Universal polyethylene glycol linkers for attaching receptor ligands to quantum dots

Article abstract of DOI:10.1016/j.bmcl.2006.09.031

Biologically active small molecule derivatives that can be conjugated to quantum dots have the promise of revolutionizing fluorescent imaging in biology. In order to achieve this several technical hurdles have to be surmounted, one of which is non-specifi

Full text of DOI:10.1016/j.bmcl.2006.09.031

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6262–6266
Universal polyethylene glycol linkers for attaching receptor
ligands to quantum dots
Ian D. Tomlinson,a Anthony P. Gies,a Paul J. Gresch,b Joel Dillard,a Rebecca L. Orndorff,a
Elaine Sanders-Bush,b David M. Herculesa and Sandra J. Rosenthala,b,
*
aDepartment of Chemistry, Vanderbilt University, Station B, 351822, Nashville, TN 37235-1822, USA
bDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA
Received 4 August 2006; revised 2 September 2006; accepted 7 September 2006
Available online 26 September 2006
Abstract—Biologically active small molecule derivatives that can be conjugated to quantum dots have the promise of revolutionizing
fluorescent imaging in biology. In order to achieve this several technical hurdles have to be surmounted, one of which is non-specific
adsorption of quantum dots to cell membranes. Pegylating quantum dots has been shown to eliminate non-specific binding. Con-
sequently it is necessary to develop a universal synthetic methodology to attach small molecule ligands to polyethylene glycol. These
pegylated small molecules may then be conjugated to the surfaces of quantum dots. Ideally this universal strategy should be adapt-
able and be applicable to PEG chains of varying lengths. This paper describes the development of one such methodology and the
synthesis of a pegylated derivative of the known 5HT2 agonist 1-(2-aminopropyl)-2,5-dimethoxy benzene. This compound was test-
ed and found to be an agonist for the 5HT2A and 5HT2C receptor having EC50 values of 250 and 50 nM, respectively.
Ó 2006 Elsevier Ltd. All rights reserved.
Since their introduction into biological imaging in
19981,2 quantum dots have shown great promise as fluo-
rescent imaging agents. Their unique fluorescent proper-
ties include narrow emission spectra, photostability, and
high quantum yields making them superior to conven-
tional organic fluorescent dyes.3–9 To have biologically
specificity the surfaces of quantum dots are modified
with biologically active molecules such as proteins,10,11
nucleic acids,12–14 peptides,15–18 small molecules,19–23
and antibodies.24–29 Studies have also reported their
use in vitro30 and in vivo. For example, quantum dots
have been used as medical imaging agents to label senti-
nel lymph nodes thus eliminating the requirement for
radioactive tracers.31
silinization,33 encapsulation in amphiphilic polymers,26
and encapsulation in proteins such as avidin and strep-
tavidin.26 Quantum dots encapsulated in modified
amphiphilic polyacrylamide (AMP) are extensively used.
This polymer has carboxylic acid functionalities that can
be conjugated to ligands using coupling reagents such as
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDC). We have reported that the non-specific
adsorption of AMP dots to cellular membranes may be
reduced by pegylating the surfaces of the AMP dots.34
In order to take advantage of this PEG/AMP strategy we
have developed a synthetic methodology to synthesize
PEG derivatives that may be attached to small organic
molecules or peptides and subsequently conjugated to
AMP dots. We have designed this methodology to be
generic and applicable to a wide range of PEG lengths
as we have reasoned that the length of the PEG chain
may be important in ligand binding. In this letter we re-
port the synthesis and characterization of a universal
PEG linker. We demonstrate this strategy for 1-(2-
aminopropyl)-2,5-dimethoxy benzene (1), a compound
that has high affinity for 5HT2A and 5HT2C receptors.
In order to use quantum dots as imaging agents in biol-
ogy a number of physical properties are required. These
include colloidal stability in saline, high quantum yields,
and low non-specific adsorption to cellular membranes.
A number of strategies have been developed to achieve
these goals; these include encapsulating in micelles,32
Keywords: Nanocrystals; Quantum dots; Polyethylene glycol; 5HT2A
agonist.
*
In early work we synthesized a derivative of the known
5HT2A agonist (1). An alkyl linker arm was attached to
Corresponding author. Tel.: +1 615 322 2633; fax: +1 615 343
1234; e-mail: sandra.j.rosenthal@vanderbilt.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.031

I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6262–6266
6263
NH2
NH2
O
O
O
O
(1)
(2)
SH
the aromatic ring resulting in 6-(2,5-dimethoxy-4-(2-
aminopropyl)phenyl)hexylthiol (2). The ability of (2)
to initiate the production of inositol pyrophosphate in
stably transfected LLC cells was measured and found
to be 88 nM for the 5HT2A receptor. The synthetic route
used to synthesize this ligand is described in an earlier
publication.35
tert-butyl 1-(4-(6-aminohexyl)-2,5-dimethoxyphenyl)-
propan-2-ylcarbamate (4) was synthesized by converting
the intermediate (5) to (4) in a 73% yield using hydrazine
monohydrate. tert-Butyl 1-(4-(6-(1,3-dioxoisoindolin-
2-yl)hexyl)-2,5-dimethoxyphenyl)propan-2-ylcarbamate
(5) was synthesized using two different methods: in one
method tert-butyl 1-(4-(6-hydroxyhexyl)-2,5-dimethoxy-
phenyl)propan-2-ylcarbamate (6) was converted to the
intermediate (5) using a Mitsonubi reaction giving (5)
in a 67% yield. Alternatively 6-(2,5-dimethoxy-4-(2-
[N-(tert-butoxycarbonyl)aminopropyl]phenyl)hexylbro-
mide (7) was reacted with potassium phthalimide giving
(5) in a 40% yield. These compounds were characterized
To reduce non-specific adsorption to cell membranes
we have designed an analog of (2) that incorporates
a PEG chain (3). This PEG chain is amino terminated
and may be conjugated to the AMP surface of quan-
tum dots.
1
by H NMR and 13C NMR.
NH2
The polyethylene glycol linker arm was synthesized from
a commercially available polyethylene glycol and the
synthetic methodology used is shown in Scheme 2. All
the pegylated derivatives were characterized by NMR
and MALDI mass spectrometry. For our purposes we
selected PEG 600 which was initially reacted with
phthalimide using a Mitsonubi reaction giving the mono
substituted product (8) in a 40% yield. The terminal
hydroxyl group was oxidized using 70% nitric acid at
80 °C giving the desired carboxylic acid (9) in a 99%
yield.
O
O
HN
O
Compound 4 was coupled to the PEG chain and the
methodology used is outlined in Scheme 3.
O
NH2
n
O
(3)
The monophthalimide derivative of the PEG 600 car-
boxylic acid (9) was coupled to 0.85 g of (4) using a
DCC coupling in methylene chloride to give 1.1 g of
the phthalimido protected PEG derivative of (10). The
phthalimide was removed by dissolving (10) in ethanol
and adding hydrazine monohydrate. This gave 0.85 g
of (11). The t-Boc-protecting group was removed by stir-
ring a solution containing 0.1 g of (11) dissolved in
The synthetic methodology used to synthesize our func-
tionalized PEGs and attach them to our ligand is outlined
in Schemes 1–3. The synthesis of the t-Boc-protected
5HT2 agonist derivative is shown in Scheme 1.

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I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6262–6266
O
O
O
O
O
O
NH
O
NH
O
NH
O
O
O
(ii)
(i)
O
(7)
N
O
O
OH
(6)
Br
(5)
(iii)
O
O
O
NH
O
(4)
NH
2
Scheme 1. Reagents: (i) Phthalimide, DIAD, PPh3; (ii) Potassium phthalimide; (iii) hydrazine hydrate.
˚
tum dot attachment to ligand of ꢀ46 A, indicating that
when the ligand is bound to the AMP dot the ligand is
well removed from the AMP surface.36 Shorter and long-
er PEG chains were also observed in MALDI however
these were not the predominant species.
O
O
O
OH
N
O
(i)
O
OH
+
NH
n
HO
O
O
n
(8)
(ii)
O
Compound 3 was tested for its ability to initiate Phos-
phoinositide hydrolysis, which was determined in
NIH3T3 cell lines stably expressing 5-HT2A or 5-HT2C
receptors as described by Barker et al.37 Compound 3
stimulated phosphoinositide hydrolysis in NIH 3T3 cells
expressing the rat 5-HT2A receptor with an EC50 of
250 nM. In NIH 3T3 cells expressing the rat 5-HT2C
receptor, the EC50 of compound 3 was 50 nM. Thus,
compound 3 demonstrates agonist properties at the
5-HT2A and 5-HT2C receptor.
O
O
OH
N
O
n
O
O
(9)
Scheme 2. Reagents: (i) PPh3, DIAD; (ii) 70% HNO3.
methylene chloride with trifluoroacetic acid resulting in
0.09 g of (III).
One concern when conjugating positively charged small
molecules to the surface of negatively charged AMP
dots is the possibility of electrostatic interactions caus-
ing the ligand to fold over. We have studied similar
systems where this was not any apparent problem. In
one instance serotonin was conjugated to carboxylic
acid coated dots through a short linker.20 Electrostatic
The MALDI spectrum of compound 3 is shown in
Figure 1. The peaks shown indicate that there are differ-
ent length PEG chains in the polydispersed PEG 600 con-
jugated to the agonist. As can be seen the predominant
masses are 902.6, 946.6, and 990.7 which correspond to
n = 11, 12, and 13. An AM1 optimization of an entirely
trans isomer with n = 12 renders a total length from quan-

I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6262–6266
6265
ligand that is further removed on a longer PEG should
have weaker interactions with the dots’ surface.
O
O
H2N
O
OH
N
O
O
+
n
O
N
H
O
O
In conclusion we have developed a universal strategy for
synthesizing amino-terminated PEGs that may be at-
tached to agonists and antagonists and conjugated to
the surfaces of AMP-coated quantum dots. This chemis-
try is generic and may be used with a wide range of PEG
chain lengths and a number of different agonists or
antagonists. When 1-(2-aminopropyl)-2,5-dimethoxy
benzene was conjugated to PEG chains biological activ-
ity was retained and we hope to use such systems in fu-
ture imaging applications.
O
(4)
(9)
(i)
O
H
N
O
O
N
O
n
O
O
O
O
(10)
N
O
H
(ii)
H
N
O
O
H2N
O
n
Acknowledgments
O
(11)
O
O
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 help-
ful advice during the course of this study. This work was
supported by grants from the National Institutes of
Health (RO1EB003728-02 and GM72048-02) and an
EBS Grant (RO1 MH34007-25).
N
O
H
(iii)
H
O
N
O
H2N
O
n
O
O
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NH2
(3)
Scheme 3. Reagents: (i) DCC, NHS; (ii) Hydrazine; (iii) trifluoroacetic
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