Cryptophane-Folate Biosensor for 129Xe NMR

Article abstract of DOI:10.1021/bc5005526

Folate-conjugated cryptophane was developed for targeting cryptophane to membrane-bound folate receptors that are overexpressed in many human cancers. The cryptophane biosensor was synthesized in 20 nonlinear steps, which included functionalization with folate recognition moiety, solubilizing peptide, and Cy3 fluorophore. Hyperpolarized ¹²⁹Xe NMR studies confirmed xenon binding to the folate-conjugated cryptophane. Cellular internalization of biosensor was monitored by confocal laser scanning microscopy and quantified by flow cytometry. Competitive blocking studies confirmed cryptophane endocytosis through a folate receptor-mediated pathway. Flow cytometry revealed 10-fold higher cellular internalization in KB cancer cells overexpressing folate receptors compared to HT-1080 cells with normal folate receptor expression. The biosensor was determined to be nontoxic in HT-1080 and KB cells by MTT assay at low micromolar concentrations typically used for hyperpolarized ¹²⁹Xe NMR experiments.

Full text of DOI:10.1021/bc5005526

Article
pubs.acs.org/bc
129
Cryptophane-Folate Biosensor for Xe NMR
Najat S. Khan, Brittany A. Riggle, Garry K. Seward, Yubin Bai, and Ivan J. Dmochowski*
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104, United States
S
* Supporting Information
ABSTRACT: Folate-conjugated cryptophane was developed for targeting
cryptophane to membrane-bound folate receptors that are overexpressed in
many human cancers. The cryptophane biosensor was synthesized in 20
nonlinear steps, which included functionalization with folate recognition
moiety, solubilizing peptide, and Cy3 fluorophore. Hyperpolarized 129Xe NMR
studies confirmed xenon binding to the folate-conjugated cryptophane.
Cellular internalization of biosensor was monitored by confocal laser scanning
microscopy and quantified by flow cytometry. Competitive blocking studies
confirmed cryptophane endocytosis through a folate receptor-mediated
pathway. Flow cytometry revealed 10-fold higher cellular internalization in
KB cancer cells overexpressing folate receptors compared to HT-1080 cells
with normal folate receptor expression. The biosensor was determined to be
nontoxic in HT-1080 and KB cells by MTT assay at low micromolar
concentrations typically used for hyperpolarized 129Xe NMR experiments.
accessible, in liter quantities at near-unity polarization levels.18
INTRODUCTION
■
Several HP 129Xe MRI studies imaging the lungs and brain have
Magnetic resonance imaging (MRI) is a noninvasive medical
imaging technique most useful for obtaining high contrast in
been published, for both rodents and humans.19−24
Our laboratory has developed water-soluble tris(triazole
ethylamine) and tris-carboxylate derivatives of cryptophane-A
vivo images of tissues, organs, and bone at high spatial
1
resolution. H MRI is limited by low sensitivity, in part due to
that bind xenon with usefully large association constants, KA =
17 000−42 000 M−1 in buffer at 293 K.25,26 Additionally, one
tris-carboxylate cryptophane was shown to bind Xe in human
plasma with appreciable affinity, KA = 22 000 M−1 at 310 K.25
The chemical shift of 129Xe-bound cryptophane can be
modulated stereoelectronically, e.g., by ruthenating the
aromatic rings,27 varying the size of the cavity28 or appending
water-solubilizing moieties.25,26,29 We and others have synthe-
sized cryptophane biosensors that are conjugated to various
ligands via a hydroxyl or propargyl group. These include biotin-
modified cryptophane biosensors for detecting streptavi-
din,30−34 an enzyme-responsive biosensor for matrix metal-
loproteinase-7 (MMP-7),35 and a series of benzenesulfona-
mide-functionalized cryptophane-A derivatives that exhibited
isozyme-specific chemical shift changes, upon binding carbonic
anhydrases I or II.36 Another example included a peptide-
labeled 129Xe biosensor by Schlundt et al. that produced a 1
ppm downfield shift upon binding to a major histocompatibility
complex (MHC) class II protein.37 The delivery of
cryptophanes using cell-penetrating peptides or targeting cell-
surface αvβ3 integrin receptors has also been demonstrated with
cancer and normal cell lines.38,39 Recent efforts have high-
lighted the potential for using xenon biosensors in cellular HP
129Xe magnetic resonance spectroscopy and imaging.34,40−42
high background from endogenous proton signals. In order to
increase signal, especially when imaging vascular tissues or
analyzing brain perfusion, gadolinium- or iron-oxide-based
contrast agents are commonly used. However, early and
accurate diagnoses of human disease increasingly rely upon
information gleaned from molecular imaging of protein
biomarkers or metabolic processes. There are now many
examples using PET and SPECT imaging agents with readily
detected radioactive nuclei.1 By comparison, current MRI
contrast agents have limited ability to detect proteins or
metabolites of low abundance in cells.2−4 The goal of making
“smart” MRI contrast agents that produce readily measured
signals in response to environmental cues has led to intense
investigation of nuclei that can be hyperpolarized (HP) to
achieve a majority of unpaired nuclear spins, most commonly
5−8
3
13C, He, 129Xe, and 38Kr.
Recent applications of HP 13C
pyruvate in human and small animal MRI highlight the great
potential of hyperpolarization techniques for evaluating
metabolites associated with prostate cancer and cardiac
dysfunction.9−11
129Xe is a nontoxic gas with high water solubility (4.2 mM
atm−1 at 300 K),12 and unique physical−chemical properties
that motivate the development of a new class of versatile MRI
contrast agents. Xenon’s significant polarizability contributes to
its affinity for void spaces in natural13 and synthetic
materials,14−17 as well as chemical-shift sensitivity to its
molecular environment. Recent advances in 129Xe hyper-
polarization technology make this agent now much more
Received: October 13, 2014
© XXXX American Chemical Society
A
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Scheme 1. Five-Step Synthesis of [2-(Trimethylsilyl)ethoxy]-2-N-[2-(trimethylsilyl)-ethoxycarbonyl]folic Acid
a
Scheme 2. Synthesis of Unlabeled and Cy3-Labeled Biosensors 24 and 25, Respectively
a
Monopropargyl cryptophane was joined to the folate-conjugated azidopeptide on solid support via Cu(I)-catalyzed [3 + 2] azide−alkyne
cycloaddition.
Folic acid has been investigated over the past two decades as
a means for targeted delivery of payloads to tumor cells. In
cells, there are three types of transporters that are responsible
for the uptake of folate. These include reduced folate carrier
(RFC),43 proton-coupled high affinity folate transporter,44 and
folate receptor (FR, also known as high affinity folate binding
protein).45 Folate receptors are cell surface glycosylphosphati-
dylinositol (GPI)-linked membrane glycoproteins with molec-
ular weights ranging from 38 to 45 kDa.46 RFC is ubiquitously
expressed throughout normal adult tissue, but as the name
implies, this low affinity folate carrier is specific for the
physiological form of reduced folic acid, 5-methyl tetrahy-
drofolate, which binds RFC with a micromolar dissociation
constant. The high affinity FR (Kd ≈ 0.1−1 nM, 1:1
stoichiometry) binds the nonphysiological folic acid as well as
5-methyltetrahydrofolate. FR exhibits narrow tissue distribu-
tion, being predominately expressed on the apical surface of
polarized epithelial cells and thus not in contact with circulating
folate. In humans, FR has 4 different isoforms: α, β, γ, and δ,
where α and β are membrane-bound.45 Among them, FRα is
overexpressed in non-mucinous adenocarcinomas of the ovary,
cervix, uterus, and ependymal brain tumors;45,47,48 FRα is
B
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overexpressed in 90% of ovarian carcinomas; and higher levels
of expression are generally associated with poorly differentiated
and aggressive tumors.49,50 It is believed that FRα is
overexpressed because the fast growth rate of cancer cells
requires more folic acid.45 FR expression is generally absent
from normal tissues except in the choroid plexus, the placenta,
and at low levels in lung, thyroid, and kidney. These FRs do not
present a problem when using folic acid to target cancer
because of localizations inaccessible to circulation; the brain
side of the blood-brain barrier (choroid plexus), on the luminal
side (lungs and gut), and in the proximal tubule lumen
(kidney). FR functions via receptor-mediated endocytosis. FR
is largely recycled back to the cell surface. Additionally, folate
remains stable for several hours after endocytosis by cancer
cells.51 These characteristics make FRα an attractive target for
the development of a new cryptophane biosensor. Here, we
explored bioconjugation strategies for synthesizing a folate−
cryptophane biosensor that targets cancer cells overexpressing
FRα.
The FR−cryptophane biosensor was designed with four
functional components: First was a monopropargyl derivative
of cryptophane-A, which is known to bind xenon in organic and
aqueous solvents.25,26,29,52 Second was a solubilizing poly-
cationic (RKR-repeat) peptide where the positive charges were
deliberately interrupted by a polyethylene glycol (PEG) unit:
This avoided the problem that polycationic sequences of five or
more peptide units can induce nonspecific cell uptake.53 Third,
a folate-linker moiety with high affinity for FRα was synthesized
and conjugated via an orthogonally protected lysine on the
peptide sequence. This final synthetic step involved reaction of
the N-terminal azide of the peptide-folate conjugate with
monopropargyl cryptophane via Cu(I)-catalyzed [3 + 2]
azide−alkyne cycloaddition (CuAAC).54−56 Finally, a Cy3
fluorescent dye was conjugated via maleimide linkage to a
cysteine residue on the peptide. The dye assisted in
fluorescence imaging and quantitation of biosensor uptake in
both FR+ and FR− cells. Cell uptake and cytotoxicity studies
were performed using a combination of confocal laser scanning
microscopy, flow cytometry, and MTT assays for FR+ and FR−
cancer cells.
carboxypeptidase-G to give pteroic acid 2. Carbonyldiimidazole
(CDI) and 2-trimethylsilylethanol in dry dimethyl sulfoxide
(DMSO) were added to the crude pteroic acid to produce the
protected pteroic acid, 1-(2-N-teoc-pteroyl)imidazole 3 in 62%
yield. In order to synthesize the second intermediate, the α-
carboxylate group in N-Boc-L-Glu (OBn)-OH 4 was protected
by treating it with CDI and 2-trimethylsilylethanol while the γ-
carboxylate group was selectively deprotected using Pd−C to
give 5 in 88% yield. The N-Boc protecting group was
subsequently removed using TsOH to give α-(2-TMS-ethyl)
glutamate 6 in 51% yield. Finally, 3 was treated with 1.5 equiv
of 6 and N-methyl-1,5,9-triazabicyclo[4.4.0]-decene (MTBD)
in dry DMSO to give the folate recognition moiety 7 (41%
yield).
Monopropargyl Cryptophane (21). A monopropargyl
derivative of cryptophane-A was synthesized in 12 nonlinear
steps in 3% overall yield (Supporting Information, Schemes
S1−S3).35
Synthesis of Fluorescent Folate−Cryptophane Con-
jugate (25). Final steps in biosensor synthesis are shown in
Scheme 2. The azidopeptide 22 was synthesized by standard
solid-phase synthesis using Fmoc-substituted reagents in 85%
yield and consisted of three polyethylene glycol units and
lysine-arginine units to help solubilize the cryptophane in
water.58 3-Azido propionic acid was prepared according to
literature procedures and incorporated as the N-terminal
residue.59 The cysteine was incorporated in the peptide to
enable site-specific fluorescent labeling of the biosensor for cell
studies. The azidopeptide was orthogonally deprotected using
4% hydrazine in water and readily coupled to the γ-folate
conjugate 7 in dry DMF to yield 23 in 75% yield. In order to
monitor the reaction, a portion of the reaction mixture was
cleaved from the resin and purified by reverse-phase HPLC.
Once product formation was confirmed, 23 (still on solid
support) was coupled to monopropargyl cryptophane 21 to
give 24 by copper(I)-catalyzed [3 + 2] cycloaddition. Although
the yield for 24 was initially low (∼20%), it was subsequently
improved to approximately 80% by using a large excess of
sodium ascorbate (40 equiv) to ensure that copper remained
reduced as Cu(I) to catalyze the reaction. The product was
cleaved from solid support, purified by reverse-phase HPLC
and was determined to be readily soluble in water. To
fluorescently label the conjugate 24, the cysteine was
deprotected using TCEP and coupled with the maleimide-
functionalized Cy3 dye (λex = 550 nm, λem = 575 nm). 25 was
purified by reverse-phase HPLC. Cy3 labeling efficiencies were
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Folate−Crypto-
phane Conjugate. The fluorescent, folate−cryptophane
conjugate was synthesized in 20 nonlinear steps from four
commercially available starting materials. Folate recognition
moiety 7 (5 steps, Scheme 1), monopropargyl cryptophane 21
(12 steps), and solubilizing azido-peptide 22 were joined in two
steps to form biosensor 24 and finally conjugated with Cy3 dye
(1 step) to give the fluorescent biosensor 25 (Scheme 2).
Synthesis of α-[2-(Trimethylsilyl)ethoxy]-2-N-[2-(tri-
methylsilyl)-ethoxycarbonyl] Folic Acid (7). Folic acid
has two carboxylates (α- and γ-) whose reaction with the
peptide sequence via N,N′-dicyclohexylcarbodiimide (DCC)
would produce a mixture of α-folate and γ-folate conjugates.
Because only the γ-conjugate is recognized by the FRα
receptor, a selectively protected folic acid derivative 7 was
synthesized in 5 nonlinear steps in 13% overall yield following
established protocols (Scheme 1).57 The folate recognition
moiety was prepared from two intermediates: the 2-N-teoc-
pteroic acid derivative, where teoc is 2-(trimethylsilyl)-
ethoxycarbonyl), and the α-carboxyl-protected glutamic
acid.57 Folic acid 1 underwent enzymatic hydrolysis with
determined from the ratio of dye absorbance at 550 nm (ε550
=
150 000 M−1 cm−1 with a correction factor of 0.05 at A280) to
the cryptophane absorbance at 280 nm (ε280 = 10 000 M−1
cm−1).60 Cy3-labeling yields were only 20−30%, likely a result
of steric hindrance from both the bulky cryptophane and folate
group appended to the peptide. Removal of excess TCEP using
a gel filtration column, prior to Cy3 addition, did not improve
the yield.
Visualization of Cell Delivery by Confocal Microscopy.
In order to determine whether 25 could be selectively delivered
to FR+ cells, confocal laser scanning microscopy (CLSM) was
performed as shown in Figure 1. Human nasopharyngeal
epidermoid carcinoma cells (KB) and human cervical
carcinoma cells (HeLa) were used as receptor positive cell
lines, with KB strongly overexpressing FRα and HeLa
moderately expressing FRα.61 Human fibrosarcoma (HT-
1080) was used as a negative control cell line (FR−) because
C
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Figure 2. Cytotoxicity assays for folate-conjugated cryptophane 24 in
KB (blue) and HT-1080 cells (red). Percent viability was determined
via MTT assay after 24 h incubation with increasing concentrations of
24 as compared to untreated cells.
highest concentration of 24 (100 μM). Cell viabilities
determined when 0−10 μM of the biosensor was added to
KB cells were comparable to those seen previously with
fluorescent contrast agents such as folate-substituted poly(p-
phenyleneethynylene).63 At concentrations ∼2 μM, 25
exhibited sufficient fluorescence intensity to be detected
intracellularly via both confocal microscopy and flow cytometry
and was also minimally cytotoxic. The viability of HT-1080
cells with 24 ranged from 100% at 0 μM to 82% at 100 μM.
The greater cytotoxicity observed for KB cells was likely due to
the higher levels of FRα that are expressed on KB cells versus
HT-1080 cells, which in turn caused increased intracellular
accumulation of the folate−cryptophane conjugates. Similar
trends were seen in a previous study where a cyclic RGD
peptide-conjugated cryptophane was determined to be more
toxic in cell lines overexpressing the targeted αvβ3 integrin
receptors (60% toxicity in ASPC-1 cells versus 30% toxicity in
HFL-1 cells) after 24 h incubation at 100 μM concentration.39
Quantifying Cellular Internalization with Flow Cy-
tometry. In an effort to quantify the selective cellular
internalization of 25 in KB and HT-1080 cells, flow cytometry
was performed (Figure 3). After 4 h incubation in both KB and
Figure 1. Confocal micrographs and corresponding brightfield images
of 4 μM Cy3-labeled biosensor 25 targeting FRα. Uptake occurred in
(A) KB, (B) HeLa, and (C) HT-1080 cells after 4 h incubation at 37
°C in folic acid-depleted media. Uptake was blocked in (D) KB, (E)
HeLa, and (F) HT-1080 cells preincubated in folic acid containing
media.
these cells exhibit relatively little folate uptake.62 After 4 h
incubation with Cy3-labeled 25, fluorescence was seen evenly
distributed in the perinuclear region of KB cells as expected for
receptor-mediated endocytosis (Figure 1A). This was desirable
as nuclear internalization of imaging agents can cause potential
mutagenic effects on healthy cells. Uptake of Cy3-labeled 25 in
HeLa cells was also confirmed by CLSM after 4 h incubation
(Figure 1B). The fluorescence intensity was lower in HeLa cells
than in KB cells, which was in agreement with previous
studies.61,62 Uptake of 25 was negligible in HT-1080 (FR−)
cells, thereby indicating that biosensor 25 was able to
discriminate between FR+ and FR− cells (Figure 1C). To
analyze whether the uptake of 25 was facilitated by folate
receptor-mediated endocytosis, 25 was coincubated with folic
acid rich medium for all three cell lines. Because folic acid is
known to have a very high affinity for FRα, excess folic acid was
expected to outcompete 25, thereby blocking uptake. The
reduction in fluorescence in Figure 1D−F indicated that 25 was
specifically recognized by the FR receptor. Indeed, the folate
recognition moiety was critical for cellular uptake of this
biosensor.
Figure 3. Flow cytometry quantified cell uptake of 4 μM Cy3-labeled
25. Uptake in (A) KB (FR+, red) and (B) HT-1080 (FR-, blue) cells
was compared to cells inhibited by folic acid (black).
HT-1080 cells, there was a dramatic increase in cell-associated
fluorescence. When biosensor 25 was coincubated with excess
folic acid, the increase in median fluorescence intensity was
much lower than when folic acid was absent. This was in
agreement with the results from the CLSM studies, where
uptake was significantly reduced when excess folic acid was
present. In the absence of folic acid, the median cell-associated
fluorescence intensity was approximately 45-fold higher in KB
Cytotoxicity Studies. The cytotoxicity of folate−crypto-
phane conjugate 24 was evaluated by incubating KB (FR+) and
HT-1080 (FR−) cells with increasing concentrations (0 to 100
μM) of 24 for 24 h, as shown in Figure 2. In KB cells, the
viability (scaled to 100% at 0 μM) decreased from 80% at 10
μM to 50% at 38 μM. Maximum toxicity in KB cells (23%
viability) was found when the cells were incubated with the
D
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cells and 3-fold higher in HT-1080 cells. Based on flow
cytometry data, it was determined that the median fluorescence
intensity in KB cells was approximately 10-fold higher than in
HT-1080 cells, consistent with the expected levels of FRα
expression.62,64 These data confirmed that 25 selectively
targeted cancer cells overexpressing the intended folate
receptors.
5.0 with and without 10% glycerol; vortex mixed and incubated
30 min). The 129Xe NMR peaks for 24 in the presence of FBP
(at 66.7 and 68.0 ppm, Figure S11) were essentially identical to
those shown for 24 in the absence of FBP (Figure 4). However,
it is important to emphasize that the biosensor need not induce
a chemical shift change to be useful in vivo. As we have
demonstrated with the confocal microscopy and flow cytometry
studies, the biosensor should preferentially localize in FR+ cells
as the free biosensor is diluted in circulation. Thus, detection of
HP 129Xe NMR signal corresponding to the cryptophane
biosensor will indicate FR+ tissue. Recent improvements in
129Xe hyperpolarization methods and HP 129Xe cellular NMR
techniques should make it possible using similar folate-
cryptophane biosensors in the future to discriminate between
FR+ and FR− cells by 129Xe NMR.34,41,67
Hyperpolarized 129Xe NMR. Hyperpolarized 129Xe NMR
spectra of 24 were acquired at 60 μM in acetate buffer at pH
5.0 using a 10 mm NMR probe and BURP-shaped soft pulse.
Sample temperature was controlled by a VT unit on the NMR
spectrometer to 300 1 K. Chemical shifts were referenced
relative to 129Xe gas at 0 ppm when extrapolated to 0 atm.
CONCLUSIONS
■
In summary, a water-soluble, folate- and Cy3-conjugated
cryptophane biosensor was synthesized in 20 nonlinear steps
and fully characterized. During conjugation, the folate moiety
was selectively protected to ensure that only the α-carboxylate
group was available to bind to FRα. Confocal imaging with FR+
and FR− cells confirmed the selective uptake of biosensor via
folate receptor-mediated endocytosis by cells overexpressing
FRα. Flow cytometry analysis quantified that uptake was 45-
fold higher in KB cells and 3-fold higher in HT-1080 cells than
when competing excess folate was in solution. Also, the median
fluorescence intensity was approximately 10-fold higher in KB
cells than HT-1080 cells, which motivates the use of folate-
conjugated cryptophane for in vivo biosensing in cells
overexpressing FRα. Cytotoxicity assays indicated that in the
relevant concentration range required for confocal and flow
cytometry analysis (1−10 μM), the viability was greater than
80% in both cell lines. Further advances in efficient
cryptophane synthesis68,69 will make it more practical in the
future to generate folate-cryptophane biosensors in larger
quantities needed for cell or in vivo 129Xe NMR studies.
Figure 4. Hyperpolarized 129Xe NMR spectrum of 60 μM 24 in
acetate buffer at pH 5.0 (40 scans; S/N = 30:1 with 50 Hz line
broadening).
Figure 4 shows a HP 129Xe-aq NMR peak at 195.0 ppm and
129Xe NMR chemical shifts of 64.8 and 66.0 ppm
corresponding to diastereomers of 24. This was due to the
chirality of the three componentsfolate, peptide, and
cryptophane (LRL and LRR). Cryptophane-A is a chiral
molecule and the racemic mixture of two enantiomers
encapsulating xenon is known to give rise to a single 129Xe
NMR resonance in water.65 However, upon conjugation of
chiral moieties to racemic cryptophane, diastereomers are
formed. Previous examples of xenon biosensor diastereomers
include the biotin-conjugated cryptophane developed by
Spence et al.30 The biosensor consisted of four diastereomers
due to the chiral cryptophane, L-amino acids, and maleimide
center formed when the biotin was conjugated to the rest of the
biosensor (RLR, RLL, LLR, LLL).30 This resulted in 4 distinct
peaks in the 129Xe NMR spectrum. Similarly, the enzyme
MMP-7 biosensor developed in our laboratory indicated the
presence of two diastereomers that were separated by 0.6
ppm.35 This was due to the chirality of the two components,
peptide and cryptophane-A (RL and LL).35,52,65
EXPERIMENTAL PROCEDURES
■
Reagents. Sigma-Aldrich. Dimethyl sulfoxide (DMSO),
dimethylformamide (DMF), folate binding protein (FBP), folic
acid, methanol, triisopropylsilane (TIPS, 99%), 2,6-lutidine,
piperidine, 3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide (MTT).
Fisher. Sodium chloride, copper(II) sulfate, trifluoroacetic
acid (TFA), diethyl ether (Et2O).
Alfa Aesar. Cesium carbonate, L-glutathione.
Novabiochem. 2-(1H-benzotriazole-1-yl) 1,1,3,3-tetrame-
thyluronium hexafluorophosphate (HBTU), N-hydroxybenzo-
triazole (HOBt), N-methylmorpholine (0.4 M), Fmoc-15-
amino-4,7,10,13 tetraoxapentadecanoic acid ((PEG)3), rink
amide resin, Fmoc-protected amino acids including Fmoc-L-
Lys(ivDde)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Arg(Pbf)-OH,
Fmoc-Cys(tButhio)-OH, N-methylmorpholine (0.4 M).
GE Healthcare. Cy3 monoreactive dye pack.
Our HP 129Xe NMR studies with folate binding protein
(FBP) and 24 resulted in no observable protein bound signal
(Supporting Information, Figure S11). FBP is a membrane-
bound protein and is known to aggregate at low micromolar
concentrations.66 It was necessary, therefore, to investigate
experimental conditions where the protein is maximally stable
(30 μM FBP, 30 and 60 μM biosensor 24 in acetate buffer, pH
Calbiochem. Tris(2-carboxyethyl)phosphine hydrochloride
(TCEP-HCl).
Invitrogen. RPMI-1640 medium, folate-depleted RPMI-
1640 medium, Dulbecco’s phosphate buffered saline (DPBS).
For biological assays, all solutions were prepared using
deionized water purified by Mar Cor Premium grade Mixed
Bed Service Deionization.
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General Methods. All organic reactions were carried out
allowed to swell in DMF (500 μL) in a 10 mL peptide vessel
for 10 min. The solution was filtered and 1 mL of 4% hydrazine
in DMF was added to deprotect the ivDde group on the
orthogonally protected lysine. This was repeated 5 times and
the absorption of the filtrate at 290 nm was monitored by UV−
vis spectroscopy to ensure that deprotection had taken place.
The resin was dried under vacuum. 22 was then added to a
mixture of 7 (0.0402 mmol, 2 equiv), HBTU (0.0603 mmol, 3
equiv), HOBt (0.0905 mmol, 4.5 equiv), and DIEA (0.1206
mmol, 6 equiv) in dry DMF. The reaction was stirred overnight
at rt under nitrogen. Once the reaction was complete, the resin
was carefully transferred to a fritted reaction vessel and washed
sequentially with DMF, CH2Cl2, MeOH, 1:1 MeOH/CH2Cl2,
and MeOH before drying under vacuum. The peptide coupled
to folate 23 was cleaved from the resin using a mixture of TFA,
TIPS, and water (90/5/5) at rt for 4 h. The reaction mixture
was filtered using a peptide vessel, concentrated, and the
peptide was precipitated by the addition of ether. The cleavage
cocktail removed side chain protecting groups from all amino
acids except for the t-butylthiol-protected cysteine. Semi-
preparative HPLC purification of 23 was accomplished using
the following gradient: time 0, A/B = 95/5; 0−45 min, linear
increase to A/B = 50/50; 45−47 min, linear increase to A/B =
20/80; 47−56 min, linear change to A/B = 20/80; 56−57 min,
linear increase to A/B =95/5; 57−72 min, linear change to A/B
= 95/5 (SI, Figure S5). MALDI-MS calculated for peptide−
folate conjugate 23, C91H158N38O21S2 (M + H+) 2184.19;
found 2184.05 (SI, Figure S6).
1
under nitrogen atmosphere. H NMR (500.14 MHz) and 13C
NMR (125.77 MHz) spectra were obtained on a Bruker AMX
500 spectrometer at the University of Pennsylvania Chemistry
Department NMR facility. Electrospray ionization (ESI) mass
spectrometry was performed in low-resolution mode on a
Micromass LC Platform and in high-resolution mode on a
Micromass Autospec. Matrix-assisted laser desorption/ioniza-
tion mass spectrometry (MALDI-MS) was performed on a
Bruker Daltonic Ultraflex III MALDI-TOF/TOF spectrometer
at the Mass Spectrometry Center in the Chemistry Department
at the University of Pennsylvania. Column chromatography was
performed using 60 Å porosity, 40−75 μm particle size silica gel
from Sorbent Technologies. Thin layer chromatography (TLC)
was performed using silica gel plates with UV light at 254 nm
for detection. HPLC analysis was performed on an Agilent
1100 system equipped with a quaternary pump and diode array
detector using a Varian Microsorb-MV 300−5 C8 column (4.6
× 250 mm, 5 μm). The gradient eluent was composed of two
solvents: 0.1% aqueous TFA (solvent A) and a 0.1% solution of
TFA in CH3CN (solvent B). UV−visible spectra were
measured using a diode-array Agilent 89090A spectropho-
tometer.
Synthesis of 7. α-[2-(Trimethylsilyl)ethoxy]-2-N-[2-(tri-
methylsilyl)-ethoxycarbonyl]folic acid (7) was prepared accord-
ing to literature procedures57 (Scheme 1) and matched the
reported physical constants and NMR spectra (SI, Figures S1
and S2).
Synthesis of 21. Monopropargyl cryptophane was prepared
according to literature procedures35 and matched the reported
physical constants and NMR spectra.
Synthesis of 24. Monopropargyl cryptophane 21 (20 mg,
0.02186 mmol, 2 equiv) in 900 μL dry DMSO was added to 23
(18.2 mg on solid support, maximum 0.01093 mmol
azidopeptide, 1 equiv) and allowed to stir for 10 min. 2,6-
Lutidine (0.0219 mmol, 1 equiv) was added and the reaction
mixture was degassed. Sodium ascorbate (0.4372 mmol, 40
equiv) was added dropwise, the mixture was degassed, and
finally an aqueous solution of copper(II) sulfate (0.0054 mmol,
0.5 equiv) was added. The suspension was degassed with N2
and stirred at rt for 24 h. The resin was then carefully
transferred to a fritted reaction vessel and washed sequentially
with CH2Cl2, MeOH, water, and 1:1 MeOH/CH2Cl2 before
drying under vacuum. The [3 + 2] cycloaddition reaction
between the azide-terminated folate-peptide 23 and monop-
ropargyl cryptophane 21 generated the folate-peptide-crypto-
phane conjugate 24 which was cleaved from the resin using a
mixture of TFA, TIPS, and water (90/5/5) at rt for 4 h. The
reaction mixture was filtered using a peptide vessel,
concentrated, and the peptide was precipitated by the addition
of ether. The cleavage cocktail removed side chain protecting
groups from all amino acids except for the t-butylthiol-
protected cysteine. Semipreparative HPLC purification of 24
was accomplished using the following gradient: time 0, A/B =
95/5; 0−65 min, linear increase to A/B = 30/70; 65−68 min,
linear increase to A/B = 20/80; 68−70 min, linear increase to
A/B = 5/95 (SI, Figure S7). MALDI-MS calculated for 24,
Synthesis of 22. Peptide 22 (Scheme 2) was prepared by
solid-phase synthesis using standard Fmoc amino acid
protection chemistry on Rink Amide resin (0.1 mmol scale).
Couplings of Fmoc-protected amino acids to the resin were
carried out with HBTU and N-methylmorpholine to generate
the activated ester. The resin was swelled in DMF (10 min)
prior to synthesis. Amino acids were then added sequentially
until 3-azidopropionic acid was attached at the N-terminus as
the final step. All residues were coupled onto resin by the
following procedure: removal of Fmoc group (20% piperidine
solution in DMF, 2 × 5 min), wash (DMF, 6 × 30 s), activation
(amino acid/HBTU/N-methylmorpholine, 1 × 30 s), coupling
(amino acid/HBTU/N-methylmorpholine, 1 × 60 min), rinse
(DMF, 3 × 30 s). The resin was swelled in DMF for 10 min
and the orthogonal lysine was deprotected by washing the resin
five times with 4% hydrazine in DMF. The resin was dried and
the peptide was cleaved using a mixture of TFA, TIPS, and
water (90/5/5) at rt for 4 h. The reaction mixture was filtered
using a peptide vessel, concentrated and the peptide was
precipitated by the addition of ether. The cleavage cocktail
removed side chain protecting groups from all amino acids
except for the t-butylthiol-protected cysteine. Semipreparative
HPLC purification of 22 was accomplished using the following
gradient: time 0, A/B = 95/5; 0−45 min, linear increase to A/B
= 50/50; 45−47 min, linear increase to A/B = 20/80; 47−56
min, linear change to A/B = 20/80; 56−57 min, linear increase
to A/B = 95/5; 57−72 min, linear change to A/B = 95/5 (SI,
Figure S3). MALDI-MS calculated for peptide 22,
C72H141N31O16S2 (M + H+) 1761.06; found 1760.91 (SI,
Figure S4).
C
147H211N38O32S2 (M + H+) 3102.61; found 3103.08 (SI,
Figure S8).
Synthesis of 25. Cys-protected folate-peptide-cryptophane
conjugate 24 was dissolved in PBS buffer (100 mM, pH 7.1) at
a concentration of 60 μM. The GE protocol was followed to
deprotect the t-butylthiol group and label the cysteine with the
Cy3-maleimide construct. TCEP (0.0006 mmol, 10 equiv) was
added to a 1 mL PBS solution containing 24 and degassed. The
solution was stirred for 40 min to which was added Cy3 dye
Synthesis of 23. Peptide 22 on the Rink Amide resin (30
mg, maximum, 0.0201 mmol azidopeptide 22, 1 equiv) was
F
dx.doi.org/10.1021/bc5005526 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
dissolved in 50 μL dry DMSO. The reaction was degassed and
stirred under nitrogen at rt for 16 h. The reaction mixture was
purified by HPLC using the following gradient: time 0, A/B =
95/5; 0−65 min, linear increase to A/B = 30/70; 65−68 min,
linear change to A/B = 20/80; 68−70 min, A/B = 5/95 (SI,
Figure S9). MALDI-MS calculated for C180H253N42O42S3 (M+
H+) 3771.81; found 3771.59 (SI, Figure S10). Extinction
coefficients used to determine solution concentrations of Cy3-
labeled 25 were ε280 = 38 000 M−1 cm−1 and ε552 = 150 000
M−1 cm−1 in water.
Cell Culture. KB, HeLa, and HT-1080 cells were obtained
from Dr. Jerry Glickson (University of Pennsylvania, Perelman
School of Medicine, Philadelphia, PA). All cells were grown in
25 cm2 tissue culture flasks in RPMI-1640 with 25 mM HEPES
supplemented with 2 mM L-glutamine, 15% fetal calf serum,
100 units penicillin, and 100 units streptomycin. Cells were
subcultured on a weekly basis.
Cell Viability (MTT) Assay. In 96 well plates 25 000 KB or
HT-1080 cells were plated per well and allowed to grow
overnight. A stock solution of nonfluorescently labeled folate-
peptide-cryptophane conjugate 24 was added to wells in
triplicate at final concentrations of 0, 2, 10, 25, 50, 75, and 100
μM and incubated for 24 h in the dark. The medium was
aspirated and the cells were washed thrice with DPBS before
being treated with 20 μL of MTT (5 mg/mL) for 3 h. The
medium was removed and DMSO was added to solubilize the
resulting crystals. A Labsystems Fluoroskan II microplate reader
was used to record the absorbance at 540 nm. Absorbance
readings were subtracted from the value of wells containing
untreated cells, and the reduction in cell growth was calculated
as a percentage of control absorbance in the absence of any
treatment. Data show the mean of at least three independent
experiments SD.
the hyperpolarizer. 129Xe was hyperpolarized to 10−15% after
having been cryogenically separated, accumulated, thawed, and
collected in CAV NMR tubes (New Era). After Xe collection,
NMR tubes were shaken vigorously to mix cryptophane
solutions with Xe. All 129Xe NMR measurements were carried
out on a 500 MHz Bruker BioDRX NMR spectrometer. RF
pulse frequency for 129Xe was 138.12 MHz. Samples were
observed using a 10 mm PABBO NMR probe. 129Xe NMR
spectra were acquired using the exchange signal averaging
(ESA) method. 54 Selective pulses (90 degree flip angle,
EBURP-1 shaped) were generated at the Xe@cryptophane
resonance frequencies. Each pulse lasted 5 ms, which gave a
designated excitation region 1 kHz (7.2 ppm). All spectra were
signal averaged by 40 scans. A delay of 0.15 s was given
between scans to allow polarized Xe to exchange in and
depolarized Xe to exchange out of the cryptophane cavity. The
natural line widths of Xe@cryptophane peaks are around 80 Hz
(fwhm, Lorentzian fitted). The spectra shown above are
exponentially broadened by 100 Hz, to give a larger signal/
noise ratio. Sample temperature was controlled by a VT unit on
the NMR spectrometer to 27 1 °C.
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC, MALDI-MS, and NMR characterization. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ivandmo@sas.upenn.edu. Phone: 215-898-6459.
Notes
The authors declare no competing financial interest.
Cell Uptake Studies. KB, HeLa, and HT-1080 cells were
grown to confluence on LabTek 8-well microscope slides with
glass coverslip bottoms at a density of 50 000 cells per plate.
The cells were grown in folate-depleted media for 24 h prior to
incubation with 4 μM solution of Cy3-labeled 25 for 4 h at 37
°C. For blocking studies, cells were grown in media containing
folic acid for 24 h prior to incubation with 25 under the same
conditions mentioned above. The medium was removed and
the cells were washed three times with DPBS. Cells were
visualized using an Olympus FV1000 confocal laser scanning
microscope with 543 nm (HeNe) laser excitation and Cy3
emission filter under 40× magnification (Olympus UApo/340,
1.15 NA water objective).
ACKNOWLEDGMENTS
■
We thank Dr. George Furst for assistance with NMR, Dr. Jerry
Glickson for providing cell lines, and Dr. Qian Wei, Dr. Aru
Hill, and Tara Kaufmann for their early contributions to this
work. This research was supported by NIH CA110104,
GM097478, and DOD BC061527. Bruker Ultraflex III
MALDI-TOF/TOF and Waters LCT Premier XE LC/MS
ESI-TOF were supported by NSF CHE-0820996 and NIH
1S10RR023444. Olympus FV1000 confocal microscope was
supported by NIH 1S10RR021113.
REFERENCES
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PBS and immediately analyzed on a BD LSRII machine at the
Flow Cytometry Laboratory, Abramson Cancer Center, at the
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