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Biomimetic studies on iodothyronine deiodinase intermediates: modeling the reduction of selenenyl iodide by thiols

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Biomimetic studies on iodothyronine deiodinase intermediates: modeling the reduction of selenenyl iodide by thiols
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  440  ¹ WILEY-VCH-Verlag GmbH, 69451 Weinheim, Germany, 2002 1439-4227/02/03/05 $ 20.00+.50/0 ChemBioChem  2002  , 3, 440±447  Biomimetic Studies on Iodothyronine DeiodinaseIntermediates: Modeling the Reduction of Selenenyl Iodide by Thiols Govindasamy Mugesh, Wolf-Walther du Mont,* Cathleen Wismach, andPeter G. Jones [a] Enzyme mimetic studies on the crucial intermediate (E   SeI) of theiodothyronine deiodinase cycle have been carried out by using anareneselenenyl iodide stabilized by intramolecular Se¥¥¥N interac-tions. Treatment of this compound with aromatic thiols and thiobenzoxazole in the presence of NEt  3  affords areneselenenyl sulfides that are stable towards disproportionation reactions. Thestructures of three of the areneselenenyl sulfides were determined by X-ray crystallography. In one case, in the absence of NEt  3  , adiselenide can be formed rather than the selenenyl sulfide. Theareneselenenyl iodide also reacts with a related selenol to producethe corresponding diselenide, and this reaction is found to be muchfaster than that with thiols. The high reactivity of the selenenyl iodide with the selenol suggests that a reduced selenol group(R   SeH) may react with the E   SeI intermediate to produce adiselenide (E   Se  Se  R   ) without any thiol cosubstrate. The inter-mediacy of selenenyl sulfides during the reduction of selenenyl iodide by thiols and its possible relevance to the iodothyroninedeiodinase catalytic cycle is also described. KEYWORDS: cofactors  ¥  iodothyronine deiodinase  ¥  selenium  ¥ selenoenzymes  ¥  selenenyl iodides Introduction The monodeiodination of the prohormone thyroxin ( T4 ) toafford the biologically active hormone 3,5,3  -triiodothyronine( T3 ) (Scheme 1) is the first step in thyroid hormone action. Type Iiodothyronine deiodinase (ID-1), an enzyme containing seleno-cysteine in its active site, is responsible for most of thisconversion. [1] ID-1 is an integral membrane protein present inits highest amounts in liver, kidney, thyroid, and pituitary. [2] Scheme 1.  Monodeiodination of thyroxin (  T4  ) by ID-1 to afford 3,5,3  -triiodo-thyronine (  T3  ). The 5  -deiodination catalyzed by ID-1 has been proposed tobe a ping-pong, bisubstrate reaction in which the selenolategroup of the enzyme (E  Se  ) first reacts with thyroxin ( T4 ) toform a selenenyl iodide (E  SeI) with release of the deiodinatedcompound triiodothyronine ( T3 ). Subsequent reaction betweenthe selenenyl iodide and an unidentified cytoplasmic thiolcofactor (RSH) releases I  and regenerates the active site. Theanti-thyroid thiourea drug, 6- n -propyl-2-thiouracil (PTU), reactswith the selenenyl iodide and thus inhibits enzyme regeneration(Scheme 2). [3] Scheme 2.  Proposed mechanism for type I iodothyronine deiodination and inhibition by PTU. According to the mechanism shown in Scheme 2, thecomplete reaction requires two substrates: tetraiodothyronine( T4 ) and a cellular thiol or another cofactor. [4] A number of substrates have been proposed as suitable cofactors for thereduction of the E  SeI intermediate. Although it is customary touse dithiothreitol (DTT) as the second substrate in in vitroexperiments, [1d] the identity of the physiological second sub-strate is still uncertain. The tripeptide glutathione (GSH) can alsoact as a thiol cosubstrate, but GSH is a much less potent cofactorthan DTT for ID-1. [1a,d] In addition to GSH, other native thiols suchas dihydrolipoic acid or dihydrolipoamide may serve as cofactors [a]  Prof. Dr. W.-W. du Mont, Dr. G. Mugesh, Dipl.-Chem. C. Wismach,Prof. Dr. P. G. JonesInstitut f¸r Anorganische und Analytische ChemieTechnische Universit‰t BraunschweigPostfach 3329, 38023 Braunschweig (Germany)Fax:(   49)531-391-5387 E-mail: w.du-mont@tu-bs.de  Iodothyronine Deiodinase Intermediates ChemBioChem  2002  , 3, 440±447   441 for ID-1. [1a, 5] Interestingly, there is also evidence that the quantityof the enzyme present in the tissues is sufficient to renderregeneration of ID-1 by thiol cosubstrates unnecessary fornormal conversion of   T4  into  T3 . [6] Therefore, Scheme 2 may bean incomplete or incorrect representation of the catalyticmechanism of ID-1, since evidence for the cofactor systemsmentioned above has only been presented for in vitro studiesand not for in vivo analysis. It is well known in the case of glutathione peroxidase (GPx) that the active site selenol isregenerated from the oxidized selenium species (E  SeOH)through the formation of selenenyl sulfide (E  Se  S  G) byreaction with GSH. [7] Surprisingly, no such selenenyl sulfideintermediate has been proposed for the in vivo ID-1 mechanism,due to the lack of sufficient information about the physiolog-ically relevant thiol or other cofactors that can reduce the E  SeIintermediate. However, in an in vitro study, Sun et al. proposedthat one of the cysteine residues near the active center forms aninternal Se  S bond that could be reduced by DTT. [8] In contrast,Croteau et al. reported that the conserved cysteines near the ID-1 active site are not essential for catalytic activity. [9] In view of these conflicting reports, we have undertakenmodel studies to evaluate whether the selenenyl iodide canreact with thiols to produce the related selenol through theformation of selenenyl sulfides, or whether the selenenyl iodidemay be directly reduced by selenol in a single step without anythiol cosubstrates. Reactions between organoselenenyl iodidesand thiols or selenols as models for the deiodinase cycle havenot been studied previously, since areneselenenyl iodides suchas PhSeI are themselves generally unstable; they dispropor-tionate in solution. [10d] Even the sterically hindered arenesele-nenyl iodides 1 ± 3 have been found to exist in equilibria with thecorresponding diselenides  4 ± 6  in solution (Scheme 3). [10a,c] The Scheme 3.  Examples of unstable (  1 ± 6  ) and stable (  7   ,  8  ) organoselenenyl iodides. ™nonexistence∫ of stable binary Se  I compounds has beenassociated with the very similar electronegativities of these twoelements (that is, the lack of ionic±covalence resonance energyinSe  I bonds). [11] However, therecent observationsthat covalentSe  I bonds may be stabilized against dismutation reactionsthrough the introduction of sterically more demanding sub-stituents [10b,d] ( such as those in  7 ) or internally chelatinggroups [12] (as shown, for example, in  8 ) have provided oppor-tunities for studying the reactivity of pure selenenyl iodides.Recently, we found that compounds  7  and  8  react rapidly withthe thiouracil drugs 6- n -propyl-2-thiouracil (PTU) and 6-methyl-2-thiouracil (MTU) in the presence of base to give thecorresponding selenenyl sulfides. [13] Results and Discussion The internally chelated selenenyl iodide  8  was selected for thisstudy for four reasons: 1) compound  8  is not involved indismutation equilibria in solution, 2) the Se  I bond in thiscompound is kinetically activated by a strong Se¥¥¥N interaction(Se¥¥¥N distance: 2.133(4) ä), [12a] 3) the Se¥¥¥N interaction wouldbe expected to stabilize the resulting selenenyl sulfides againstdismutation reactions, and 4) the five-membered oxazolinemoiety in compound  8  resembles one of the active site featuresof ID-1, as the basic histidine residues present near the seleniumatom have been shown to play important roles in thedeiodination. [14] To investigate the possible intermediacy of aselenenyl sulfide (E  Se  S  R) in the deiodinase cycle experimen-tally, model reactions were carried out with various aromaticthiols. When 8 was treated with a stoichiometric amount of PhSH( 9 ) in an inert solvent, we were not able to observe the formationof the expected selenenyl sulfide  10 , but we did observe theformation of the diselenide  12  (Scheme 4). This may appear Scheme 4.  Reaction between areneselenenyl iodide  8  and PhSH (  9  ) in theabsence of NEt  3 . somewhat surprising, since it has been shown that an electro-philic selenium species, the selenenic acid  11 , readily reacts withPhSH to produce the selenenyl sulfide  10  and water. [15] However,in contrast to the reaction between the selenenic acid and PhSH,the HI liberated during the reaction between  8  and  9  may act ascatalyst for the formation of   12 . Acid-catalyzed formation of   12 from  10  was confirmed by  77 Se NMR spectroscopy, when asample of pure  10  in chloroform was treated with an excessamount of aqueous HCl in a two-phase system.Interestingly, the reaction between  8  and PhSH did afford theexpected selenenyl sulfide  10  when the HI produced during thereaction was trapped by addition of excess triethylamine to  8 before treatment with  9 . This allowed compound  10  to be  W.-W. du Mont et al. 442  ChemBioChem  2002  , 3, 440±447  isolated by column chromatography. Similarly, reactions be-tween  8  and  p -thiocresol ( 13 ),  p -chlorobenzenethiol ( 14 ),  p -bromobenzenethiol ( 15 ), 2,5-dimethylbenzenethiol ( 16 ), andthiobenzoxazole ( 17 ) in the presence of NEt 3  afforded thecorresponding selenenyl sulfides  18 ± 22  in good yields(Scheme 5). In the absence of NEt 3 , compound  8  reacted withall of the above-mentioned thiols to afford the diselenide  12 ,indicating that HI plays a major role in these reactions. Scheme 5.  Reactions between areneselenenyl iodide  8  and various thiols in the presence of NEt  3 . Whereas the reactions of   8  with  9  or  13 ± 16  were found to bemuch slower than those with the thiourea drugs PTU andMTU, [13] the reaction between  8  and  17  proceed-ed at a rate comparable to that with PTU andMTU. This, however, is not surprising, since it isknown that thiobenzoxazole ( 17 ) exists in solu-tion in its thione form, [16] which can react muchmore rapidly than the corresponding thiol formwith the Se  I bond. Similarly to PTU and MTU,thiobenzoxazole was inactive towards the disele-nide  12 . In contrast, all other aromatic thiols ( 9 , 13 ± 16 ) used in this study readily cleaved thediselenide bond to form the correspondingselenenyl sulfides. The high reactivity of   17 towards the selenenyl iodide  8  suggests that thiscompound might also act as an antithyroid drug.However, compound  17  has been shown to be aweak inhibitor of rat and human liver ID-1, [17] which indicates the absence of any structuralrequirements other than the reactive thiol group.The low inhibitory effect of   17  has been ascribedto the hydrophobic property of the oxazolemoiety, which lacks any polar substituents. [17b] The internally chelated selenenyl sulfides  10 and  18 ± 22  were characterized by  1 H,  13 C, and 77 Se NMR spectroscopy and mass spectral studies,and some of them were studied by X-ray crystalography. Thesynthesis and characterization of selenenyl sulfides (R  Se  S  R  )are of great interest from the biological point of view, asillustrated by a number of reports that postulate the interme-diacy of these species in the catalytic cycle of GPx and itssynthetic model compounds. [18] More recently, it has beenreported that the selenocysteine active site of mammalianthioredoxin reductase forms a selenenyl sulfide intermediateduring the catalytic cycle. [19] Moreover, the synthetic organo-selenium compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2 H  )-one) inhibits the activity of several enzymes by reactingwith the critical SH groups of the enzymes to form stableselenenyl sulfide (Se  S) adducts. [20] Although several selenenylsulfides such as  23 ± 33  (Scheme 6) have been proposed asintermediates or models in biologically relevant processes, [18, 21] very few of them (for example,  25 ± 27 ) have been isolated andfully characterized. [18c±e] Similarly to the selenenyl iodides, certain selenenyl sulfides arealso known to be unstable compounds that undergo dispro-portionation reactions to give the corresponding diselenides(R  Se  Se  R) and disulfides (R   S  S  R  ). [22] For example,2-NO 2 C 6 H 4 SeSCH 2 Ph disproportionates in solution to give anequilibrium mixture containing the diselenide (2-NO 2 C 6 H 4 Se) 2 and the disulfide (PhCH 2 S) 2 . [22] In the first mechanistic study of the GPx behavior of ebselen and related compounds, Fischerand Dereu proposed a similar disproportionation equilibriumbetween selenenyl sulfide  34  and the corresponding diselenide 35  (Scheme 7). [23] In this case, the equilibrium constant wasreported to be strongly influenced by the pH value of themedium.Compounds  10  and  18 ± 22  were found to be very stable insolution, and no dismutation reactions were observed. Whiletheseselenenylsulfides werevery slowlyreduced by thiols tothe Scheme 6.  Examples of biologically important organoselenenyl sulfides.  Iodothyronine Deiodinase Intermediates ChemBioChem  2002  , 3, 440±447   443 Scheme 7.  Dismutation equilibrium between a selenenyl sulfide and a diselenide. corresponding selenol 36 ,they showedhigher reactivitytowardsthiol exchange reactions. For example, compound  18  reactedvery slowly with  13  to produce the selenol  36 , but reactedrapidly with  9  to produce  10  by a thiol exchange reaction(Scheme 8). Similar thiol exchange reactions were observed for Scheme 8.  Thiol exchange reaction facilitated by Se¥¥¥N interactions. some selenenyl sulfides in the glutathione peroxidase likecatalytic cycle, and it was proposed that these reactions werefacilitated by Se¥¥¥N interactions. [15, 18d,e, 24] The  77 Se NMR chem-ical shifts indicate that compounds  10  and  18 ± 22  also exhibitintramolecular Se¥¥¥N interactions in solution. In general, the  77 SeNMR signals of arylselenenyl sulfides are known to appeardownfield from those of the corresponding diaryl diselenidesand upfield from those of the corresponding arylselenenylhalides. [25] As was to be expected, a large deshielding of thesignals was observed for the selenenyl sulfides  10  and  18 ± 22 (   557±633 ppm), with respect to the diselenide 12  (   454 ppm), and a large shielding of thesignals was observed with respect to the selenenyliodide  8  (   762 ppm).Despite the biological importance of selenenylsulfides containing coordinating amino/imino/ether groups, [15, 18, 20, 21] none has been structurallycharacterized. As part of our research program onmodeling the iodothyronine deiodinase cycle, werecently reported the structural characterization of a selenenyl sulfide stabilized by the stericallydemanding tris(trimethylsilyl)methyl group. [13] Inthis study, we report the X-ray crystal structures of three selenenyl sulfides ( 10 ,  18 , and  22 ), which allexhibit significant Se¥¥¥N interactions in the solidstate. The structures of compounds  10 ,  18 , and  22 ,together with significant bond lengths and angles,are shown in Figures 1, 2, and 3, respectively. TheSe¥¥¥N atomic distances of 2.617(2) ä for  10 ,2.590(3) ä for  18 , and 2.543(2) ä for  22  are largerthan that in the iodo derivative  8  (2.133(4) ä), [12a] but are significantly smaller than the sum of the Figure 1.  Molecular structure of   10  showing the Se¥¥¥N interaction. Selected bond lengths [ä] and angles [    ]: Se¥¥¥N 2.617(2), Se  S 2.2176(6), Se  C17 1.943(2),S  C1 1.786(2), O  C11 1.370(2), O  C7 1.458(3), N   C11 1.263(3), N   C8 1.493(3);C17   Se  S 100.70(6), C1  S  Se 104.61(7), N   Se  S 176.59(4). van der Waals radii (3.54 ä). [26] The Se¥¥¥N interactions observedhere are slightly stronger than that of diselenide  12 , in which thetwo Se¥¥¥N atomic distances were 2.819(5) and 2.705(5) ä. [15, 27] The N¥¥¥Se  S moieties in  10 ,  18 , and  22  adopt nearly lineararrangements with bond angles of 176.59(4), 177.18(7), and173.69(4)  , respectively.The  77 Se NMR chemical shifts of the compounds used in thisstudy are summarized in Table 1 along with some literaturevalues. From Table 1, it is evident that the  77 Se NMR chemicalshift values for  10  and  18 ± 22  are comparable to those of otherinternally chelated selenenyl sulfides. However, the  77 Se NMRsignals of the compounds with  ortho -chelating substituents areshifted downfield from those of the unsubstituted derivative(PhSeSPh); this indicates the existence of Se¥¥¥N/Se¥¥¥O inter-actions in solution. Figure 2.  Molecular structure of   18  showing the Se¥¥¥N interaction. Selected bond lengths [ä] and angles [    ]: Se¥¥¥N 2.590(2), Se  S 2.2272(8), Se  C1 1.948(3), S  C12 1.781(3), O  C7 1.360(3), O  C81.454(3), N   C7 1.255(3), N   C9 1.486(3); C1  Se  S 100.78(8), C12  S  Se 102.42(9), N   Se  S 177.17(6).  W.-W. du Mont et al. 444  ChemBioChem  2002  , 3, 440±447  Figure 3.  Molecular structure of   22  showing the Se¥¥¥N interaction. Selected bond lengths [ä] and angles [    ]: Se¥¥¥N1 2.543(2), Se  S 2.2416(6), Se  C11.937(2),S  C12 1.745(2), O1  C7 1.355(3), O1  C9 1.452(3), O2  C12 1.363(3), O2  C181.383(3), N1  C7 1.265(3), N1  C8 1.485(3), N2  C12 1.291(3), N2  C13 1.403(3);C1  Se  S 98.55(6), C12  S  Se 100.98(8), N   Se  S 173.69(4). The second objective of this study was to evaluate thepossible reactions between selenols and selenenyl iodides.When the areneselenenyl iodide  8  was treated with selenol  36  inthe presence or absence of NEt 3 , the corresponding diselenide( 12 ) was formed quantitatively. To the best of our knowledge,this is the first example of a reaction between a selenenyl iodideand a selenol. The reaction between  8  and selenol  36  was foundto be much faster than reactions between  8  and thiols  9  and 13 ± 17 . Interestingly, when  8  was added to a mixture containingthe selenol  36  and glutathione (GSH) in the presence of NEt 3 ,only the formation of the diselenide  12  was observed by  77 SeNMR spectroscopy, with no indication of the formation of selenenyl sulfide  37  (Scheme 9). This suggests that the selenenyliodide  8  reacts with selenol  36  much more rapidly than withGSH.From our model studies, two possible mechanisms for thereduction of E  SeI intermediates can be proposed. The isolationof stable selenenyl sulfide intermediates in the reaction betweenselenenyl iodide and thiols suggests that the catalytic mecha-nism of iodothyronine deiodinase (Scheme 2) proceeds throughthe formation of an E  Se  S  R intermediate when a thiolreducing agent (RSH) is used by the enzyme as a cosubstrate.The catalytic mechanism may, therefore, involve an initial attack of the nucleophilic selenol (E  SeH) or selenolate (E  Se  ) atiodothyronine to form a selenenyl iodide (E  SeI), followed by anucleophilic attack of RSH at the selenium atom of the E  SeI toproduce another intermediate, a selenenyl sulfide (E  Se  S  R),which in turn reacts with a second equivalent of RSH toregenerate the selenol as shown in Scheme 10 (pathway A). Scheme 10.  Proposed catalytic pathways of ID-1. On the other hand, the higher reactivity of the selenenyliodide  8  with selenol  36  compared with thiols suggests thatanother mechanism involving the reduction of E  SeI by anyselenol group present in other enzymes may also be consideredwhen the formation of an Se  Se bond is sterically feasible(Scheme 10, pathway B). While the reduction of the Se  Se bondwould become a limiting factor of the catalytic efficiency of theenzyme, the active site (E  SeH) must be regenerated since thethiourea drug PTU clearly inhibits the production of   T3  in vivo.The cleavage of the diselenide bond in compound  12  by  9  and 13 ± 16  in this study and similar observations reported in theliterature [15, 18c,d,f, 23] suggest that GSH or some other native thiolsmay act in vivo as reducing substrates towards the Se  Se bond.Under these circumstances, the thiol cofactors reduce thediselenide bond rather than the selenenyl iodide, and this maypossibly account for the lower potency of monothiols relative todithiols such as DTT. Conclusions The mechanism of the reduction of selenenyl iodideintermediate in the iodothyronine deiodinase cycle ismodeled by use of an internally chelated arenesele-nenyl iodide. Our model study (1) provides furtherevidence that the intermediacy of a selenenyl iodideas a product of step I of the deiodinase cycle plays acrucial role in the catalytic cycle, (2) suggests that theconcept of a selenenyl sulfide intermediacy is morereasonable than a one-step, two-electron reductionof E  SeI to E  SeH when a thiol co-substrate is used Table 1.  77  Se NMR chemical shifts (    ) of some internally chelated arenesele-nenyl sulfides. [a] Compd   [ppm] Compd   [ppm]PhSeSPh 526 [25] 21  557 10  576  22  633 18  591  26  572 [18d] 19  587  33  565 [23] 20  586  34  590 [23] [a] Chemical shifts from this work unless another reference is given. Scheme 9.  Reaction between the internally chelated selenenyl iodide  8  and selenol   36 .
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