Regioselective deiodination of thyroxine by iodothyronine deiodinase mimics: An unusual mechanistic pathway involving cooperative chalcogen and halogen bonding

Article abstract of DOI:10.1021/ja210478k

Iodothyronine deiodinases (IDs) are mammalian selenoenzymes that catalyze the conversion of thyroxine (T4) to 3,5,3′-triiodothyronine (T3) and 3,3′,5′-triiodothyronine (rT3) by the outer- and inner-ring deiodination pathways, respectively. These enzymes also catalyze further deiodination of T3 and rT3 to produce a variety of di- and monoiodo derivatives. In this paper, the deiodinase activity of a series of peri-substituted naphthalenes having different amino groups is described. These compounds remove iodine selectively from the inner-ring of T4 and T3 to produce rT3 and 3,3′-diiodothyronine (3,3′-T2), respectively. The naphthyl-based compounds having two selenols in the peri-positions exhibit much higher deiodinase activity than those having two thiols or a thiol-selenol pair. Mechanistic investigations reveal that the formation of a halogen bond between the iodine and chalcogen (S or Se) and the peri-interaction between two chalcogen atoms (chalcogen bond) are important for the deiodination reactions. Although the formation of a halogen bond leads to elongation of the C-I bond, the chalcogen bond facilitates the transfer of more electron density to the C-I σ* orbitals, leading to a complete cleavage of the C-I bond. The higher activity of amino-substituted selenium compounds can be ascribed to the deprotonation of thiol/selenol moiety by the amino group, which not only increases the strength of halogen bond but also facilitates the chalcogen-chalcogen interactions.

Full text of DOI:10.1021/ja210478k

Article
pubs.acs.org/JACS
Regioselective Deiodination of Thyroxine by Iodothyronine
Deiodinase Mimics: An Unusual Mechanistic Pathway Involving
Cooperative Chalcogen and Halogen Bonding
Debasish Manna and Govindasamy Mugesh*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India
S
* Supporting Information
ABSTRACT: Iodothyronine deiodinases (IDs) are mamma-
lian selenoenzymes that catalyze the conversion of thyroxine
(T4) to 3,5,3′-triiodothyronine (T3) and 3,3′,5′-triiodothyr-
onine (rT3) by the outer- and inner-ring deiodination
pathways, respectively. These enzymes also catalyze further
deiodination of T3 and rT3 to produce a variety of di- and
monoiodo derivatives. In this paper, the deiodinase activity of a
series of peri-substituted naphthalenes having different amino
groups is described. These compounds remove iodine selectively from the inner-ring of T4 and T3 to produce rT3 and 3,3′-
diiodothyronine (3,3′-T2), respectively. The naphthyl-based compounds having two selenols in the peri-positions exhibit much
higher deiodinase activity than those having two thiols or a thiol−selenol pair. Mechanistic investigations reveal that the
formation of a halogen bond between the iodine and chalcogen (S or Se) and the peri-interaction between two chalcogen atoms
(chalcogen bond) are important for the deiodination reactions. Although the formation of a halogen bond leads to elongation of
the C−I bond, the chalcogen bond facilitates the transfer of more electron density to the C−I σ* orbitals, leading to a complete
cleavage of the C−I bond. The higher activity of amino-substituted selenium compounds can be ascribed to the deprotonation of
thiol/selenol moiety by the amino group, which not only increases the strength of halogen bond but also facilitates the
chalcogen−chalcogen interactions.
the other hand, catalyzes the conversion of T4 to rT3 by an
inner-ring (5) deiodination.2,3 It is known that ID-3 plays a
crucial role in the maintenance of serum T3 concentration by
converting T3 to 3,3′-T2, which is believed to be a key step in
the protection of tissues from excess thyroid hormone.2−4
Although deiodinases are essential for the function of thyroid
gland, the mechanism of the deiodination reactions remains
unknown.
The interesting regioselective deiodinations of thyroid
hormones by different deiodinases have sparked interest in
the development of simple selenium compounds as mimics of
these redox enzymes.5,6 Goto et al. reported a synthetic selenol
that converts N-butyrylthyroxine methyl ester to the corre-
sponding triiodo derivative by 5′-deiodination.6 We have shown
that compound 1, having two thiol moieties, and compound 2,
having a thiol−selenol pair in the peri-positions, convert T4 and
T3 to rT3 and 3,3′-T2, respectively, by 5-deiodination.7 Very
recently, we reported, in a preliminary communication, that
compound 3, bearing two selenol moieties, is more active than
1 and 2 in the deiodination of T4 under physiologically relevant
conditions.8 However, the mechanism by which these
compounds remove iodine selectively from the 5-position of
T4 or T3 is not clear. Herein we report the synthesis,
deiodinase activity, and mechanism of action of a series of peri-
INTRODUCTION
■
Iodothyronine deiodinases (IDs) are mammalian selenoen-
zymes that are involved in the activation and inactivation of
thyroid hormones.1 Type-1 and -2 deiodinases (ID-1 and ID-2)
catalyze the outer-ring (5′) deiodination of thyroxine (3,5,3′,5′-
tetraiodothyronine, T4) to produce 3,5,3′-triiodothyronine
(T3). These two enzymes also convert 3,3′,5′-triiodothyronine
(reverse T3 or rT3) to 3,3′-diiodothyronine (3,3′-T2) by 5′-
deiodination (Scheme 1).2 The type-3 deiodinase (ID-3), on
Scheme 1. Activation and Inactivation of Thyroid Hormones
by Three Iodothyronine Deiodinases (ID-1, ID-2, and ID-3)
Received: November 8, 2011
Published: February 22, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Article
128.8, 129.1, 130.8, 137.7, 138.3, 149.4, 154.9, 187.5; 77Se NMR
(CDCl3) δ (ppm): 773; ESI-MS (m/z) calcd for C11H6OSSe [M]+
265.93, found 265.92.
substituted naphthalene derivatives. We also show for the first
time that the peri-interactions between two chalcogen atoms
(chalcogen bond) facilitate the halogen-bond-assisted activation
of the carbon−iodine bond in T4 and related compounds.
Synthesis of 25. This compound was synthesized by following the
procedure given for compound 24 and purified from the other isomers
by silica gel column chromatography using toluene as mobile phase.
1H NMR (CDCl3) δ (ppm): 7.47 (t, J = 7.6 Hz, 1H), 7.54 (d, J = 8
Hz, 1H), 7.61−7.69 (m, 2H), 7.72 (d, J = 7.2 Hz, 1H), 10.18 (s,1H);
13C NMR (CDCl3) δ (ppm): 122.8, 123.4, 125.1, 129.5, 130.4, 130.8,
139.5, 146.8, 155.2, 188.6; 77Se NMR (CDCl3) δ (ppm): 408, 673;
ESI-MS (m/z) calcd for C11H6OSe2 [M]+: 313.87, found 313.91.
Synthesis of 26. To a solution of naphthalene-1,8-disulfide-2-
carboxaldehyde (500 mg, 2.29 mmol) in 30 mL of dry acetonitrile was
added dropwise a 30% solution of methyl amine (1.8 g, 50.84 mmol).
It was stirred at 60 °C for 8 h. The progress of the reaction was
monitored by TLC. After completion of the reaction, the solvent was
evaporated under reduced pressure. The resulting Schiff base was
purified by column chromatography using petroleum ether/ethyl
EXPERIMENTAL SECTION
General Procedure. n-Butyllithium (n-BuLi) was purchased from
Acros Chemical Co. (Belgium). Selenium powder, DTT, T4, rT3, and
T3 were obtained from Aldrich Chemical Co. Liquid state NMR
■
1
spectra were recorded in CDCl3 or d4-MeOH as a solvent. H (400
MHz), 13C (100.56 MHz), and 77Se (76.29 MHz) NMR spectra were
obtained using a Bruker 400 MHz NMR spectrometer. Chemical shifts
are cited with respect to SiMe4 as internal (1H and 13C) and Me2Se as
external (77Se) standard. Thin-layer chromatography analyses were
carried out on precoated silica gel plates (Merck), and spots were
visualized by UV irradiation. Column chromatography was performed
on glass columns loaded with silica gel or on an automated flash
chromatography system (Biotage) by using preloaded silica cartridges.
High-performance liquid chromatography (HPLC) experiments were
carried out on a Waters Alliance System (Milford, MA) consisting of a
2695 separation module and a 2996 photodiode-array detector. The
assays were performed in 1.8 mL sample vials, and a built-in
autosampler was used for sample injection. The HPLC system was
controlled with EMPOWER software (Waters Corporation, Milford,
MA).
1
acetate as eluent to give 26 as a red solid in 87% yield: H NMR
(CDCl3) δ (ppm): 3.75 (s, 3H), 7.39−7.44 (m, 2H), 7.45−7.47 (m,
1H), 7.49 (s, 2H), 8.58 (s, 1H); 13C NMR (CDCl3) δ (ppm): 43.8,
118.3, 120.4, 122.8, 125.9, 127.2, 128.7, 135.5, 135.7, 147.5, 148.9,
157.4; ESI-MS (m/z) calcd for C12H9NS2 [M]+: 231.02, found 230.84.
Synthesis of 27. To a solution of naphthalene-1,8-selenenylsulfide-
2-carboxaldehyde (300 mg, 1.13 mmol) in 20 mL of dry acetonitrile
was added dropwise a 30% solution of methyl amine (0.7 g, 22.62
mmol). The resulting reaction mixture was stirred at 60 °C for 14 h.
The progress of the reaction was monitored by TLC. After completion
of the reactions, the solvent was evaporated under reduced pressure.
The resulting Schiff base was purified by column chromatography
using petroleum ether/ethyl acetate as eluent to give 27 as a brown
Deiodination Assay. The deiodination reactions were carried out in
100 mM phosphate buffer (pH 7.5) with 10 mM dithiothreitol (DTT)
at 37 °C. Selenols and thiols were freshly prepared by reducing the
corresponding selenenyl sulfides, diselenides, or disulfides with NaBH4
prior to use. The reaction products were analyzed by reverse-phase
HPLC (Lichrospher C18 column, 4.6 μm, 150 mm × 5 mm) with
gradient elution using acetonitrile/ammonium acetate (pH 4.0) as the
mobile phase. The formation of rT3 was monitored at λ= 275 nm, and
the amounts of deiodinated products formed in the reactions were
calculated by comparing the peak areas.
Synthesis of 4. Compound 4 was synthesized by following a
literature procedure.9c 1H NMR (CDCl3) δ (ppm): 7.15 (d, J = 8 Hz,
2H), 7.28 (t, J = 7.2 Hz, 2H), 7.35 (d, J = 8 Hz, 2H); 13C NMR
(CDCl3) δ (ppm): 116.4, 122.1, 128.4, 135.2, 136.2, 144.5; ESI-MS
(m/z) calcd for C10H6S2 [M]+: 189.99, found 189.87.
Synthesis of 5. Compound 5 was synthesized by following a
literature procedure.9d 1H NMR (CDCl3) δ (ppm): 7.18 (d, J = 7.2
Hz, 1H), 7.23−7.31 (m, 3H), 7.34 (d, J = 8 Hz, 1H), 7.45 (d, J = 8 Hz,
1H); 13C NMR (CDCl3) δ (ppm): 118.8, 119.8, 122.7, 123.8, 128.0,
128.3, 136.6, 137.2, 141.1, 145.1; 77Se NMR (CDCl3) δ (ppm): 556;
ESI-MS (m/z) calcd for C10H6SSe [M]+: 237.93, found 237.80.
Synthesis of 6. Compound 6 was synthesized by following a
literature procedure.9c 1H NMR (CDCl3) δ (ppm): 7.23 (d, J = 7.6
Hz, 2H), 7.35 (d, J = 7.6 Hz, 2H), 7.47 (d, J = 8 Hz, 2H); 13C NMR
(CDCl3) δ (ppm): 121.5, 124.2, 128.1, 137.9, 138.3, 141.2; 77Se NMR
(CDCl3) δ (ppm): 415. ESI-MS (m/z) calcd for C10H6Se2 [M]+:
285.88, found 285.56.
1
solid in 78% yield: H NMR (CDCl3) δ (ppm): 3.83 (s, 3H), 7.46−
7.48 (m, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.70−
7.72 (m, 1H); 77Se NMR (CDCl3) δ (ppm): 770.
Synthesis of 28. To a solution of naphthalene-1,8-selenenylsulfide-
2-carboxaldehyde (300 mg, 1.13 mmol) in 20 mL of dry acetonitrile
was added dropwise a 70% solution of ethyl amine (1.01 g, 22.62
mmol). The resulting reaction mixture was stirred at 60 °C for 14 h.
The progress of the reaction was monitored by TLC. The solvent was
evaporated under reduced pressure. The resulting Schiff base was
purified by column chromatography using petroleum ether/ethyl
1
acetate as eluent to give 28 as a brown solid in 72% yield: H NMR
(CDCl3) δ (ppm): 1.51 (t, J = 7.2 Hz, 3H), 4.03 (q, J = 6.4 Hz, 2H),
7.42−7.46 (m, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.52, (d, J = 8.4 Hz, 1H),
7.68−7.70 (m, 1H), 8.79 (s, 1H); 13C NMR (CDCl3) δ (ppm): 18.3,
50.1, 120.1, 122.3, 125.0, 126.2, 127.8, 128.5, 135.8, 150.1, 150.7,
154.6; 77Se NMR (CDCl3) δ (ppm): 762.
Synthesis of 29. To a solution of naphthalene-1,8-diselenide-2-
carboxaldehyde (300 mg, 0.96 mmol) in 20 mL dry acetonitrile was
added dropwise a 30% solution of methyl amine (595 mg, 19.2 mmol).
The resulting reaction mixture was stirred at 60 °C for 12 h. The
progress of the reaction was monitored by TLC. The solvent was
evaporated under reduced pressure. The resulting Schiff base was
purified by column chromatography using petroleum ether/ethyl
Synthesis of 24. This compound was synthesized by Vilsmeier−
Haack formylation by following a procedure reported for the
corresponding disulfide, naphthalene-1,8-disulfide-2-carboxalde-
hyde.7,10 The 2-formyl naphthalene selenenyl sulfide was synthesized
by adding POCl3 (0.9 g, 0.6 mL, 12.9 mmol) dropwise to a stirred
solution of selenenyl sulfide 5 (0.8 g, 3.4 mmol) in dry DMF (10 mL)
at 0 °C under a nitrogen atmosphere. The reaction mixture was
allowed to warm to room temperature, and the stirring was continued
for 24 h under nitrogen. To this, water was added to give a yellow
solid, which was extracted with EtOAc (200 mL) and washed three
times with brine. The organic layer was separated and dried over
anhydrous Na2SO4. After filtration, the solvent was removed under
reduced pressure. The desired compound 24 was purified from the
other isomers by silica gel column chromatography using toluene as
1
acetate as eluent to give 29 as a red solid in 82% yield: H NMR
(CDCl3) δ (ppm): 3.82 (s, 3H), 7.39 (t, J = 7.6 Hz, 1H), 7.56−7.59
(m, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.85−7.87 (m, 1H), 8.8 (s, 1H);
13C NMR (CDCl3) δ (ppm): 42.0, 122.0, 125.3, 125.5, 126.9, 128.2,
129.4, 137.1, 138.4, 146.1, 151.6, 158.0; 77Se NMR (CDCl3): 339, 696;
ESI-MS (m/z) calcd for C12H9NSe2 [M + H]+: 327.91, found 327.92.
Synthesis of 30. To a solution of naphthalene-1,8-diselenide-2-
carboxaldehyde (300 mg, 0.96 mmol) in 20 mL of dry acetonitrile was
added dropwise a 70% solution of ethyl amine (865 mg, 19.2 mmol).
The resulting reaction mixture was stirred at 60 °C for 12 h. The
progress of the reaction was monitored by TLC. The solvent was
evaporated using reduced pressure. The resulting Schiff base was
purified by column chromatography using petroleum ether/ethyl
1
1
mobile phase. H NMR (CDCl3) δ (ppm): 7.45 (d, J = 6.8 Hz, 1H),
acetate as eluent to give 30 as a red solid in 85% yield: H NMR
7.56 (1H, J = 7.6 Hz, 1H), 7.59−7.64 (m, 2H), 7.77 (d, J = 8.4 Hz,
(CDCl3) δ (ppm): 1.56 (t, J = 6.8 Hz, 3H), 4.05 (q, J = 7.2 Hz, 2H),
7.38 (t, J = 8 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.72 (d, J = 8.4, 1H),
1H), 10.30 (s, 1H); 13C NMR (CDCl3) δ (ppm): 120.5, 121.3, 124.6,
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Journal of the American Chemical Society
Article
7.84 (d, J = 7.2 Hz, 1H), 8.83 (s, 1H); 13C NMR (CDCl3) δ (ppm):
18.1, 50.6, 122.0, 125.4, 125.5, 127.0, 128.2, 129.4, 138.4, 146.1, 146.1,
151.4, 155.9; 77Se NMR (CDCl3) δ (ppm): 340, 690; ESI-MS (m/z)
calcd for C13H11NSe2 [M + H]+: 341.93, found 341.94.
2.73−2.78 (q, J = 7.2 Hz, 2H), 3.94 (s, 1H), 7.04 (d, J = 8 Hz, 1H),
7.17−7.21 (m, 1H), 7.26−7.29 (m, 2H), 7.42 (d, J = 8.4 Hz, 1H); 13
C
NMR (CDCl3) δ (ppm): 15.2, 44.9, 52.5, 120.0, 121.2, 124.4, 126.5,
127.6 132.1, 136.0, 137.8, 139.5, 146.2; 77Se NMR (CDCl3) δ (ppm):
562; ESI-MS (m/z) calcd for C13H13NSSe [M + H]+: 296.00, found
295.80.
Synthesis of 31. To a solution of naphthalene-1,8-diselenide-2-
carboxaldehyde (300 mg, 0.96 mmol) in 20 mL of dry acetonitrile was
added dropwise n-propyl amine (1.13 g, 19.2 mmol). The resulting
reaction mixture was stirred at 60 °C for 12 h. The progress of the
reaction was monitored by TLC. The solvent was evaporated under
reduced pressure. The resulting Schiff base was purified by column
chromatography using petroleum ether/ethyl acetate as eluent to give
31 as a red solid in 92% yield: 1H NMR (CDCl3) δ (ppm): 1.06 (t, J =
7.4 Hz, 3H), 1.95 (m, 2H), 3.96 (t, J = 7.4 Hz, 2H), 7.39 (t, J = 7.7 Hz,
1H), 7.57 (d, J = 7.8 Hz, 2H), 7.72 (d, J = 8 Hz, 1H), 7.84 (d, J = 8
Hz, 1H), 8.78 (s, 1H); 13C NMR (CDCl3) δ (ppm): 12.45, 25.9, 58.2,
122.0, 125.3, 125.5, 127.0, 128.2, 129.3, 137.0, 138.4, 146.1, 151.4,
156.4; 77Se NMR (CDCl3) δ (ppm): 341, 690; ESI-MS (m/z) calcd
for C14H13NS2 [M + H]+: 355.94, found 355.73.
Synthesis of 32. To a solution of naphthalene-1,8-diselenide-2-
carboxaldehyde (300 mg, 0.96 mmol) in 20 mL of dry acetonitrile was
added dropwise isopropyl amine (1.13 g, 19.2 mmol). The resulting
reaction mixture was stirred at 60 °C for 12 h. The progress of the
reaction was monitored by TLC. The solvent was evaporated under
reduced pressure. The resulting Schiff base was purified by column
chromatography using petroleum ether/ethyl acetate as eluent to give
32 as a red solid in 90% yield: 1H NMR (CDCl3) δ (ppm): 1.53 (d, J
= 6.4 Hz, 6H), 4.05−4.10 (m, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.48 (d, J
= 8.4 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 8 Hz, 1H), 7.84
(d, J = 7.6 Hz, 1H), 8.73 (s, 1H); 13C NMR (CDCl3) δ (ppm): 26.1,
58.0, 122.1, 125.3, 125.4, 127.2, 128.2, 129.4, 136.9, 138.4, 146.0,
150.9, 154.2; 77Se NMR (CDCl3) δ (ppm): 341, 678; ESI-MS (m/z)
calcd for C14H13NS2 [M + H]+: 355.94, found 355.76.
Synthesis of 36. Compound 29 (200 mg, 0.62 mmol) was dissolved
in 30 mL of MeOH−CHCl3 (5:1). NaBH4 (468 mg, 12.4 mmol) was
added to the above solution. The red solution turned to pale yellow
immediately. The reaction mixture was stirred at room temperature for
3 h. The resulting red solution was poured into 100 mL of water and
extracted with 40 mL of dichloromethane. The dichloromethane
extract was then dried and evaporated to give a yellow solid, which was
then purified by column chromatography using petroleum ether/ethyl
1
acetate as eluent to give 36 as a red solid in 78% yield: H NMR
(CDCl3) δ (ppm): 2.51 (s, 3H), 3.95 (s, 2H), 7.06 (d, J = 8 Hz, 1H),
7.16−7.20 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8 Hz, 1H), 7.46−7.48 (m, 2
H); 13C NMR (CDCl3) δ (ppm): 36.3, 55.5, 123.0, 123.5, 124.9,
127.0, 127.6, 133.6, 136.8, 139.6, 140.7, 141.8. 77Se NMR (CDCl3) δ
(ppm): 342, 480; ESI-MS (m/z) calcd for C12H11NSe2 [M]+: 327.14,
found 327.66.
Synthesis of 37. Compound 30 (200 mg, 0.59 mmol) was dissolved
in 30 mL of MeOH−CHCl3 (5:1). NaBH4 (446 mg, 11.8 mmol) was
added to the above solution. The red solution turned to pale yellow
immediately. The reaction mixture was stirred at room temperature for
3 h. The resulting red solution was poured into 100 mL of water and
extracted with 40 mL of dichloromethane. The dichloromethane
extract was then dried and evaporated to give a red solid, which was
then purified by column chromatography using petroleum ether−ethyl
1
acetate as eluent to give 37 as a red solid in 82% yield: H NMR
(CDCl3) δ (ppm): 2.21−2.23 (t, J = 6.8 Hz, 3H), 2.68−2.73 (q, J =
6.8 Hz, 2H), 3.94 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.17 (t, J = 8 Hz,
1H), 7.38 (d, J = 8 Hz, 1H), 7.47 (t, J = 8 Hz, 2H); 13C NMR
(CDCl3) δ (ppm): 15.2, 44.5, 53.5, 123.0, 123.5, 124.8, 126.6, 127.6,
134.2, 136.8, 139.6, 140.6, 141.7; 77Se NMR (CDCl3) δ (ppm): 345,
466; ESI-MS (m/z) calcd for C13H13NSe2 [M + H]+ = 343.93, found
343.67.
Synthesis of 33. Compound 26 (150 mg, 0.65 mmol) was dissolved
in 30 mL of MeOH−CHCl3 (5:1). NaBH4 (737 mg, 19.5 mmol) was
added to the above solution. The color of the solution turned to pale
yellow immediately. The reaction mixture was stirred at room
temperature for 2 h. The resulting yellow solution was poured into
100 mL of water and extracted with dichloromethane. The
dichloromethane extract was then dried and evaporated to give a
yellow solid, which was then purified by column chromatography using
petroleum ether−ethyl acetate as eluent to give 33 as a yellow solid in
Synthesis of 38. Compound 31 (180 mg, 0.51 mmol) was dissolved
in 30 mL of MeOH−CHCl3 (5:1). NaBH4 (385 mg, 10.20 mmol) was
added to the above solution. The red solution turned to pale yellow
immediately. The reaction mixture was stirred at room temperature for
3 h. The resulting red color solution was poured into 100 mL of water
and extracted with 40 mL of dichloromethane. The dichloromethane
extract was then dried and evaporated to give a red solid, which was
then purified by column chromatography using petroleum ether−ethyl
1
78% yield: H NMR (CDCl3) δ (ppm): 2.43 (s, 3H), 3.89 (s, 2H),
7.12−7.18 (m, 2H), 7.21−7.26 (m, 1H), 7.30−7.33 (2H); 13C NMR
(CDCl3) δ (ppm): 36.0, 54.6, 111.7, 121.3, 122.4, 127.7, 128.0, 130.1,
135.3, 136.5, 142.7, 144.7; ESI-MS (m/z) calcd for ESI calcd for
C12H11NS2 [M]+: 233.04, found 233.82.
1
acetate as eluent to give 38 as a red solid in 85% yield: H NMR
Synthesis of 34. Compound 27 (100 mg, 0.38 mmol) was dissolved
in 20 mL of MeOH−CHCl3 (5:1). NaBH4 (408 mg, 10.8 mmol) was
added to the above solution. The color of the solution turned to pale
yellow immediately. The reaction mixture was stirred at room
temperature for 4 h. The resulting yellow solution was poured into
100 mL of water and extracted with 20 mL of dichloromethane. The
dichloromethane extract was then dried and evaporated to give a
yellow solid, which was then purified by column chromatography using
petroleum ether/ethyl acetate as eluent to give 34 as a brown solid in
68% yield: 1H NMR (CDCl3) δ (ppm): 2.53 (s, 3H), 3.91 (s,3H), 7.06
(d, J = 8 Hz, 1H), 7.17−7.21 (m, 1H), 7.27−7.29 (m, 2H), 7.44 (d, J =
8 Hz, 1H); 13C NMR (CDCl3) δ (ppm): 36.7, 54.5, 120.0, 121.2,
124.4, 126.6, 127.6, 131.5, 136.0, 139.5, 146.2; 77Se NMR (CDCl3) δ
(ppm): 577; ESI-MS (m/z) calcd for C12H11NSSe [M]+: 280.98,
found 280.92.
(CDCl3) δ (ppm): 0.94 (t, J = 8 Hz, 3H), 1.64−1.70 (m, 2H), 2.68 (t,
J = 7.2 Hz, 2H), 3.95 (s, 2H), 7.08 (d, J = 8 Hz, 1H), 7.17 (t, J = 8 Hz,
1H), 7.38−7.44 (m, 2H), 7.48 (d, J = 8 Hz, 1H); 13C NMR (CDCl3) δ
(ppm): 12.1, 22.7, 51.5, 53.4, 123.1, 123.4, 124.9, 127.1, 127.6, 133.0,
136.9, 139.4, 141.2, 141.7; 77Se NMR (CDCl3) δ (ppm): 345, 470;
ESI-MS (m/z) calcd for C14H15NSe2 [M]+: 355.95, found 356.97.
Synthesis of 39. Compound 32 (220 mg, 0.62 mmol) was dissolved
in 30 mL of MeOH−CHCl3 (5:1). NaBH4 (471 mg, 12.45 mmol) was
added to the above solution. The red solution turned to pale yellow
immediately. The reaction mixture was stirred at room temperature for
3 h. The resulting red solution was poured into 100 mL of water and
extracted with 40 mL of dichloromethane. The dichloromethane
extract was then dried and evaporated to give a red solid, which was
then purified by column chromatography using petroleum ether−ethyl
acetate as eluent to give 39 as a dark red solid in 85% yield: 1H NMR
(CDCl3) δ (ppm): 1.22 (s, 6H), 2.95−3.01 (m, 1H), 3.97 (s, 2H),
7.04 (d, J = 8 Hz, 1H), 7.17 (t, J = 8 Hz, 1H), 7.39 (d, J = 8 Hz, 1H),
7.44−7.47 (m, 2H); 13C NMR (CDCl3) δ (ppm): 22.8, 49.7, 51.2,
123, 123.5, 124.8, 126.7, 127.6, 134.6, 136.8, 139.6, 141.1, 141.7; 77Se
NMR (CDCl3) δ (ppm): 350, 445; ESI-MS (m/z) calcd for
C14H15NSe2 [M]+: 356.95, found 356.98.
Synthesis of 35. Compound 28 (100 mg, 0.34 mmol) was dissolved
in 20 mL of MeOH−CHCl3 (5:1). NaBH4 (388 mg, 10.26 mmol) was
added to the above solution. The reaction mixture was stirred at room
temperature for 4 h. The resulting yellow color solution was poured
into 100 mL of water and extracted with 20 mL of dichloromethane.
The dichloromethane extract was then dried and evaporated to give a
yellow solid, which was then purified by column chromatography using
petroleum ether−ethyl acetate as eluent to give 35 as a brown solid in
Single Crystal X-ray Crystallography. X-ray crystallographic
studies were carried out on a Bruker CCD diffractometer with
graphite-monochromatized Mo Kα radiation (λ = 0.71073 Å)
1
65% yield: H NMR (CDCl3) δ (ppm): 1.23 (t, J = 7.2 Hz, 3H),
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Kuiper et al.18 They showed that the substitution of Sec by a
cysteine (Cys) significantly reduces the catalytic efficiency and
that the substrate turnover numbers for the deiodinations of T4
and T3 by the Cys mutant were 6- and 2-fold lower,
respectively, than for the wild-type enzyme.
controlled by a Pentium-based PC running the SMART software
package.11 Single crystals were mounted at room temperature on the
ends of glass fibers, and data were collected at room temperature. The
structures were solved by direct methods and refined using the
SHELXTL software package.12 All non-hydrogen atoms were refined
anisotropically, and hydrogen atoms were assigned idealized locations.
Empirical absorption corrections were applied to all structures using
SADABS.13,14 The structures were solved by direct methods (SIR-92)
and refined by full-matrix least-squares procedures on F2 for all
reflections (SHELXL-97).
Crystal Data for 30. C H NSe ; M = 339.15, orthorhombic P1, a
̅
13 11
2
r
= 7.6025(8) Å, b = 15.0667(17) Å, c = 10.5922(10) Å, α = 90°, β =
90°, γ = 90°, V = 1213.3(2) Å3, Z = 4, Mo Kα radiation (λ = 0.71073
Å), T = 293(2) K, R1 = 0.0400, wR2 = 0.1018 (I > 2σ(I)), R1 =
0.0507, wR2 = 0.1114 (all data).
Crystal Data for 31. C H NSe , M = 353.17, monoclinic P1, a =
̅
14 13
2
r
The deiodination of T4 by compounds 1−3 occurred even in
the absence of DTT. When T4 was treated with an excess
amount of compound 2 or 3 (5 equiv), complete conversion of
T4 into rT3 was observed. Analysis of the reaction products by
HPLC and mass spectrometry indicated that compounds 1, 2,
and 3 were oxidized to the dichalcogenides 4, 5, and 6,
respectively. As the formation of 4−6 was observed even under
nitrogen atmosphere, the second thiol/selenol group may act as
a cofactor not only for the deiodination reaction but also for the
release of iodide (I−) from the intermediates. Therefore, the
rate of deiodination depends on the nature of the groups
present in the 1,8-positions. It has been proposed that the
release of iodide from iodothyronines occurs during the first
half of the ID-1 cycle19 and that one of the Cys residues in the
active site of ID-1 may interact with the selenium center to
generate an intramolecular selenenyl sulfide (-Se−S-) bond.20
These observations suggest that the thiol cofactor (DTT) may
interact with the enzyme to regenerate the active site only after
the release of iodide.
10.8085(11) Å, b = 11.5763(11) Å, c = 10.5415(10) Å, α = 90°, β =
93.079(4)°, γ = 90°, V = 1317.1(2) Å3, Z = 4, Mo Kα radiation (λ =
0.71073 Å), T = 293(2) K, R1 = 0.0406, wR2 = 0.1124 (I > 2σ(I)), R1
= 0.0818, wR2 = 0.1395 (all data).
Crystal Data for 35. C13H13NSSe; Mr = 294.3, triclinic P1, a =
7.9434(7) Å, b = 8.0334(8) Å, c = 19.9028(2) Å, α = 80.665(6)°, β =
80.049(6)°; γ = 82.737(7)°, V = 1228.0(2) Å3, Z = 4, Mo Kα radiation
(λ = 0.71073 Å), T = 293(2) K, R1 = 0.0655, wR2 = 0.1275 (I >
2σ(I)); R1 = 0.2531, wR2 = 0.1917 (all data).
Crystal Data for 37. C H NSe ; M = 341.17, triclinic P1, a =
̅
13 13
2
r
9.0033(15) Å, b = 10.0187(14) Å, c = 14.8110(19) Å, α = 74.780(8)°,
β = 82.647(9)°, γ = 89.847(9)°, V = 1277.8(3)Å3, Z = 4, Mo Kα
radiation (λ = 0.71073 Å), T = 293(2) K, R1 = 0.0558, wR2 = 0.1564
(I > 2σ(I)); R1 = 0.0558, wR2 = 0.1564 (all data).
Computational Methods. All calculations were performed using
the Gaussian03 and Gaussian09 suites of quantum chemical
programs.15 The hybrid Becke 3−Lee−Yang−Parr (B3LYP) exchange
correlation functional was employed to predict the minimum energy
molecular geometries of the compounds.16 Geometries were fully
optimized in the gas phase at the B3LYP level of theory by using the 6-
31+G* basis set for all atoms except iodine, for which it does not exist.
Therefore, the 6-311G** basis set was used for iodine. Frequency
calculations were performed on each optimized structure using the
same basis set to ensure that it was a minimum on the potential energy
surface. NBO calculations17 were performed with the 6-311G** basis
set for iodine and 6-311+G** for all other atoms and the same level of
theory. Electrostatic potential maps of thyroid hormones were mapped
on the surface of molecular electron density 0.01 au by using Gauss
View 5.0 software.
It has been proposed previously that the 5′-deiodination of
thyroxine by ID-1 or synthetic selenol (RSeH) may involve
nucleophilic attack of the selenol (or selenolate) on the iodine
center of the keto form (Scheme 2). In this case, the
Scheme 2. Proposed Mechanism for the 5′-Deiodination by
Enol−Keto Tautomerism6
RESULTS AND DISCUSSION
■
The deiodination of T4 and T3 by compounds 1−3 in the
presence of dithiothreitol (DTT) was studied by using a HPLC
method.7,8 Compounds 1−3 were obtained by reducing the
corresponding dichalcogenides 4−6 with NaBH4 in phosphate
buffer. The amounts of rT3 and 3,3′-T2 formed in these
reactions were calculated from the respective peak areas.
Interestingly, compounds 1−3 remove iodine exclusively from
the 5-position of T4 to produce rT3 as the only deiodinated
product. However, these compounds do not mediate the 5′-
deiodination, as no 3,3′-T2 formation was observed when pure
rT3 was treated with 1, 2, or 3. In contrast, treatment of T3
with compounds 1−3 produced 3,3′-T2, indicating that
compounds 1−3 mediate 5-deiodination of both T4 and T3.
A comparison of the deiodinase activities indicated that
compound 3, having two selenol moieties, is almost 75-fold
more active than compound 1. On the other hand, only a 13-
fold enhancement in the activity was observed when one of the
thiols in compound 1 was replaced by a selenol (compound 2).
The importance of the selenocysteine (Sec) residue in the
catalytic center of ID-3 has been demonstrated previously by
deiodination is accompanied by the formation of a selenenyl
iodide (RSeI) species. In agreement with this mechanism,
compound 7, which cannot undergo tautomerization to the
keto form, did not undergo deiodination upon treatment with
RSeH (Scheme 2).6 ID-2, which mediates the 5′-deiodination
exclusively, may also follow a similar mechanism, although the
formation of a selenenyl iodide intermediate has not been
proposed. Kuiper et al.21 proposed a mechanism for the 5′-
deiodination of T4 by ID-2 in which the nucleophilic Sec
residue attacks at the 2′-position of T4 to form an intermediate
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having a carbon−selenium covalent bond. The abstraction of
iodonium ion (I+) by thiol cofactor leads to the formation of an
ID-2−T3 complex and a cofactor sulfenyl iodide. While the
rapid disproportionation of the sulfenyl iodide produces iodide
(I−) and disulfide, a thiol attack at the enzyme intermediate
regenerates the enzyme active site with the elimination of T3.
However, it is not clear whether ID-3 uses any additional
functional groups for the selective removal of iodine from the
inner ring of T4 (5-deiodination). It should be noted that ID-1,
which mediates the conversion of T4 to T3, is also capable of
removing iodine from T3 to produce 3,3′-T2 by 5-deiodination.
As all three enzymes contain Sec in their active sites and share
many common features in their amino acid sequences, they are
expected to follow a similar mechanism for the 5- and 5′-
deiodinations.
It is known that aryl halides in which the halogen substituent
is ortho or para to an electron-withdrawing group are activated
for nucleophilic aromatic substitutions via the addition/
elimination mechanism (SNAr).22 For example, the reactions
of o- and p-nitroaryl iodides with thiolates have been shown to
follow such a mechanism.22,23 Tiecco et al. reported that
alkylselenium groups can be introduced into an aromatic ring
by nucleophilic displacement of unactivated halogen atoms.24
The reaction of compound 2 with T4 in the absence of DTT
produces the selenenyl sulfide 5 and rT3. This reaction can
follow the SNAr mechanism, in which the selenium substituted
compound 9 and iodide can be generated through formation a
Meisenheimer complex (8) (Scheme 3). The free thiol group
follow the SNAr mechanism shown in Scheme 3. Furthermore,
the formation of rT3 was not observed even after the addition
of DTT (Scheme 4). Similarly, no deiodination was observed
Scheme 4. Attempted Deiodination of T4 by Compound 10
in the Presence of Dithiothreitol (DTT)
when compound 11, which has a tert-amino group capable of
deprotonating the selenol moiety, or compound 12, which
contains an amide group in the close proximity of selenium, was
employed. A large upfield shift in the 77Se NMR signal for
compound 11 (54 ppm) relative to that of PhSeH (145 ppm)
suggests that the selenol moiety in 11 is more nucleophilic than
that in PhSeH. These observations indicate that the presence of
an additional thiol or selenol group in proximity to the
selenium atom is more important for the 5-deiodination than
basic amino groups.
The mechanism of deiodination of T4 by compounds 1−3
may involve the formation of a halogen bond25 between a
thiol/selenol group and an iodine atom. Halogen bonds play
key roles in the recognition of thyroid hormones. It has been
shown that T4 forms short I···O interactions with its transport
protein transthyretin and that T4 can bind to RNA sequences
through halogen bonds.26 Recently, Bayse and Rafferty studied
S···I and Se···I interactions in model compounds by using
density functional theory (DFT) calculations to understand the
mechanism of thyroid hormone deiodination.27 On the basis of
detailed theoretical investigations, they proposed that the
halogen bonding between selenium and iodine may play an
important role in the deiodination. The activation barriers for
deiodination of an aromatic iodide by MeSeH and MeSH were
calculated to be 17.6 and 19.8 kcal·mol−1, respectively,
indicating that the substitution of selenol by thiol marginally
increases the activation barrier. It has also been shown that
deprotonation of the selenol moiety in MeSeH significantly
strengthens the halogen bond and shortens the Se···I
distance.27 To understand the nature of iodine atoms present
in thyroid hormones, we have carried out DFT calculations of
T4, T3, rT3, and 3,3′-T2 at the B3LYP/6-311G** (for iodine)
and 6-31+G* (for other atoms) levels.15 The electrostatic
potential maps obtained from these calculations indicate that
the iodine atoms have significant positive potential regions (σ-
hole)28 for possible halogen bonding with sulfur or selenium
(Figure 1). The role of σ-hole in halogen bonding has been
studied extensively for small molecules.29
Scheme 3. Possible Mechanism for the Deiodination of T4
by Compound 2
present in the 8-position can then attack at the electrophilic
selenium center to produce the selenenyl sulfide 5 with the
elimination of rT3. This mechanism, in principle, may account
for the initial rapid release of iodide from T4 in the absence of
DTT. However, we could not detect the formation of any
substituted products. The formation of rT3 and 5 was observed
even at −20 °C, indicating that the reaction does not produce
any stable intermediates.
To understand whether a rapid nucleophilic attack of thiol at
the selenium center is responsible for the facile formation of
rT3, we have studied the reactions of T4 with compounds
10−12, which do not have any additional thiol or selenol
When the selenolate form of compound 10 (Figure 2a)
interacts with iodobenzene (Figure 2b, a model compound), a
halogen bonded adduct (Figure 2c, 15) is formed. The charges
on the selenium and iodine atoms in 15 indicate that the
formation of a halogen bond between the selenolate and iodine
leads to a considerable decrease in the electron density around
the selenium atom and an increase in the electron density
around the iodine atom. A further decrease in the negative
charge on selenium was observed when the strength of the
halogen bond was increased by introducing fluorine atoms in
moieties. The 77Se NMR chemical shift observed for compound
10 (−34 ppm) indicates that the selenol moiety in this
compound is highly nucleophilic. Therefore, the reaction of T4
with 10 is expected to produce the substituted compound 13
via the SNAr mechanism. However, no such compound was
detected by 77Se NMR spectroscopy or mass spectral analysis,
indicating that the deiodination of T4 by compound 3 may not
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Figure 3. Halogen bonding between thiolate/selenolate and iodine.
The δ+ and δ− signs in the structures do not represent the actual
positive or negative charges; instead, they are intended to indicate the
decrease and increase in the electron density, respectively, relative to
that of free thiolate/selenolate and iodine atom before forming
halogen bonding.
Figure 1. Electrostatic potential maps of thyroid hormones, showing
regions of positive potential (σ-hole) on iodine atoms.
Table 1. Halogen Bonding Energy and Bond Order
Calculated for Different Thiolate/Selenolate−Iodobenzene
a
Adducts
bond order
compd
ES/Se···I (kcal·mol−1
)
S/Se···E
E···I
C−I
14
15
16
17
18
19
20
21
21.73
23.43
52.90
68.00
0.208
0.236
0.400
0.454
0.180
0.197
0.419
0.473
0.855
0.835
0.625
0.522
0.877
0.860
0.610
0.511
0.048
0.083
0.101
0.077
0.047
0.079
Figure 2. (a) Charges obtained from natural bond orbital (NBO)
analysis for the selenolate form of compound 10, (b), iodobenzene,
and (c) halogen-bonded adduct 15. (d) A downfield shift in the 77Se
NMR spectra of compound 10 upon addition of iodobenzene (0 (i), 5
(ii), 10 (iii), 20 (iv), 30 (v), 50 (vi), 70 (vii) equiv), indicating the
formation of complex 15.
b
5.04
b
20.71
55.72
74.32
a
The structures were optimized at the DFT level by using B3LYP/6-
the phenyl ring (Figure S46 of the Supporting Information). As
previously observed for a variety of halogen bonded
compounds,30 the Se···I−C angle in 15 is almost 180°,
indicating the donation of a Se lone pair to the σ* orbital of
the C−I bond. The formation of a σ-hole on selenium was
clearly observed for the model selenenyl iodide (Figure S46 of
the Supporting Information).31 In agreement with the
theoretical data, addition of iodobenzene to compound 10
led to a significant downfield shift in the 77Se NMR spectrum
(Figure 2d), indicating the formation of the halogen bonded
adduct (15). An increase in the strength of the halogen bond
was observed when 1-iodo-4-nitrobenzene was used instead of
iodobenzene (Figure S41 of the Supporting Information). A
marginal decrease in the electron density upon interaction with
iodine is sufficient for the selenium atom to accept electrons
from donor groups to form a chalcogen bond.32 Therefore, the
introduction of a thiolate/selenolate at the 8-position in
compound 10 is expected to favor the formation of chalcogen
bonds through peri-interactions.33 As the thiolate/selenolate at
the 1,8-positions can form a covalent bond upon oxidation of
one of these substituents, the interaction between two
chalcogen atoms leads to the formation of a covalent bond
with a release of iodide.
311G** for iodine and 6-31+G* for other atoms. The NBO and
Wiberg bond order analyses were performed with the 6-311G** basis
set for iodine and 6-311+G** for all other atoms and at the same level
of theory. The decrease in S/Se···I interaction energy is due to the
movement of electron density from thiolate/selenolate to the
undissociated thiol.
b
energy. On the other hand, the halogen bonding interaction in
complexes 16 and 17 having a thiolate−selenolate pair and two
selenolates, respectively, is much stronger than that of 14 and
15, which lack the additional selenolate group. Furthermore,
the stronger halogen bonding in complex 17 as compared to
that of 16 is due to the formation of a chalcogen bond between
the two selenium atoms. As a result, the Se−Se bond order in
17 was found to be much higher than the Se−S bond order in
16. Similarly, a significant decrease in the C−I bond order was
observed for 17 as compared to that for 16 (Table 1).
Therefore, simple selenols such as PhSeH and 10, which lack
any additional thiol or selenol groups, do not exhibit deiodinase
activity, as the strength of halogen bond is not sufficient to
cleave the C−I bond. Furthermore, the formation of a stable
selenenyl iodide does not appear to be a driving force for the
deiodination reaction, as compounds 11 and 12, capable of
forming stable selenenyl iodides,34 do not remove iodine from
T4 or T3.
As can be seen from Figure 3 and Table 1, the strength of the
halogen bond depends not only on the chalcogen atom that
interacts with iodine but also on the second chalcogen that is
involved in the peri-interactions. A comparison of the halogen
bonding energies obtained for different S···I and Se···I
complexes (14−17) indicates that the replacement of a thiolate
in complex 14 by a selenolate moiety (complex 15) leads to
only a marginal increase (∼1.7 kcal·mol−1) in the halogen bond
A comparison of the halogen bonding interactions in
complexes 18−21 shows some interesting features. Complexes
18 and 19, having the thiol in its protonated form, exhibit weak
halogen bonding. The halogen bond energies for 18 (5.04
kcal·mol−1) and 19 (20.71 kcal·mol−1) were found to be lower
than those of 14 (21.73 kcal·mol−1) and 15 (23.43 kcal·mol−1),
respectively, indicating that the introduction of a thiol group in
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compounds 14 and 15 decreases the strength of halogen bonds.
This is probably due to the donation of electrons by the
thiolate/selenolate to the sulfur atom of the protonated thiol.
Interestingly, when a thiolate instead of thiol was employed for
the calculations, a dramatic increase in the strength of the
halogen bond was observed. The halogen bond energies for 20
(55.72 kcal·mol−1) and 21 (74.32 kcal·mol−1) were found to be
remarkably higher than those of 18 and 19, respectively. The
energy for compound 21, having a thiolate−selenolate pair, is
significantly higher than that of compound 17, which contains
two selenolate moieties in the 1,8-positions.35 Therefore, the
differences in the deiodinase activity between different
naphthyl-based compounds can be ascribed to various degrees
of ionization of the thiol and selenol moieties. While the selenol
group in compound 3 is predominantly in its anion selenolate
form at physiological pH, the thiol moieties in compounds 1
and 2 are expected to be only partly ionized. Interestingly, the
deiodinase activity of compound 2 was found to be similar to
that of 3 at pH 11.0 (Figure S47 of the Supporting
Information), which is in agreement with the theoretical
observations.
deiodinase activity is observed when both the thiol groups in
compound 1 are replaced by selenols, but only a marginal
increase in the activity is observed when one of the thiol groups
in 1 is replaced by a selenol. It should be noted that the selenol
(selenolate) moiety abstracts iodine from T4 or T3 as
iodonium ion (I+) and eliminates as iodide (I−). Although
the electrons required for such conversion are provided by one
of the selenol (selenolate) moieties, the donation of electrons is
facilitated by the peri-interactions. Although thiolate is a better
donor than selenolate for the formation of a chalcogen bond
and selenolate is a slightly better donor than thiolate for the
formation of a halogen bond (Table 1), the deprotonation of
both the selenols in compound 3 is probably responsible for its
higher activity as compared to that of 2 at physiological pH.35
Furthermore, the selenium center in the halogen-bonded
adducts 17 and 21 is more electrophilic than the sulfur center
in compounds 16 and 20, which may account for the higher
activity of compounds 2 and 3 as compared to that of 1.36
Although the phenolic ring (5′) deiodination of T4 can take
place by a mechanism similar to the one shown in Scheme 5,
the reason for the selectivity of compounds 1−3 toward the
tyrosyl ring (5) deiodination is not clear. As the chemical
reactivity of the phenolic ring iodines is expected to be
somewhat different from that of the tyrosyl ring iodines, the
compounds that can form stronger halogen bonds with iodines
may mediate both 5- and 5′-deiodinations. Similarly, such
compounds may mediate further deiodination of rT3 and 3,3′-
T2 to produce 3′,5′-T2 and 3′-T1 by tyrosyl ring deiodination
under appropriate conditions. It should be noted that both ID-1
and ID-3 are capable of converting rT3 to 3′,5′-T2 by tyrosyl
ring deiodination. However, such reactions may require longer
reaction time and, therefore, are believed to lie off the main
catalytic pathway. In compounds 1−3, the rigidity of the
naphthalene backbone does not allow a closer approach of the
two chalcogen atoms, which may disfavor the formation of
stronger halogen bonds with iodothyronines.37 Our preliminary
studies show that the strength of the halogen bond decreases
upon removal of one of the iodines from the tyrosyl ring.
Therefore, T4 may form stronger halogen bonded adducts with
compounds 1−3 as compared to rT3.
On the basis of the experimental observations and theoretical
data, a mechanism for the deiodination of T4 by compound 3
can be proposed (Scheme 5). According to this mechanism, the
Scheme 5. Proposed Mechanism for the Deiodination of
Thyroxine (T4) by Compound 3 Involving Chalcogen and
Halogen Bonding
In the deiodination of T4 and T3 catalyzed by ID-3, the thiol
group of the conserved Cys residue (Figure 4) may interact
initial interaction of one of the selenol (selenolate) moieties
with an iodine atom leads to the formation of a halogen bond.
The transfer of electron density from selenium to the σ* orbital
of the C−I bond generates a σ-hole or partial positive charge on
the selenium atom, which facilitates an interaction between the
halogen-bonded selenium atom and the free selenol (seleno-
late) moiety (intermediate 22). The selenium−selenium
interaction (chalcogen bond) strengthens the halogen bond,
leading to a heterolytic cleavage of the C−I bond. The
protonation of the resulting carbanion leads to the formation of
rT3. On the other hand, the formation of an Se−Se bond
produces the diselenide 6 with elimination of iodide as HI. The
reductive cleavage of the Se−Se bond in compound 6
regenerates the diselenol 3. However, the reduction of
compound 6 by DTT is not a favored process, as the Se−Se
bond in this compound is conformationally restricted. It should
be noted that the endogenous cofactor used by the deiodinases
for the deiodination of T4 and T3 is not known, and therefore,
DTT has been used extensively for in vitro experiments.1−3
The formation of chalcogen- and halogen-bonded adducts
(Scheme 5) also explains why a dramatic increase in the
Figure 4. Amino-acid sequences at the active sites of ID-3 from
different species indicating the conserved Cys (C) and Sec (U)
residues.
with selenium to activate the C−I bond. Sun et al. have
proposed that the active site Cys in ID-1 may play an important
role in reducing the oxidized selenium species (possibly
selenenyl iodide) to form an intramolecular selenenyl sulfide
bond.20 A further reduction by DTT then regenerates the
selenol. However, it is not clear whether the conserved Cys
residue in ID-3 forms a selenenyl sulfide with Sec upon
deiodination. The mechanism shown in Scheme 5 and the
requirement of high DTT concentrations for an effective
deiodination suggest that the ID-3-mediated deiodination may
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involve the formation a selenenyl sulfide intermediate, which is
reduced by DTT. This is in agreement with the observation
that the release of iodide from T4 can occur even in the
absence of DTT. However, this depends on the redox states of
Sec and Cys residues in the native protein. When these two
residues form an intramolecular selenenyl sulfide linkage upon
auto-oxidation before the deiodination experiments, strong
reducing agents such as DTT are required to activate the
enzyme.
the amino group abstracts the proton from the selenol moiety.
Similarly, the signal for one of the selenol groups in compounds
43−46 (δ = 76−85 ppm) was shifted upfield significantly
relative to that for compound 3 (δ = 155 ppm). These
observations suggest that the secondary amino group in
compounds 40−46 can increase the nucleophilic reactivity by
deprotonating the adjacent thiol or selenol moiety (Figure 5).
In addition to Sec and Cys residues, one or more histidines
(His) at the active sites of deiodinases appear to play an
important role in the deiodination reaction. Kohrle and others
̈
have proposed that one of the His residues at the active site of
ID-1 may activate the Sec residue toward nucleophilic attack by
forming a selenolate−imidazolium zwitterion.38 The important
role of the His residue in the ID-1-mediated deiodination has
been proposed on the basis of experiments with histidine-
directed reagents, pH-dependent kinetic properties, and site-
directed mutagenesis studies.2b,38 In agreement with this, Goto
et al.6 reported that the 5′-deiodination of N-butyrylthyroxine
methyl ester by an organoselenol (Scheme 2) takes place only
in the presence of triethylamine. They have proposed that the
efficiency of the deiodination process largely depends on the
nucleophilicity of the selenol functionality. It should be noted
that the His residues corresponding to positions 158 and 174 in
human ID-1 are conserved in ID-2 and ID-3.2a However, the
formation of a selenolate−imidazolium ion pair has not been
proposed yet for the ID-3 enzyme. However, the inactivity of
compound 11 bearing a reactive selenolate moiety indicates
that additional interactions at the active site of ID-3 may be
required for the deiodination.
Figure 5. Deprotonation of thiol or selenol by an N-alkylamino group
in compounds 40−46.
Theoretical calculations also reveal that the amino group
interacts with the adjacent thiol/selenol group in compounds
40−46 (Table S1, Supporting Information). Interestingly, the
77Se NMR signal for one of the selenol groups in compounds
43−46 (δ = 76−85 ppm) was shifted upfield drastically relative
to that of the other selenol in the same compounds (δ = 214−
217 ppm). Furthermore, these signals were shifted ∼60 ppm
downfield relative to that of compound 3, indicating that the
deprotonation of one of the selenols in each compound by the
amino group may decrease the nucleophilicity of the other
selenol.
A comparison of the deiodinase activity of the amino-
substituted compounds 40 and 43−46 to that of compounds 1
and 3 indicates that the introduction of an amino group
significantly enhances the activity (Figure 6). The activity of
To understand the role of basic amino groups in the
deiodination reactions, we have synthesized compounds 33−39
starting from the dichalcogenides 4−6. The formylation of
compounds 4−6 by POCl3/DMF (Vilsmeier−Haack reac-
tion)9,10 afforded the corresponding aldehydes (23−25), which
upon reaction with primary amines produced the Schiff bases
26−32. The 77Se NMR spectra of 27−32 (678−770 ppm)
show strong noncovalent Se··· N interactions. Single crystal X-
ray studies of some of these dichalcogenides (30, 31, 35, 37)
also indicate the formation of chalcogen bonds between the
imine nitrogen and selenium atom. The desired secondary
amine-based dichalcogenides 33−39 were obtained by reducing
the corresponding Schiff bases with NaBH4 (Scheme 6).
Figure 6. Comparison of the rate of deiodination of T4 by compounds
1−3 and 40−46. The reactions were carried out in phosphate buffer
(pH 7.5) at 37 °C. The final assay mixture contained 600 μM of 1−3
and 40−46, 300 μM of T4, and 10 mM of DTT.
Scheme 6. Synthesis of the sec-Amine-Based Dichalcogenides
33-39 by Using the Vilsmeier−Haack Formylation Method
compound 40 was found to be almost 7-fold higher than that of
compound 1. Similarly, compounds 43−46 were found to be
∼1.5−2-times more active than 3 under identical conditions.
Unexpectedly, compounds 41 and 42, having a thiol−selenol
pair, were found to be ∼2-times less active than compound 2,
indicating that the introduction of an N-methyl- or N-ethyl-
amino group in close proximity of the selenol decreases the
activity of the parent compound. This is probably due to the
introduction of steric hindrance around the selenol, which
decreases the possibility of an interaction between the selenol
and T4. Therefore, in compounds 41 and 42, the thiol group is
expected to interact with iodine to form a halogen bond. In
contrast, the selenol moiety in compound 2 interacts with
The thiols/selenols 40−46 required for the deiodinase assays
were obtained by treating the corresponding dichalcogenides
(33−39) with NaBH4. The 77Se NMR spectra of compounds
41 and 42 showed that the signals for these compounds are
significantly shifted upfield (δ = 109 and 110 ppm, respectively)
relative to that for compound 2 (δ = 162 ppm), indicating that
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iodine. When two thiols (compound 40) or two selenols
(compounds 43−46) are present, the increase in the reactivity
of one of the thiol or selenol groups may enhance the
interactions between these compounds and T4. Although the
activated selenol moiety is unlikely to participate in the halogen
bonding directly due to steric hindrance, such activation should
strengthen the chalcogen bonding, which in turn facilitates the
cleavage of the C−I bond.
When the amino-substituted thiols/selenols 40−46 were
employed for the deiodination instead of 3 (Scheme 5), the
corresponding dichalcogenides 33−39 were obtained. The X-
ray crystallographic and theoretical studies reveal that the
secondary amino group in compounds 34−39 interacts
noncovalently with the selenium to form chalcogen bonds
(Figure 7 and Table 2).39 The Se···N distances (2.591−2.623
for the selenium atoms that do not interact with nitrogen show
significant upfield shift relative to that of 10. These
observations suggest that the nitrogen atom may start
interacting with selenium along the course of a diselenide
bond formation, which may facilitate the release of iodide from
selenium during the deiodination (Scheme 5).
It has been shown that the antithyroid drug 6-n-propyl-2-
thiouracil (PTU) inhibits ID-1 by reacting with the selenenyl
iodide intermediate to form a stable selenenyl sulfide (Scheme
7).1−3,40 Interestingly, PTU and related thiourea-based
Scheme 7. Inhibition of ID-1 by the Thiourea-Based
Antithyroid Drug, n-Propyl-2-thiouracil (PTU)
compounds do not inhibit the activity of ID-2 or ID-3. In
experiments involving ID-3, no effect on deiodinase activity was
observed up to 1 mM concentration of PTU at different DTT
levels.1−3,41 The mechanism shown in Scheme 5 suggests that
the iodine is eliminated as iodide even before the formation of a
well-defined selenenyl iodide intermediate, due to the
formation of a chalcogen bond between the S/Se atoms at
the peri-positions. As the formation of a stable Se−I bond is
required for the formation of a selenenyl sulfide,42 PTU does
not inhibit the activity of ID-3. In agreement with this, no
inhibition of activity was observed when the deiodination of T4
was carried out with compound 3 in the presence of PTU up to
0.45 mM concentration (Figure S44 of the Supporting
Information). In contrast to thiols such as DTT or glutathione
that can form intermolecular selenenyl sulfides upon reaction
with diselenides, PTU is inactive toward diselenides.
Figure 7. Single crystal X-ray structures of compounds 30, 31, 35, and
37 showing strong Se···N interaction. The thermal ellipsoids were
drawn at 50% probability.
Table 2. Se···N Distances, 77Se NMR Chemical Shifts, and
Chalcogen Bond Energies Obtained for Compounds 34-39
a
b
a
77Se NMR (Se1, Se2)
ESe···N (kcal·mol−1
)
While thiourea drugs such as PTU react with the selenenyl
iodide intermediate of ID-1 to form a selenenyl sulfide as a
dead-end product (Scheme 7), the other known inhibitors of
ID-1, such as gold thioglucose (GTG) and iodoacetic acid
(IAA), may react with the selenol group of the enzyme. In
contrast, ID-2 and ID-3 are not inhibited by GTG and IAA.1,2
Although IAA irreversibly inhibits the activity of several Cys
peptidases,43 the Sec-containing glutathione peroxidase
(GPx),44 and thioredoxin reductase (TrxR)45 by forming the
corresponding alkylated Cys or Sec derivatives, the reason for
the insensitivity of ID-3 toward IAA is not clear. When
compound 3 was treated with IAA, the reaction did not
produce the expected Se-carboxymethylated derivative (47) but
instead produced diselenide 6 with the elimination of acetic
acid. The formation of acetic acid in this reaction indicates that
IAA is rapidly deiodinated by compound 3. Similarly,
iodoacetamide, bromoacetic acid, and bromoacetamide were
dehalogenated by 3, although the debromination was found to
be much slower than the deiodination. The mechanism of
dehalogenation may involve a cooperative chalcogen and
halogen bonding as shown in Scheme 5. As bromine is a
weaker halogen bond donor than iodine, the debromination is
slower than the deiodination. In contrast to the reactivity of
compound 3, compound 10, which lacks the second selenol
compd
Se···N (Å)
34
35
36
37
38
39
2.587
2.560
2.623
2.594
2.583
2.591
577
16.51
18.34
14.94
16.37
16.94
16.84
562
341, 480
344, 467
345, 470
350, 445
a
The structures were optimized at the DFT level by using the B3LYP/
6-31+G* basis set, and NBO analyses were performed at B3LYP/6-
311+G** level of theory. The NMR chemical shifts were obtained
experimentally in CDCl3 and are cited with respect to Me2Se.
b
Å) in these compounds are significantly shorter than the sum of
the van der Waal’s radii of nitrogen and selenium (3.45 Å). The
interaction energies (ESe···N) obtained by NBO analysis also
indicate that the nitrogen strongly interacts with the selenium
atom upon formation of an intramolecular diselenide or
selenenyl sulfide bond. Furthermore, the 77Se NMR chemical
shifts obtained experimentally for compounds 36−39 indicate
that the signal for the selenium atom that interacts with
nitrogen is shifted downfield (δ = 445−480 ppm) relative to
that of the other selenium atom (δ = 341−350 ppm) in the
same compound and that of the diselenide obtained from
compound 10 (δ = 426 ppm). On the other hand, the signals
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moiety, underwent facile carboxymethylation by IAA to
produce compound 48 as the major product. These
observations suggest that IAA may undergo rapid deiodination
in the presence of ID-3, which may account for the insensitivity
of this enzyme toward IAA.
ACKNOWLEDGMENTS
■
This study was supported by the Department of Science and
Technology (DST), New Delhi. G.M. acknowledges the DST
for the award of a Swarnajayanti Fellowship, and D.M. thanks
the Indian Institute of Science for a fellowship.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, and 77Se NMR spectra for all the new sulfur/selenium
compounds, HPLC traces obtained for the deiodination of T4
by 2 and 3, effect of PTU on the deiodination of T4 by 3,
archive entries for the optimized geometries, NBO charges, and
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AUTHOR INFORMATION
■
(20) Sun, B. C.; Harney, J. W.; Berry, M. J.; Larsen, P. R.
Endocrinology 1997, 138, 5452−5458.
Corresponding Author
mugesh@ipc.iisc.ernet.in
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