 4.
(a) Scope of small molecular initiators for the ROP of ProNCA in mixed ACN/H2O. (b) The SEC trace of MPT-PLP50. (c) Scheme of PLP-EGFP synthesis via the site-specific NCL of MPT-PLP and Cys-EGFP. (d) Native (left) and SDS-PAGE (right) analysis of the purified PLP-EGFP conjugate.
Figure 5.
Figure 5.
Protein-initiated ROP of ProNCA and stability of the Protein-PLP conjugates. (a) Cartoon illustration of the protein-initiated ROP of ProNCA. (b) SDS-PAGE characterization; inset: snapshots of the EGFP-PLP conjugates. (c) SDS-PAGE characterization of the DHFR-PLP conjugates. (d) DHFR enzymatic assay under normal or extreme conditions for wt-DHFR (black bars) and the DHFR-PLP conjugate (purple bars). P value was determined by ANOVA (ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001).
Figure 6.
Figure 6.
Pharmacological properties and immunogenicity of the ASNase-PLP conjugate. (a) SDS-PAGE and (b) aqueous SEC characterization of ASNase-PLP prepared by ASNase-mediated ROP of ProNCA. (c) In vitro cytotoxicity of ASNase-PLP on a human NK/T lymphoma cell line NKYS; n = 3. (d) Pharmacokinetic profile of intravenously infused ASNase-PLP in SD rats; n = 4; the enzymatic activity, obtained using Nessler's reagent (Merck, Germany), was employed to measure the plasma concentration of ASNase. (e) NKYS tumor growth curves and (f) immunohistochemistry (anti-human ki67) of tumor sections after receiving biweekly intraperitoneal treatments of PBS, wt ASNase (15 U/mouse) or ASNase-PLP (15 U/mouse). Scale bars, 50 μm. Six-week-old B-NDG mice were subcutaneously inoculated with NKYS cells (6.0 × 106) and randomized (n = 8 for each group) when the tumors reached ∼300 mm3 (day 0). (g) ELISA analysis of the titers of anti-ASNase IgG in the week-4 antisera drawn from wt ASNase-, ASNase-PEG- or ASNase-PLP-infused SD rats. (h, i) ELISA analysis of the changes of anti-polymer (h) IgG and (i) IgM levels in the antisera drawn from wt ASNase-, ASNase-PEG- or ASNase-PLP-infused SD rats. For the immunization, SD rats (n = 4 for each group) were subcutaneously infused with wt ASNase, ASNase-PEG or ASNase-PLP (200 U/kg) once every week for totally four weeks. Antisera were drawn every week before each injection. For the ELISA assay, the plates were coated with (g) wt ASNase, a PLP-interferon conjugate (to detect anti-PLP antibodies) and (h, i) a PEG-interferon conjugate (to detect anti-PEG antibodies). Data are represented as mean ± SD. P values are determined by ANOVA (ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Best RB, Merchant KA, Gopich IVet al. . Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Proc Natl Acad Sci USA 2007; 104: 18964–9.10.1073/pnas.0709567104 - DOI - PMC - PubMed
    1. Schuler B, Lipman EA, Steinbach PJet al. . Polyproline and the “spectroscopic ruler” revisited with single-molecule fluorescence. Proc Natl Acad Sci USA 2005; 102: 2754–9.10.1073/pnas.0408164102 - DOI - PMC - PubMed
    1. Kay BK, Williamson MP, Sudol P. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J 2000; 14: 231–41.10.1096/fasebj.14.2.231 - DOI - PubMed
    1. Mahoney NM, Janmey PA, Almo SC. Structure of the profilin-poly-L-proline complex involved in morphogenesis and cytoskeletal regulation. Nat Struct Biol 1997; 4: 953–60.10.1038/nsb1197-953 - DOI - PubMed
    1. Yu HT, Chen JK, Feng SBet al. . Structural basis for the binding of proline-rich peptides to SH3 domains. Cell 1994; 76: 933–45.10.1016/0092-8674(94)90367-0 - DOI - PubMed