Porphodilactones represent the porphyrin analogues, in which the peripheral bonds of two pyrrole rings are replaced by lactone moieties. They provide an opportunity to investigate how β-substituent orientation of porphyrinoids modulates the electronic structures and optical properties, in a manner similar to what is observed with naturally occurring chlorophylls. In this work, a comprehensive description of the synthesis, characterization, and optical properties of meso-tetrakispentafluorophenylporphodilactone isomers is first reported. The β-dilactone moieties are found to lie at opposite pyrrole positions (trans- and cis-configurations are defined by the relative orientations of the carbonyl group when one lactone moiety is fixed), in accordance with earlier computational predictions (Gouterman, M. J. Am. Chem. Soc. 1989, 111, 3702). The relative orientation of the β-dilactone moieties has a significant influence on the electronic structures and photophysical properties. For example, the Qy band of trans-porphodilactone is red-shifted by 19 nm relative to that of the cis-isomer, and there is a 2-fold increase in the absorption intensity, which resembles the similar trends that have been reported for natural chlorophyll f and d. An in depth analysis of magnetic circular dichroism spectral data and TD-DFT calculations at the B3LYP/6-31G(d) level of theory demonstrates that the trans- and cis-orientations of the dilactone moieties have a significant effect on the relative energies of the frontier π-molecular orbitals. Importantly, the biological behaviors of the isomers reveal their different photocytotoxicity in NIR region (>650 nm). The influence of the relative orientation of the β-substituents on the optical properties in this context provides new insights into the electronic structures of porphyrinoids which could prove useful during the development of near-infrared absorbing photosensitizers.

A β-oxazolone moiety on porpholactone plays an important role in stabilizing such hydroporphyrin structures through tuning energy gaps between the frontier π-molecular orbitals, which is verified by MCD studies combined with TD-DFT calculations.

The aim of this study was to perform an association study between two single nucleotide polymorphisms (SNPs) rs2910164 G>C and rs3746444 T>C in pre-miRNA (hsa-mir-146a and hsa-mir-499) and rheumatoid arthritis (RA) in the Han Chinese population. 208 Han Chinese patients with RA and 240 healthy controls were recruited in this study. The SNPs was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Anti-cyclic citrullinated peptide (anti-CCP) antibody was measured by enzyme linked immunosorbent assay and rheumatoid factor (RF) was measured by rate nephelometry. The genotype frequencies between cases and controls were compared by χ(2) analysis. No significant association between the SNPs (rs2910164 and rs3746444) and RA was observed (P = 0.631 and 0.775, respectively), and the SNPs did not show any association with the RF-positive (P = 0.631 and 0.775, respectively). However, there was a significant difference on the level of anti-CCP antibody between different genotypes in rs3746444 (P = 0.007). The heterozygote CT had significantly higher level of anti-CCP antibody compared with homozygote CC and TT (P = 0.054 and 0.003, respectively). We first investigated the association between the SNPs (rs2910164 G>C and rs3746444 T>C) in the pre-miRNA (hsa-mir-146a and hsa-mir-499) and RA in a Han Chinese population. We did not find a significant association between the SNPs and the susceptibility to RA, while the SNP rs3746444 may affect anti-CCP antibody production.

We demonstrate that incorporation of MnSalen into a protein scaffold enhances the chemoselectivity in sulfoxidation of thioanisole and found that both the polarity and hydrogen bonding of the protein scaffold play an important role in tuning the chemoselectivity.Metalloenzymes have set a golden standard for carrying out reactions with high reactivity and selectivity. Understanding how proteins confer such reactivity and selectivity is important not only to providing deeper insight in biological functions, but also to its application in chemical transformations. [1][2][3][4][5][6][7][8][9][10][11] Toward this goal, much work has focused on the study of native metalloenzymes, such as cytochrome P-450s, a metalloenzyme with high chemoselectivity in the oxidation of C-H bonds. [12][13][14] These studies indicate that the protein scaffold is capable of creating the proper environment to modulate the reactive pathways of active intermediates so as to inhibit side reactions such as over oxidization. In contrast to the tremendous progress made in biochemical and biophysical studies of native metalloenzymes and their variants, much less has been reported regarding the application of the insight gained from such studies for designing artificial enzymes. In addition to testing our knowledge of metalloenzymes, designing artificial enzymes can provide new information that otherwise may be difficult to obtain from studying native enzymes. 1-8, 12, 15 By carefully choosing protein scaffolds that are small, stable and easy to produce, such artificial enzymes may find interesting applications in chemical transformations to generate fine chemical intermediates. An emerging area in artificial enzyme design is the incorporation of non-native metal catalysts into proteins to expand the reactivity and functionality of metalloenzymes, thus transforming achiral and water-insoluble metal catalysts into asymmetric aqueous solution catalysts for reactions such as sulfoxidation, hydrogenation, and cycloaddition (Diels-Alder reaction). 7,8,[16][17][18][19][20][21][22][23][24] A notable bonus to such an approach is the opportunity to compare how the selectivity of the metal catalyst can be fine-tuned using biological and chemical approaches. 7 Understanding how such systems control catalysis can enrich our knowledge of catalyst design, generating more selective catalysts. The majority of artificial metalloenzyme design studies have been devoted to exploring the use of the protein scaffold to tune enantioselectivity. 7,8,[16][17][18][19] However, learning to control chemoselectivity in these artificial biocatalysts, especially in catalytic oxidation, is equally important. To demonstrate the ability of the protein scaffold to tune the oxidative reactivity of metal catalysts and to discover factors involved in tuning such chemoslectivity, we report here that introducing manganese salen (salen=N,N′-bissalicylidene-1,2-ethanediamino anion, MnSalen, 1) as a non-native metal cofactor into apo NIH-PA Author ManuscriptNIH-PA Author Manuscri...

To demonstrate protein modulation of metal cofactor reactivity through non-covalent interactions, pH-dependent sulfoxidation and ABTS oxidation reactivity of a designed myoglobin (Mb) containing non-native MnSalen complex (1) was investigated using H2O2 as the oxidant. Incorporation of 1 inside the Mb resulted in increase in turnover numbers through exclusion of water from the metal complex and prevention of MnSalen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen bonding interactions resulted in no increase in turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen bond interactions resulted in increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the MnSalen complex. Nonlinear least-squares fitting of rate vs. pH plots demonstrates that 1·Mb(H64X, X= H, R and F) and the control MnSalen 1 exhibit pKas varying from pH 6.4 to 8.3, and that the lower pKa of the distal ligand in 1·Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1·Mb displays another pKa at low pH, with pKa of 5.0±0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen bonding interactions by the distal ligand to bind, orient and activate H2O2 is very important for designing artificial catalysts with dramatic different and tunable reactivity from catalysts without proteins.

Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds, and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over the single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +14% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta- and para- substituted substrates. The 1•Mb(T39C/L72C) showed the highest reactivity (TON up to 2.31 min-1) and selectivity (ee% up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.

Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.

No abstract