Porphyrin-Cored Polymer Nanoparticles: Macromolecular Models

Created using collapsible heme-centered star polymers containing photodimerizable anthracene units, these systems afford model heme cofactors buried w...
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Porphyrin-Cored Polymer Nanoparticles: Macromolecular Models for Heme Iron Coordination Kyle J. Rodriguez,† Ashley M. Hanlon,† Christopher K. Lyon,† Justin P. Cole,† Bryan T. Tuten,‡ Christian A. Tooley,† Erik B. Berda,*,†,‡ and Samuel Pazicni*,† †

Department of Chemistry and ‡Materials Science Program, University of New Hampshire, 23 Academic Way, Durham, New Hampshire 03824, United States S Supporting Information *

not offer the tunability necessary to systematically replicate the various microenvironments found in proteins. The field of single-chain nanoparticles (SCNPs) has emerged as a viable option for generating intramolecularly folded macromolecules with tunable scaffolds that can mimic naturally occurring macromolecular environments.21−24 Recently, Tooley et al. reported the design and synthesis of a [FeFe]hydrogenase model that was covalently attached to a SCNP, generating the first bioinspired SCNP active site.25 This achievement has inspired the design and synthesis of novel porphyrin-cored polymer nanoparticles (PCPNs) as macromolecular models for heme proteins. PCPNs were designed as tetrafunctionalized porphyrin-cored star polymers (PCSPs) possessing reactive monomer units that cross-link to generate nanoparticles (i.e., collapse) upon postpolymerization modification (Figure 1). We report systems

ABSTRACT: Porphyrin-cored polymer nanoparticles (PCPNs) were synthesized and characterized to investigate their utility as heme protein models. Created using collapsible heme-centered star polymers containing photodimerizable anthracene units, these systems afford model heme cofactors buried within hydrophobic, macromolecular environments. Spectroscopic interrogations demonstrate that PCPNs display redox and ligand-binding reactivity similar to that of native systems and thus are potential candidates for modeling biological heme iron coordination.

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eme proteins are responsible for a wide variety of functions throughout biology. Their functions consist of O2 activation, redox catalysis, electron transfer, gasotransmitter sensing, and transport.1−6 While the primary coordination sphere of the heme cofactor predominately dictates function, the hydrogen-bonding-rich secondary coordination sphere afforded by the protein heavily influences the reactivity of heme proteins.5,7−11 Precisely how these secondary effects influence the heme reactivity continues to intrigue researchers; however, synthetic model complexes have provided valuable insight. In the 1970s, Collman and co-workers designed and synthesized a porphyrin model, the picket-fence porphyrin (TpivPP), a monooxygenase model with enough steric hindrance to reversibly bind O2 and prevent further undesired reactions.12 The success of TpivPP as a functional model leads this system to be the foundation for many subsequent studies.13−15 Recently, Pluth and co-workers used TpivPP to study HS− binding to heme iron by probing the system’s absorbance features.16 While TpivPP systems have provided a fundamental understanding of heme iron coordination by incorporating elements of the secondary coordination sphere, their design does not wholly replicate the enveloping macromolecular environment afforded by a folded polypeptide. Replicating protein environments with macromolecular systems is quite challenging: function in well-defined protein structures is difficult to reproduce synthetically. Wang reported a heme dipyridyl copolymer stabilized by poly(L-lysine) that catalyzed the oxidation of cytochrome c by air;17 others have synthesized porphyrin-cored dendrimers18,19 to introduce the heme group into macromolecular environments. These latter systems, while functional, are synthetically challenging20 and do © XXXX American Chemical Society

Figure 1. Collapsible PCSP used to generate Fe-PCPNs. The presence of photodimerizable anthracene units in the four poly(methacrylate) arms permits intramolecular covalent cross-linking to form nanoparticles. For FeIII-PCPN1 discussed herein, the anthracene incorporation, y, is 24.7%.

of two different experimental molecular weight ranges (Table S1): 12−15 kDa (1, detailed herein) and 32−49 kDa (2, detailed in the Supporting Information). PCSPs were prepared by reversible addition−fragmentation chain transfer (RAFT) polymerization using a porphyrin-cored chain-transfer agent as the macroinitiator (Scheme S3).26 Simulations suggest that this “core-first” approach ensures encapsulation of the porphyrin Received: May 9, 2016

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DOI: 10.1021/acs.inorgchem.6b01113 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry upon collapse of the star polymer.27−29 RAFT polymerizations are well-controlled and allow for high-molecular-weight polymers with uniform chain length (i.e., a polydispersity index, Đ, near unity). RAFT is also compatible with a variety of monomers, permitting access to a range of tunable scaffolds and collapse chemistries. 9-Anthracenylmethyl methacrylate (AMMA) was used as the reactive monomer unit in this work, given its RAFT compatibility and unique near-UV signatures, which can be used to monitor nanoparticle formation.30 PCSP1 was synthesized using methyl methacrylate and AMMA as comonomers, yielding a system robust to oxidative31,32 and reductive (Figure S40) damage. PCSP1 was characterized by 1H NMR, indicating 24.7% AMMA incorporation (Figure S4), and size-exclusion chromatography (SEC; Mn = 14.7 kDa and Đ = 1.06; Figure S5). Insertion of FeIII into PCSP1 using FeBr2 was confirmed by both the contact shift of the pyrrole 1H NMR signal (from 8.89 to 81 ppm; Figure S6) and the anticipated changes in the Soret and α and β absorbance signatures33 (Figure S8) for FeIII-PCSP1. SEC confirmed that FeII insertion and subsequent air oxidation resulted in no degradation/aggregation of the PCSP due to putative reactive oxygen species generated during this process (Figures S9, S14, and S19). Taken together, these data indicate that the PCSP heme is protected from μ-oxo dimer formation upon FeII insertion and subsequent air oxidation. FeIII-PCPN1 was generated by photodimerization (λ = 350 nm) of the FeIII-PCSP1 anthracene units; this process was monitored by absorbance spectroscopy and SEC (multiangle light scattering, MALS;Figure 2). We observed changes in the

Figure 3. Absorbance spectra of ∼25 μM FeIII-PCPN1 in 4:1 DMSO/ H2O (red) and upon equilibration with 5.0 mM sodium dithionite to yield (DMSO)2FeII (green). The addition of 5.0 mM imidazole to (DMSO)2FeII yielded a bis(imidazole) complex (blue). The addition of CO to (Im)2FeII yielded the Im/CO FeII adduct (black).

those of porphyrin systems reported previously.12,35−37 In a 4:1 dimethyl sulfoxide (DMSO)/H2O solution, FeIII-PCPN1 exhibits an absorbance spectrum featuring a broad Soret band (425 nm) and one α,β-region signature (527 nm); we assign this spectrum to a high-spin six-coordinate bis(DMSO) species, based on comparison to previous reports.36,38,39 Reduction of the [(DMSO)2FeIII]+ adduct with 5.0 mM sodium dithionite resulted in a spectrum consistent with a low-spin (DMSO)2FeII species: a sharpened, red-shifted Soret band (429 nm) and three well-defined α,β-region features at 530, 566, and 606 nm. Imidazole (5.0 mM) was then added to the (DMSO)2FeII adduct. This addition resulted in a red shift of the Soret band to 431 nm and shifts in the α,β-region bands to 533 and 565 nm. These signatures are in good agreement with those reported for [FeII(TpivPP)(1-MeIm)2].35 The [(Im)2FeIII]+ adduct was also obtained by adding 5.0 mM imidazole directly to the [(DMSO)2FeIII]+ complex (Figure S36). Finally, the (Im)2FeII species was exposed to CO to yield a (Im)(CO)FeII complex. In comparison to the (Im)2FeII species, this Im/CO adduct displayed a blue-shifted Soret band at 426 nm; α,β-region bands were shifted to 541 and 585 nm. The order of CO/ imidazole addition to the (DMSO)2FeII species did not appreciably affect the spectrum of the Im/CO adduct (Figure S51). The addition of CO directly to the (DMSO)2FeII species resulted in a spectrum that we assign to a six-coordinate CO/ DMSO adduct (Figure S46).35,40 PCPN2, the other PCSPs, and the control system all exhibited behavior similar to that of FeIIIPCPN1 when subjected to the same binding conditions, with only minor fluctuations in spectral signatures (Table S2). For further 1H NMR characterization,41 the bis(cyano) adduct of FeIII-PCPN1 was generated by adding 5.0 mM tetrabutylammonium cyanide to the [FeIII(Br)]+ species. Conversion to the low-spin [FeIII(CN)2]− adduct was confirmed by a shift in the pyrrole proton signal from +81 to −16 ppm (Figures S55 and S56).42 In addition, we observed no pyrrole proton signal shifts for either the [FeIII(Br)]+ or [FeIII(CN)2]− species upon nanoparticle formation, indicating that the heme electronic state was not perturbed by PCSP collapse. In this report, we have described the design and synthesis of the first PCPN, a system that envelopes a model heme moiety in a protein-like, macromolecular environment that is heretofore unaccounted for in small-molecule heme model complexes. Indeed, the polymer environment does appear to protect the model heme group, given that μ-oxo dimer formation is not observed. PCPN exhibited reactivity toward reductant and exogenous heme ligands similar to that of native heme proteins.

Figure 2. Collapse of FeIII-PCSP1 to form FeIII-PCPN1, as monitored by (a) absorbance spectroscopy and (b) SEC (MALS detector traces).

SEC retention time and hydrodynamic radius (Rh) and a decrease in the intrinsic viscosity ([η]) from 5.3 to 4.6 mL/g upon conversion of FeIII-PCSP1 to FeIII-PCPN1 (Table S1). This decrease in [η] is consistent with intramolecular polymer folding.34 Similar observations were made with the other PCSPs/ PCPNs investigated in this study (Table S1). We did not observe spectral changes or a decrease in [η] upon irradiation of a control system that lacked AMMA units (Table S1 and Figure S29), indicating that the porphyrin core did not influence nanoparticle formation. SEC traces revealed monomodal peaks for all polymers with no signs of aggregation, low-molecular-weight polymers, or interchain cross-linking. The Mn value for FeIIIPCPN1 was 12.1 kDa, and Đ = 1.20. We investigated the utility of FeIII-PCPN1 to serve as a model for heme iron coordination by comparing the spectral characteristics of complexes derived from FeIII-PCPN1 (Figure 3) with B

DOI: 10.1021/acs.inorgchem.6b01113 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

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The various species generated from PCPN display absorbance and 1H NMR features that are in good agreement with those of previously reported heme model complexes. This work opens potential avenues utilizing PCPNs and their inherent tunability to explore how various protein microenvironments (hydrophobic, hydrophilic, hydrogen-bonding-rich, etc.) affect heme spectroscopic attributes and heme iron reactivity toward relevant biological ligands (O2, HS−, etc.).

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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.inorgchem.6b01113. Materials and instrumentation, experimental procedures, NMR spectra, SEC traces, absorbance spectra, and supporting figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Data were collected on the 400 and 500 MHz NMR within the University Instrumentation Center at the University of New Hampshire in Durham, NH. S.P. and E.B.B. graciously acknowledge financial support from the University of New Hampshire. E.B.B. additionally acknowledges the Army Research Office for support through Award W911NF-14-1-0177 and the NIST for support through Award 70NANB15H060. Finally, the authors thank Alex Vorrilas for designing the Table of Contents graphic.

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DOI: 10.1021/acs.inorgchem.6b01113 Inorg. Chem. XXXX, XXX, XXX−XXX