Communication pubs.acs.org/bc
Monolithic Peptide−Nucleic Acid Hybrid Functioning as an Artificial Microperoxidase Koji Nakano,* Junichi Tanabe, Ryoich Ishimatsu, and Toshihiko Imato Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, Japan S Supporting Information *
ABSTRACT: A new peptide nucleic acid (PNA) with an installed peroxidase function has been developed. Fmoc solid phase peptide synthesis prepared a PNA hybrid (VQKCAQCHTVE-(C2H4O)2CH2-[PNA(T)]6-G) that renders the microperoxidase backbone, followed by reconstitution with hemin. The resulting holocompound catalyzed the oxidation of 3,3′,5,5′-tetramthylbenzidine by H2O2 to 50% that of natural microperoxidase-11, whereas the apo-form and hemin gave no responses. The peroxidase domain was found to be active toward direct electrochemistry and the PNA hybrid served for gene sensor; in the presence of the target DNA (5′CATGTATAAAAAA-3′), an electrode-attached DNA probe (5′-TsTsTsTsTsTCTCATACATG-3′) showed the ferric-toferrous quasi-reversible wave (−276 mV vs Ag/AgCl) through sandwich hybridization. Moreover, the hybridization product could accept H2O2 as an oxidant to enhance the reduction current, which occurred likely based on the iron(II)-center-recycling with specific rate constant of 0.19 s−1.
N
ucleic acid analogues have evolved rapidly,1−8 with particular compounds now used in PCR.9,10 Peptide nucleic acids (PNAs)11 are a type of synthetic nucleic acid that consists of a poly[N-(2-aminoethyl)-glycine] instead of the sugar−phosphate linkage characteristic of DNA and RNA. With this unique structure, they possess greater hybridization affinity and are resistant to enzyme degradation and, therefore, are well suited for in vivo studies. With this view in mind, PNAs have been diversely functionalized, with examples including oligopeptides,12,13 fluorophores,14 and metal complexes,15,16 but enzymatic functions remain elusive. In the future, it is expected that in vitro selection could generate PNA sequences that perform catalysis. However, currently solid-phase peptide synthesis (SPPS) is the primary tool for diverse polypeptides. Here we present a totally synthetic approach to obtain a catalytic PNA. In designing a catalytic PNA, we focused on microperoxidases (MPs). MPs are a series of metalloporphyrinyl peptides that are obtained by enzymatic cleavage of cytochrome c. The undecamer (MP11) was chosen in this study as a potential candidate. The host PNA used was an uncomplicated 6-base thymine recognition sequence.17,18 Preparation of MP11 conjugates has remained limited to post-synthesis routes because nature has been the only source of MP11. In contrast, we took the strategy of one-pot SPPS using microwave heating. The resulting pseudopeptide−peptide hybrid, VQKCAQCHTVE-(C2H4O)2CH2-[PNA(T)]6-G, was subsequently reacted with hemin for reconstitution. We found that the thiol−ene click reaction19 with coexisting reducing agents20 reproducibly gave the corresponding holo-compound, MP-PNA(T6) © XXXX American Chemical Society
(Figure 1). N-Acetylated MP11, NAcMP (AcVQKCAQCHTVE), that possesses weak aggregability21 was also prepared for comparison. The detailed synthetic procedure
Figure 1. Synthetic scheme of MP-PNA(T6) and a 3D model of the expected structure of the PNA hybrid. The microperoxidase domain (Gaussian 09/LanL2DZ) and the PNA domain (molecular-mechanics/UFF) were independently optimized. Received: April 14, 2017 Revised: July 7, 2017 Published: July 11, 2017 A
DOI: 10.1021/acs.bioconjchem.7b00216 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Communication
Bioconjugate Chemistry
in a 5-coordination, high-spin-state embedded by particular amino acid sequences that exhibit considerable homology. Spectral measurements depicted the necessary structure for either type of artificial hemepeptides. Therefore, we concluded that our synthetic peroxidases use the same enzyme reaction mechanism that is commonly found in nature. The initial rates obtained for NAcMP and MP-PNA(T6) were 2.6 × 10−8 M−1 s−1 and 3.0 × 10−8 M−1 s−1, respectively, which are ∼50% that of native MP11 (v0 = 5.9 × 10−8 M−1 s−1). The specific activities obtained were MP11 = 1.6 mol min−1 mg−1, NAcMP = 0.69 mol min−1 mg−1, and MP-PNA(T6) = 0.41 mol min−1 mg−1. The peroxidase reaction is described by a three-step mechanism involving the formation of compound (cpd) I and II, both of which are intermediate-catalyst states:
with data analysis (Figures S1−S4) is provided as Supporting Information. The synthesized materials were first characterized by ultraviolet−visible (UV−vis) spectroscopy (Figure S5). As a standard reference substance, native MP11 dissolved in phosphate buffer (pH 7) showed a Soret band at 404 nm, which followed the Beer’s Law over the concentration range of 1.6−52 μM. The peak moved to a longer wavelength (416 nm) upon reduction by dithionite. NAcMP showed an absorption peak at 408 nm that shifted to 416 nm after dithionite treatment. Comparison with reference data showed that the UV−vis peaks represent ferric- and ferrous-NAcMP species, respectively. Results for MP-PNA(T6) were also consistent with those of the native substances; the corresponding ferricand ferrous-pseudopeptide gave Soret bands at 408 and 420 nm, respectively. Circular dichroic (CD) spectral measurements were recorded to analyze the secondary structure (Figure S6). All hemepeptides showed a pair of broad n → π* transitions centered around 220 nm. We did not confirm the entire π → π* transition because measuring data
