Glutathione peroxidase mimics based on conformationally-restricted, peri -like 4,5-disubstituted fluorene dichalcogenides

Glutathione peroxidase (GPx) regulates cellular peroxide levels through glutathione oxidation. GPx-mimics based on 4,5-disubstituted fluorene diselenides, their oxides, and ditellurides show catalytic activities comparable to related, conformationally-restricted, 1,8-naphthalene dichalcogenides.

Fluorene diselenides 3a (R = Me) and 3b (R = Bu) were synthesized from fluorenes 4a and 4b, through quenching the 4,5-dilithiofluorene species, generated using BuLi-TMEDA, 9   with elemental selenium (Scheme 1).Diselenide oxidation was investigated, in view of selenium oxides showing potential GPx-like activity, and to compare their behaviour with the analogous 5-membered ring naphthalene bis-selenium species reported by Kice (2a) 10 and Back (2b). 7Oxidation with 1.2 equivalents of mCPBA in Et 2 O gave selenolselinates 5a and 5b along with recovered starting material.We did not see any evidence of formation of the symmetrical selelenic anhydride in these mono-oxidations, in contrast to the oxidation of 2a, where a mixture of isomeric monoxides is observed. 10Use of a larger excess (3.5 equivalents) of mCPBA resulted in the precipitation of seleninic anhydrides 6 as single stereoisomers in excellent 85-95% yields.These were assigned as the trans, C 2 -symmetric stereoisomers, rather than the alternative cis, meso structures, on the basis of the equivalent Me groups at C-9 in the 1 H and 13 C NMR of 6a.Treatment of 6a with KOH in CD 3 OD formed the dipotassium salt of the bis-seleninic acid 7a, evidenced by Se NMR, which upon acidification returned the same stereoisomer 6a in 86% yield, suggesting the transisomer is thermodynamic preferred.In contrast, naphthalenes 2 give mixtures of diastereomeric seleninic anhydrides in both selenium oxidation and in base-mediated ring-openingacidification. 7,10 The ditellurides 8a and 8b were also prepared from fluorenes 4a and 4b using tellurium as the quench for the dilithio species.
14 This C-Se-Se-C dihedral angle is still much smaller than in the conformationally unconstrained diphenyl diselenide (85.4(2), -85.5(3)) 15 and in the less constrained biaryl diselenide, dibenzo[c,e][1,2]diselenine (9, Scheme 1) (59.0(3), -59.0(4), -57.0(4)).The GPx-like catalytic activities of diselenides 3, selenolseleninates 5, seleninic anhydrides 6 and ditellurides 8 were determined using Iwoka's NMR assay, 16 which monitors the drop in concentration of dithiotheritol (DTT red ) as it is oxidized to the disulfide DTT ox over time (Figure 3).A solvent system of 2:1 CD 3 OD:CDCl 3 was used to maintain solubility of all components and hence compare catalytic activity under homogenous conditions, although rates in this solvent system are much slower than in the original report of D 2 O. 17 The times taken for the initial concentration of DTT red to halve (T 50 ), after addition of H 2 O 2 are shown in Table 2. T 50 allows catalysts to be compared where there is a rapid initial reaction, as is the case herein for selenolseleninates and seleninic anhydrides, prior to addition of H 2 O 2 .Back's naphthalene diselenide 2b, wherein the electron-donating ortho-OMe groups were shown to increase catalytic activity over the non-substituted 2a, was also included, along with a background reaction (no catalyst).In order to gain further mechanistic insight into the catalytic cycle, stoichiometric reactions of selenium-containing catalysts were carried out (Scheme 2).Treatment of diselenide 3a with a large excess (10 equiv.) of H 2 O 2 in 2:1 MeOH:CH 2 Cl 2 at room temperature gave slow oxidation to monoxide 5a (Scheme 3, equation 2).No higher oxides were detected, and independent treatment of selenolseleninate 5a or seleninic anhydride 6a with H 2 O 2 under these conditions gave no reaction, suggesting 6a is not an intermediate in the catalytic cycle.
Diselenide 3a does not react with 4-t-butylbenzylthiol (10) in CH 2 Cl 2 /MeOH at room temperature (Scheme 2, equation 2).However reaction of selenolseleninate 5a with 10 equivalents of 10 gave an essentially instantaneous and quantitative transformation to diselenide 3a and disulfide 11 (94% based on 5a) along with 75% recovery of the thiol, showing 5a consumes two equivalents of thiol (equation 3).Under the same conditions, seleninic anhydride 6a underwent a similarly rapid and high-yielding transformation to 3a and 11 (equation 4), where the 90% yield of 11 is based on theoretical consumption of molar equivalents of thiol 10 and recovery of 4 equivalents of 10 (proposed intermediates and stoichiometries in the reactions of 5a and 6a with thiols are shown in the ESI, schemes S1-S2).
No intermediate bis-selenium species were observed in the reactions of 5a and 6a with 10.However, reaction of 5a with the bulkier thiol, t-BuSH, gave the bis-selenenyl sulfide 12a (equation 5), a potential intermediate in the formation of 3a (ESI, Scheme S1).Indeed, isolated 12a is slowly transformed over 24 h in solution to diselenide 3a and di-tert-butyldisulfide (13).This rate of this reaction is not changed by addition of 3 equivalents of 4-t-butylbenzylthiol (10), and no disulfides derived from 10 were formed, only 13.The breakdown of bisselenenyl sulfide 12a to diselenide 3a and disulfide 13 is thus presumably intramolecular, but given the steric hindrance provided by the t-Bu group, care should be taken in extrapolating these observations to all thiols.Kice   The reaction of seleninic anhydride 6a with t-BuSH also gave 12a (equation 6), though clearly there are multiple intermediates preceding its formation (ESI, Scheme S2).These intermediates account for the formation of tert-butyldisulfide (13) (98% based on theoretical amount of t-BuSH consumed and recovery of exactly 4 equivalents of thiol), whereas 13 is not observed in the reaction of selenolseleninate 5a with t-BuSH.§ § Based on the above observations, a catalytic cycle directly analogous to that proposed by Back for naphthalane diselenides 2 is proposed (Scheme 3): this cycle is mechanistically distinct from catalysis by other diselenides, which involve initial Se-Se bond cleavage by reaction with thiols. 20The rate-determining step is the oxidation of diselenide 3 to selenolseleninate 5, which in turn rapidly consumes two equivalents of thiol and forms disulfides via the intermediates 14 (not observed) and 12 (observed as 12a for R = Me, R' = t-Bu).As noted above, the conversion of 12 to 3 may occur by more than one mechanism and may also be catalysed by thiol: this step is severely slowed in the case of R = t-Bu where nucleophilic attack at sulfur is restricted and where an intramolecular mechanism appears most likely.In conclusion, bay-substituted 4,5-fluorene diselenides 3 possess properties analogous to peri-substituted 1,8naphthalene diselenides 2, including increased GPx-like activity over non-conformationally constrained diselenides.Despite a greater twist in the diselenide bond, the catalytic activity of fluorenes 3a and 3b is similar to that of naphthalene 2b in a homogenous DDT redox assay, without the need for additional activation by ortho-OMe groups on the aromatic rings.Moving forward, the fluorene scaffold is anticipated to be amenable to structural variation through incorporation of different groups at C-9, for example towards water-soluble GPx mimics 19,21 and application in other enzyme mimics based on naphthalene dichalcogenides. 13,22The ease of synthesis and reactivity of seleninic anhydrides such as 6 may also hold promise in situations where rapid oxidation of thiols to disulfides may be required.
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Fig. 2
Fig. 2 Two views of the crystal structure of diselenide 3a with ellipsoids drawn at the 50 % probability level.

Scheme 3
Scheme 3 Proposed catalytic cycle for oxidation of thiols to disulfides

Table 1
GPx-like activity of chalcogen-containing catalystsAll of the selenium-and tellurium-containing compounds 3, 5, 6 and 8 catalyse the oxidation of DTT red to DTT ox .Delenides 3 have comparable activities to the naphthalene diselenide 2b in this assay, despite lacking activating ortho-OMe substituents (Fig 1 and table 1, entries 1-3).The selenolseleninates 5 have shorter T 50 than the corresponding diselenides 3 ( a After addition of 10 mol% catalyst before addition of H2O2; b T50 is the time required to halve the initial thiol concentration after the addition of H2O2; c data in parenthesis are the experimental error.This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3Please do not adjust margins Please do not adjust margins

table 1
]naphthalene, which led Back to propose bisselenenyl sulfides as intermediates in the catalytic cycle of 2b.