Inhibitors of the serotonin transporter protein (SERT): The design and synthesis of biotinylated derivatives of 3-(1,2,3,6-tetrahydro-pyridin-4-yl)-1H- indoles. High-affinity serotonergic ligands for conjugation with quantum dots

Article abstract of DOI:10.1016/j.bmcl.2005.08.030

There is a growing demand for compounds with specificity for the serotonin transporter protein (SERT) that can be conjugated to cadmium selenide/zinc sulfide core shell nanocrystals. This letter describes the design and synthesis of two different biotinylated SERT antagonists that can be attached to streptavidin-coated cadmium selenide/zinc sulfide core shell nanocrystals.

Full text of DOI:10.1016/j.bmcl.2005.08.030

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5307–5310
Inhibitors of the serotonin transporter protein (SERT):
The design and synthesis of biotinylated derivatives of
3-(1,2,3,6-tetrahydro-pyridin-4-yl)-1H-indoles. High-affinity
serotonergic ligands for conjugation with quantum dots
Ian D. Tomlinson,a John N. Mason,b Randy D. Blakelyb and Sandra J. Rosenthala,*
aThe Department of Chemistry, Vanderbilt University, Station B, 351822, Nashville, TN 37235-1822, USA
bThe Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37235, USA
Received 27 June 2005; revised 9 August 2005; accepted 10 August 2005
Available online 23 September 2005
Abstract—There is a growing demand for compounds with specificity for the serotonin transporter protein (SERT) that can be
conjugated to cadmium selenide/zinc sulfide core shell nanocrystals. This letter describes the design and synthesis of two different
biotinylated SERT antagonists that can be attached to streptavidin-coated cadmium selenide/zinc sulfide core shell nanocrystals.
Ó 2005 Elsevier Ltd. All rights reserved.
Cadmium selenide/zinc sulfide core shell nanocrystals or
Ôquantum dotsÕ are novel semiconducting crystals that
have unique physical properties that differ significantly
from those of bulk material.1 They have fluorescence
line widths in the visible region of the electromagnetic
spectra and do not rapidly photobleach. Our research
efforts are based on applications of quantum dots as
fluorescent tags for receptors and transporter proteins:
Quantum dots have several distinct advantages over
conventional fluorescent dyes, such as narrower
emission spectra and increased brightness.2–4 Several
groups have reported attaching antibodies, nucleic
acids, and proteins to quantum dots.5–9
periods of time than is currently achievable with conven-
tional dyes.
Specifically, we are developing systems to study the sero-
tonin transporter protein based on derivatives of known
compounds that can be attached to quantum dots. We
hope to use such conjugates to gain greater insights into
the biological mode of action of selective serotonin reup-
take inhibitors. Also by dynamic and static fluorescent
imaging, we hope to gain more knowledge about the
distribution and localization of the serotonin reuptake
protein within neuronal cell cultures.
In our early studies, we synthesized a derivative of sero-
tonin (1).14 This ligand was attached directly to the sur-
face of a quantum dot via an acid–base interaction
between a thiol in the ligand and the zinc on the surface
of the nanocrystal. This ligand was shown to be biolog-
ically active and had an IC50 of 115 lM, when assayed
against HeLa cells transiently transfected with human
serotonin transporters (hSERT) (see Fig. 1).
Our approach has been to attach neurotransmitter and
drug derivatives to nanocrystals.10–12 We hope to be
able to use such conjugates to image biological systems
in vitro.10,13 The emission spectra of quantum dots are
size-tunable thus enabling the possibility of experiments
where several biological targets are studied simulta-
neously and their increased photostability should enable
experiments that can be run over significantly longer
Unfortunately, (1) lacked high affinity and specificity for
hSERT. For our studies we required ligands with bio-
logical activities in the nanomolar region and specificity
for SERT. Therefore, we designed and synthesized new
ligands with high affinity and specificity for SERT that
could be attached to quantum dots.
Keywords: Serotonin; Antagonist; Serotonin transporter; Nanocrystal;
Quantum dots.
*
Corresponding author. Tel.: +1 61 53 22 26 33; fax: +1 61 53 43 12
34; e-mail: sjr@femto.cas.vanderbilt.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.030

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I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5307–5310
NH2
O
N
O
O
N
HS
O
O
N
H
(1)
(a), (b)
Figure 1. A pegylated derivative of serotonin.
(6)
N
H
N
H
A review of the literature showed that the indole15 deriv-
ative 3-(1,2,3,6-tetrahydropyrindin-4-yl)-1H-indole (2)
can be derivatized giving a biologically active compound
3.16–18 From these results, we reasoned that the pyridi-
nyl nitrogen in 3-(1,2,3,6-tetrahydropyrindin-4-yl)-1H-
indole (2) occupies a region of steric tolerance. We
decided to attach linker arms to the nitrogen atom in
the tetrahydropyridinyl indole derivative (2) resulting
in 4. X in compound 4 is a functionality that can be
attached to the quantum dot (see Fig. 2).
(c)
NH2
O
N
H
(d)
N
N
H
N
O
(7)
We have observed that the fluorescence of quantum dots
decreases (quenches) when thiols are attached directly to
the surface of the quantum dot. We hoped that a ligand
that contained another functionality may not quench
the fluorescent emission of the dot. For this reason, we
decided to attach biotin to the end of our linker arm
as the point of attachment to quantum dots.
NH
(5)
N
H
S
N
H
Scheme 1. Reagents: (a) 4-piperidone monohydrate monohydrochlo-
ride, KOH, MeOH; (b) N-(2-bromoethylene)phthalimide;
(c) H2NNH2; (d) biotin, DCC, NHS.
methylene chloride by stirring for two days at room
temperature, resulting in 5 in a 21% yield.
Our first biotinylated ligand was N-(2-(4-(1H-indol-
3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl-5-(2-oxohexahy-
dro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (5) and
this was synthesized as shown in Scheme 1. Initially, in-
dole was reacted with 4-piperidone monohydrate mono-
hydrochloride in refluxing methanolic KOH for 6 h to
give 2 in a 81% yield. The tetrahydropyridinyl nitrogen
We thought that the short linker arm in (5) may reduce
the biological activity of the compound when it was
bound to streptavidin. There may be considerable steric
interactions between the surface of the dot and the
SERT transporter protein. In order to determine if this
was happening, we synthesized 8. The synthetic route
of (8) is outlined in Scheme 2. In this route, 2-(4-(1H-
indol-3-yl)-5,6-dihydropyridin-1-(2H)-yl)ethanamine (7)
was dissolved in methylene chloride and stirred at room
temperature for 18 h with a NHS-biotinylated PEG
derivative (9) that was purchased from the Nektar
Therapeutics Corporation of HuntsvilleAlabama. The
polyethylene glycol in this linker arm was large and
the molecular mass of 9 was 3400 g wt. The methylene
chloride was removed under reduced pressure and the
crude biotinylated material was attached to quantum
dots.
was alkylated by refluxing
2
in acetone with
N-(2-bromoethyl)phthalimide in the presence of potassium
carbonate for 18 h resulting in 2-(2-(4-(1H-indol-
3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)isoindoline-1,3-
dione (6) in a 20% yield. The phthalimide group was
removed using hydrazine monohydrate in ethanol at
room temperature giving (7) in a 40% yield and
this was coupled to biotin using DCC and NHS in
O
S
O
H
N
N
N
Streptavidin-conjugated quantum dots were obtained
from the Quantum Dot Corporation of Haywood Cali-
fornia, and compounds 5 and 8 were attached to these
dots. Compound (5) was dissolved in DMSO and added
to a borate buffer solution containing quantum dots at a
known concentration. This mixture was stirred over-
night at room temperature and purified via column
chromatography on a Sephadex column. The fluorescent
band was collected and assayed against HEK293 cells
transfected with hSERT. The concentration of the dots
was measured using a UV–vis spectrophotometer19 and
the IC50 value for the conjugate is based upon the con-
centration of dots. The conjugate of 8 was prepared in a
similar manner to the conjugate of 5; however, the DMF
(3)
N
H
N
(2)
H
O
X
N
H
N
N
H
N
O
NH
(5)
N
H
N
H
(4)
S
Figure 2. SERT selective ligands.

I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5307–5310
5309
NH2
O
S
PEG O
N
H
N
+
O
NH
HN
O
N
O
O
(7)
(9)
O
N
H
(a)
O
S
PEG O
N
H
O
NH
HN
HN
O
N
(8)
N
H
Scheme 2. Reagent and conditions: (a) CH2Cl2, rt, 24 h.
was replaced with borate. Where appropriate the IC50 of
the free ligand was also determined.
activity of the conjugate. These ligands have higher
affinities for hSERT than our first ligand and we ob-
served no quenching when the ligand was bound to
the dot. We hope to be able to use these conjugates in
fluorescent imaging assays in future studies.
Table 1 shows the biological activities of the conjugated
ligands 5 and 8 as well as free ligand 5. IC50 values for
the parent drug (2) and our initial compound (1) in free
solution and conjugated to dots are also shown. When
ligands were conjugated to the streptavidin conjugated
dots, there was no observable decrease in the fluorescent
intensity of the dots. However, when the ligand 1 was at-
tached to quantum dots, there was a significant reduc-
tion in fluorescence that could be observed by the
naked eye. These observations indicated that the thiol
in 1 may be quenching the dots and consequently
ligands with a thiol attached to a linker arm should be
avoided whenever possible. Also, the proximity of the
ligand to quantum dots does not appear to have a detri-
mental effect on the biological activity of the conjugate.
Acknowledgments
We thank Quantum Dot Corporation for supplying the
core shell nanocrystals used in this study. We would also
like to thank Dr. Marcel Bruchez of Quantum Dot Cor-
poration for helpful advice during the course of this
study. This work was supported by grants from the
National Institutes of Health and Quantum Dot
Corporation.
References and notes
In conclusion, we have prepared two biotinylated li-
gands (5 and 8) that may be attached to streptavidin-
conjugated quantum dots. These ligands have high affin-
ity for hSERT when in solution and bound to dots, and
the length of the linker arm between the ligand and the
dot doesnot appear to significantly affect the biological
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