High affinity inhibitors of the dopamine transporter (DAT): Novel biotinylated ligands for conjugation to quantum dots

Article abstract of DOI:10.1016/j.bmcl.2006.05.097

Compounds capable of inhibiting the dopamine transporter protein (DAT) that can be conjugated to cadmium selenide/zinc sulfide/core shell nanocrystals may be used to image the location and distribution of the DAT in neuronal cell membranes. This letter de

Full text of DOI:10.1016/j.bmcl.2006.05.097

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4664–4667
High affinity inhibitors of the dopamine transporter (DAT): Novel
biotinylated ligands for conjugation to quantum dots
Ian D. Tomlinson,a,* John N. Mason,b Randy D. Blakelyb,c and Sandra J. Rosenthala,b
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 37232-8548, USA
cVanderbilt Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA
Received 9 May 2006; revised 25 May 2006; accepted 30 May 2006
Available online 19 June 2006
Abstract—Compounds capable of inhibiting the dopamine transporter protein (DAT) that can be conjugated to cadmium selenide/
zinc sulfide/core shell nanocrystals may be used to image the location and distribution of the DAT in neuronal cell membranes. This
letter describes the synthesis of biotinylated analogs of the DAT antagonists GBR 12909 and GBR 12935 that can be attached to
streptavidin coated cadmium selenide/zinc sulfide/core shell nanocrystals.
Ó 2006 Elsevier Ltd. All rights reserved.
Quantum dots are a new type of fluorescent markers that
are increasingly finding novel applications in biology.
Many groups have reported imaging applications based
upon quantum dots conjugated to peptides,1–6 nucleic
acids,7–13 proteins,14,15 and antibodies.16–21 Our research
focus involves conjugating ligands that bind to neuronal
proteins to quantum dots.22–26 We are particularly inter-
ested in drugs that have high affinity for neuronal trans-
porter proteins including the serotonin transporter
protein (SERT) and the dopamine transporter protein
(DAT). Both SERT and DAT exhibit regulated traffick-
ing that controls transport capacity. Imaging transporter
movements in neuronal membranes is a challenging but
important objective.
Abnormalities in the dopaminergic system have been
observed in the etiology of a wide range of diseases
including Alzheimer’s and Parkinson’s diseases. The
dopaminergic system is also thought to have a role in
the reinforcing effects of cocaine addiction. We hope
to be able to use DAT antagonist quantum dot conju-
gates to provide more information about the biological
mode of action of dopamine reuptake inhibitors. In
addition by using static and dynamic biological imaging
techniques it is hoped that these conjugates may reveal
information about the location and distribution of the
DAT within neuronal cells.
In our initial studies we synthesized derivatives of the
high affinity DAT inhibitors GBR 12909 and GBR
12935.
Quantum dots have the potential to significantly im-
prove the sensitivity and duration of in vitro biological
imaging experiments that are currently performed with
fluorescent dyes. Their increased photostability, bright-
ness, and narrower emission spectra should enable the
development of imaging studies that can be performed
for longer durations and with lower concentrations of
analyte.
X
O
N
N
X
GBR 12909 X = F
GBR 12935 X = H
Keywords: Dopamine transporter; Quantum dot; Cadmium selenide;
Reuptake inhibitor.
*
Ligands 1 and 2 were selected for their ease of synthesis,
potency, and specificity for the DAT over SERT. They
Corresponding author. Tel.: +1 615 322 2633; fax: +1 615 343
1234; e-mail: iand@femto.cas.vanderbilt.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.097

I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4664–4667
4665
X
were bound to the surface of the quantum dot via an
acid–base interaction between a thiol and the zinc on
the surface of the quantum dot.25
O
NH2
N
N
(6) X = H
(7) X = F
X
X
H
O
O
N
N
N
(i)
X
X
H
N
(1) X = H
(2) X = F
O
O
N
N
SH
X
HN
S
We observed that when the ligands 1 and 2 were
bound to the surface of quantum dots the fluorescence
of the dots diminished. This reduction in quantum
yield could be due to the thiols acting as traps. In
addition to reduced quantum yields these conjugates
had a limited shelf life. The thiols were not irreversibly
bound to the surfaces of quantum dots and they disso-
ciated from the surface over time. This dissociation
caused the dots to aggregate and precipitate from
solution.
(3) X = H
(4) X = F
O
N
H
Scheme 1. Reagents: (i) Biotin, CDI.
The synthetic route used to prepare compound 5 is out-
lined in Scheme 2. 1-[2-[4,40-Flourobenzhydryloxy]
ethyl]-4-(3-(4-aminophenyl)propyl)piperazine 7 was
coupled to the phthalimide protected 11-aminoundeca-
noic acid 8 using two different methods. In the first
method the acid was converted to the acid chloride with
thionyl chloride and reacted it with 7 in dry dichloro-
methane resulting in a 69% yield of 9. The second meth-
od coupled 8–7 using CDI in dry DMF resulting in a
33% yield of 9.29 The phthalimide protecting group
was removed using hydrazine in ethanol giving 1030 in
a 72% yield. Biotin was coupled to the terminal amino
functionality using DCC in dry DMF resulting in a
12.4% yield of 5.31
As biotin has
a
high affinity for avidin
(Ka ꢀ 1015 molꢁ1) and streptavidin, we decided to syn-
thesize a new generation of DAT ligands that incorpo-
rated a biotin moiety for use with streptavidin coated
quantum dots. Our initial ligands had biotin bound
directly to the GBR derivatives 3 and 4. Steric hin-
drance between the cell surface and the dot’s surface
may reduce the affinity of these conjugates. Therefore
we also designed a GBR derivative that incorporated
an alkyl linker between the GBR derivative and the bio-
tin 5 to reduce steric effects.
Compounds 3, 4, and 5 were conjugated to strepatvidin
coated quantum dots as follows; 0.1 ml of a 8.5 lM
solution of streptavidin coated quantum dots that had
a maximum fluorescence emission of 605 nm was added
X
H
O
O
N
N
N
X
X
N
X
HN
S
O
OH
X
N
X
(3) X = H
(4) X = F
O
N
O
H
O
(i)
N
N
+
X
H
H
N
(8)
(7)
(9)
N
O
O
O
O
N
N
O
NH2
N
O
HN
N
O
O
(5) X = F
X
HN
S
(ii)
O
N
H
X
X
X
N
X
(iii)
The synthesis of compounds 3 and 4 is outlined in
Scheme 1. Either 1-[2-[bisphenylmethoxy]ethyl]-4-(3-(4-
aminophenyl)propyl)piperazine 6 or 1-[2-[4,40-flour-
obenzhydryloxy]ethyl]-4-(3-(4-aminophenyl)propyl)pi-
perazine 7 was biotinylated in dry DMF using CDI.
This resulted in a 29.7% yield of 327 and a 16.4% yield
of 4.28 The synthesis of the amino analogs of GBR
12909 and GBR 12935 has previously been described
in an earlier publication.25
O
O
N
N
N
(10)
(5) X = F
S
H
N
NH
O
HN
O
NH2
HN
O
N
O
H
Scheme 2. Reagents: (i) Method A SOCl2, CH2Cl2; Method B CDI;
(ii) a—hydrazine, ethanol; b—CH2Cl2; (iii) Biotin, NHS, DCC, DMF.

4666
I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4664–4667
to 0.9 ml of borate buffer at pH 8.4. Compounds 3, 4,
and 5 were dissolved in DMSO to give an 850 lM solu-
tion of each compound. 0.1 ml of this solution was add-
ed to the solution of quantum dots in borate buffer and
the mixture was stirred at 25 °C for 18 h. Excess ligand
was removed from this solution by dialyzing the borate
solution for 18 h. The concentration of the resultant
solution was determined by UV/visible spectrophotome-
try by measuring the absorbance at 600 nm and using an
extinction coefficient of 650,000.
significant open space leading to the binding site. This
methodology may also be applied to ligands that have
specificity for other transporters such as SERT.
Acknowledgments
We thank Quantum Dot Corporation for supplying the
core shell nanocrystals used in this study. We thank
Dr. Marcel Bruchez of Quantum Dot Corporation for
helpful advice during the course of this study. This work
was supported by grants from the National Institutes of
Health (RO1EB003728-02 and GM72048-02)
The ability of compounds 3, 4, and 5 to inhibit the up-
take of dopamine was measured using a competitive
transport assay. Briefly, 12 concentrations of each com-
pound (10ꢁ5 and 10ꢁ12 M) were competed against triti-
ated dopamine (50 nM). Nonspecific uptake was
defined with 1 mM GBR and subtracted from total radi-
olabeled uptake. Subtracted values were plotted versus
logarithmic values of each compound and fitted with a
one-site competition curve to determine IC50 values
using Graphpad prism.
References and notes
1. Vu, T. Q.; Maddipati, R.; Blute, T. A.; Nehilla, B.
J.; Nusblat, L.; Desai, T. A. Nano Lett. 2005, 5,
603.
´
˚
2. Akerman, M. E.; Chan, W. C. W.; Laakkonen, P.; Bhatia,
S. N.; Ruoslahti, E. PNAS 2002, 99, 12617.
3. Chen, F.; Gerion, D. Nano Lett. 2004, 4, 1827.
4. Pinaud, F.; King, D.; Moore, H. P.; Weiss, S. J. Am.
Chem. Soc. 2004, 126, 6115.
5. Lidke, D. S.; Nagy, P.; Heintzmann, R.; Arndt-Jovin, D.
J.; Post, J. N.; Grecco, H. E.; Jares-Erijman, E. A.; Jovin,
T. M. Nat. Biotechnol. 2004, 22, 198.
6. Ian D Tomlinson, John Mason, Randy D Blakely, Sandra
J. Rosenthal. Bionanotechnology Protocols, Totowa, New
Jersey; 2005, p 51.
7. Mason, J. N.; Farmer, H.; Tomlinson, I. D.; Schwartz, J.
W.; Savchenko, V.; DeFelice, L. J.; Rosenthal, S. J.;
Blakely, R. D. J. Neurosci. Methods 2005, 143, 3.
8. Gerion, D.; Parak, W. J.; Williams, S. C.; Zanchet, D.;
Micheel, C. M.; Alivisatos, A. P. J. Am. Chem. Soc. 2002,
124, 7070.
9. Parak, W. J.; Gerion, D.; Zanchet, D.; Woerz, A. S.;
Pellegrino, T.; Micheel, C.; Williams, S. C.; Seitz, M.;
Bruehl, R. E.; Bryant, Z.; Bustamante, C.; Bertozzi, C. R.;
Alivisatos, A. P. Chem. Mater. 2002, 14, 2113.
10. Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Bnin,
U.; Willner, I. J. Am. Chem. Soc. 2003, 125, 1318.
11. Gerion, D.; Chen, F.; Kannan, B.; Fu, A.; Parak, W. J.;
Chen, D. J.; Majumdar, A.; Alivisatos, A. P. Anal. Chem.
2003, 75, 4766.
12. Dubertret, B.; Skourides, P.; Norris, D. J.; Noirex, V.;
Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759.
13. Alivisatos, P. Nat. Biotechnol. 2004, 22, 47; Murphy, C. J.
Anal. Chem. 2002, 520; Xiao, Y.; Barker, P. E. Nucleic
Acids Res. 2004, 32, 28.
Table 1 shows the potencies of the ligands 1–5 against
dopamine transport as well as the potency of conjugated
nanocrystals. When ligands 3–5 were conjugated to
quantum dots, no significant reduction in fluorescence
was observed; however when ligands 1–2 were conjugat-
ed to quantum dots, a reduction in the fluorescent inten-
sity of the dot solution was observed. No attempts were
made to quantify this reduction. The proximity of the
quantum dot to compounds 3 and 4 appears to reduce
the affinity of these conjugated nanocrystals for the
DAT. When a spacer is introduced between the biotin
and the drug as in compound 5, no significant reduction
in potency was observed. The measured IC50s of the un-
conjugated ligands were between 5 and 36 nM, whilst
the IC50s of the conjugates differed by a factor of a hun-
dred-fold, indicating that the ligand is bound tightly to
the surface of the quantum dot and not dissociating in
solution. These conjugates were stable and could be kept
at 4 °C for a period of several weeks with no precipita-
tion of aggregates.
In conclusion we have synthesized three new DAT li-
gands that have high affinity for hDAT. These ligands
may be conjugated to the surfaces of streptavidin coated
quantum dots with little or no reduction in affinity. In
addition, unlike earlier conjugates, no significant reduc-
tion in quantum yield was observed when these ligands
were conjugated to quantum dots. The unbound ligands
have high affinity for the DAT suggesting that there is
14. Hoshino, A.; Hanaki, K.; Suzuki, K.; Yamamoto, K.
Biochem. Biophys. Res. Commun. 2004, 314, 46.
15. Ba¨umle, M.; Stamou, D.; Segura, J.; Hovius, J.; Vogel, H.
Langmuir 2004, 20, 3828.
16. Watson, A.; Wu, X.; Bruchez, M. Bioimaging 2003, 34,
296.
Table 1. Inhibition of dopamine reuptake by compounds 1–5 and their
nanocrystal conjugates
´
17. Dahan, M.; Levi, S.; Luccardini, C.; Rostaing, P.; Riveau,
B.; Triller, A. Science 2003, 302, 442.
Compound
IC50 of unbound
ligand (nM)
IC50 of ligand
conjugatesa (nM).
18. Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.;
Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat.
Biotechnol. 2003, 21, 41.
19. Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M.
Nat. Biotechnol. 2003, 21, 47.
20. Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda,
H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Anal.
Chem. 2004, 76, 684.
1
2
3
4
5
10
18
11
36
5
32
140
637
300
1
a IC50 was measured relative to quantum dot concentration.

I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4664–4667
4667
21. Gao, X.; Cui, Y.; Levensonson, R. M.; Chung, L. W. K.;
Nie, S. Nat. Biotechnol. 2004, 22, 969.
22. Rosenthal, S. J.; Tomlinson, I. D.; Adkins, E. M.;
Schroter, S.; Adams, S.; Swafford, L.; McBride, J.; Wang,
Y.; DeFelice, L. J.; Blakely, R. D. J. Am. Chem. Soc. 2002,
124, 4586.
23. Tomlinson, I. D.; Kippeny, T.; Swafford, L.; Siddiqui, N.
H.; Rosenthal, S. J. J. Chem. Res. 2002, 527.
24. Tomlinson, I. D.; Grey, J.; Rosenthal, S. J. Molecules
2002, 7, 777.
25. Tomlinson, I. D.; Burton, J.; Mason, J.; Blakely, R. D.;
Rosenthal, S. J. Tetrahedron 2003, 59, 8035.
29. 1H NMR (CDCl3) d 1.12–1.15 (m, 10H), 1.53–1.67 (m,
6H), 2.21–2.44 (m, 12H), 2.53 (t, J = 5.58 Hz, 2H), 3.44 (t,
J = 5.58 Hz, 2H), 3.53 (t, J = 7.11 Hz, 2H), 5.22 (s, 1H),
6.82–6.88 (m, 4ArH), 6.96 (d, 2ArH), 7.16–7.18 (m,
4ArH), 7.40 (d, 2ArH), 7.51–7.55 (m, 2ArH), 6.63–7.67
(m, 2ArH), 8.70 (br s, NH); 13C NMR (CDCl3) d 25.22,
26.25, 28.01, 28.54, 28.73, 28.78, 28.82, 30.28, 32.48, 36.91,
37.41, 52.61, 53.06, 57.26, 57.32, 66.28, 81.83, 114.50,
114.79, 119.52, 122.51, 128.01, 128.11, 131.51, 133.34,
135.91, 137.04, 137.42, 137.46, 159.87, 163.12, 167.79,
171.44, 206.41; m/z ESI (M+H) 779.42864, (M+Na)
801.41052.
26. Tomlinson, I. D.; Mason, J. N; Blakely, R. D.; Rosenthal,
S. J. Bioorg. Med. Chem. Lett. 2005, 15, 5307.
30. 1H NMR (CDCl3)
d 1.16–1.32 (m, 12H), 1.60 (t,
J = 6.12 Hz, 2H), 1.68 (t, J = 7.41 Hz, 2H), 2.21–2.62 (m,
22H), 3.45 (t, J = 5.73 Hz, 2H), 6.85–6.91 (m, 4ArH), 6.97
(d, 2ArH), 7.16–7.22 (m, 4ArH), 7.35 (d, 2ArH), 8.50 (br s,
NH); 13C NMR (CDCl3) d 25.43, 26.52, 28.25, 28.98,
29.09, 29.20, 32.77, 32.91, 37.13, 41.61, 52.81, 53.26, 57.52,
57.57, 66.47, 82.15, 114.78, 115.06, 119.82, 128.21, 128.32,
135.96, 137.35, 137.51, 137.55, 160.15, 163.40, 171.66; m/z
ESI (M+H) 649.425188, (M+Na) 671.407087.
1
27. Mp = 139–140 °C. H NMR (CDCl3) d 1.4–1.50 (m, 4H),
1.51–1.56 (m, 6H), 2.37–2.81 (m, 17H), 3.01–3.1 (m, 1H),
3.48–3.53 (m, 2H), 4.21 (br s, 1H), 4.40 (br s, 1H), 5.37 (s,
1H), 6.18 (s, 1H), 6.70–7.08 (m, 4H), 7.24–7.34 (m, 8ArH),
7.54 (d, 2ArH), 8.99 (s, 1H); 13C NMR (CDCl3) d 15.10,
25.64, 27.86, 28.04, 32.79, 36.58, 40.31, 52.44, 52.81, 55.59,
57.58, 60.08, 61.49, 65.64, 66.66, 83.71, 119.89, 126.77,
127.24, 128.16, 128.46, 136.43, 137.08, 141.97, 164.31,
172.02; m/z ESI (M+H) 656.35990.
31. Mp = 147–149 °C. 1H NMR (CD3OD) d 1.23–1.29 (m,
12H), 1.32–1.37 (m, 4H), 1.52–1.53 (m, 4H), 1.70–1.65 (m,
2H), 2.09 (t, J = 7.26 Hz, 2H), 2.25 (t, J = 7.26 Hz, 2H),
2.49–2.69 (m, 18H), 2.78–2.80 (m, 1H), 3.02–3.08 (m, 4H),
3.19–3.21 (m, 1H), 3.49 (t, J = 5.34 Hz, 2H), 4.16–4.19 (m,
1H), 4.35–4.39 (m, 1H), 5.32 (s, 1H), 6.94 (m, 4ArH), 7.05
(d, 2ArH), 7.24 (m, 4ArH), 7.36 (d, 2ArH), 7.85 (br s,
NH); 13C NMR (CDCl3) d 9.24, 26.95, 27.96, 28.41, 29.50,
29.76, 30.29, 30.35, 30.35, 30.41, 30.50, 30.57, 33.60, 36.83,
37.95, 40.35, 41.06, 47.86, 53.14, 53.22, 57.01, 58.30, 61.59,
63.35, 67.19, 83.77, 115.96, 116.24, 121.45, 129.72, 129.83,
129.94,137.98, 138.32, 139.46, 139.50, 161.94, 165.19,
166.05, 174.61, 175.90; m/z ESI (M+Na) 897.492111.
28. Mp = 146–147 °C. 1H NMR (CDCl3) d 1.44–1.51 (m, 2H),
1.62–1.78 (m, 7H), 2.34–2.70 (m, 16H), 2.87 (dd,
J = 4.5 Hz, 1H), 3.12 (q, J = 7.11 Hz, 1H), 3.57 (t,
J = 5.85 Hz, 2H), 4.29 (m, 1H), 4.84 (m, 1H), 5.34 (s,
1H), 5.80 (s, 1H), 6.91 (s, 1H), 7.0 (m, 4ArH), 7.09 (d,
2ArH), 7.27 (m, 4ArH), 7.51 (d, 2ArH), 8.55 (br s, NH);
13C NMR (CDCl3) d 15.23, 25.76, 27.97, 28.16, 33.00,
36.78, 40.50, 53.10, 53.43, 55.73, 57.75, 60.22, 61.65, 65.81,
66.83, 82.47, 115.09, 115.37, 119.97, 128.49, 128.59,
128.68, 136.34, 137.52, 137.74, 137.78, 160.47, 163.73,
164.29, 171.91; m/z ESI (M+Na) 714.32345.