Carboxylation of phenols and asymmetric nucleophile addition across C=C bond

The regioselective carboxylation of electron-rich (hetero)aromatics employing decarboxylases in the redox-neutral (reverse) carboxylation reaction using bicarbonate or CO2(g) is currently exploited for the biocatalytic synthesis of carboxylic acids.1 Three enzyme classes exert complementary regioselectivities through diverse mechanisms: (i) Whereas the o-carboxylation of phenols (an equivalent to the Kolbe-Schmitt reaction) is mediated by Zn2+-dependent o-benzoic acid (de)carboxylases,2 (ii) the -carboxylation of hydroxystyrenes is catalysed by phenolic/ferulic acid (de)carboxylases acting via a pair of Tyr-Arg residues.3 (iii) Surpringly, these enzymes also exhibit a catalytic promiscuity for the nucleophile addition of H2O,4 NH2-OMe, cyanide and n-Pr-SH across the vinyl C=C bond via a quinone-methide intermediate, which yields the corresponding (S)-configurated adducts in up to 91% e.e.5 (iv) In search of ATP-independent regio-complementary p-benzoic acid (de)carboxylases, we discovered that 3,4-dihydroxybenzoic acid decarboxylase from Enterobacter cloacae6 (DHBDC_Ec) surprisingly depends on prenylated FMN7 as cofactor. In an attempt to propose a mechanism for the carboxylation of catechol by DHBDC_Ec, QM calculations revealed that the transient formation of a 1,3-dipolar cycloaddition product (as suggested for the decarboxylation of cinnamic acid with ferulic acid decarboxylase from S. cerevisiae8) was highly disfavored (\u3e30 kcal/M). As an alternative, we propose a mono-covalent nucleophile adduct involving a prFMN iminium electrophile (~14 kcal/M). Please click Additional Files below to see the full abstract

Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase

The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4′-radical of a 3′-keto-2′-deoxycytidine 5′-diphosphate. To identify the structure of the nucleotide radical [1′-[superscript 2]H], [2′-[superscript 2]H], [4′-[superscript 2]H], [5′-[superscript 2]H], [U−[superscript 13]C, [superscript 15]N], [U−[superscript 15]N], and [5,6 -[superscript 2]H] CDP and [β-[superscript 2]H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of [superscript 2]H at C4′ and C1′ altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5′-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.National Institutes of Health (U.S.) (GM29595)(EB002804)(EB002026

Synthesis of carboxylated derivatives of poly(isobutylene-co-isoprene) by azide–alkyne “click” chemistry

The final publication is available at Springer via https://dx.doi.org/10.1038/s41428-018-0130-yThe synthesis of carboxylated derivatives of poly(isobutylene-co-isoprene) (isobutylene–isoprene rubber, IIR) with substitution levels ranging from 1 to 4 mol% and different spacer lengths was accomplished through azide–alkyne Huisgen cycloaddition. Azido-functionalized IIR was first prepared by reacting brominated IIR with sodium azide to full conversion in a 90:10 tetrahydrofuran/N,N-dimethylacetamide mixture. The click reaction of azido-functionalized IIR with acetylenic acids, which was carried out using the copper(I) bromide/N,N,N′,N″,N″-pentamethyldiethylenetriamine catalyst system in tetrahydrofuran, yielded carboxylated IIRs. The products were characterized by 1H NMR and FT-IR spectroscopy, and their molecular weight was determined by size exclusion chromatography analysis. The conversion to carboxylated groups reached up to 100% as determined by NMR spectroscopy but was highly dependent on the type of solvent and the amounts of catalysts and reactants used in the procedures.ARLANXEO Canada Inc.Natural Sciences and Engineering Research Council (NSERC) of Canad

HF-EPR, Raman, UV/VIS Light Spectroscopic, and DFT Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus

Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g1-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm−1) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe2+ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class

Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g1-value of 2.0090 for the tyrosyl radical was extracted. This g1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν7a = 1500 cm−1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-sensitive Fe-O-Fe symmetric stretching (483 cm−1) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity