Halogen bonding controls the regioselectivity of the deiodination of thyroid hormones and their sulfate analogues

Article abstract of DOI:10.1002/chem.201405442

The type 1 iodothyronine deiodinase (1D-1) in liver and kidney converts the L-thyroxine (T4), a prohormone, by outer-ring (5′) deiodination to biologically active 3,3′,5-triiodothyronine (T3) or by inner-ring (5) deiodination to inactive 3,3′,5′'-triiodothronine (rT3). Sulfate conjugation is an important step in the irreversible inactivation of thyroid hormones. While sulfate conjugation of the phenolic hydroxyl group stimulates the 5-deiodination of T4 and T3, it blocks the 5′-deiodination of T4. We show that thyroxine sulfate (T4S) undergoes faster deiodination as compared to the parent thyroid hormone T4 by synthetic selenium compounds. It is also shown that ID-3 mimics, which are remarkably selective to the inner-ring deiodination of T4 and T3, changes the selectivity completely when T4S is used as a substrate. From the theoretical investigations, it is observed that the strength of halogen bonding increases upon sulfate conjugation, which leads to a change in the regioselectivity of ID-3 mimics towards the deiodination of T4S. It has been shown that these mimics perform both the 5′- and 5-ring deiodinations by an identical mechanism.

Full text of DOI:10.1002/chem.201405442

DOI: 10.1002/chem.201405442
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Enzyme Mimics |Hot Paper|
Halogen Bonding Controls the Regioselectivity of the
Deiodination of Thyroid Hormones and their Sulfate Analogues
Debasish Manna, Santanu Mondal, and Govindasamy Mugesh*[a]
Abstract: The type 1 iodothyronine deiodinase (1D-1) in
liver and kidney converts the l-thyroxine (T4), a prohormone,
by outer-ring (5’) deiodination to biologically active 3,3’,5-
triiodothyronine (T3) or by inner-ring (5) deiodination to in-
active 3,3’,5’-triiodothronine (rT3). Sulfate conjugation is an
important step in the irreversible inactivation of thyroid hor-
mones. While sulfate conjugation of the phenolic hydroxyl
group stimulates the 5-deiodination of T4 and T3, it blocks
the 5’-deiodination of T4. We show that thyroxine sulfate
(T4S) undergoes faster deiodination as compared to the
parent thyroid hormone T4 by synthetic selenium com-
pounds. It is also shown that ID-3 mimics, which are remark-
ably selective to the inner-ring deiodination of T4 and T3,
changes the selectivity completely when T4S is used as
a substrate. From the theoretical investigations, it is ob-
served that the strength of halogen bonding increases upon
sulfate conjugation, which leads to a change in the regiose-
lectivity of ID-3 mimics towards the deiodination of T4S. It
has been shown that these mimics perform both the 5’- and
5-ring deiodinations by an identical mechanism.
Introduction
Iodothyronine deiodinases (IDs) are mammalian selenoen-
zymes that play an important role in the thyroid hormone ho-
meostasis.[1] Type 1 deiodinase (ID-1) can catalyse both the
phenolic ring (5’) and tyrosyl ring (5) deiodination of prohor-
mone 3,3’,5,5’-tetraiodothyronine (T4) to produce biologically
active 3,3’,5-triiodothyronine (T3) and biologically inactive
3,3’,5’-triiodothyronine (rT3), respectively. In contrast, type 2
deiodinase (ID-2) and type 3 deiodinase (ID-3) mediates specif-
ic 5’- and 5-deiodination of T4 to produce T3 and rT3, respec-
tively. Subsequent deiodination of T3 and rT3 by all three
types of deiodinases produces biologically inactive metabolite
3,3’-T2 as shown in Scheme 1.[2] Apart from the enzymatic dei-
odination, sulfation of the 4’-hydroxyl group has been shown
to be a major metabolic pathway of thyroid hormones. In gen-
eral, sulfate conjugation is a natural detoxification mechanism
to increase the water solubility of a variety of endogenous and
exogenous lipophilic compounds and to facilitate their excre-
tion in bile and/or urine.[3] However, sulfated thyroid hormones
show very low affinity towards the thyroid hormone receptors,
which indicates that the sulfation of thyroid hormones is an in-
activation pathway. Cytosolic sulfotransferases (SULTs), which
use 3’-phosphoadenosine-5’-phosphosulfate (PAPS) as a sulfate
donor, catalyze the sulfate conjugation. Sulfotransferases, locat-
Scheme 1. Regioselective deiodinations of thyroid hormones and their sul-
fate analogues by iodothyronine deiodinases.
ed in different tissues, such as liver, kidney, and brain, exist as
homodimers with a molecular weight of 33 kDa.[4] Depending
on the amino acid sequence and substrate specificity, SULTs
are classified into four different subfamilies, such as SULT1,
SULT2, SULT3 and SULT4. Among them only the SULT1 catalyz-
es the sulfate conjugation of iodothyronines.[3a,5] Isoforms of
SULT1 show different affinity and substrate specificity towards
thyroid hormones.
[a] Dr. D. Manna, S. Mondal, Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science
Bangalore 560012 (India)
E-mail: mugesh@ipc.iisc.ernet.in
Homepage: http://ipc.iisc.ernet.in/~mugesh/
Similarly to the enzymatic deiodination of thyroid hormones,
iodothyronine sulfates (T4S, T3S, rT3S, 3,3’-T2S) also undergo
deiodination by different deiodinases (Scheme 1).[6] Surprising-
ly, while the 5-deiodination of T4S by rat ID-1 is enhanced by
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405442.
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~200 fold relative to that of T4, ID-1 does not catalyse the 5’-
deiodination of T4S.[7] Similarly, the sulfate conjugation has
been shown to enhance the 5-deiodination of T3S (~40-fold)
and 3’-deiodination of 3,3’-T2S by rat and human ID-1.[7] In
contrast, ID-2 and ID-3 do not accept T4S and/or T3S as sub-
strates. Although the sulfate-conjugates of thyroid hormones
undergo faster deiodination by IDs, the mechanism by which
sulfate conjugation enhances the deiodination remains
unknown.
In earlier reports, our group has shown that 1 and 2 bearing
a thiol/selenol pair or two selenol moieties, respectively, at the
1,8-position of the naphthyl ring mediates the 5-deiodination
of thyroid hormones and its en-
dogenous metabolite iodothyrona-
mines.[8] The chalcogen bonding
between sulfur and selenium (1) or
between two selenium atoms (2)
at the peri positions of the naph-
thyl ring facilitates the halogen-
bond-assisted cleavage of the CÀI bond. In the present study,
we show that iodothyronine sulfates can be deiodinated readi-
ly by synthetic deiodinase mimics. We also show for the first
time that the strength of halogen bonding controls the regio-
selectivity and the rate of the deiodination of both thyroid hor-
mones and their sulfate analogues.
Figure 1. HPLC chromatogram obtained for the deiodination of T4S by 2.
The chemical structures of T4S and the deiodinated products are given on
the respective peaks. Assay conditions: T4S (0.3 mm), 2 (0.6 mm), DTT
(10 mm), NaBH4 (15 mm), phosphate buffer (100 mm, pH 7.5), 378C.
for the 5-deiodination of T4 (Figure 2). Similarly, 2 mediates
a 7.6 times faster deiodination of T4S compared to T4. It
should be noted that DTT does not mediate 5- or 5’-deiodina-
tion of T4S (Figure S1, Supporting Information).
Results and Discussion
The sulfated thyroid hormones (T4S, T3S, rT3S, 3,3’-T2S) were
synthesized from the corresponding thyroid hormones by
using chlorosulfonic acid. The products of the deiodination re-
actions of thyroid hormones and their sulfate-conjugates by
deiodinase mimics were monitored and analysed by HPLC. The
deiodinated products were quantified by comparing the peak
area with that of standard samples. As reported earlier in our
laboratory,[8a,b] 1 and 2 mediate the deiodination of T4 exclu-
sively in the 5- ring to produce rT3. Interestingly, when T4S
was treated with 1 or 2 in the presence of dithiothreitol (DTT)
under identical experimental conditions, a decrease in the
HPLC peak area for T4S was observed with three new peaks
appearing at 6.4, 5.6 and 4.5 min (Figure 1). A comparison of
the HPLC chromatograms obtained with that of the authentic
samples of iodothyronine sulfates and mass spectral analyses
indicate that 1 and 2 mediate both 5- and 5’-deiodinations of
T4S to produce rT3S, T3S and 3,3’-T2S (Figure 1). Therefore,
sulfate conjugation of the phenolic hydroxyl group alters the
regioselectivity of deiodination by deiodinase mimics. Al-
though ID-1 catalyzes the 5-deiodination of T4S to produce
rT3S, this enzyme does not catalyze the 5’-deiodination of T4S.
These observations indicate that the synthetic compounds can
mediate the 5’-deiodination of T4S, which is in contrast to the
enzymatic reactions in which the 5’-deiodination of T4 by ID-
1 is blocked upon sulfation.
Figure 2. A comparison of the rate of deiodination of T4 and T4S by 1, 2
and 3–6. The reactions were carried out in phosphate buffer (pH 7.5) at
378C. The final assay mixture contained 600 mm of 1, 2 and 3–6, 300 mm of
T4 or T4S and 10 mm of DTT.
T4S undergoes much faster 5’-deiodination than 5-deiodina-
tion by both compounds 1 and 2. Interestingly, after one
iodine atom has been removed from the tyrosyl or phenolic
ring of T4S, the second iodine is removed from the other ring.
Therefore, rT3S and T3S undergo only 5’- and 5-deiodination,
respectively, by both 1 and 2 to produce 3,3’-T2S. However,
the 5’-deiodination of rT3S is 4–5 times faster than the 5-deio-
Interestingly, the sulfate conjugation not only changes the
regioselectivity of deiodination, but also enhances the overall
deiodination of T4. The initial rate for the overall deiodination
of T4S by 1 was found to be almost 5.4 times higher than that
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Table 1. NBO charge of iodine in different iodothyronines and their sul-
fate conjugates.[a]
Compound
5-Iodine
5’-Iodine Compound
5-Iodine
5’-Iodine
T4
T3
rT3
3,3’-T2
0.207
0.206
0.192
0.189
0.174
0.192
0.178
0.198
T4S
T3S
rT3S
3,3’-T2S
0.216
0.213
0.200
0.200
0.239
0.219
0.235
0.184
[a] The structures were optimized at DFT level by using B3LYP/6-311G**
for iodine and 6-31+G* for other atoms. The NBO analyses were per-
formed with 6-311G** basis set for iodine and 6-311+G** for all other
atoms and at the same level of theory.
tions, the natural bond orbital (NBO) analysis indicates that the
5-iodine in T4 is more positively charged (0.207) relative to the
5’-iodine (0.174) (Table 1). In contrast, the 5’-iodine atom of
T4S is more positively charged (0.239) than the 5-iodine
(0.216). Therefore, 5’-iodine is expected to form much stronger
halogen bond with selenium than the iodine at the 5-position.
The 5’-iodine of rT3S has more positive charge than the 5-
iodine, whereas the 5-iodine of rT3 has more positive charge
than the 5’-iodine. This may explain the reason for the facile
5’-deiodination of rT3S by 1 and 2, and the inability of rT3 to
undergo deiodination under similar experimental conditions.
A model selenol (MeSeH) was employed to understand the
ability of the 5- and 5’-iodine atoms to form a halogen bond.
As a selenol generally exists in its deprotonated selenolate
form at a physiological pH of 7.4, it is more appropriate to use
methyl selenolate (MeSeÀ) in the theoretical calculations. How-
ever, owing to the difficulty in estimating the interaction in-
volving the selenolate anion correctly, we used the selenium
compound in its protonated form for the calculations. The hal-
ogen-bonded geometries of the thyroid hormones and their
sulfated analogues were optimized by using the same level of
theory and basis sets as discussed earlier. As expected, MeSeH
forms halogen bonding with both the 5- and 5’-iodine atoms
of T4 and T4S. In all the geometries, the non-bonded distance
between the selenium and iodine atom is shorter than the
sum of their van der Waal radii (3.88 ꢁ). However, the 5-iodine
atom of T4 forms a Se···I halogen bond that is a little shorter
(3.741 ꢁ) than the 5’-iodine (3.780 ꢁ) (Figure 5). In contrast, the
Se···I halogen bond formed by the 5- iodine atom of T4S
(3.697 ꢁ) is almost comparable to that formed by the 5’-iodine
(3.727 ꢁ). In all the halogen-bonded geometries formed by the
5- and 5’-iodine atoms of T4 and T4S, the CÀI···Se bond angle
is ~1808 whereas the I···SeÀC bond angle is ~908. These theo-
retically predicted geometries are quite similar to the observed
Se···I halogen-bonded geometries obtained from single-crystal
X-ray diffraction. It has been shown that tetramethylammoni-
um selenocyanate (Me4NSeCN) form quite strong Se···I halogen
bonding with 1,2-diiodotetrafluorobenzene and 1,4-diiodote-
trafluorobenzene.[10a] These halogen bonds have Se···I distances
ranging from 3.394–3.552 ꢁ, which indicates that these interac-
tions are stronger than those observed in the present study.
While the CÀI···Se bond angles vary from 161.7 to 178.38, the
I···SeÀC bond angles range from 80.9 to 111.78. The halogen
bonded adduct between 1,4-diiodotetrafluorobenzene and tri-
Figure 3. A comparison of the rate of deiodination of T3S and rT3S by com-
pounds 1, 2 and 3–6. The reactions were carried out in phosphate buffer
(pH 7.5) at 378C. The final assay mixture contained 600 mm of 1, 2 and 3–6,
300 mm of T3S or rT3S and 10 mm of DTT.
dination of T3S by 1 and 2 (Figure 3). These observations sug-
gest that the removal of iodine from the 5’-position contrib-
utes significantly to the overall deiodinase activity observed
with T4S. It should be noted that rT3 does not undergo 5’-dei-
odination by 1 and 2 even at higher concentrations. However,
as observed earlier for 3,3’-T2, the corresponding sulfate ana-
logue, 3,3’-T2S, does not undergo further deiodination by
1 and 2.
In agreement with the enzymatic deiodination, the deiodina-
tion of sulfated thyroid hormones by synthetic compounds is
faster than that of the parent thyroid hormones. However, the
mechanism by which sulfation enhances the deiodination of
the thyroid hormones remains unknown. Also, the synthetic
mimics of deiodinases lose their regioselectivity of deiodina-
tion upon sulfation of the thyroid hormones. To address these
questions, we carried out detailed DFT calculations. Geometries
were fully optimized in gas phase at the B3LYP level of theory
by using the 6-311G** and 6-31+G* basis sets for iodine and
other atoms, respectively. The electrostatic potential map indi-
cates that the 5-iodine in T4 have more positive potential (s-
hole)[9] than the 5’-iodine (Figure 4 and Table 1). Therefore, the
iodine atoms at the 5-position of T4 are expected to form
stronger halogen bond with the selenium than the iodine at
the 5’-position. On the other hand, sulfate conjugation of T4
significantly increases the positive potential on the iodines at
the 5’-position (Figure 2). In agreement with these observa-
Figure 4. Electrostatic potential map of T4 and T4S.
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gen bonds (5.25 kcal.molÀ1) than
the 5’-iodine of T4S. This may
explain the selective phenolic
ring deiodination of rT3S by
1 and 2. However, these com-
pounds do not deiodinate rT3
even at the higher concentra-
tions. This may be explained
from the weak halogen-bond in-
teraction energy (4.12 kcalmolÀ1
)
between 5’-iodine of rT3 and
methyl selenol. The higher inter-
action energy for the 5-iodine
(4.95 kcal.molÀ1) of T3S com-
pared to the 5’-iodine (4.43 kcal.
molÀ1) may explain the selective
5-ring deiodination of T3S by
1 and 2. However, it is not clear
why 3,3’-T2S, with a reasonably
strong Se···I interaction, does not
undergo further deiodination.
Recently, we reported that the
introduction of a basic amino
group in the close proximity of
Figure 5. Comparison of the Se····I distances (ꢁ) for the 5-ring and phenolic ring iodine atoms in the halogen-
bonded adducts of T4S (a,b) and T4 (c,d), respectively, with methyl selenol.
phenylphosphine selenide reveals Se···I distances of 3.394–
Table 2. The Se···I interaction energies for various iodothyronines and
3.639 ꢁ, CÀI···Se angles of 154.5–171.28 and I···SeÀP angles of
89.6–1128.[10b] A similar adduct between 1,2-diiodotetrafluoro-
benzene and triphenylphosphine selenide also exhibits Se···I
distances of 3.435 ꢁ, CÀI···Se angle of 170.98 and I···SeÀP angle
of 100.18. The crystal packing of iododiisopropylphosphine sel-
enide shows an Se···I halogen bonding with an Se···I distance
of 3.642 ꢁ.[10c] Similar types of compound iododi-tert-butyl-
phosphine selenide shows two types of Se···I interactions with
Se···I distances 3.69 and 3.844 ꢁ.[10d]
their sulfates with methyl selenolate.[a]
Compound
ESe··M.>I [kcalmolÀ1
]
Compound ESe···I [kcalmolÀ1
5-iodine 5’-iodine
]
5-iodine
5’-iodine
T4
T3
rT3
3,3’-T2
4.18
3.96
3.17
3.20
3.83
3.03
4.12
3.61
T4S
T3S
rT3S
3,3’-T2S
4.98
4.95
4.36
4.23
5.19
4.43
5.25
4.44
[a] The structures were optimized at DFT level by using B3LYP/6-311G**
for iodine and 6-31+G* for other atoms. The NBO analyses were per-
formed with 6-311G** basis set for iodine and 6-311+G** for all other
atoms and at the same level of theory.
To calculate the Se···I halogen-bonding interaction energies,
NBO analysis was performed on the optimized halogen-
bonded geometries using the same level of theory and basis
sets as described earlier. The strength of halogen bonding
may, at least partly, account for the differences in the reaction
rates for the deiodination of thyroid hormones and their sul-
fate analogues. The Se···I interaction energy of the 5-iodine in
T4 with methyl selenol (4.18 kcalmolÀ1) is higher than that of
the 5’-iodine (3.83 kcalmolÀ1; Table 2). Therefore, stronger halo-
gen-bond energy may facilitate the selective removal of iodine
from the 5-position. On the other hand, the 5’-iodine of the
T4S forms a stronger halogen bond (5.19 kcal.molÀ1) with
methyl selenol compared to the 5-iodine (4.98 kcalmolÀ1). As
both the 5- and 5’-iodine of T4S forms much stronger halogen
bond with methyl selenol relative to T4, T4S may undergo
both 5- and 5’-deiodination by 1 and 2. The high interaction
energies may also account for the faster deiodination of T4S
compared to T4. After the removal of first iodine from the 5-
position of T4S, the second iodine in the same ring of rT3S in-
teracts much more weakly (4.36 kcalmolÀ1) with methyl sele-
nol. However, the 5’-iodine of rT3S forms even stronger halo-
one of the selenol moieties in 2 increases its reactivity (see
compound 3).[8b,c] As in the case of T4 deiodination, 3 signifi-
cantly enhances the deiodination of T4S and rT3S (Figure 2
and 3). Introduction of an additional amino group (4) or hy-
droxyl group (5) further enhances the deiodination. The deiodi-
nase activity of 6, which has a hydroxyl group instead of an
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amino group, was found to be much higher than that of 3;
this indicates that substituents other than the amino group
can also enhance the rate of deiodination. The thiols/selenols
4–6 required for the deiodinase assays were obtained by treat-
ing the corresponding dichalcogenides 10–12. Compounds 10
and 11 were prepared by following the literature procedure
(Scheme 2) and 12 was prepared by reducing 7 with NaBH4.
due in peptides occurs at pH values as low as 3 and that Sec
residues in different environments can exist completely in their
selenolate forms at pH>6.0.[12] However, the pKa of the selenol
in 2 could not be determined after several attempts mainly
due to the difficulty in generating the diselenol from disele-
nide at acidic pH. When selenol is deprotonated to produce
a selenolate, an upfield shift in 77Se NMR spectra is expected.
However, no such upfield shift in the 77Se NMR spec-
tra of 2 was observed between pH 7 and 11 (Fig-
ure S24, Supporting Information). These results indi-
cate that the deprotonation of the selenol in 2
occurs below pH 7, which is consistent with the re-
sults reported by Mobli et al.
A recent crystal structure of the catalytic domain
of mouse ID-3 indicates that Sec170 is responsible for
the deiodination of thyroid hormones. Based on an
Arg-T3-His clamp in T3Rb, a model structure of ID-3
in complex with T4 has been generated. His202,
Arg275, Glu259 residues have been found to play an
important role in the binding of T4 to the ID-3 active
site.[13] This binding mode placed the 5-iodine atom
3–4 ꢁ away from the Sec residue. At this position, the selenol
may form halogen-bonding contact with the s-hole of iodine
atom to elongate the CÀI bond. The role of Se···I halogen
bonding in the activation of CÀI bond has been supported ear-
lier by DFT studies.[14] After the iodine atom is removed as sele-
nenyl iodide, the protonation of the 5-position occurs from the
back side of the tyrosyl ring. The His219-Glu200-Ser167 triad
along with Tyr197 and Thr169 residues have been shown to
play an important role in proton transfer.[13]
Scheme 2. Synthesis of dichalcogenides 10–12.
Kçhrle and others have reported that one of the histidine
(His) residues may play an important role in the catalysis by
forming an imidazolium–selenolate ion pair with the selenocys-
teine (Sec) residue at the active site of ID-1.[11] To understand
the nature of selenol moieties in 4–6, we have studied the
77Se NMR spectral behaviour of these compounds. The
77Se NMR spectra of compounds 4, 5 and 6 showed that the
signals for one of the selenol moieties are significantly shifted
upfield (d=90, 85 and 51 ppm, respectively) relative to the
other selenol (d=217, 216 and 208 ppm, respectively) (see Fig-
ures S7, S10 and S14, Supporting Information) in the same
compound, which indicates that the sec-amino or hydroxyl
groups form a hydrogen bond with the proximal selenol
group as shown in the chemical structures of the compounds.
The formation of a hydrogen bond between one of the sele-
nols and the hydroxyl moieties in 6 is further supported by
theoretical calculations. The SeH···O distance obtained from op-
timized geometry of 6 is 2.240 ꢁ, which is within the range of
moderately strong hydrogen-bonding distance. From the AIM
analysis, a bond critical point was observed between SeH···O
(Figure S23, Supporting Information). Formation of this kind of
H-bond may ultimately lead to deprotonation of the selenol to
produce selenolate. However, hydrogen bonding between a se-
lenol and a nearby basic amine moiety should not interfere
with the formation of Se···I halogen bonding because selenium
forms a halogen bond through its lone pairs. Optimizing the
geometry of iodobenzene with a model selenol, which is H-
bonded to a nearby basic secondary amino group, leads to for-
mation of moderately strong halogen-bonded adduct
(Table S1, Supporting information).
In comparison to the Se···I halogen bond mediated 5-deiodi-
nation, tautomerisation of the enol form of T4 (phenolic ring)
to the corresponding keto form has been proposed to be cru-
cial for the 5’-deiodination of T4 by ID-1 (Scheme 3).[15] The 5-
Scheme 3. Proposed mechanism of phenolic ring deiodination of T4
through enol–keto tautomerism.
ring deiodination of T4 by synthetic compounds involves co-
operative halogen bonding and chalcogen bonding.[8c] A simi-
lar mechanism involving halogen-bond-assisted activation of
the CÀI bond is also possible for the removal of 5’-iodine. To
understand the effect of the enol–keto tautomerism on the
charge distribution of iodine, we carried out DFT calculations
on both the keto tautomers of T4. It is known that the
strength of halogen bond also depends on the hybridization
of the carbon centre to which the halogen atom is attached.
The halogen atom attached to an sp-hybridized carbon atom
The deprotonation of a selenol, to form a selenolate, is ex-
pected to form a stronger halogen bond with iodine. In our
previous reports, we have shown that the initial rate of deiodi-
nation of T4 by 2 increases with pH.[8b] This has been explained
by deprotonation of the selenol at basic pH. Recently, Mobli
and co-workers showed that the deprotonation of a Sec resi-
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forms the strongest halogen bond, followed by the ones that
are attached to an sp2 and sp3 carbon atoms, respectively.[16]
As the enol–keto tautomerism in T4 leads to the formation of
a partially sp3-hybridized carbon atom (Figure 6), the phenolic
ring iodines may become less reactive than the 5- ring iodines.
which the synthetic compounds perform the 5- and 5’-deiodi-
nations are identical and an enol–keto tautomerism is probably
not required for the 5’-deiodination of T4 or T4S. It appears
that halogen bonding is one of the crucial factors that control
the regioselectivity of the deiodination of both thyroid hor-
mones and their sulfate ana-
logues. While the synthetic com-
pounds reported in this paper
function as ID-3 mimics with re-
spect to thyroid hormones, their
regioselectivity is altered when
the sulfate analogues are used
as substrates.
Experimental Section
Synthesis
Synthesis of T4S: l-Thyroxine (T4)
Figure 6. a) Electrostatic potential map of the keto form of T4. b) Effect of enol–keto tautomerism on the charge
of iodine in T4 (grey: charge in keto form; black: charge in enol form).
(50 mg, 0.06 mmol) was dissolved
in TFA (2 mL) and the solution was
cooled to 08C. ClSO3H (100 mL)
was added very slowly to this solu-
tion. A white precipitate appeared immediately. The reaction mix-
ture was stirred at this temperature for 10 min. The resulting white
suspension was poured to a beaker containing ice-cold water
(5 mL). The resulting white suspension was centrifuged for 5 min
at 8000 rpm and the solid obtained was washed thoroughly with
water to remove excess TFA. The product (T4S) was purified by re-
verse-phase HPLC by using MeOH/H2O as the mobile phase. As
this reaction produced a poor yield in higher molar scale, the reac-
tion was repeated several times to obtain the desired amount of
product. Yield: 40%; 1H NMR (D2O): d=2.56–2.62 (dd, J=5.6 Hz,
1H), 2.79–2.84 (dd, J=8 Hz, 1H), 3.31–3.34 (m, 1H), 7.30 (s, 1H),
7.78 ppm (s, 1H); 13C NMR (D2O): d=36.5, 57.7, 90.4, 90.5, 127.4,
140.8, 141.5, 148.2, 151.6, 153.9, 182.2 ppm; ESI-MS: m/z: calcd for
C15H11I4NO7S: 857.65 [M+H]+; found: 857.72.
This is further supported by NBO analysis of both the enol and
keto form of T4. The NBO analysis indicates that the positive
charge of the iodine in the keto form (0.080 and 0.085) is con-
siderably lower than the enol form (0.174 and 0.207). As
a result, the average charge on the outer ring iodine atoms de-
creases from enol (0.191) to keto form (0.157). Therefore, the
attack of selenol, as shown in Scheme 3, will be less favoured
on the iodine in the keto form than in the enol form of T4.
Blocking of the 4’-hydroxyl group by sulfate conjugation does
not inhibit the phenolic ring deiodination by deiodinase en-
zymes. These observations indicate that the keto–enol tauto-
merism is not required to remove the phenolic ring iodine
atoms of the thyroid hormones. However, under suitable con-
ditions, phenolic ring iodine can also form a halogen bond
with selenol and can undergo deiodination similar to the 5-
iodine. Although T4S cannot undergo enol–keto tautomerism,
the synthetic compounds are capable of mediating the phe-
nolic ring deiodination. Also, the identical change in the activi-
ty of compounds 1–6 on moving from T3S to rT3S suggests
that both the 5- and 5’-deiodinations occurs through a similar
mechanism.
Synthesis of T3S: 3,3’,5-Triiodothyronine (T3) (50 mg, 0.076 mmol)
was dissolved in TFA (2 mL) and the resulting solution was cooled
to 08C. ClSO3H (100 mL) was added very slowly to the solution. A
white precipitate appeared immediately. The reaction mixture was
stirred at this temperature for 10 min. The resulting white suspen-
sion was poured into ice-cold water (5 mL). The white suspension
was centrifuged for 5 min at 8000 rpm. The white precipitate was
washed thoroughly with water to remove excess TFA. Then it was
purified by reverse-phase HPLC analysis using MeOH/H2O as the
mobile phase. The reaction was repeated multiple times to get de-
1
sired amount of product. Yield: 25%; H NMR (D2O): d=2.56–2.62
(dd, 1H), 2.78–2.83 (dd, 1H), 3.32–3.35 (m,1H), 6.78–6.82 (dd, J=
6.4 Hz, 1H), 7.17 (s, 1H), 7.25 (d, J=8.6, 1H), 7.68 ppm (s, 2H);
13C NMR (D2O): d=39.7, 57.7, 90.7, 91.5, 117.2, 122.8, 126.1, 140.6,
141.4, 147.1, 151.9, 154.1, 182.1 ppm; ESI-MS: m/z: calcd for
C15H12I3NO7S: 731.75 [M+H]+; found: 731.79.
Conclusion
We showed that iodothyronine sulfates can be rapidly deiodi-
nated by synthetic deiodinase mimics. In contrast to the selec-
tive 5-deiodination of T4, the synthetic compounds mediate
both 5’- and 5-deiodinations of T4S. The overall rate of deiodi-
nation by the synthetic deiodinase mimics is increased upon
sulfate conjugation. However, 5’-deiodination of T4S by ID-1 is
completely blocked upon sulfation. For enzymatic deiodinase
activity, besides the Se···I interaction, the binding affinity of the
substrates to the active site of the enzyme also plays an impor-
tant role. This study also reveals that the mechanisms by
Synthesis of rT3S: 3,3’,5’-Triiodothyronine (rT3) (30 mg,
0.046 mmol) was dissolved in TFA (1 mL) and the resulting solution
was cooled to 08C. ClSO3H (50 mL) was added very slowly to the
solution. The resulting white suspension was poured into ice-cold
water (3 mL). The white suspension was centrifuged for 5 min at
8000 rpm. The white precipitate was washed thoroughly with
water to remove excess TFA. It was then purified by reverse-phase
HPLC by using MeOH/H2O as the mobile phase. The reaction was
Chem. Eur. J. 2015, 21, 2409 – 2416
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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repeated multiple times to get desired amount of product. Yield:
21%; 1H NMR (D2O): d=2.59–2.64 (dd, 1H), 2.78–2.82 (dd, 1H),
3.31–3.34 (m, 1H), 6.65 (d, J=8.4 Hz, 1H), 7.01–7.03 (dd, 1H), 7.31
(s, 2H), 7.64 ppm (1H); 13C NMR (D2O): d=40.1, 57.8, 89.3, 90.4,
121.4, 129.2, 131.7, 138.1, 140.9, 148.6, 153.6, 155.2, 182.5 ppm;
ESI-MS: m/z: calcd for C15H12I3NO7S: 731.75 [M+H]+; found: 731.76.
Synthesis of 12: Naphthalene-1,8-diselenide-2-carboxaldehyde
(300 mg, 0.96 mmol) was dissolved in MeOH/CHCl3 (5:1, 30 mL).
NaBH4 (726 mg, 19.2 mmol) was added to the above solution. The
reaction mixture was stirred at room temperature for 2 h. The re-
sulting brown solution was poured into water (100 mL) and ex-
tracted with dichloromethane (40 mL). The organic layer was
washed with water. The dichloromethane extract was then dried
and evaporated to give pure 12 as a deep-brown solid in 95%
Synthesis of 3,3’-T2S: 3,3’-Diiodothyronine (3,3’-T2) (30 mg,
0.057 mmol) was dissolved in TFA (1 mL) and it was cooled to 08C.
ClSO3H (50 mL) was added very slowly to the solution. The resulting
white suspension was transferred into ice-cold water (3 mL). The
white suspension was centrifuged for 5 min at 8000 rpm. The
white precipitate was washed thoroughly with water to remove
excess TFA. It was then purified by reverse-phase HPLC by using
MeOH/H2O as the mobile phase. The reaction was repeated multi-
1
yield. H NMR (CDCl3): d=4.75 (s, 2H); 7.20–7.27 (m, 2H), 7.40 (d,
J=7.2 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H), 7.51 ppm (d, J=7.2 Hz, 1H);
13C NMR(CDCl3): d=66.3, 122.0, 123.7, 124.9, 127.2, 127.8, 133.9,
137.5, 138.8, 140.6, 140.8 ppm; 77Se NMR (CDCl3): d=406, 411 ppm;
ESI-MS: calcd for C11H8OSe2: 338.88 [M+Na]+; found: 338.96.
1
ple times to get desired amount of product. Yield: 16%; H NMR
Computational Studies
(D2O): d=2.98–3.04 (dd, 1H), 3.15–3.23 (dd, 1H), 3.96–3.99 (dd,
1H), 6.92–6.98 (m, 2H), 7.19 (d, J=8.4 Hz, 1H), 7.31–7.34 (m, 2H),
7.77 ppm (s, 1H); 13C NMR (D2O): d=35.8, 56.3, 89.5, 91.4, 119.4,
121.0, 122.8, 128.5, 131.6, 134.1, 140.9, 147.7, 154.9, 155.2,
174.4 ppm; ESI-MS m/z: calcd for C15H13I2NO7S: 605.85 [M+H]+;
found: 605.80.
All calculations were performed by using the Gaussian 03 and 09
suites of quantum chemical programs.[17] The hybrid B3LYP ex-
change correlation functional was employed to predict the mini-
mum energy molecular geometries of the compounds.[18] Geome-
tries 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 calculations[19]
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.
Synthesis of 10: N,N-Dimethylethylenediamine (1.76 g, 19.2 mmol)
was added dropwise to a solution of naphthalene-1,8-diselenide-2-
carboxaldehyde (7) (300 mg, 0.96 mmol) in dry acetonitrile (20 mL).
The resulting reaction mixture was stirred at 608C for 12 h. The
progress of the reaction was monitored by TLC analysis. The sol-
vent was evaporated under reduced pressure after completion of
the reaction. The resulting solid was dissolved in MeOH/CHCl3 (5:1,
30 mL). NaBH4 (726 mg, 19.2 mmol) was added to the above solu-
tion. The reaction mixture was stirred at room temperature for 3 h.
The resulting red solution was poured into water (100 mL) and ex-
tracted with dichloromethane (40 mL). The dichloromethane ex-
tract was then dried and evaporated to give a red liquid, which
was then purified by column chromatography using petroleum
ether/ethyl acetate as eluent to give 10 as a red semisolid in 72%
Acknowledgements
1
This study was supported by the Science and Engineering Re-
search Board (SERB), Department of Science and Technology
(DST) and Department of Biotechnology (DBT), New Delhi.
D.M. and S.M. thank the Indian Institute of Science for a re-
search fellowship.
yield. H NMR (CDCl3): d=2.24 (s, 6H), 2.52 (t, J=4.8 Hz, 2H), 2.68
(t, J=6.0 Hz, 2H), 3.4 (s, 2H), 7.03 (d, J=8.0 Hz, 1H), 7.18 (t, J=
7.6 Hz, 1H), 7.38 (d, J=7.6 Hz, 1H), 7.45–7.49 ppm (m, 2H);
13C NMR(CDCl3): d=45.9, 47.3, 53.6, 58.4, 123.0, 123.5, 124.8, 126.9,
127.5, 134.3, 136.8, 139.6, 140.6, 141.8 ppm; 77Se NMR (CDCl3): d=
344, 475 ppm; ESI-MS: m/z: calcd for C15H18N2Se2: 386.98 [M+H]+;
found: 386.93.
Keywords: enzyme mimics · halogen bonding · iodothyronine
deiodinase · sulfate conjugatation · thyroid hormones
Synthesis of 11: Ethanolamine (1.17 g, 19.2 mmol) was added
dropwise to a solution of naphthalene-1,8-diselenide-2-carboxalde-
hyde (7) (300 mg, 0.96 mmol) in dry acetonitrile (20 mL). The result-
ing reaction mixture was stirred at 608C for 12 h. The progress of
the reaction was monitored by TLC analysis. The solvent was
evaporated under reduced pressure after completion of the reac-
tion. The resulting solid was dissolved in MeOH/CHCl3 (5:1, 30 mL).
NaBH4 (726 mg, 19.2 mmol) was added to the above solution. The
reaction mixture was stirred at room temperature for 3 h. The re-
sulting red solution was poured into water (100 mL) and extracted
with dichloromethane (40 mL). The dichloromethane extract was
then dried and evaporated to give a red liquid, which was then pu-
rified by column chromatography using petroleum ether/ethyl ace-
tate as the eluent to give 11 as a red sticky oil in 75% yield.
1H NMR (CDCl3): d=2.81 (t, J=4.8 Hz, 2H), 3.87 (t, J=4.8 Hz, 2H),
4.00 (s, 2H), 7.02 (d, J=8.1 Hz, 1H), 7.18 (t, J=7.72 Hz, 1H), 7.40 (d,
J=8.0 Hz, 1H), 7.44 (d, J=7.4 Hz, 1H), 7.49 ppm (d, J=8.4 Hz, 1H);
13C NMR(CDCl3): d=51.3, 53.7, 61.7, 123.1, 123.4, 124.9, 127.2,
127.6, 134.1, 136.9, 139.5, 140.4, 141.4 ppm; 77Se NMR (CDCl3): d=
356, 455 ppm; ESI-MS: m/z: calcd for C13H13NOSe2: 358.93 [M]+;
found: 358.92.
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Received: September 28, 2014
Published online on December 8, 2014
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