The synthesis of 13C9-15N-labeled 3,5-diiodothyronine and thyroxine

Article abstract of DOI:10.1080/00397911.2011.639005

Thyroid hormones undergo extensive metabolism to regulate hormone activity. A labeled thyroid hormone would be useful to track hormone metabolism through various pathways. While radiolabeled thyroid hormones have been synthesized and used for in vivo studies, a stable isotope labeled form of thyroid hormone is required for studying thyroid hormone metabolism by LC-MS/MS, an analytical technique that has certain advantages without the complications of radioactivity. Here we report the synthesis of ¹³C₉- ¹⁵N-T₂ and ¹³C₉-¹⁵N- T₄, two labeled thyroid hormone derivatives suitable for in vivo LC-MS/MS studies. Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications to view the free supplemental file.

Full text of DOI:10.1080/00397911.2011.639005

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The Synthesis of 13C9-15N-Labeled The
3,5-Diiodothyronine and Thyroxine
Sarah A. Hackenmueller a & Thomas S. Scanlan a
a Department of Physiology and Pharmacology, Oregon Health and
Science University, Portland, Oregon, USA
Accepted author version posted online: 05 Jun 2012.Version of
record first published: 05 Mar 2013.
To cite this article: Sarah A. Hackenmueller & Thomas S. Scanlan (2013): The Synthesis of 13C9-15N-
Labeled The 3,5-Diiodothyronine and Thyroxine, Synthetic Communications: An International Journal
for Rapid Communication of Synthetic Organic Chemistry, 43:10, 1439-1446
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Synthetic Communications1, 43: 1439–1446, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2011.639005
THE SYNTHESIS OF 13C9-15N-LABELED THE
3,5-DIIODOTHYRONINE AND THYROXINE
Sarah A. Hackenmueller and Thomas S. Scanlan
Department of Physiology and Pharmacology, Oregon Health and Science
University, Portland, Oregon, USA
GRAPHICAL ABSTRACT
Abstract Thyroid hormones undergo extensive metabolism to regulate hormone activity. A
labeled thyroid hormone would be useful to track hormone metabolism through various
pathways. While radiolabeled thyroid hormones have been synthesized and used for in vivo
studies, a stable isotope labeled form of thyroid hormone is required for studying thyroid
hormone metabolism by LC-MS/MS, an analytical technique that has certain advantages
without the complications of radioactivity. Here we report the synthesis of 13C9-15N-T2 and
13C9-15N-T4, two labeled thyroid hormone derivatives suitable for in vivo LC-MS/MS
studies.
Supplemental materials are available for this article. Go to the publisher’s online edition
of Synthetic Communications1 to view the free supplemental file.
Keywords 13C9-15N-T2; 13C9-15N-T4; thyroid hormone; thyroxine
INTRODUCTION
Thyroid hormones (THs) are important endocrine signaling molecules that
regulate a variety of physiological functions, including body temperature, cardiac
function, metabolism, and mental status. THs are synthesized and secreted by the
thyroid gland predominantly as 3,30,5,50-tetraiodothyronine (T4, thyroxine), which
Received September 15, 2011.
Address correspondence to Thomas S. Scanlan, Department of Physiology and Pharmacology,
Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., L334, Portland, OR
97239-3098 USA. E-mail: scanlant@ohsu.edu
1439

1440
S. A. HACKENMUELLER AND T. S. SCANLAN
Figure 1. Structures of T4, 3,5-T2, and T1AM.
undergoes extrathyroidal deiodination to 3,30,5-triiodothyronine (T3). T3 is largely
considered to be the active form of the hormone and exerts its effects by binding
to TH nuclear receptors and regulating transcription of TH-responsive genes. In
addition to the commonly known transcriptional actions of TH, there are rapid,
nontranscriptionally mediated effects of TH that remain less well understood,
suggesting the existence of additional biologically active TH metabolites.[1,2]
T1AM is an endogenous compound present in serum and various tissues of
rats, hamsters, and humans.[3–6] Acute administration of T1AM in vivo results in
induction of a torpor-like state characterized by hypothermia, bradycardia, a shift
in respiratory quotient from carbohydrate to lipid utilization, hyperglycemia, and
hypoinsulinemia.[3,5,7] In an ex vivo perfused rat heart, T1AM decreases cardiac out-
put.[3,8] These effects tend to be oppose those normally attributed to T3 and suggest
that T1AM may also play a role in TH signaling by modulating the effects of T3.
Structural similarities between T4 and T1AM (Fig. 1) have led to speculation
that T1AM is a deiodinated and decarboxylated derivative of TH, but this relation-
ship has not been directly investigated. To test this metabolic question, it is necessary
to definitively trace the fate of a suspected precursor, which can be accomplished
through incorporation of a label in T4. Several methods for analyzing T1AM by
liquid chromatography coupled with tandem mass spectrometry (LC-MS=MS) have
been reported.[4,9,10] This technique is ideal for analysis of isotopically labeled com-
pounds, because any metabolites arising from a labeled precursor would contain a
mass signature distinct from endogenous compounds. T4, as the predominant form
of TH produced endogenously, is the first candidate to test as a suspected precursor
to T1AM. Based on in vitro data showing that T3 is not a substrate for outer ring
deiodination, it remains unclear if 3,5-diiodothyroinine (3,5-T2), another endogen-
ous TH, arises from extrathyroidal metabolism of T4 or some alternate biosynthetic
pathway.[10] This indicates a second potential candidate for a biosynthetic precursor
to T1AM. Herein we describe the novel syntheses of 13C9-15N-3,5-T2 and 13C9-15N-
L-thyroxine (13C9-15N-T4) that can be used to study TH metabolism by liquid
chromatography–tandem mass spectroscopy (LC-MS=MS).
RESULTS AND DISCUSSION
Synthesis of 13C9-15N-3,5-T2
The synthesis of 13C9-15N-L-T2 was carried out in parallel with unlabeled T2
according to the route shown in Scheme 1. Commercially available 13C9-15N-L-
tyrosine 1 was N-Boc and O-methyl ester protected and bis-iodinated with
N-iodosuccinimide in dichloromethane (DCM), a modification of the method of
Bovonsombat et al., to give 13C9-15N-3,5-diiodo-L-tyrosine 3 in 59% yield over the

SYNTHESIS OF 13C9-15N-LABELED 3, 5-T2 AND T4
1441
Scheme 1. Reagents and conditions: (a), HCl, 2,2-dimethoxypropane; (b), BOC2O, NaHCO3, THF=H2O;
(c), NIS, DCM; (d), 4-(triisopropyl)silyloxyphenyl boronic acid, Cu(OAc)2, DIPEA, pyridine, DCM; (e),
TBAF, THF; (f), LiOH, MeOH=H2O; and (g), HCl, dioxane. An asterisk (ꢀ) indicates 13C or 15N.
three steps.[11,12] 4-(Triisopropyl)silyloxyphenyl boronic acid was synthesized as
previously described and coupled to 3 via a copper(II)-mediated biaryl ether
formation.[13,14] Deprotection with tetrabutylammonium flurodie (TBAF) gave
N-boc-3,5-diiodo-13C9-15N-L-thyronine-OMe 4 in 30% yield over two steps. Sequen-
tial deprotection with lithium hydroxide to cleave the methyl ester and hydrochloric
acid to cleave the t-Boc gave 13C9-15N-3,5-T2 5, in 28% yield as the trifluoroacetic
acid (TFA) salt after purification by preparative high-performance liquid chromato-
1
graphy (HPLC).[15] Because of the difficulty in interpreting H NMR spectra as a
result of 13C coupling effects, labeled intermediates and 13C9-15N-3,5-T2 were
characterized by high-resulution mass spectrometry (HRMS) and chromatographic
comparison to unlabeled standards, which were synthesized in parallel to 13C9-15N-
labeled compounds.
Synthesis of 13C9-15N-L-T4
The synthesis of 13C9-15N-L-T4 was carried out in parallel with unlabeled T4
according to the route shown in Scheme 2. Biaryl ether 4 was bis-iodinated on the
outer ring with iodine monochloride and butylamine to give N-Boc-13C9-15N-T4-
OMe 6 in 37% yield.[13] Sequential deprotection of 6 as described previously pro-
duced 13C9-15N-T4 7, which was purified by preparative HPLC as the TFA salt in
23% yield from 6. As described for 13C9-15N-3,5-T2, synthetic intermediates and
13C9-15N-L-T4 were characterized by HRMS and chromatographic comparison to
unlabeled standards, which were synthesized in parallel.
125I, 131I, and 14C labeled THs have been used to study TH metabolism and
pharmacokinetics but the use of radiolabels in metabolism studies is limited to tra-
cing the fate of the radioactivity and does not provide information on metabolic
pathways that do not involve radiolabeled portions of the hormones.[16–25] In
addition, the use of outer ring labeled 125I-T4, the most commonly employed labeled
Scheme 2. Reagents and conditions: (a), ICl, BuNH2, 4:1 DCM=DMF; (b), LiOH, MeOH=H2O; and (c),
HCl, dioxane. An asterisk (ꢀ) indicates 13C or 15N.

1442
S. A. HACKENMUELLER AND T. S. SCANLAN
form of T4, could not be used to follow T4 metabolites that are extrathyroidally deio-
dinated in the outer ring, such as 3,5-T2 and T1AM. The incorporation of nonra-
dioactive isotopes such as 13C or 15N into T4 and other THs allows for more
in-depth investigations of alternate pathways of TH metabolism by LC-MS=MS.
The syntheses of unlabeled and of 13C-labeled T4 and T3 have been reported, but
here we report an alternate synthetic route to produce a novel labeled T4 containing
15N and 13C.[14,26–29] Although the synthesis of 125I-3,5-T2 has been reported, to our
knowledge this is the first reported synthesis of a stable isotope labeled T2.[30]
EXPERIMENTAL
All chemicals used for synthesis were purchased from Aldrich, Sigma-Aldrich,
Fluka, Acros or TCI. Anhydrous solvents were obtained from an in-house solvent
distillation system. Intermediates were purified by flash chromatography (HPFC)
on a Biotage SP1 purification system with a fixed wavelength ultraviolet detector
(Biotage, Charlotte, NC). Syntheses of unlabeled and 13C-15N labeled compounds
were carried out in parallel. The synthetic scheme was prospected using unlabeled
material, which also served as standards to match 13C-15N-labeled compounds by
thin-layer chromatography (TLC). NMR spectra for 13C -labeled compounds were
difficult to interpret due to 13C J-couplings. For this reason, 13C labeled compounds
1
were characterized by high-resolution mass spectrometry (HRMS) only. H NMR
were taken for unlabeled compounds only. 1H NMR spectra were taken on a Bruker
400 (400 MHz), and spectra were processed using ACD=NMR Processor Academic
Edition software. HRMS and MS=MS fragmentation (contained in the Supplemen-
tal Figures, available online) using electrospray ionization (ESI) in positive polarity
was performed at the Mass Spectrometry Laboratory at the University of Illinois at
Urbana–Champaign. HRMS are reported for all novel compounds, and MS=MS
fragmentation for 13C9-15N-labeled compounds are available in the Supplementary
Material, available online.
Boc-OMe-L-tyrosine
Concentrated hydrochloric acid (2.7 ml) was added dropwise to a stirring
solution of L-tyrosine (0.500 g, 2.76 mmol) in 2,2-dimethyoxypropane (33 ml). The
reaction was stirred at room temperature for 24 h, and the crude reaction mixture
was used in the next step.
NaHCO3 (7.5 ml, 1.1 M in water) was added to crude OMe-L-tyrosine and
Boc2O (0.620 g, 2.85 mmol) in THF (12 ml). The reaction was stirred at room tem-
perature for 24 h. The reaction was diluted with ether (20 ml), and the aqueous layer
was extracted twice with ether. The combined organic layers were washed with 0.5 M
HCl and brine and dried over MgSO4. The crude material was purified by HPFC
(10–80% ethyl acetate = hexanes over 10 column volumes) to give Boc-OMe-L-tyro-
1
sine (0.787 g, 2.66 mmol, 96.5% yield). H NMR (400 MHz, chloroform-d)d ppm
1.41 (s, 9 H) 2.86–3.21 (m, 2 H), 3.75 (s, 3 H), 4.47–4.63 (m, 1 H), 5.13 (br. s., 1 H),
5.48 (br. s., 1 H), 7.10 (s, 2 H), 7.64 (s, 2 H).
Boc-OMe-13C9-15N-L-tyrosine 2 was synthesized using the same procedure
with 13C9-15N-l-tyrosine (0.500 g, 2.62 mmol) as starting material, in 87.2% yield

SYNTHESIS OF 13C9-15N-LABELED 3, 5-T2 AND T4
1443
(0.702 g, 2.30 mmol). HRMS (ESIþ) for C613C9H2215NO5 [M þ H] calculated
306.1770, found 306.1776. MS=MS fragmentation spectrum is available in Supple-
mental Fig. S1, available online.
3,5-Diiodo-Boc-OMe-L-tyrosine
N-Iodosuccinimide (1.2 g, 5.32 mmol) was added to a stirring solution of
Boc-OMe-L-tyrosine (0.787 g, 2.66 mmol) in DCM (17.7 ml) at 0 ꢁC. The reaction
was monitored by TLC (40% ethyl acetate = hexanes) for consumption of starting
material, which occurred in ꢂ40 min. A solution of 10% Na2S2O3 was added drop-
wise, and the reaction mixture was diluted with water and extracted three times with
ethyl acetate. The ethyl acetate layers were combined and dried over MgSO4. The
crude material was purified by HPFC (10–80% ethyl acetate = hexanes over 10 col-
umn volumes) to give 3,5-diiodo-Boc-OMe-L-tyrosine as a white solid (0.908 g,
1
1.66 mmol, 67.5% yield). H NMR (400 MHz, chloroform-d)d ppm 1.45 (s, 9 H),
2.96 (s, 2 H), 3.68–3.79 (m, 3 H), 4.49 (d, J ¼ 7.07 Hz, 1 H), 5.03 (d, J ¼ 7.58 Hz,
1 H), 5.70 (s, 1 H), 7.44 (s, 2 H).
3,5-Diiodo-Boc-OMe-13C9-15N-L-tyrosine 3 was synthesized following the
same procedure from Boc-OMe-13C9-15N-L-tyrosine (0.702 g, 2.30 mmol) starting
material, in 67.5% yield (0.864 g, 1.55 mmol). HRMS (ESIþ) for C613C9H19I215N-
NaO5 [M þ Na] calculated 579.9523, found 579.9528. MS=MS fragmentation
spectrum is available in Supplemental Fig. S2, available online.
3,5-Diiodo-Boc-OMe-L-thyronine
˚
Molecular sieves 4 A were added to a round-bottom flask that was then flame
dried and flushed with dry air. 4-(Triisopropyl)silyloxyphenyl boronic acid (0.612 g,
2.08 mmol), DCM (6.3 ml), diisopropylethylamine (4.15 mmol), and pyridine
(4.15 mmol) were added under argon. Dry copper(II) acetate (0.151 g, 0.83 mmol)
was added, and the reaction mixture was stirred under argon for 10 min.
3,5-Diiodo-Boc-OMe-L-tyrosine (0.452 g, 0.83 mmol) was added in DCM (2 ml) over
5 min. The reaction was stirred at room temperature under dry air for 48 h. The reac-
tion was diluted with ether; filtered through silica=celite; sequentially washed with
HCl, H2O, and brine, and dried over MgSO4. The crude reaction material was taken
into the next reaction.
Crude biaryl ether was dissolved in THF and cooled to 0 ꢁC. Tetra-N-butyl-
ammonium fluoride (3.15 mmol) was added dropwise over 5 min. The reaction was
stirred at room temperature for 15 min. The reaction was diluted with ethyl acetate
and washed with HCl. The aqueous layer was washed with ethyl acetate, and the
combined organic layers were washed sequentially with H2O and brine and dried
over MgSO4. The crude product was purified by HPFC (10–80% ethyl acetate = hex-
anes over 10 column volumes) to yield 3,5-diiodo-Boc-OMe-13C9-15N-L-thyronine as
a solid (0.196 g, 0.31 mmol, 37.0% yield over two steps). 1H NMR (400 MHz,
chloroform-d)d ppm 1.45 (s, 9 H), 2.83–3.19 (m, 2 H), 3.66–3.88 (m, 3 H), 4.54 (br.
s., 1 H), 4.69 (br. s., 1 H), 5.10 (br. s., 1 H), 6.62–6.92 (m, 4 H), 7.63 (s, 2 H). HRMS
(ESIþ) for C21H23I2NNaO6 [M þ Na] calculated 661.9512; found 661.9512.

1444
S. A. HACKENMUELLER AND T. S. SCANLAN
3,5-Diiodo-Boc-OMe-13C9-15N-L-thyronine 4 was synthesized following the
same procedure from 3,5-diiodo-Boc-OMe-13C9-15N-L-tyrosine (0.547 g, 0.98 mmol)
starting material, in 29.9% yield (0.190 g, 0.29 mmol). HRMS (ESIþ) for
C1213C9H24I215NO6 [M þ H] calculated 649.9965; found 649.9966. MS=MS fragmen-
tation spectrum is available in Supplemental Fig. S3, available online.
3,5-Diiodo-13C9-15N-L-thyronine (13C9-15N-T2) 5
To cleave the methyl ester, lithium hydroxide (0.39 mmol, 100 ml of 3.9 M in
H2O) was added to a stirred solution of 3,5-diiodo-Boc-OMe-13C9-15N-L-thyronine
(0.051 g, 0.078 mmol) in methanol (300 ml) at 4 ꢁC. The reaction was stirred at 4 ꢁC
for 90 min. Solvent was removed under argon, and the crude reaction product was
dried in a modified Abderhalden drying apparatus for use in the next step.
The dried crude reaction product from the methyl ester deprotection was dis-
solved in 4 M HCl in dioxane (300 ml). The reaction was stirred under argon at room
temperature overnight. The reaction mixture was diluted with 500 ml of 1:1 water=
acetonitrile and purified by preparatory HPLC using a Rainin HPXL solvent deliv-
ery system and a Prostar PDA detector (Varian Inc., Paolo Alto, CA). Reaction pro-
duct was injected onto a Varian Dynamax Microsorb C18 21.4 ꢃ 250 mm column
and monitored at wavelength 254 nm. Mobile phases were A (water þ 0.1% TFA)
and B (acetonitrile þ 0.1% TFA) with gradient conditions as follows: 5% B
0–5 min, 5–95% B 5–50 min, 95% B 50–65 min, 95–5% B 65–75 min, and 5% B
75–90 min. Commercially available 3,5-T2 was used to determine the retention time
of the product, 31.5 min. HRMS (ESIþ) for C613C9H14I215NO4 [M þ H] calculated
535.9284; found 535.9291. MS=MS fragmentation spectrum is available in Supple-
mental Fig. S4, available online.
3,3’,5,5’-Tetraiodo-N-Boc-OMe-L-thyronine
Iodine monochloride in DCM (0.26 mmol, 260 ml) was added dropwise to a
stirring solution of 3,5-diiodo-Boc-OMe-L-thyronine (0.072 g, 0.11 mmol) and butyl-
amine (0.64 mmol, 64 ml) in 4:1 DCM=DMF (3.7 ml) at 0 ꢁC. The reaction was stirred
at 0 ꢁC for 20 min. The reaction was diluted with ethyl acetate; washed sequentially
with HCl, 10% sodium thiosulfate, H2O, and brine; and dried over MgSO4. The
crude product was purified by HPFC (10–80% ethyl acetate = hexanes over 10 col-
umn volumes) to yield 3,30,5,50-tetraiodo-N-Boc-OMe-L-thyronine as a brown solid
(0.022 g, 0.025 mmol, 22.9% yield). 1H NMR (400 MHz, chloroform-d)d ppm 1.41 (s,
9 H), 2.86–3.21 (m, 2 H), 3.75 (s, 3 H), 4.47–4.63 (m, 1 H), 5.13 (br. s., 1 H), 5.48 (br.
s., 1 H), 7.10 (s, 2 H), 7.64 (s, 2 H). HRMS (ESIþ) for C21H21I4NNaO6 [M þ Na] cal-
culated 913.7445; found 913.7437.
3,30,5,50-Tetraiodo-N-Boc-OMe-13C9-15N-L-thyronine 6 was synthesized fol-
lowing the same procedure using 3,5-diiodo-Boc-OMe-13C9-15N-L-thyronine
(0.150 g, 0.23 mmol) as starting material, in 36.9% yield (0.076 g, 0.085 mmol).
HRMS (ESIþ) for C1213C9H21I415NNaO6 [M þ Na] calculated 923.7718; found
923.7715. MS=MS fragmentation spectrum is available in Supplemental Fig. S5,
available online.

SYNTHESIS OF 13C9-15N-LABELED 3, 5-T2 AND T4
1445
3,3’,5,5’-Tetraiodo-13C9-15N-L-thyronine (13C9-15N-T4) 7
To cleave the methyl ester, lithium hydroxide (0.43 mmol, 110 ml of 3.9 M in
H2O) was added to a stirred solution of 3,30,5,50-tetraiodo-Boc-OMe-13C9-15N-L-
thyronine (0.076 g, 0.085 mmol) in methanol (330 ml) at 4 ꢁC. The reaction was stirred
at 4 ꢁC for 90 min. Solvent was removed under argon and the crude reaction product
was dried in a modified Abderhalden drying apparatus for use in the next step.
The dried crude reaction product from the methyl ester deprotection was dis-
solved in 4 M HCl in dioxane (320 ml). The reaction was stirred under argon at room
temperature overnight. To the reaction mixture, 500 ml of 1:1 water=acetonitrile was
added, and the solution was filtered through a 0.22-mm filter prior to purification by
preparatory HPLC as described previously. Commercially available T4 was used
to determine the retention time of the product, 37.7 min. HRMS (ESIþ) for
C613C9H11I415NO4 [M þ H] calculated 787.7217; found 787.7218. MS=MS fragmen-
tation spectrum is available in Supplemental Fig. S6, available online.
ACKNOWLEDGMENT
This work was supported by a grant from the National Institutes of Health
(DK-52798, T. S. S.).
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