Controlling the Architecture, Coordination, and Reactivity of Nanoparticle Coating Utilizing an Amino Acid Central Scaffold

Article abstract of DOI:10.1021/jacs.5b10359

We have developed a versatile strategy to prepare a series of multicoordinating and multifunctional ligands optimized for the surface-functionalization of luminescent quantum dots (QDs) and gold nanoparticles (AuNPs) alike. Our chemical design relies on the modification of l-aspartic acid precursor to controllably combine, through simple peptide coupling chemistry, one or two lipoic acid (LA) groups and poly(ethylene glycol) (PEG) moieties in the same ligand. This route has provided two sets of modular ligands: (i) bis(LA)-PEG, which presents two lipoic acids (higher coordination) appended onto a single end-functionalized PEG, and (ii) LA-(PEG)₂ made of two PEG moieties (higher branching, with various end reactive groups) appended onto a single lipoic acid. These ligands are combined with a new photoligation strategy to yield hydrophilic and reactive QDs that are colloidally stable over a broad range of conditions, including storage at nanomolar concentration and under ambient conditions. AuNPs capped with these ligands exhibit excellent stability in various biological conditions and improved resistance against NaCN digestion. This route also provides compact nanocrystals with tunable surface reactivity. As such, we have covalently coupled QDs capped with bis(LA)-PEG-COOH to transferrin to facilitate intracellular uptake. We have also characterized and quantified the coupling of dye-labeled peptides to QD surfaces using fluorescence resonance energy transfer interactions in QD-peptide-dye assemblies.

Full text of DOI:10.1021/jacs.5b10359

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Article
Controlling the Architecture, Coordination and Reactivity of
Nanoparticle Coating Utilizing an Aminoacid Central Scaffold
Naiqian Zhan, Goutam Palui, Anshika Kapur, Valle Palomo, Philip E. Dawson, and Hedi Mattoussi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b10359 • Publication Date (Web): 30 Nov 2015
Downloaded from http://pubs.acs.org on December 3, 2015
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Controlling the Architecture, Coordination and Reactivity of Nanoparticle
Coating Utilizing an Aminoacid Central Scaffold
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Naiqian Zhan,1 Goutam Palui,1 Anshika Kapur,1 Valle Palomo,2 Philip E. Dawson,2 and Hedi
Mattoussi1
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1Florida State University, Department of Chemistry and Biochemistry, Tallahassee, FL 32306
2The Scripps Research Institute, Department of Chemistry, La Jolla, CA 92037
Keywords: quantum dots, gold nanoparticles, surface functionalization, aspartic acid, coupling
chemistry, lipoic acid, polyethylene glycol, multi-coordination, imaging
Abstract
We have developed a versatile strategy to prepare a series of multicoordinating and
multifunctional ligands optimized for the surface-functionalization of luminescent quantum
dots (QDs) and gold nanoparticles (AuNPs) alike. Our chemical design relies on the modification
of L-aspartic acid precursor to controllably combine, through simple peptide coupling
chemistry, one or two lipoic acid (LA) groups and poly(ethylene glycol) (PEG) moieties in the
same ligand. This route has provided two sets of modular ligands: bis(LA)-PEG which presents
two lipoic acids (higher coordination) appended onto a single end-functionalized PEG, and (ii)
LA-(PEG)2 made of two PEG moieties (higher branching, with various end reactive groups)
appended onto a single lipoic acid. These ligands were combined with a new photoligation
strategy to yield hydrophilic and reactive QDs that are colloidally stable over a broad range of
conditions, including storage at nanomolar concentration and under ambient conditions. AuNPs
capped with these ligands exhibited excellent stability in various biological conditions and
improved resistance against NaCN digestion. This route also provided compact nanocrystals
with tunable surface reactivity. As such, we have covalently coupled QDs capped with bis(LA)-
PEG-COOH to transferrin to facilitate intracellular uptake. We have also characterized and
quantified the coupling of dye-labeled peptides to QD surfaces using fluorescence resonance
energy transfer interactions in QD-peptide-dye assemblies.
Email: mattoussi@chem.fsu.edu
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Introduction
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In the past decade, inorganic nanocrystals, specifically gold nanoparticles (AuNPs) and
semiconductor quantum dots (QDs), have generated great interest for applications in several
areas of biology and medicine.1-18 This stems from their unique sets of physical and chemical
properties that exhibit size-, shape- and composition-dependence. For example, metallic AuNPs
show size- and shape-dependent Surface Plasmon Resonance (SPR) absorption ranging from
the visible to the near-infrared (NIR).19-24 Similarly, some of the properties of semiconductor
QDs including broad excitation, narrow and tunable emission across the visible and near-IR
spectrum, high two-photon action cross-section and superior photochemical stability account
for their widespread applications as biological tagging and sensing agents.3,7,25-29 However,
typical synthesis of high quality QDs (via “hot injection” routes) with narrow size distribution
and control over size and core crystallinity provides nanocrystals that are capped with
hydrophobic organic ligands.30-36 These materials are exclusively soluble in hydrophobic
solvents (such as toluene or hexane); this limits one’s ability to integrate them with
biomolecules, or introduce them into live cells. Therefore, an additional surface-modification
with tailor-made ligands is required to render the nanocrystals stable in buffer media and
biocompatible.14,37-39
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Several strategies including silica coating, encapsulation and ligand exchange have been
reported for preparing biocompatible QDs.40-53 Among those routes, ligand exchange which
relies on the substitution of the native surface cap with hydrophilic coordinating ligands offers a
few key advantages. This strategy is easy to implement and provides compact nanocrystals in
aqueous media. It also permits easy introduction of specific reactive functionalities on the
nanocrystal surfaces, for further modification with target biomolecules.41,45,46,49,54-57 Several
modular ligands bearing thiol, amine, pyridine and imidazole as anchoring groups have been
recently documented in the literature.58-61 Among these, multidentate thiolated ligands, such
as derivatives of dihydrolipoic acid (DHLA), provide enhanced colloidal stability of QDs (e.g.,
CdSe-ZnS) in aqueous media compared with those presenting monodentate coordinating
groups, due to the strong affinity of thiol to the zinc-rich QD surface and higher coordination of
dithiol groups. Over the past decade, a variety of DHLA-based ligands have been synthesized
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and tested, confirming the benefits of cooperative coordination onto the ZnS-overcoated
QDs.46,50,51,60,62-65 The enhanced binding affinity of multithiol-appended ligands to AuNPs and
AuNRs have also been reported.66-68 To further exploit these effects, several groups have
explored the possibility of using polymeric ligands instead, even though these can increase the
hydrodynamic size of nanoparticles in buffer media.41,46,48,55,61 One of the challenges in
designing the ligands (either polymeric or molecular scale) is the versatility and scalability of the
synthetic scheme. Specifically, factors that need to be taken into consideration include the
design of ligands with multiple functionalities and the use of versatile and scalable reaction
schemes.
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Here, we report the design and synthesis of two sets of multicoordinating ligands that
are suitable for capping both QDs and AuNPs. The present synthetic scheme starts from L-
aspartic acid to develop a versatile platform that allows controllable coupling of one or more
lipoic acid (LA) groups, one or more polyethylene glycol (PEG) moieties, along with terminal
reactive groups, yielding molecular-scale ligands with various architectures and selective
reactivity. We have prepared a series of ligands presenting either one PEG chain appended with
two lipoic acid (e.g., bis(LA)-PEG), or two PEG chains attached onto one lipoic acid (e.g., LA-
(PEG)2). The chemical structures of the ligands described in this study are shown in Figure 1.
This synthetic route provides high reaction yield at each step, and the ligand synthesis can be
easily scaled up. We have applied these ligands to cap AuNPs and luminescent QDs.
Additionally, these ligands are fully compatible with a mild photoligation strategy to promote
the in-situ ligand exchange and phase transfer of hydrophobic QDs to buffer media.59,69,70 The
nanocrystals ligated with bis(LA)-PEG exhibit remarkable colloidal stability over a broad range of
biological conditions; for instance, AuNPs capped with bis(LA)-PEG show improved resistance to
sodium cyanide digestion, compared to dithiol-capped nanoparticles. Incorporation of acid or
amine groups in the ligand coating permits covalent conjugation of a specific protein or dye-
labeled peptide to the QDs using common bioconjugation strategies. In particular, we show
that QDs coupled to transferrin facilitate efficient intracellular uptake of QDs, while QD-peptide
conjugation has been confirmed by quantifying the fluorescence resonance energy transfer
interactions measured for QD-peptide-dye assemblies.
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Results and Discussion
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Ligand Design. One key feature of our design is the use of a single precursor, the aminoacid L-
aspartic acid to prepare the various ligands described in this report. The ability to selectively
activate one or two groups in the aspartic acid allows one to build up the desired structure,
while controlling the nature and number of coordinating groups as well as the number of
hydrophilic PEG moieties introduced in the same ligand. Additionally, the various synthetic
steps rely mainly on conventional peptide coupling chemistry (e.g., BOC protection and
deprotection under acidic conditions along with carbodiimide chemistry). Thus, our design
allows the introduction of several reactive groups (e.g., -N3, -NH2,-COOH) in the ligands and
provides high reaction yield at each step. Several ligand architectures, including bis(LA)-PEG
(higher coordination ligands) and LA-(PEG)2 (ligands with higher PEG branching) have been
prepared (see Figure 1). The present route for preparing bis(LA)-appended ligands is simpler
than the Michael addition reaction we have previously employed and is more effective for
introducing terminal reactive groups in the ligands.64 Furthermore, comparison between NP
coating with either bis(LA)-PEG or LA-(PEG)2 has allowed us to explore the effects of
coordination vs steric hindrance on the surface ligand density. For LA-(PEG)2, one can combine
terminally-inert PEG and PEG-appended with a reactive group within the same ligand by
coupling the NH2-PEG-OCH3 and NH2-PEG-N3 sequentially onto the Boc-aspartic acid. A few
additional precautions have to be applied, nonetheless. The use of Boc-anhydride as an amine
protection eliminates issues of cross coupling, while selective mono coupling is achieved by
controlling the molar ratio of DCC (coupling reagent) and Boc-aspartic (e.g., a molar ratio of
1.1:1 was used in the scheme shown in Figure 1A, step ii). The use of LA-ethylenediamine
permits the introduction of lipoic acid via DCC coupling onto COOH groups in the aspartic acid
(Figure 1A, step iii). To functionalize the bis(LA)-PEG ligands, NH2-PEG1000-N3 is substituted for
NH2-PEG750-OCH3; transformation of the azide to amine and acid follows conventional chemical
modifications.54 The synthesis of LA-(PEG)2 ligands is slightly simpler, as coupling of two PEG
moieties could be carried in the same step as shown in Figure 1C and D. Finally, we should note
that the chiral nature of the L-aspartic acid, with its off plane arrangements of the three
reactive arms, yields ligands where the anchoring groups and the hydrophilic/reactive
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functionalities do not lay in the same plane. This offers reduced steric hindrance and may
improve the ligand packing on the NP surfaces.
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It should be noted that some of the intermediate compounds (precursors), such as
compound 4’ and compound 7’ can be used as capping ligands for QDs or AuNPs. Indeed these
two compounds provide two commonly used reactive groups, amine and azide, that can be
further modified. For example, compound 4’ can potentially allow the attachment of target
small molecules (redox complexes) very close to the NP surface. Similarly, compound 7’
combines both azide and amine on the same ligand, which can permit dual targeting of the NPs
with different biomolecules, or transformation of the amine to acid, yielding azide/acid
functionalized NPs.
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Ligand Exchange and Optical Characterization of the Nanocrystals. The prepared ligands are
compatible with photoligation as means of promoting in-situ cap exchange and phase transfer
of TOP/TOPO-QDs to polar and buffer media.69 This strategy exploits the photochemical
transformation of lipoic acid in the presence of UV irradiation (~ 330-360 nm) and starts with
the oxidized form of the ligands, eliminating the need for chemical reduction of the LA groups
prior to phase transfer.69 Furthermore, this scheme is mild towards sensitive groups such as
azide and aldehyde, two functions that are highly desirable in bio-orthogonal chemistry. It is
also compatible with other LA-based hydrophilic ligands such as LA-zwitterion.65,69
The photoligation was applied to cap exchange a few different size QDs with the new
ligands, as shown in Figure 2. The absorption and PL spectra collected from the four sets of QDs
before and after phase transfer to DI water were essentially identical, indicating that the
photoligation strategy did not alter the photophysical properties of the nanocrystals (Figure 2).
Similar observations were collected for QDs photoligated with LA-(PEG-OCH3)2 ligands. Phase
transfer of AuNPs was more straightforward as it did not require chemical reduction or photo-
irradiation of the ligand prior to coating of the NPs. The oxidized ligands (i.e., bis(LA)-PEG and
LA-(PEG)2) were used; reduction of the dithiolanes upon coordination onto the Au surfaces is
expected to take place.71 Figure 2d shows that the SPR peak in the absorption spectra collected
from dispersions of AuNPs before and after phase transfer are essentially identical, indicating
no change in the size or integrity of the nanoparticles after ligand exchange.
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Colloidal Stability Tests Applied to QDs and AuNPs. We focused on the colloidal stability of
both types of materials phase transferred using bis(LA)-PEG as these ligands are expected to
provide stronger binding onto the metal-rich surfaces of the nanocrystals. Tests carried out
using LA-(PEG)2 were used as reference. Colloidal stability tests of QDs photoligated with
bis(LA)-PEG-OCH3 were carried out in the presence of added excess NaCl, cell growth media,
acidic and basic pHs; additionally, we probed the long term stability of dispersions at very low
concentrations stored at room temperature and under light exposure. Colloidal stability is most
critical for applications in biology where media rich in reducing agents and salts are used, and
rather low nanocrystal concentrations are needed for imaging and sensing.64 The fluorescence
images in Figure 3a indicate that QDs photoligated with bis(LA)-PEG-OCH3 stayed
homogeneously dispersed with no sign of aggregation over the pH range 3-14 for at least 6
months of storage at 4 ˚C (duration of the test). These dispersions also remained stable in the
presence of 1M, 2M NaCl and when dispersed in 50%, 100% cell growth media. Stability tests of
these bis(LA)-PEG-OCH3-QD dispersions at nanomolar concentration were carried by visual
examination combined with tracking of the PL emission with storage time. The progression of
the fluorescence intensity collected from 50 nM and 100 nM QD dispersions was tracked over a
four-week period (see Figure 3d). Both dispersions stayed homogeneous and remained
fluorescent for around 2 months (test period), though the PL signal progressively decayed to ~
50 % of its initial value. This is promising compared to data collected for dispersions of QDs
capped with monothiol-PEG and even DHLA-PEG ligands, where PL is strongly reduced under
similar storage conditions.45,70 The result is attributed to the higher coordination of the
photoligated bis(LA)-PEG ligands onto the QDs.
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Similarly, bis(LA)-PEG-OCH3 was found to provide AuNPs with excellent long-term
colloidal stability over the pH range 2 to 14, in the presence of excess NaCl and in cell growth
media for over 1 year of storage under ambient conditions (Figure 4). Additional tests
compared the stability of AuNPs ligated with either LA-(PEG)2 or bis(LA)-PEG ligands (lower
coordination and larger spatial extension, and vice versa) against NaCN digestion. Cyanide
anions (CN-1) are highly reactive towards metal surfaces and can digest AuNP cores, forming
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Au(CN)2 complexes in the medium.68,72 This results in progressive loss of the plasmonic
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absorption feature.68 This test was applied to AuNPs (RH = 10 nm) ligated with LA-PEG-OCH3,
LA-(PEG-OCH3)2 and bis(LA)-PEG-OCH3, and provided a side-by-side comparison of the effects of
coordination, ligand size and spatial extension of the PEG moieties on the NPs stability to NaCN
digestion. Aliquots of 6.2 M NaCN solution (5 µL) were added to dispersions of all three ligated-
AuNPs (using final AuNP and NaCN concentrations of 6.3 nM and 62 mM, respectively) and the
absorption spectra were collected every 20 min for periods ranging from 1.5 to 10 hours. Figure
4d shows that the absorption rapidly decreased for LA-PEG-OCH3-AuNPs to nearly baseline
values after 1.5 hours; the dispersion progressed from pinkish-red to completely colorless,
indicating the near complete digestion of the AuNP cores. In comparison, a slightly slower loss
in the plasmonic absorption (corresponding to a slower digestion) was measured for LA-(PEG-
OCH3)2-AuNPs, where background level was reached after 3.5 hours. The strongest resistance
was measured for dispersions of bis(LA)-PEG-OCH3-AuNPs, where nearly complete digestion of
the AuNPs was reached after 10 hours (see Figure 4f). We further assessed the rate of
decomposition by measuring the time-dependent decrease of the surface plasmon peak at 520
nm and fitting it to a first order exponential decay function of the form:
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t
tD
y  y0 e
,
(1)
Where tD designates the decay time and y0 is the absorbance value at t = 0, as shown in Figure
4g. The decay time (tD) extracted from fits to the data collected from the various AuNP samples
are: 56 min for LA-PEG-OCH3-AuNPs, 75 min for LA-(PEG-OCH3)2-AuNPs and 526 min for bis(LA)-
PEG-OCH3-AuNPs.68 We found that tD (bis(LA)-PEG-OCH3) is ~ one order of magnitude longer
than tD (LA-PEG-OCH3).
Colloidal Stability Against Ligand Desorption. It is understood that coordination of the ligands
on the QDs is not irreversible.14,41 There is equilibrium between bound and free ligands in the
dispersions, with stronger coordination producing lower dissociation constant and vice versa.
Phase transfer performed via ligand exchange is usually carried out in the presence of large
excess of the new ligands because the process is driven by mass action. Thus, following ligand
exchange dispersions are routinely purified by removing as much as possible the fraction of free
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solubilized ligands by applying precipitation using a solvent mixture when organic media are
used. For dispersions in buffer media, free ligands are removed by applying a few rounds of
concentration/dilution using a membrane centrifugal filtration device (with a defined cutoff
molecular weight, as described in the experimental section). This procedure relies on the
ability of centrifugal forces to extrude the solvent along with solubilized small molecules,
including ligands, through the membrane filter to counter the osmotic pressure in the
dispersion (due to a lower chemical potential of the mixture). If the procedure is excessively
applied, ligand desorption can shift the adsorption/desorption equilibrium between surface-
bound and free ligands, resulting in instability buildup and eventually aggregation of the
nanoparticles. When using DHLA and DHLA-based ligands for stabilizing QDs, we often apply
the above procedure 3-4 times, while finding that stickiness to membrane can take place when
more than 5 rounds are applied using ~ 2000 g for ~ 7-10 minutes.71 We tested the colloidal
stability of QDs photoligated with bis(LA)-PEG-OCH3 and AuNPs capped with the same ligand,
by extending the rounds of centrifugation/dilution applied to dispersions of both materials. We
found that the bis(LA)-PEG-OCH3 ligands significantly reduced the ligand desorption rate and
provided homogeneous and aggregate-free QD dispersions in buffer media even after 8 and 9
rounds of purification. Figure 5 shows that the absorption and emission spectra of QDs were
essentially unchanged between the first and eighth round. When the test was carried out using
AuNPs, additional 10 rounds of membrane centrifugal filtration were applied for LA-PEG-AuNPs
and bis(LA)-PEG-AuNPs dispersions following ligand exchange (data not shown). In both cases,
the AuNP dispersions stayed homogeneous, with no aggregate-buildup. This can be attributed
to the strong coordination between thiol/sulfur and Au atoms in the AuNP dispersions.
The above results combined are promising and further confirm that higher coordination
ligands bind stronger onto the QD surfaces, thus greatly improving the nanocrystal colloidal
stability in buffers under several conditions. They also clearly demonstrate that higher
coordination provides better resistance to AuNPs against sodium cyanide digestion. Our data
also show that higher PEG branching (i.e., LA-(PEG-OCH3)2) yielded slightly better protection of
the NPs compared to LA-PEG-OCH3 ligands, attributed to the denser PEG packing on the
nanoparticle surfaces.
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Intracellular Delivery of QD-Transferrin Conjugates. Our design can be combined with the use
of mixed ligand exchange to prepare QDs that present varying numbers of reactive groups. This
can be achieved by introducing (during the ligation step) a controllable fraction of -COOH, -NH2,
or N3-modified ligands along with the inert-terminated ones. The gel electrophoresis image in
Figure 6a shows that the gel mobility shift of the QDs depends on the nature and fraction of
terminally-modified ligands introduced during the phase transfer step.49 We utilized 540 nm-
emitting QDs photoligated with 15 % bis(LA)-PEG-COOH to carry out covalent coupling of
transferrin to the QDs via EDC/NHS coupling. The coupling reaction targeted available amines
on the protein surface. The formed conjugates were further tested for their biological activity
by incubating the QD-transferrin conjugates (150 nM) with HeLa cells at 37 ˚C for 1 hour;
transferrin is a glycoprotein that binds to specific receptors on the cell surface, promoting its
transport inside the cell via receptor-mediated endocytosis.73 Cells incubated with non-
conjugated QDs or with Texas Red-Transferrin respectively provided negative and positive
control experiments. A representative set of epifluorescent images, shown in Figure 6c (panels
A-D), indicate that an efficient intracellular uptake of the QD-transferrin conjugates has taken
place. The green QD fluorescence was mostly distributed in the perinuclear region of the cells;
the nuclei were counterstained with DAPI. Additionally, the fluorescence pattern of the QDs
was co-localized with that of Texas Red dye-labeled transferrin, indicating that the nanocrystals
were primarily distributed within the endosomal compartments. Conversely, we did not
observe any detectable fluorescence signal from the culture incubated with QDs only (control),
indicating the absence of nonspecific interactions of the QDs with the cell membranes (see
Figure 6c, panels E-H).
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QD-peptide-Cy3 Conjugates and FRET Analysis. Several QD-based sensors using energy or
charge transfer interactions (as transduction mechanisms) have been designed over the past
decade using QD-conjugates, to detect properties such as changes in the environment pH, the
presence of metal ions and for monitoring enzymatic activity.74-77 Designing of QD sensors
greatly benefits from the ease of stable surface and ease of coupling with biomolecules of
interest.
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We have probed the assembly of QD-peptide-dye conjugates formed by coupling the
COOH (on bis(LA)-PEG-QDs) with the peptide sequence shown in Figure 7a. The C-terminal
cysteine of the peptide was first reacted with maleimide-Cy3, yielding Cy3-labeled peptide,
then the N-terminal amine was further conjugated with COOH-functionalized QDs, via
EDC/NHS coupling; two fractions of carboxylic acid modified QDs (5% and 10%) were used. The
composite absorption spectra collected from dispersions of the purified conjugates show
contributions from the QDs and Cy3 dye; additionally, the dye contribution to the spectra
varied with the fraction of COOH-modified ligands used in the ligand exchange step, indicating
that the number of dyes per QD tracked the fraction of bis(LA)-PEG-COOH ligands used. Figure
7c also shows the emission spectra collected from the same dispersion. There is a progressive
loss in QD emission combined with enhancement in the dye PL. Since the samples were excited
at 400 nm where direct excitation of the dye is minimal, we attribute the observed fluorescence
data to efficient resonance energy transfer between the QDs and bound Cy3, producing strong
quenching of the QD signal along with sizable sensitization of the dye fluorescence (see Figure
7c). This was further supported by the pronounced shortening in the QD PL radiative decay time
(see Supporting Information). Conversely, the absorption and fluorescence spectra collected
from dispersions of Cy3-peptide mixed with methoxy-functionalized-QDs (after purification)
showed only contribution from the QDs and none from the dye (data not shown). This
indicates that no conjugation to the dye-labeled peptide has taken place in the absence of
COOH groups on the QD surfaces.
The absorption data were combined with the extinction coefficients of Cy3 (1.5×105 M-
1cm-1 at λ = 555 nm) and green-emitting QDs (3.348×105 M-1cm-1 at λ = 350 nm), deduced from
size and cross-section absorption measurements reported in previous studies,78,79 to extract an
estimate for the number of Cy3 (n) attached to a QD. We measured n 4 and 12 for
conjugates prepared with 5% and 10% COOH-modified ligands, respectively.79 Similarly, analysis
of the deconvoluted fluorescence spectra within the Förster FRET model provided additional
estimate for the valence. Assuming a centro-symmetric QD-peptide-dye configuration where
acceptors are arrayed around the central donor at a fixed separation distance, the expression
for the quenching efficiency, En, is given by:80
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nR0
En 
(2)
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nR0  r6
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Where r represents the separation distance from the donor (QD) to the acceptors and R0 is the
Förster radius corresponding to En=1 = 0.5. Additional details on the FRET analysis is provided in
the Supporting Information.
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For our system, we used R0  52 Å, extracted from the experimental spectral overlap
and a QD value of ~18 %. We also used estimates for the QD radius 27 Å (core-shell), the
capping layer including a coiled PEG chain in good solvent conditions (end-to-end distance of
∼23 Å), a peptide segment of ∼11 Å and the size of the maleimide-dye ~ 5 Å, to extract a value
for the center-t-center separation distance r of  66 Å.70,81 Using this information and equation
2, we estimated that n 4 for 5% COOH-QDs and 11 for 10% COOH-QDs. These values are in
reasonable agreement with the values obtained from absorption spectra. The high FRET
efficiencies measured for our QD-peptide conjugates proves that our ligand design provides
compact QDs and QD-conjugates.
We should note that the peptide structure shown in Figure 7a includes a sequence
expected to be specifically recognized and cleaved by the enzyme matrix metalloproteinase
(MT1-MMP), an extremely important indicator of cancer in cell cultures and tissue. We will
pursue measurements of the kinetics of enzyme digestion of the dye-peptide substrate on the
QDs both in solution and in cancer cell lines, and hope to report on those findings in future
publications.
Conclusion
Starting from the aspartic acid, as a precursor, we synthesized several molecular scale multi-
functional PEG-based ligands that present two and four coordinating groups and varying
architectures.
Ligands
prepared
and
tested
include
bis(LA)-PEG
and
LA-(PEG)2. This design exploits the availability of two carboxyl and one amine groups in the
chiral L-aspartic acid and combines that with the use of BOC (tert-butyloxycarbonyl) protective
and carbodiimide chemistries to synthesize several capping ligands with controlled
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architecture, coordination and reactivity. The ligands were applied to cap AuNPs and QDs and
transfer them to buffer media. The resulting nanoparticles exhibited great long-term colloidal
stability over a broad range of conditions. We have also shown that the synthetic strategy
permits the attachment of reactive groups including azide, amine and carboxylic acid, on the
same ligand. This allowed conjugation of QDs with biomolecules (transferrin protein and
peptide). These were tested for cellular uptake and energy transfer interactions.
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QDs or AuNPs coated with these ligands would be greatly useful in sensing applications,
based on FRET and charge transfer (CT) interactions including sensing of soluble ions and
enzymatic activity. The hydrophilic NPs described here are also promising for intracellular
sensing and imaging where colloidal stability at very low concentrations combined with multi-
functionality is highly desired. The bis(LA)-PEG and LA-(PEG)2 ligands provide the means to
probe the effects of coordination versus steric interactions on the ligand density on inorganic
nanocrystals. We should emphasize that aminoacids are promising platforms for developing
novel organic ligands and the synthetic strategy applied here can be potentially applied to other
precursors, such as lysine, to provide ligands for stabilizing other inorganic nanoparticles.
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Materials. Poly(ethylene glycol) with molecular weight 600 and 1000 were purchased from
Acros Organics (Morris Plains, NJ). Poly(ethylene glycol) methyl ether (molecular weight of 750),
L-aspartic acid, lipoic acid (LA), N,N-dicylohexylcarbodiimide (DCC), 4-(N,N-
dimethylamino)pyridine (DMAP), di-tert-butyl dicarbonate (Boc2O), triphenylphosphine,
succinic anhydride, 4M HCl in dioxane, triethylamine, tetramethylamonium hydroxide (TMAH),
and organic solvents (chloroform, methanol, hexane, ethyl acetate, etc.) were purchased from
Sigma Chemicals (St. Louis, MO). Phosphate salts used for buffer preparation, NaCl, Na2CO3,
and Na2SO4 were also purchased from Sigma Chemicals. Hydroxybenzotriazole (HOBtˑH2O) was
purchased from Alfa Aesar (Ward Hill, MA). Column chromatography purification was
performed using silica gel (60 Å, 230−400 mesh, from Bodman Industries, Aston, PA). Sulfo−Cy3
maleimide dye and PD-10 column were purchased from GE Healthcare (Piscataway, NJ).
Deuterated solvents used for NMR experiments were purchased from Cambridge Isotope
Laboratories Inc. (Andover, MA). The chemicals and solvents were used as received unless
otherwise specified. All synthetic reactions described here were carried out under nitrogen
atmosphere, unless otherwise specified. Standard nitrogen vacuum manifold technique was
used to carry out chemical reactions when needed, and air sensitive materials were handled in
an MBraun Labmaster 130 glovebox (Stratham, NH).
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Instrumentation. H NMR spectra were collected using a Bruker SpectroSpin 600 MHz
spectrometer (Bruker SpectroSpin, Billerica, MA). A Shimadzu UV−Vis absorption
spectrophotometer (UV 2450 model, Shimadzu, Columbia, MD) was used to measure the UV-vis
absorption spectra from the various dispersions, while the fluorescence spectra were collected
on a Fluorolog-3 spectrometer (Jobin Yvon Inc., Edison, NJ) equipped with PMT and CCD
detectors. Solvent evaporation (to concentrate or dry samples) was carried out using a lab-scale
Buchi rotary evaporator R-215 (New Castle, DE). The photoligation experiments were carried
out using a UV photo-reactor Model LZC-4V (Luzchem Research Inc., Ottawa, Canada). Gel
electrophoresis experiments were performed using a 1% agarose gel. Samples were prepared
by diluting dispersions of QDs or QD-conjugates in a TBE buffer (100 mM Tris, 83 mM boric acid,
1 mM EDTA, pH8.4), then mixing with loading buffer (2.5% ficoll 400, 1.6 mM Tris-HCl, 8.3 mM
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EDTA, pH7.4). Aliquots of these dispersions were loaded into the agarose gel and run for 20 min
using an applied voltage of 8.0 V/cm. Gel images were captured in the fluorescence mode
using a Gel Doc XR+ System.
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Ligand Synthesis. The set of ligands prepared in this study, either made of one PEG moiety
appended with two lipoic acid anchoring groups (bis(LA)-PEG), or made of two PEG moieties
attached onto one lipoic acid (LA-(PEG)2) were all prepared starting from the aminoacid aspartic
acid as precursor. Our synthetic route also allows easy functionalization of the ligands with
various terminal reactive functions such as azide, amine and acid groups. The poly(ethylene
glycol) precursors used for the synthesis, namely NH2-PEG750-OCH3 and NH2-PEG1000-N3 were
prepared and purified following protocols described in our previous reports.54,71 These ligands
were applied without pre-reduction of dithiolane moiety to cap exchange with oleylamine-
AuNPs. They were, however, combined with the photochemical modification of lipoic acid to
achieve in-situ ligand exchange and phase transfer of TOP/TOPO-capped QDs. Figure 1 shows
the chemical structures along with the synthetic steps used for preparing of the various ligands.
Below, we detail the synthetic protocols used for preparing these ligands.
Compound 1 (Boc-Asp). In a 500 mL one-neck round bottom flask, aspartic acid (Asp, 4 g, ~30
mmol), 1,4-dioxane (120 mL) and H2O (60 mL) were mixed yielding a heterogeneous solution.
An aqueous solution of NaOH (1 M) was added to the mixture with constant stirring until the
solution became homogeneous and clear indicating that the aspartic acid was completely
dissolved. The solution was cooled using an ice-bath, then di-tert-butyl dicarbonate (Boc2O, 7.2
g, ~33 mmol) dissolved in 1,4-dioxane (20 mL) was added dropwise. The reaction mixture was
then stirred at room temperature overnight. Once the reaction was complete, the solvent was
partially evaporated, using a rotary evaporator, to a final volume of ~30 mL. Then, EtOAc (20
mL) was added and the water layer was acidified under ice-cold conditions using an aqueous
solution of KHSO4 to pH2. The solution mixture was transferred to a separatory funnel and the
product was extracted using EtOAc (40 mL, 3 times). The organic layers were combined and
dried over Na2SO4. The solvent was evaporated using rotary evaporator, and further vacuum
drying was applied overnight to yield the compound as white solid (~6 g); reaction yield ~85%.
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1H NMR (600 MHz, DMSO-d6): δ 1.38 (s, 9H), 2.51-2.55 (m, 1H), 2.65-2.69 (m, 1H), 4.24-4.28 (m,
1H), 7.05-7.07 (d, 1H, J = 12 Hz), 12.5 (s, 2H).
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Compound 2 (Boc-Asp-PEG-OCH3). Boc-Asp, compound 1 (3.1 g, 13.3 mmol) was dissolved in
EtOAc (30 mL) in a 100 mL two-neck round bottom flask equipped with a magnetic stirring bar.
The solution was purged with N2 and cooled to ~ 0 ˚C using an ice bath, then a solution of DCC
(3.01 g, 14.6 mmol) in EtOAc (30 mL) was added dropwise through an additional funnel over
~20 minutes. Once the addition was complete, the mixture was gradually warmed up to room
temperature and was continuously stirred for another 4 hrs. The white solid byproduct was first
removed using a filter paper and the solvent was evaporated to provide the DCC activated Boc-
aspartic acid compound as a white solid. This compound was mixed with NH2-PEG750-OCH3
(14.7 g, 20 mmol) and THF (100 mL) in a 250 mL three-neck round bottom flask and the solution
was refluxed at 66 ˚C overnight. The reaction mixture was cooled to room temperature, filtered
through a filter paper and chromatographed on a silica column (230-400 mesh). A one round
elution with CHCl3 was first applied to remove impurities, and the product was collected using a
mixture of 40:1 (vol/vol) CHCl3:MeOH as the eluent. After evaporating the solvent, light
yellowish oil was collected (~11 g); reaction yield ~90%. We should note that the mono coupling
of NH2-PEG-OCH3 and NH2-PEG-N3 onto Boc-aspartic acid yields a mixture of two isomers (one is
from amine coupling to the alpha-carboxylic acid and the other is from amine coupling to the
beta-carboxylic acid); these isomers are eluted together from a gel column, because they have
similar polarity. The final products were also collected as isomer mixtures and directly applied
for the attachment lipoic acid when synthesizing compound 3 and compound 6. In the synthesis
scheme detailed above, only one representative isomer is shown.
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1H NMR (600 MHz, CDCl3): δ 1.39 (s. 9H), 2.56-2.60 (m, 1H), 2.91-2.95 (m, 1H), 3.33 (s, 3H),
3.49-3.51 (m, 3H), 3.59-3.60(m, 64H), 4.43-4.47 (m, 1H), 5.75 (br, 1H), 6.97 (br, 1H).
Compound 3 (LA-PEG-OCH3). In a round bottom flask, compound 2 (7.1 g, 7.5 mmol) was mixed
with THF (50 mL) and cooled under ice cold condition. A mixture of DCC (2 g, 9.7 mmol) and
HOBtˑH2O (1.5 g, 9.8 mmol) dissolved in THF (15 mL) was added to the reaction flask dropwise
under N2 atmosphere with constant stirring. After the addition was complete, the solution was
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gradually warmed up to room temperature and stirred for 1 hour. LA-ethylenediamine (2.8 g,
11.3 mmol) dispersed in CHCl3 (5 mL) was added dropwise via a syringe; LA-ethylenediamine
was pre-synthesized following previous literature protocols.82 The reaction mixture was left
stirring at room temperature for two days, then filtered through a filter paper to remove the
white solid byproduct. The compound was then purified on a silica gel column in 2 steps:
impurities were first removed by elution with CHCl3.Then the compound was eluted using 30:1
(vol:vol) CHCl3:MeOH solvent mixture. Following evaporation of the solvent the product was
collected as a yellow oil (~ 6.7 g); reaction yield ~76%.
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1H NMR (600 MHz, CDCl3): δ 1.42-1.48 (m, 11H), 1.61-1.73 (m, 4H), 1.87-1.93 (m, 1H), 2.21-2.23
(t, 2H, J = 6Hz), 2.42-2.48 (m, 1H), 2.57-2.60 (m, 1H), 2.71-2.74 (m, 1H), 3.08-3.12 (m, 1H), 3.14-
3.18 (m, 1H), 3.37 (s, 3H), 3.39-3.45 (m, 4H), 3.52-3.55 (m, 5H), 3.63 (m, 65H), 4.45-4.49 (m,
1H), 6.20 (br, 1H), 6.91 (br, 1H), 6.99 (br, 1H).
Compound 4 (bis(LA)-PEG-OCH3). In a round bottom flask, 4 M HCl in 1,4-dioxane (15 mL) was
added to compound 3 (6.7 g, 5.7 mmol) under ice cold conditions and left stirring for 4 hours at
room temperature. The solvent was evaporated using rotary evaporator followed by dispersion
in H2O (70 mL). This aqueous solution was transferred to a separation funnel and washed with
diethyl ether (50 mL, two times). The aqueous layer was collected, basified using saturated
Na2CO3 to ~pH 9, and the compound was extracted using CHCl3 (50 mL, three times). The
solvent was finally evaporated to obtain the Boc-deprotected product (compound 4’, 5.5 g,
yield ~90 %). This Boc-deprotected compound (5.5 g, 5.1 mmol) was mixed with LA (1.6 g, 7.8
mmol) and DMAP (0.2 g, 1.6 mmol) in CHCl3 (50 mL) and the mixture was cooled to ~ 0 ˚C under
ice cold conditions. DCC (1.6 g, 7.8 mmol) dissolved in CHCl3 (15 mL) was added dropwise, then
the reaction was stirred for two days at room temperature. The white solid byproduct was
removed by filtration, and the chloroform layer was further washed with saturated sodium
carbonate solution (30 mL, two times) to remove excess unreacted lipoic acid. The solution was
concentrated and purified on a silica gel column using a 30:1 (vol:vol) CHCl3:MeOH as eluent.
After solvent evaporation, the product was finally collected as a yellow solid (~4.5 g); reaction
yield ~70%.
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1H NMR (600 MHz, CDCl3): δ 1.46-1.51 (m, 4H), 1.65-1.73 (m, 8H), 1.9-1.95 (m, 2H), 2.22-2.24 (t,
2H, J=6Hz), 2.26-2.28 (t, 2H, J=6Hz), 2.45-2.85 (m, 2H), 2.53-2.57 (m, 1H), 2.67-2.7 (m, 1H), 3.11-
3.18 (m, 2H), 3.18-3.22 (m, 2H), 3.39 (s, 3H), 3.44-3.47 (m, 4H), 3.55-3.59 (m, 8H), 3.65 (m,
65H), 4.74-4.77 (m, 1H), 6.80 (br, 1H), 6.90 (br, 1H), 7.40 (br,1H).
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Compound 5 (Boc-Asp–PEG-N3). Boc-aspartic acid (compound 1, 2.1 g, 9.0 mmol) was
dissolved in EtOAc (30 mL) using a 100 mL two-neck round bottom flask equipped with a
magnetic stirring bar. The solution was cooled using an ice bath, purged with N2, and DCC (2.04
g, 9.9 mmol) dissolved in EtOAc (20 mL) was added dropwise. The reaction was gradually
warmed up to room temperature and left stirring for 4 hours. The white solid byproduct was
first removed using a filter paper, followed by solvent removal using a rotary evaporator. The
obtained white powder was mixed with NH2-PEG1000-N3 (14 g, 13.5 mmol, dissolved in 100 mL
THF) and refluxed at 66 ˚C overnight. The product mixture was cooled to room temperature,
filtered through filter paper, dried over Na2SO4, concentrated using rotary evaporator and
chromatographed on a silica gel column. The pure product was collected using a solvent
mixture of 20:1 (vol:vol) CHCl3:MeOH for elution. A light yellow oil was collected after
evaporating the solvent (~ 10 g); reaction yield ~90 %.
1H NMR (600 MHz, CDCl3): δ 1.38 (s, 9H), 2.62-2.66 (m, 1H), 2.82-2.94 (m, 1H), 3.33-3.34 (m,
5H), 3.47-3.5 (m, 5H), 3.59 (m, 90H), 4.39-4.43 (m, 1H), 5.56 (br, 1H), 6.80 (br, 1H).
Compound 6 (LA-PEG-N3). Compound 5 (5 g, 4 mmol) and THF (40 mL) were mixed in a 100 mL
two-neck round bottom flask equipped with a magnetic stirring bar. A solution mixture of DCC
(1.1 g, 5.3 mmol), HOBtˑH2O (0.8 g, 5.2 mmol) and THF (10 mL) was added to the flask dropwise
under ice cold conditions. The mixture was stirred at room temperature for 1 hour, then LA-
ethylenediamine (1.48 g, 6 mmol) dissolved in CHCl3 (5 mL) was subsequently added dropwise.
The reaction mixture was left stirring at room temperature and under N2 atmosphere for two
days. Once the reaction was completed, the mixture was filtered using a filter paper (to remove
the white solid byproduct), and the solution was concentrated under vacuum using a rotary
evaporator, then purified on a silica gel column using a mixture of 30:1 (vol:vol) CHCl3:MeOH as
eluent. The product was collected as a yellow oil (~4.7 g); reaction yield ~79%.
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1H NMR (600 MHz, CDCl3): δ 1.42-1.46 (m, 11H), 1.63-1.69 (m, 4H), 1.86-1.92 (m, 1H), 2.29-2.31
(t, 2H, J = 6Hz), 2.42-2.47 (m, 1H), 2.71-2.74 (m, 1H), 2.75-2.78 (m, 1H), 3.07-3.11 (m, 1H), 3.14-
3.18 (m, 1H), 3.37-3.4 (m, 6H), 3.54-3.55 (m, 4H), 3.62 (m, 90H), 4.52-4.55 (m, 1H).
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Compound 7 (bis(LA)-PEG-N3). De-protection of the BOC group was applied to compound 6: 4
M HCl in 1,4-dioxane (15 mL) was added to 4 g (2.7 mmol) of compound 6 and the mixture was
stirred for 4 hours at room temperature. The purification was carried out following the same
procedure used in the synthesis of compound 4, as detailed above. This de-protected product
(compound 7’, 3 g, 2.17 mmol), LA (0.67 g, 3.25 mmol) and DMAP (0.08 g, 0.66 mmol) were
dissolved in CHCl3 (20 mL) followed by dropwise addition of a DCC solution in CHCl3 (0.67 g, 3.25
mmol, 10 mL) under ice cold conditions. The reaction mixture was purged with N2 and stirred
for 2 days at room temperature. A white solid byproduct was removed by filtration, the CHCl3
layer was washed with saturated Na2CO3 solution (15 mL, two times), then the solution mixture
was purified on a silica gel column using 30:1 (vol:vol) CHCl3:MeOH mixture as the eluent to
obtain the compound 7 as a yellow solid (~2.3 g); reaction yield ~ 67%.
1H NMR (600 MHz, CDCl3): δ 1.42-1.47 (m, 4H), 1.64-1.69 (m, 8H), 1.86-1.92 (m, 2H), 2.21-2.23
(t, 2H, J = 6 Hz), 2.24-2.26 (t, 2H, J = 6 Hz), 2.42-2.48 (m, 2H), 2.65-2.69 (m, 1H), 2.78-2.82 (m,
1H), 3.08-3.12 (m, 2H), 3.15-3.19 (m, 2H), 3.4-3.43(m, 5H), 3.51-3.56 (m, 6H), 3.63 (m, 90H),
4.70-4.75 (m, 1H), 6.90 (br, 1H), 7.10 (br, 1H), 7.40 (br, 1H).
Compound 8 (bis(LA)-PEG-NH2). Compound 7 (2.9 g, 1.86 mmol) was dissolved in THF (50 mL)
at room temperature with constant stirring; a slight heating may be required to ensure that the
compound is completely dissolved. Triphenylphosphine (0.73 g, 2.8 mmol) was added (at room
temperature) and the reaction mixture was stirred for 40 minutes under N2, followed by
addition of H2O (0.33 mL, 18.3 mmol), and the reaction mixture was further left stirring
overnight. Once the reaction was complete, the solvent was evaporated using a rotary
evaporator, then EtOAc was added to the residue and stirred with slight heating (~ 60 °C) to
dissolve the compound. The solution was transferred to a separatory funnel, to which 1 M HCl
(50 mL) was added. The organic layer was removed and the aqueous layer was further washed
with EtOAc (40 mL, 1 time) to remove the remaining impurities. Saturated Na2CO3 solution was
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added to the aqueous layer to basify the solution (~pH 9). The final product was extracted with
CHCl3 (50 mL, three times), dried over Na2SO4 and collected after evaporating the solvent as
yellow oil (~1.8 g); reaction yield ~ 62%.
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1H NMR (600 MHz, CDCl3): δ 1.42-1.47 (m, 4H), 1.63-1.68 (m, 8H), 1.85-1.89 (m, 2H), 2.18-2.20
(t, 2H, J = 6 Hz), 2.22-2.24 (t, 2H, J = 6 Hz), 2.42-2.48 (m, 2H), 2.53-2.56 (m, 1H), 2.75-2.79 (m,
1H), 2.83-2.87 (t, 2H, J = 6Hz), 3.08-3.12 ( m, 2H), 3.15-3.19 (m, 2H), 3.33-3.39 (m, 6H), 3.51-
3.56 (m, 6H), 3.63 (m, 90 H), 4.71-4.75 (m, 1H).
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Compound 9 (bis(LA)-PEG-COOH). bis(LA)-PEG-NH2 (compound 8, 1 g, 0.65 mmol), succinic
anhydride (0.13 g, 1.3 mmol), triethylamine (0.23 mL, 1.69 mmol) and CHCl3 (20 mL) were
mixed in a 100 mL one-neck round bottom flask. The mixture was stirred at room temperature
overnight under N2 atmosphere. The solvent was removed under vacuum and 1 M HCl (20 mL)
was added to the residue. The product was further extracted using CHCl3 (30 mL, three times).
The organic layers were combined, dried over Na2SO4, filtered through a filter paper, and the
solvent was evaporated, yielding the final product (compound 9) as a yellow oil (~0.63 g);
reaction yield ~ 60%.
1H NMR (600 MHz, CDCl3): δ 1.4-1.46 (m, 4H), 1.64-1.7 (m, 8H), 1.88-1.93 (m, 2H), 2.24-2.29 (m,
4H), 2.44-2.49 (m, 2H), 2.54-2.56 (m, 1H), 2.61-2.65 (m, 4H), 2.74-2.78 (m, 1H), 3.09-3.12 (m,
2H), 3.14-3.19 (m, 2H), 3.34-3.4 (m, 6H), 3.50-3.55 (m, 6H), 3.64(m, 90 H), 4.72-4.75 (m, 1H).
Compound 10 (Boc-Asp-(PEG-OCH3)2). Compound 1 (1 g, 4.29 mmol), NH2-PEG750-OCH3 (7.6 g,
10.34 mmol) and DMF (20 mL) were mixed in a 100 mL two-neck round bottom flask equipped
with a magnetic stir bar. The solution was stirred at room temperature until all the solid
materials were fully dissolved. DCC (1.9 g, 9.22 mmol) and HOBtˑH2O (1.41 g, 9.22 mmol)
dissolved in DMF (10 mL) were further added dropwise using a syringe under ice cold
conditions. Once the addition was complete, the mixture solution was warmed up to room
temperature and left stirring for two days under N2 atmosphere. The reaction mixture was
filtered through a filter paper (to remove white solid byproduct), and then CHCl3 (40 mL) was
added. This solution was further washed with 1 M HCl (20 mL, 1 time) and saturated Na2CO3 (20
mL, 1 time), dried by adding Na2SO4, and the solvent was evaporated using rotary evaporator.
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The residue was chromatographed on silica gel column with 25:1 (vol:vol) CHCl3:MeOH mixture
as eluent, yielding the product as a yellow oil (~6.2 g); reaction yield ~86 %.
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1H NMR (600 MHz, CDCl3): δ 1.39 (s, 9H), 2.51-2.54 (m, 1H), 2.8-2.83 (m, 1H), 3.33 (s, 6H), 3.4-
3.42 (m, 4H), 3.49-3.59 (m, 9H), 3.60 (m, 140H), 4.39 (m, 1H), 6.14 (br, 1H), 6.65 (br, 1H), 7.14
(br, 1H).
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Compound 11 (LA-(PEG-OCH3)2). The de-protection of compound 10 and purification of the
product were carried out following the same procedure as detailed above for compound 7. The
de-protected compound 10 (3 g, 1.91 mmol) was mixed with LA (0.47 g, 2.28 mmol) and DMF
(15 mL) in a 100 mL two-neck round bottom flask equipped with a magnetic stirring bar. In a
separate vial, a solution of DCC (0.47 g, 2.28 mmol), HOBtˑH2O (0.35 g, 2.28 mmol) in DMF (5
mL) was added dropwise to the reaction solution under ice cold conditions with constant
stirring. The reaction solution was gradually warmed up to room temperature and left stirring
for another two days to ensure the completeness of the reaction. After filtering off the white
solid byproduct, the solution was washed with saturated Na2CO3 (10 mL, two times), dried over
Na2SO4, and concentrated using a rotary evaporator. The crude product was further purified
over silica gel chromatography using 20:1 (vol:vol) CHCl3:MeOH mixture as eluent to provide a
yellow oil paste (~2.5 g); reaction yield ~74 %.
1H NMR (600 MHz, CDCl3): δ 1.40-1.52 (m, 2H), 1.62-1.72 (m, 4H), 1.87-1.93 (m, 1H), 2.24-2.26
(t, 2H, J = 6 Hz), 2.43-2.48 (m, 1H), 2.53-2.57 (m, 1H), 2.81-2.85 (m, 1H), 3.08-3.13 (m, 1H), 3.15-
2.29 (m, 1H), 3.37 (s, 6H), 3.42-3.46 (m, 5H), 3.52-3.56 (m, 9H), 3.63 (m, 120H), 4.70-4.72 (m,
1H), 7.05 (br, 1H), 7.34 (br, 1H), 7.46 (br, 1H).
Compound 12 (Boc-Asp-(PEG-N3)2). Boc-aspartic acid (compound 1, 1.0 g, 4.29 mmol), NH2-
PEG1000-N3 (10.4 g, 10.27 mmol) and DMF (20 mL) were added to 100 mL two-neck round
bottom flask containing a magnetic stir bar. The mixture was stirred until a homogeneous
solution was formed. A solution of DCC (1.95 g, 9.46 mmol) and HOBtˑH2O (1.44 g, 9.41 mmol)
in DMF (5 mL) was added to the reaction mixture dropwise under ice cold conditions. Once the
addition was complete, the reaction solution was gradually warmed up to room temperature
and left stirring for two days. A white solid byproduct was filtered off using a filter paper, and
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the solvent was evaporated under vacuum. The residue was purified over silica gel column
using 20:1 (vol:vol) CHCl3:MeOH mixture as eluent, yielding the product as a yellow oil (~6.2 g);
reaction yield ~ 65%.
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2.50-2.53 (m, 5H), 3.63 (m, 175H), 4.40-4.44 (m, 1H), 6.14 (br, 1H), 6.67 (br, 1H), 7.13 (br, 1H).
Compound 13 (LA-(PEG-N3)2). Compound 12 was de-protected and purified following the same
procedure as described above. The de-protected compound 12 (3 g, 1.41 mmol), LA (0.35 g, 1.7
mmol), and DMF (10 mL) were mixed in a 100 mL two-neck round bottom flask. The solution
was stirred until becoming homogeneous, then cooled to ~0 °C using an ice bath. In a separate
vial, DCC (0.32 g, 1.55 mmol) and HOBtˑH2O (0.24 g, 1.55 mmol) were dissolved in DMF (5 mL),
and then added to the above reaction mixture under N2 atmosphere. The reaction solution was
warmed to room temperature and further stirred for two days. The byproduct (as a white solid)
was filtered off using a filter paper, and the residue was purified using silica gel
chromatography using 20:1 (vol:vol) CHCl3:MeOH mixture as eluent. The product was obtained
as a yellow oil (~2.3 g); reaction yield ~70%.
1H NMR (600 MHz, CDCl3): δ 1.38-1.46 (m, 2H), 1.62-1.67 (m, 4H), 1.96-1.92 (m, 1H), 2.21-2.23
(t, 2H, J = 6Hz), 2.41-2.46 (m, 1H), 2.62-2.65 (m, 1H), 2.87-2.91 (m, 1H), 3.05-3.09 (m, 1H), 3.12-
3.16 (m, 1H), 3.38-3.41 (m, 6H), 3.52-3.55 (m, 6H), 3.62 (m, 175H), 4.73-4.76 (m, 1H), 6.81-6.89
(br, 2H)
Compound 14 (LA-(PEG-NH2)2). Compound 13 (2 g, 0.87 mmol) was dissolved in THF (40 mL) in
a 100 mL one-neck round bottom flask equipped with a magnetic stirring bar, followed by the
addition of triphenylphosphine (0.68 g, 2.6 mmol). The solution was stirred at room
temperature under N2 atmosphere for 30 min, then H2O (0.31 g, 17.3 mmol) was added. The
reaction mixture was further stirred at room temperature overnight and purified following the
same steps as described for preparing bis(LA)-PEG-NH2 (compound 8). The product was
collected as a yellow oil (~1.2 g); reaction yield ~60%.
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1H NMR (600 MHz, CDCl3): δ 1.4-1.45 (m, 2H), 1.59-1.65 (m, 4H), 1.85-1.89 (m, 1H), 2.1-2.3 (t,
2H, J = 6 Hz), 2.39-2.44 (m, 1H), 2.67-2.73 (m, 1H), 2.83-2.85 (t, 4H, J = 6 Hz), 2.84-2.87 (m, 1H),
3.04-3.08 (m, 1H), 3.11-3.15 (m, 1H), 3.37-3.4 (m, 3H), 3.48-3.52 (m, 10H), 3.62 (m, 175H), 4.43-
4.46 (m, 1H).
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Compound 15 (LA-(PEG-COOH)2). Compound 14 (1 g, 0.44 mmol), succinic anhydride (0.09 g,
0.88 mmol), triethylamine (0.16 mL, 1.15 mmol) and CHCl3 (15 mL) were mixed in a 100 mL one-
neck round bottom flask equipped with a magnetic stirring bar. The reaction was stirred at
room temperature overnight under N2, then the solvent was evaporated using a rotary
evaporator. The residue was dissolved in 1 M HCl (20 mL), and the product was extracted using
CHCl3 (40 mL, three times). After drying over Na2SO4 and evaporating the organic solvent,
compound 15 was obtained as a yellow oil (~0.6 g); reaction yield ~ 60 %.
1H NMR (600 MHz, CDCl3): δ 1.39-1.46 (m, 2H), 1.60-1.66 (m, 4H), 1.86-1.90 (m, 1H), 2.19-2.21
(t, 2H, J = 6 Hz), 2.30-2.46 (m, 1H), 2.60-2.66 (m, 8H), 2.70-2.74 (m, 1H), 2.84-2.88 (m, 1H), 3.25-
3.09 (m, 1H), 3.11-2.16 (m, 1H), 3.38-3.41 (m, 6H), 3.50-3.55 (m, 6H), 3.63 (m, 180 H), 4.70-4.74
(m, 1H).
Quantum Dot Synthesis. CdSe-ZnS core-shell QDs with different cores sizes were synthesized
by reacting organometallic precursors (e.g., cadmium acetylacetonate and trioctylphosphine
selenium) in hot coordinating solvent mixture made of alkylphosphines, alkyphosphine-
carboxyl, and alkylamine.32,83 As-prepared the QD surfaces were primarily capped with TOP/
TOPO ligands making them highly hydrophobic in nature. Ligand exchange with lipoic acid
modified ligands prepared as described above was applied to promote their transfer to water
media and render them biocompatible.45
Photoligation of QDs with LA-modified Ligands. The phase transfer of the QDs relied on the in-
situ photochemical transformation of the various LA-modified ligands. We briefly describe the
procedure applied to cap the QDs with bis(LA)-PEG-OCH3 and LA-(PEG-OCH3)2 (compound 4 and
compound 11) and transfer them to aqueous media. Stock dispersion of TOP/TOPO-capped
CdSe-ZnS quantum dots (8 µM, 163 µL) in toluene/hexane mixture was precipitated using
ethanol. The turbid mixture was centrifuged at 1900 g for 15 min, the supernatant was
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discarded, and the solid pellet was redispersed in hexane (500 µL). In a separate scintillation
vial, bis(LA)-PEG-OCH3 (68 mg) or LA-(PEG-OCH3)2 (95 mg) was dissolved in MeOH (500 µL)
mixed with a catalytic amount of tetramethylammonium hydroxide (TMAH). The contents of
the vials were combined in one vial containing a magnetic stir bar. The vial atmosphere was
switched to N2, then placed inside a UV reactor (Luzchem Research Inc., Ottawa, Canada). The
reaction mixture was irradiated with UV light (λirr peak at 350 nm, at a power of 4.5 mW/cm2)
for 30-40 min with continuous stirring. A complete transfer of the QDs from hexane layer to the
bottom methanol layer occurred, indicating ligand exchange of the native TOP-TOPO with the
bis(LA)-PEG-OCH3 or LA-(PEG-OCH3)2 has indeed taken place. The solvents were evaporated
under vacuum, then a solvent mixture (made of 1:1:10 in volume MeOH:CHCl3:C6H14) was
added followed by centrifugation at 1900 g for 6 minutes. The top solvent layer was decanted,
the precipitate was mildly dried under vacuum. The resulting QD pellet was readily dispersed in
DI water. The water dispersions of QDs were further purified using three rounds of
centrifugation using a membrane filtration device (Amicon Ultra, 50 kD) to remove the free
unbound ligands. The same protocol can be applied to prepare QDs that are functionalized with
reactive groups such as -N3, -NH2 and –COOH. Here we simply mix a small fraction of bis(LA)-
PEG-N3, bis(LA)-PEG-NH2 or bis(LA)-PEG-COOH with the inert bis(LA)-PEG-OCH3 ligands and
follow the steps described above. Similarly, hydrophilic and reactive QDs were prepared using
a mixture of LA-(PEG-OCH3)2 and -N3, -NH2 and –COOH-appended ligands and following the
same steps. Note: the dissolution of bis(LA)-PEG-OCH3 ligands in water requires slight heating
and continuous stirring. However, once the disulfide ring is opened followed by ligation with
QDs, the obtained nanoparticles are readily dispersed in DI water.
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Ligand Exchange on AuNPs. Hydrophobic oleylamine-stabilized AuNPs with a hydrodynamic
radius of 10 nm were prepared following a previously detailed synthetic scheme and stored in
hexane.84 Cap exchange of these AuNPs with bis(LA)-PEG-OCH3 and LA-(PEG-OCH3)2 was carried
out following the same protocol. Here, we briefly describe the cap exchange using bis(LA)-PEG-
OCH3 ligands using either two-phase or one-phase configuration. A stock dispersion of
oleylamine-AuNPs (30 nM, 100 µL) in hexane was further added with hexane solvent to a total
volume of 500 µL. Bis(LA)-PEG-OCH3 (20 mg) dissolved in MeOH (500 µL) were added to the
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above dispersion of AuNPs in hexane, and the mixture was left stirring at room temperature
overnight. This produces a phase transfer of the AuNPs from top hexane layer to bottom
methanol layer, indicating that oleylamine has been replaced by bis(LA)-PEG-OCH3 ligands. The
procedure is also expected to induce a reduction of LA to DHLA.49 The bis(LA)-PEG-OCH3-AuNPs
were purified and dispersed in DI water, by first evaporating the solvent(s), re-dispersion in a
solvent mixture, and centrifugation, using the same conditions as done above for the QDs.
Finally 2-3 rounds of centrifugation using a membrane filtration device (Amicon Ultra, 50 kD)
was used to remove the remaining unbound ligands. Conversely, the single phase configuration
utilized THF as the solvent. Briefly, starting with oleylamine-AuNPs (30 nM, 100 µL) in hexane,
the solvent was evaporated under vacuum then bis(LA)-PEG-OCH3 (20mg) in THF (500 µL) was
added forming a homogeneous phase. The solution was stirred at room temperature overnight,
then THF was evaporated under vacuum. To the dry NPs, a solvent mixture of
MeOH/CHCl3/Hexane was added, similar to the one used above for the QDs, yielding a turbid
sample. Following centrifugation, the solvent was decanted, the sample was gently dried. The
resulting precipitate was readily dispersed in water, then 2-3 rounds of centrifugation using a
membrane filtration device (Amicon Ultra, 50 kD) were used to purify the AuNPs from excess
unbound ligands and solubilized organics.
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Conjugation of QDs to Transferrin via EDC Coupling. The QDs (12.5 µM, 40 µL) photoligated
with a mixture of bis(LA)-PEG-OCH3 and bis(LA)-PEG-COOH (85:15 in molar ratio), EDC (52 mM
in DI water, 14.4 µL), NHS (87 mM in DI water, 17.3 µL) were mixed with 178.3 µL of 10 mM
phosphate buffer (PB, pH 6.5) in a scintillation vial. The vial was wrapped with aluminum foil
and the mixture was stirred at room temperature for 1 hour. Then 4 mL of 10 mM PB (pH 8.7)
was added and the content was subsequently transferred to a membrane filtration device
(Amicon Ultra, 50 kD), then one round of concentration/dilution was applied to remove excess
EDC. The sample was concentrated to a final volume of ~100 µL, then NHS (87 mM in DI water,
9 µL), transferrin (2.4 mg) and 10 mM PB (pH 8.7) were added; the total volume of the reaction
was maintained at 400 µL. The mixture was left to react at room temperature for 5 hours with
constant stirring, then loaded onto a PD-10 desalting column (GE Healthcare) to remove
unreacted transferrin and excess coupling reagents. The conjugates were characterized using
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absorption spectroscopy before testing them in cellular uptake measurements. The
experimental details on cell uptake and fluorescence imaging are provided in the Supporting
Information.
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Conjugation of QDs with Peptide (A42). QDs photoligated with 5 % and 10 % bis(LA)-PEG-
COOH were used to label a peptide with the sequence of GASGRIGFLRTAACA (M.W. ~ 1449.4).
This peptide has a C-terminal cysteine (C) at one end which was coupled to a maleimide-
functionalized dye and a glycine residue (G) at the N-terminal was reacted with the COOH-QDs.
It was synthesized manually using in situ neutralization cycles for Boc-solid-phase-peptide
synthesis (Boc-SPPS) following procedures described in the literature.85 Briefly, the synthesis
was carried out using 0.2 mmol MBHA resin (4-Methylbenzhydrylamine, 0.40 mmol/g), 1.0
mmol of aminoacid, 1.0 mmol of HCTU (in a 0.4 M solution in DMF), and 1.5 mmol of DIEA.
Coupling times were 20 min. Following chain assembly, the peptide was cleaved from the resin
with HF and 10% of anisole for 1 hour at 0 °C.
The labeling of the peptide with sulfo-Cy3 dye through cysteine and maleimide reaction
has been provided in the Supporting Information. Here, we provide the details for conjugating
5% COOH-QDs and peptide-Cy3 using carbodiimide chemistry. Briefly, in a scintillation vial, 5%
COOH-QDs (6.6 µM, 38 µL) were diluted in 10 mM PB (pH 6.5, 72 µL) and mixed with EDC (5.2
mM in 10 mM pH6.5 PB, 40 µL). The reaction mixture was stirred for 1 hour at room
temperature (in the dark), followed by the addition of NHS (8.7 mM in 10 mM pH8.7 PB, 47 µL)
and a solution of Cy3-labeled peptide (529 µM, 25 µL) in DMSO. 10 mM PB (pH8.7, 278 µL) was
added to render the mixture basic. The reaction mixture was left stirring for 5 hours at room
temperature, and the conjugates were purified using a PD-10 desalting column. The first eluted
fraction (~400 μL, containing the conjugates) was collected and characterized using absorption
and fluorescence spectroscopy. Conjugation of peptide-Cy3 to 10 % COOH-QDs was carried out
following the same protocol, except that the amount of coupling reagent and peptide-Cy3 were
doubled in order to compensate for the higher number of carboxyl groups per QD and to
maintain the same molar ratio between acid groups and target peptide-Cy3 as above. We also
prepared control dispersions by reacting QDs photoligated with 100%-methoxy terminated
ligands with Cy3-labeled peptide at similar reagent concentrations. The samples were purified
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using a PD-10 desalting column, and the first collected fraction was analyzed using absorption
and fluorescence spectroscopy.
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Acknowledgements: The authors thank FSU and the National Science Foundation for financial
support (NSF-CHE #1508501 and #1058957).
Supporting Information Available: Additional experimental details, additional absorption and
1
emission of QDs, synthesis of the peptide-Cy3, FRET analysis, H NMR spectra of the
compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
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