Strategies for labeling proteins with PARACEST agents

Article abstract of DOI:10.1016/j.bmc.2010.06.026

Reactive surface lysine groups on the monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 and 10.1 chelates were added per 3G4 molecule and between 5.6 and 5.9 chelates per molecule of

Full text of DOI:10.1016/j.bmc.2010.06.026

Bioorganic & Medicinal Chemistry 19 (2011) 1106–1114
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Strategies for labeling proteins with PARACEST agents
Olga Vasalatiy a, , Piyu Zhao a, Mark Woods a,b, Andrei Marconescu c, Aminta Castillo-Muzquiz a,
Philip Thorpe c, Garry E. Kiefer a,b, A. Dean Sherry a,d,
*
a Department of Chemistry, University of Texas at Dallas, PO Box 830688, Richardson, TX 75083, United States
b Macrocyclics, 1309 Record Crossing, Dallas, TX 75235, United States
c Department of Pharmacology, University of Texas Southwestern Medical Center, 2201 Inwood Road, Dallas, TX 75390, United States
d Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, NE 4.2, Dallas, TX 75390, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 18 June 2010
Reactive surface lysine groups on the monoclonal antibody (3G4) and on human serum albumin (HSA)
were labeled with two different PARACEST chelates. Between 7.4 and 10.1 chelates were added per
3G4 molecule and between 5.6 and 5.9 chelates per molecule of HSA, depending upon which conjugation
chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent
upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only
17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water life-
Keywords:
Eu3+ chelates
Bifunctional PARACEST agents
3G4 Monoclonal antibody
HSA
time of Eu-1 increased only marginally, up from 53
conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted
in little or no change in bound water lifetime (73–75 s for both the conjugated and non-conjugated
ls for the non-conjugated chelate to 65–77 ls for
Bound water lifetimes
l
forms). These data indicate that exchange of water molecules protons between the inner-sphere site
on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment
of the agent to the protein and likely its chemical surroundings on the surface of the protein.
Ó 2010 Elsevier Ltd. All rights reserved.
ꢀ
ꢁꢀ1
1. Introduction
Mz
M0
CqT1
55:5sHM
¼
1 þ
ð1Þ
Recently a novel class of MRI contrast agents were introduced
called chemical exchange saturation transfer (CEST) agents1 that
function by an entirely different mechanism compared to more
traditional paramagnetic relaxation agents. This mechanism is
based upon the ability to selectively pre-saturate an exchanging
proton or water molecule resonance on an agent that subsequently
exchanges into the bulk water pool and thereby causes a decrease
in intensity of the bulk water signal. A number of different para-
magnetic CEST agents have now been reported and numerous
applications are being explored.2–6 The CEST efficiency of a mole-
cule is conveniently evaluated by measuring the decrease in inten-
sity of the bulk water signal after applying a long selective
irradiation pulse at various frequencies across the entire proton
spectrum. Any decrease in water intensity (Mz/M0) after applica-
tion of a presaturation pulse is related to the concentration of CEST
agent (C), the number of exchanging protons or water molecules
Of the parameters that contribute to Eq. 1, only the lifetime of the
exchanging water proton molecule on the agent is readily modified
by chemistry so this has become the key parameter of interest in
the development of newer agents. Water proton exchange in aque-
ous lanthanide chelates depends upon the ionic radius of the Ln3+
cation, the nature of ligand side-chains that contribute electrons
to the central Ln3+, the coordination geometry of the chelate, and
the extent of prototropic and whole water exchange (the rate of
water proton exchange is the sum of the rates of these last two ef-
fects). A more detailed discussion of the impact of these chemical
features on the bound water lifetime can be found in other review
articles.7–9
It has been shown that the rate of water proton exchange in T1-
based low molecular weight Gd3+ chelates is often altered by bind-
ing of the chelate to proteins.10–14 This has an important impact in
the design of high relaxivity agents because water exchange can
ultimately limit the relaxivity of such chelates when they are cova-
lently attached to macromolecules. Even non-covalent binding of a
Gd3+ chelate to albumin (typically HSA or BSA) via either electro-
static or hydrophobic interactions can affect water exchange.15
Binding of a chelate to a hydrophobic site seems to have less im-
pact on water exchange (typically slows about two-fold) than
binding via electrostatic interactions. This may reflect restricted
(q), the T1 of bulk water, and water proton lifetime (s
HM):
* Corresponding author. Tel.: +1 972 883 2907; fax: +1 214 645 2744.
E-mail addresses: dean.sherry@utsouthwestern.edu, sherry@utdallas.edu (A.
Dean Sherry).
Present address: Imaging Probe Development Center, NHLBI, NIH, 9800 Medical
Center Drive, Rockville, MD 20850, United States.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.026

O. Vasalatiy et al. / Bioorg. Med. Chem. 19 (2011) 1106–1114
1107
access of molecules in the second hydration sphere whenever a
Gd3+ chelate is bound at a hydrophobic site on a protein.10,13 Strong
electrostatic or hydrogen bonding interactions can impact water
exchange even further. For example, a dramatic lengthening of
the bound water lifetime from ꢁ8 ns to 290 ns (36-fold) was re-
ported for a gadolinium chelate with highly charged phosphonate
pedant arms when bound to HSA.16 This was ascribed mainly to
the electrostatic forces between negatively charged pendant arms
and positively charged residues on the protein.16
ert.21,22 Given that ligands derived from macrocyclic structures
are most favorable for this application, one could consider conju-
gating such ligands to proteins either via a carbon backbone func-
tionalized macrocyclic structure (ligand 1) or via attachment
through one pendant arm (ligand 2). Given that isothiocyanates
and N-hydroxysuccinimide esters as the most widely used func-
tional groups with controlled chemistries for conjugation of
bifunctional agents, dyes, haptens to protein surfaces primarily
via e
-amine lysine residues,23,24 two different versions of ligands
The efficiency of PARACEST contrast agents can also be dramat-
ically influenced by molecular interactions that alter the bound
water lifetime. For this reason it is important to have a good under-
standing of the environmental parameters that may affect their
overall performance. This report describes the changes that occur
in the residence lifetime of Eu3+-bound water molecules before
and after conjugation of the PARACEST chelate, Eu-1, to a murine
3G4 monoclonal antibody as a model targeting vector for diagnos-
tic and therapeutic applications. 3G4 has a high affinity for phos-
phatidylserine (PS) complexed with the PS-binding plasma
protein, b2-glycoprotein I. The complex is only found on cells hav-
ing exposed PS. PS is found on the inner leaflet of the plasma mem-
brane of normal cells,17 and is maintained in this position by ATP-
dependent aminophospholipid translocases. Loss in PS symmetry
is caused by inhibition of aminophospholipid translocases or acti-
vation of PS exporters, such as ABC-A1 or scramblases18. PS exter-
nalization occurs on the vascular endothelium that lines tumor
blood vessels in a variety of tumors.19,20 PS externalization is not
observed on normal vascular endothelium. PS has become an
appealing target for molecular imaging of cancer blood vessels.
Prior to attachment of PARACEST agents to 3G4, human serum
albumin (HSA) was first used as a model protein to establish the
best conditions for conjugation of the bifunctional CEST ligands
developed here. Measurement of the bound water lifetimes for
Eu-1–HSA and Eu-2–HSA conjugates and a comparison with the
value found for the Eu-1–3G4 conjugates allowed us to evaluate
how protein surface groups influence water exchange in systems
with different points of chemical attachment. HSA decorated with
Eu-1 or Eu-2 (Chart 1) also served as models for evaluating the
detection limit of these systems for molecular imaging by CEST.
appropriate for PARACEST were chosen for this study.
2.1. Preparation of agents that conjugate via the macrocyclic
backbone
p-NO2-Benzyl-cyclen, synthesized by literature methods,25,26
was chosen as the standard building block for the backbone C-
functionalized ligand system. Introduction of the N-ligating sub-
stituents was achieved by exhaustive alkylation with excess 2-bro-
mo-N-methylacetamide under standard conditions (K2CO3/MeCN)
(Scheme 1). 2-Bromo-N-methylacetamide was synthesized using
a method adapted for preparation of chloroacetoamide27 with sig-
nificantly better yields (35% higher) compared to published val-
ues.28 Excess 2-bromo-N-methylacetamide was separated from
the tetrasubstituted p-NO2 benzyl cyclen 5 by column chromatog-
raphy. Reduction of the nitro aryl group was conducted under typ-
ical hydrogenation conditions using 10% palladium on carbon and
H2 (45 psi) to yield the aniline derivative 6. The final chelate was
obtained using two different approaches: (1) Eu-6 was first pre-
pared by addition of a stoichiometric amount of europium chloride
to the aqueous solution of 6 at pH 5.5 followed by conversion of
Eu-6 to Eu-1 using thiophosgene and a two phase solvent system
(H2O and CHCl3); or (2) 6 was first converted to 1 followed by che-
late formation (as shown in Scheme 1). Eu-1 was characterized by
high resolution 1H NMR and by IR spectroscopy.
With the development of monoclonal antibody–radioisotope
conjugates, two general synthetic methods are typically used: (1)
pre-labeling involves preparation of the chelate followed by
attachment of the chelate to the protein in a second step, and (2)
post-labeling generates the antibody–ligand conjugate first fol-
2. Results and discussion
Tumor imaging relies heavily on the highest possible signal-to-
noise ratio at the site of interest so targeting of multiple PARACEST
agents so a single localized region by use of antibodies or peptides
will be important. Given our prior experience with Gd3+ chelates,
one would anticipate that the local chemical environment around
an attached agent would indeed influence proton exchange and
ultimately affect CEST efficiency. In order to examine the nature
of these interactions, we selected 3G4 to serve as the targeting
antibody model for conjugation to well characterized PARACEST
systems, particularly those that are considered kinetically in-
Scheme 1. Synthesis of Eu-1. Reagents and conditions: (i) BrCH2CONHCH3 (3)/
K2CO3/MeCN (71%); (ii) H2/10% Pd on C/EtOH (99%); (iii) SCCl2/H2O/CHCl3 (99%);
(iv) EuCl3/H2O.
Chart 1. The chemical structures of ligands 1 and 2.

1108
O. Vasalatiy et al. / Bioorg. Med. Chem. 19 (2011) 1106–1114
lowed by addition of the radionuclide in the second step.29 In the
current study, the pre-labeling method was chosen because it
eliminates non-specific binding of metal ions to the protein. In
addition, this method maintains a ligand concentration that favors
rapid kinetics of chelation which is not the case for post-chelation
conditions where concentrations of the protein–ligand conjugate
are typically no higher than 10ꢀ4–10ꢀ5 M. Conjugations of ML
chelates to proteins were conducted as previously reported for
conjugation of DTPA (diethylenetriaminepentaacetic acid)-isothio-
cyanate to human IgG.30 Given that 3G4 has twelve solvent acces-
sible lysine residues, our initial conjugation experiments used a
100-fold excess of Eu-1 per antibody (8.3-fold excess per lysine)
to achieve maximum modification while preserving the immuno-
reactivity of the antibody (Scheme 2). Although this gave an
average of ten Eu-1 chelates per antibody (Table 1), the immunore-
activity of 3G4 was severely reduced to ꢁ17% as determined by
ELISA. This loss of immunoreactivity was unexpected given that
no lysine residues are thought to be near the binding sites of the
antibody. In a second experiment, a 50-fold excess of Eu-1 gave a
product that contained 7.4 europium chelates per the antibody
(Table 1). This product retained 88% immunoreactivity as shown
by ELISA. In the ELISA assay, the monoclonal antibody is recognized
by horseradish peroxidase (HRP) as a secondary antibody and one
possible explanations for reduced immunoreactivity encountered
at the higher loading value is that site recognition between HRP
and 3G4 was disrupted by excess modification of lysine groups.
Integrity of the antibody after the conjugation reaction was ana-
lyzed by gel electrophoresis (Fig. 1). As shown, the 3G4 conjugate
with 7.4 Eu-1 chelates retains a similar electrophoresis pattern as
the unmodified antibody, while the 3G4 conjugate with 10.1 Eu-
1 chelates attached showed some decomposition. Using identical
chemical conditions, a 20-fold excess of Eu-1 over HSA resulted
in a product with an average loading value of 5.6 chelates per pro-
tein molecule (Table 1).
Figure 1. Gel electrophoresis of modified antibody containing in average 7.4 Eu-1
(1) chelates and unmodified antibody (2) used in preparation of the conjugate,
modified antibody with average 10.1 Eu-1 chelates (3) and unmodified antibody
(4).
2.2. Preparation of agents that conjugate via a pendant arm
Eu-2 was synthesized as outlined in Scheme 3. Mono-CBz-cy-
clen 7 was alkylated with 2-bromo-N-methylacetamide in the
presence of K2CO3 to produce 8 in high yield. Following column
chromatography to remove excess 2-bromo-N-methylacetamide
and deprotection of benzyloxycarbomate with H2 and 10% palla-
dium on carbon, the tris-substituted amide 9 was obtained. N-(2-
bromoacetyl)glycine benzyl ester was prepared by alkylation of
the O-benzyl protected ester of glycine with bromoacetobromide
in dichloromethane with excess of K2CO3 as a base. Compound 9
was alkylated with N-(2-bromoacetyl) glycine benzyl ester 10
and the excess alkylating agent removed by column chromatogra-
phy to obtain 11. The benzyl protecting groups were subsequently
removed by hydrogenolysis (H2 and 10% palladium on carbon) and
the europium chelate was prepared by mixing equimolar quanti-
ties of the ligand 2 and europium chloride at pH 5.5 in water.
The N-hydroxysuccinimide ester of Eu-2 was generated in situ
Scheme 2. Schematic for the conjugation of Eu-1 chelates to 3G4.
Table 1
Initial and final ratios of reactants in the conjugation reactions and immunoreactivity
of the antibody
Initial (EuL/P)i Final (EuL/P)f (R)f/(R)i Immunoreactivitya
of 3G4, %
Eu-1–3G4 (1) 100
10.1
7.4
5.6
0.10
0.15
0.28
0.06
17
88
—
2
2
Eu-1–3G4 (2)
Eu-1–HSA
50
20
Scheme 3. Synthesis of Eu-2. Reagents and conditions: (i) BrCH2CONHCH3 (3)/
K2CO3/MeCN (86%); (ii) H2/10% Pd on C/EtOH (99%); (iii) BrCH2CONHCH2COOBn
(10)/K2CO3/MeCN (79%); (iv) H2/10% Pd on C/EtOH; (v) EuCl3/H2O.
Eu-2–HSA
100
5.9
—
a
Average of duplicate experiments (ELISA assay).

O. Vasalatiy et al. / Bioorg. Med. Chem. 19 (2011) 1106–1114
1109
Figure 2. 1H NMR spectra of PARACEST conjugates. 1H NMR spectrum of 5.5 mM
Eu-1 in the HSA conjugate (top, 270 MHz) and 0.95 mM Eu-2 in the HSA conjugate
(bottom, 500 MHz) pH 7.4 25 °C, pH 7.4 (5 mM HEPES/HCl, 140 mM NaCl). The
water peak was partially suppressed.
Scheme 4. Schematic of the conjugation of Eu-2 to HSA through an active ester
coupling. The image of HSA highlights solvent accessible lysine residues (nitrogen—
blue, aliphatic chains—green). Hydrogen atoms were omitted for clarity.
according to the literature procedure31 and a 100-fold excess of
this activated ester was added to HSA (Scheme 4). After purifica-
tion by dialysis and gel permeation chromatography, ICP analysis
showed that ꢁ5.9 Eu-2 chelates were attached to each HSA. Even
through the conjugating efficiency (Table 1) was somewhat lower
than desired, the simplified purification procedure eliminated
many of the purification problems commonly encountered with
other procedures.
Figure 3. CEST spectra of a 5.5 mM solution of Eu-1–HSA conjugate recorded at
B0 = 270 MHz, 25 °C, pH 7.4 (5 mM HEPES/HCl, 140 mM NaCl) with B1 = 1370 Hz,
1075 Hz and 833 Hz using an irradiation time of 2 s.
2.3. NMR and CEST measurements
The Eu-1 and Eu-2 protein conjugates were analyzed by high
resolution 1H NMR to confirm the integrity of the chelates once at-
tached to the protein surface. The paramagnetic Eu3+ ion produces
characteristic hyperfine shifts in ligand protons and, in particular,
the characteristic H4 macrocyclic protons that appear between 23
and 31 ppm for both Eu-1 and Eu-2 are particularly useful. The
presence of these diagnostic resonances in the isolated Eu-1/Eu-
2—protein/3G4 conjugates confirmed that the Eu3+ ion remains
sequestered by the ligand even after conjugation (Fig. 2).
Figure 3 shows typical CEST spectra of the Eu-1–HSA conjugate
measured at different irradiation powers. The CEST exchange peak
at ꢁ57 ppm corresponds to the inner-sphere water molecules
associated with the ꢁ5–6 covalently attached Eu-1 chelates on
the surface of HSA. The symmetry of this exchange peak indicates
that these 5–6 chelates experience similar chemical environments
on the surface of the protein. These combined data were fitted to a
three-pool exchange model (bulk solvent water, Eu3+ coordinated
water, and a third pool representing all other exchangeable pro-
tons associated with the protein; the 5–6 Eu-1 chelates on the pro-
tein surface were all assumed to be identical) to obtain a water
proton exchange rate of ꢁ15 ꢂ 103 sꢀ1 (corresponding to a Eu3+
-
bound water lifetime of 65
6 ls). Experimentally, a 27% signal
reduction was observed for 5.5 mM Eu-1–HSA (0.98 mM HSA)
using a presaturation field (B1) of 1370 Hz. Further dilutions of this
sample with HEPES buffer to 0.35 mM Eu-1–HSA (Fig. 4) resulted
in a 3% reduction in water intensity using the same B1. Assuming
that a 3% change in signal is the lower limit for reliable detection,
then 0.35 mM could be considered the detection limit for the Eu-1–
HSA system. However, it has been reported that CEST efficiency is
optimal when the exchange rate, Cb, equals 2p
B1.9 Thus, water pro-
ton exchange in this system is may be considered somewhat faster
than optimal for a B1 of 1370 Hz.
Only small amounts of antibody were available for experimen-
tation so a final sample of Eu-1–3G4 (1 attempt, 10.1 chelates) pre-

1110
O. Vasalatiy et al. / Bioorg. Med. Chem. 19 (2011) 1106–1114
Table 2
Experimental and fitted parameters for calculation of bound water lifetimes
sMH
(ls)
T1 (s)
100 ꢀ MS/M0 (%)
[EuL] (mM)
Eu-1–3G4 (1)a
Eu-1–3G4 (2)a
Eu-1–HSAa
Eu-6a
2.34 0.09
2.25 0.09
1.95 0.07
2.91 0.12
2.67 0.12
3
8
27
44
12
8
0.32
1.26
5.5
12.5
0.95
0.95
9.0
71
77
65
53
75
80
73
2
6
2
2
2
3
Eu-2–HSAb
EuDTMAb
Eu-2b
42
a
Data was obtained at B0 = 270 MHz, B1 = 1370, 1075, 833, 641 Hz (samples 3
and 4), B1 = 1370 Hz (sample 1), B1 = 1370 and 1075 Hz (samples 2), irradiation time
2 s, 8 scans, pH 7.4 (samples 1–3 in 5 mM HEPES/HCl buffer, containing 140 mM
NaCl) and 25 °C, Eu-6 in water pH 7.4.
b
Data was measured at B0 = 500 MHz, B1 = 1412, 1075, 770, 540 Hz, irradiation
time 2 s, 8 scans, pH 7.4 (sample 5 in 5 mM HEPES/HCl buffer, containing 140 mM
NaCl) and 25 °C.
sumably positioning the Eu3+-bound water molecule somewhat
farther away from the protein surface.
Similar fittings were performed on CEST spectra of the Eu-2–
HSA conjugate to estimate a value for sM. In this case, both Eu-2
Figure 4. Determination of the detection limit of the CEST effect for Eu-1–HSA
conjugate using two power of irradiation (B1 = 1370 Hz and B1 = 1075 Hz), irradi-
ation time 2 s,
8 scans, 25 °C, pH 7.4 (5 mM HEPES/HCl, 140 mM NaCl),
and EuDTMA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(N-
methyl)acetamide) were compared as model chelates based on
their structural similarities. From Table 2 it can be seen that bound
water lifetime in Eu-2 and EuDTMA are the same as the average va-
lue found for the Eu-2–HSA conjugate. This indicates that the pro-
tein surface has little or no influence on water proton exchange
when Eu-2 is covalently attached to the protein via its sole car-
boxyl side-arm. This is in contrast to the small decrease in proton
exchange rates reported for other similar systems.10–13 While Eu-1
and Eu-2 should have nearly identical inner-sphere Eu3+-bound
water structures, the primary difference between these two che-
lates when covalently attached to a protein is their point of the
attachment to the protein surface. Although one might anticipate
a priori that the Eu3+-bound water site in a covalently attached
Eu-1 species would be situated further from the protein surface
via the rigid benzyl thiourea linkage, it is this structure that seems
to be impacted more by its attachment to the surface that Eu-2. In
the later case where Eu-2 is attached to a protein surface via its
more flexible aliphatic side-chain, this appears to have less affect
on water or proton exchange. Clearly, further experiments with
different proteins will need to be done to see if this trend holds
for other systems as well. These studies do illustrate that covalent
attachment of a lanthanide complex to a surface has much less im-
pact on water access to the lanthanide coordination sphere com-
pared to small lanthanide chelates that bind to albumin non-
covalently. This perhaps is not surprising since non-covalent bind-
ing of small molecules to albumin occur at sites buried further into
the protein structure where interactions with other protein side
chains is much more likely than for a lanthanide chelate attached
to surface moiety.
B0 = 270 MHz. Dilutions were made using HEPES buffer.
pared for CEST measurements contained only 0.32 mM Eu-1, near
the estimated detection limit based upon the HSA results. Never-
theless, the same CEST spectrum was observed for this sample
using a B1 of 1370 Hz (data not shown) as observed for Eu-1-
HSA. The Eu3+-bound water proton lifetime determined from this
single CEST spectrum was 71 ls, a value similar to that found for
Eu-1 conjugated to HSA. During the second preparation of Eu-1-
3G4 (7.4 chelates), a larger amount of material was obtained allow-
ing preparation of a sample with a higher concentration of anti-
body (ꢁ170
lM) and Eu-1 (1.26 mM) for CEST measurements.
CEST spectra acquired at 1370 and 1075 Hz were fitted to a similar
three-pool model to obtain a Eu3+-bound water proton lifetime of
77
2 ls. This suggests that water proton exchange in Eu-1 is rel-
atively insensitive to protein identity, although more systems must
be studied to substantiate this conclusion. Even though the con-
centration of 3G4 antibody in the first sample was only ꢁ32
lM,
this is still much higher than one would estimate for a local anti-
body concentration in a biological sample. Thus, we must tenta-
tively conclude that CEST imaging using a Eu-1 protein conjugate
may not be sensitive enough for detection of PS in a tumor. How-
ever, it was recently shown that thulium chelates are substantially
more sensitive than europium chelates for CEST imaging,32 so the
detection limit may be well below that estimated here for Eu-1-
HSA and Eu-1-3G4.
Although proton exchange rates were similar in Eu-1-HSA and
Eu-1-3G4, it was of interest to compare these exchange rates with
that of an appropriate model compound. Eu-6 was examined in
place of Eu-1 as discrete chelate analog to eliminate possible sam-
ple degradation through hydrolysis of the isothiocyanate group of
Eu-1 during the measurements. Interestingly, a fitting of CEST
spectra of Eu-6 collected using different B1 values yielded a slightly
3. Experimental
3.1. General remarks
shorter bound water proton lifetime (53
2 ls) than those mea-
sured for the protein conjugates (see summary in Table 2). This
suggests that proton exchange may be slowed slightly upon conju-
gation of Eu-1 to a protein. However, this increase in the bound
water proton lifetime is modest compared to the previously re-
ported changes10 but, in most cases, the previous studies have in-
volved hydrophobic interactions of Gd3+ chelates with albumin
binding pockets where the bound water molecule is likely signifi-
cantly closer to the protein surface than in this study. Here, Eu-1
likely reacts with the most solvent accessible lysine residues pre-
All organic precursors and solvents were obtained from com-
mercial sources and used as received unless otherwise stated. Hu-
man serum albumin (HSA, 66.7 kDa) was purchased from Fluka.
The monoclonal antibody (3G4) was generously provided by Pere-
grine Pharmaceuticals Inc., Tustin, CA. 1H and 13C NMR spectra
were acquired on a JEOL Eclipse 270 spectrometer operating at
270.17 and 67.93 MHz, respectively. Chemical shifts (d) are re-
ported in parts per million. CEST spectra and T1 values were mea-
sured using either a JEOL Eclipse 270 or a Varian INOVA 500

O. Vasalatiy et al. / Bioorg. Med. Chem. 19 (2011) 1106–1114
1111
spectrometer (operating at 499.99 MHz). Prior to data acquisition,
samples were warmed to room temperature and equilibrated in
the probe at 25 °C for at least 10 min before measurement. T1
was measured by using an inversion-recovery sequence with 15
different delay times and 8 averages. CEST spectra were recorded
by application of a long presaturation pulse at selected frequencies
across the spectrum followed by a single observe pulse to measure
the residual water signal. Infrared spectra were recorded on a Nico-
let Avatar 360 FT-IR spectrometer as either KBr pellets or as neat
liquids. UV–vis absorption spectra were recorded using a Shimadsu
UV-1601 double beam diode array spectrophotometer. Hydrogena-
3.1.3. 2-p-Aminobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetra(methylcarbonylamide) (6)
10% Palladium (0.4 g) on carbon was added to a solution of 5
(0.41 g, 0.69 mmol) in ethanol (10 mL). The reaction mixture was
placed in a hydrogenation vessel for 24 h with the H2 pressure
set to 45 psi. The sample was mixed twice during this 24 h period
for 10–15 min each time. The reaction mixture was filtered
through CeliteÒ545 (Aldrich) and the filtrate concentrated under
reduced pressure to afford the title compound as a pale solid
(0.39 g, 99% yield). 1H NMR (270 MHz, D2O pD 14): d = 2.60–3.67
(37H, m), 6.78 (2H, d, Ar), 7.02 (2H, d, Ar). 13C NMR (67.5 MHz,
D2O pD 14): d = 26.8–26.02 (CH3), 30.9 (CH2Ar), 51.3–52.6 (CH2
ring, br), 57.0–57.4 (CH2CO, br), 116.9 (Ar), 130.3 (Ar), 145.5 (Ar),
146.4 (Ar), 173.7–174.6 (CONH, br). FTIR (KBr): 1156, 1247, 1357,
1410, 1464, 1557, 1676, 2827, 2968, 3076, 3450 cmꢀ1. m/z (ESI+)
562 ([M+H]+), 584 ([M+Na]+).
tions were performed using a Parr hydrogenation apparatus. [Eu3+
]
was determined analytically using an Elan 6100 DRC (PE Scien) ICP
Mass Spectrometer. Electrospray ionization mass spectrometry
(ESI-MS) was performed by HT Laboratories, San Diego, California.
Fast atom bombardment mass spectrometry (FAB-MS) was per-
formed by the Mass Spectrometry Facility at University of Alabama
at Tuscaloosa. Elemental analyses were obtained from Galbraith
Labs, Knoxville, Tennessee. Gel electrophoresis was performed on
Pharmacia LKB (Phast System) using phast gel (Gradient 4–15
Amersham Bioscience). 1-Benzyloxycarbonyl-1,4,7,10-tetraazacy-
clododecane trihydrochloride salt (referred to as mono-protected
CBz-cyclen),33 2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane,26
and 1,4,7,10-tetra(methylcarbonylamide)-1,4,7,10-tetraaza cyclod
odecane (DTMA)28 were prepared using published methods.
3.1.4. 2-p-Isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetra(methylcarbonylamide) (1)
A solution of thiophosgene (0.051 g, 446
(2 mL) was added to a solution of 6 (0.065 g, 116
l
mol) in chloroform
mol) in water
l
(1 mL) and the mixture was stirred at room temperature for 2 h.
The water fraction was separated and the organic layer extracted
with water (2 ꢂ 1 mL). The water fractions were combined and
freeze-dried to give the title compound as a pale solid (0.07 g,
99% yield). 1H NMR (270 MHz, D2O pD = 2) d = 2.62–2.77 (12H,
m, CH3), 3.05–4.11 (25H, m, CH2Ar, CH2CO and ring CH2), 7.31
(4H, m, Ar). FTIR (KBr): 1158, 1236, 1362, 1410, 1471, 1571,
1689, 2116 (N@C@S), 2947, 2975, 3099, 3267. m/z (ESI+) 604
([M+H]+), 626 ([M+Na]+).
3.1.1. 2-Bromo-N-methylacetamide (3)
K2CO3 (55.3 g, 400 mmol) and methylamine hydrochloride
(13.5 g, 200 mmol) were added to dichloromethane (700 mL) and
the suspension was cooled to 0 °C. Bromoacetyl bromide
(39.75 g, 200 mmol) was then added dropwise with vigorous stir-
ring, and the reaction mixture was stirred at 0 °C for an additional
1 h followed by 2 h at room temperature. Water (70 mL) was then
added and the organic layer that separated was dried overnight
over Na2SO4. The solvent were removed under reduced pressure
and the residue was crystallized from diethylether to afford a col-
orless compound (23.4 g, 77% yield). 1H NMR (270 MHz, CDCl3):
d = 2.8 (3H, d, CH3), 3.9 (2H, s, CH2), 6.8 (1H, s br, NH); 13C NMR
(67.5 MHz, CDCl3): d = 27.0 (CH3), 29.2 (CH2), 166.1 (CONH). NMR
data is consistent with published data.28
3.1.5. 1-Benzyloxycarbonyl-1,4,7,10-tetraazacyclododecane-4,7,10-
tri(methylcarbonylamide) (8)
K2CO3 (3.37 g, 24.4 mmol) was added to a solution of mono-
protected CBz-cyclen 7 (1.1 g, 2.44 mmol) in acetonitrile (20 mL),
the suspension was heated warmed to 70 °C, and 2-bromo-N-
methylacetamide (1.15 g, 24.9 mmol) was added in one portion.
The reaction mixture was stirred at 65–70 °C for 12 h, filtered, then
concentrated under vacuum. The resulting residue was dissolved in
water, extracted with dichloromethane (3 ꢂ 50 mL), and the com-
bined organic fractions dried over Na2SO4 overnight. After filtering,
the solvent was removed under reduced pressure and the residue
purified by silica gel column chromatography, eluting with 10%
methanol in chloroform to afford the title compound as a colorless
gum (1.08 g, 85% yield). Rf = 0.3 (SiO2, MeOH/CHCl3, 1:9). 1H NMR
(270 MHz, CD3CN): d = 2.82 (8H, s br, ring CH2), 2.89 (6H, s, CH3),
2.93 (3H, s, CH3), 3.14 (4H, s br, ring CH2), 3.30 (4H, s br, ring
CH2), 3.53 (2H, s, CH2CO), 3.74 (4H, s, CH2CO), 5.3 (2H, s, OCH2Ph),
7.52 (2H, m br, NH), 7.58 (5H, m, Ph), 7.7 (1H, m br, NH). 13C NMR
(67.5 MHz, CD3CN) d = 25.4 (CH3), 25.6 (CH3), 47.0 (CH2 ring), 47.4
(CH2 ring), 53.4 (CH2 ring br), 58.3 (NCH2CO), 67.0 (OCH2Ph), 128.0
(Ph), 128.3 (Ph), 129.9 (Ph), 137.6 (Ph), 156.7 (CONH), 171.7
(CONH), 171.9.9 (COO). FTIR (NaCl pallet, CHCl3): 1049, 1119,
1157, 1219, 1417, 1533, 1673, 2827, 3013, 3056, 3352 cmꢀ1. m/z
(ESI+) 558 (100% [M+K]+).
3.1.2. 2-p-Nitrobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetra(methylcarbonylamide) (5)
K2CO3 (1.25 g, 9 mmol) was added to a solution of p-NO2 benzyl
cyclen 4 (0.6 g, 1.95 mmol) in acetonitrile (10 mL) and the suspen-
sion was heated at 70 °C for 30 min. 2-bromo-N-methylacetamide
(1.25 g, 8.21 mmol) was then added in one portion. The reaction
mixture was heated for seven days at 65–70 °C, allowed to cool
and filtered. The solvents were then removed under reduced
pressure. The resulting residue was purified by column chromatog-
raphy over silica gel. The column was first eluted with dichloro-
methane to remove impurities followed by elution of the title
compound with methanol. The residue obtained from the column
was dissolved in chloroform, filtered and solvent was removed
by vacuum to afford the title compound as a yellow solid (0.68 g,
71 % yield). Rf = 0.4 (SiO2, MeOH). 1H NMR (270 MHz, D2O, pD
14): d = 2.5 (4H, m br, ring CH2), 2.75–2.8 (12H, m, CH3), 3.01–
3.12 (4H, m br, ring CH2), 3.2–3.4 (7H, m, ring CH2), 3.7–4.14
(10H, m, CH2Ar and CH2CO), 7.5 (2H, d, Ar), 8.2 (2H, d, Ar). 13C
NMR (67.5 MHz, D2O pD 14): d = 25.7–26.1 (CH3), 32.0 (CH2Ar),
51.01–52.5 (CH2 ring br), 56.1–56.4 (CH2CO) 124.1 (Ar), 130.5
(Ar), 146.4 (Ar), 147.8 (Ar), 171.0–173.7 (CONH, br). FTIR (KBr):
1181, 1203, 1348, 1382, 1518, 1561, 1673, 2850, 2971, 3083,
3250. m/z (ESI+) 614 ([M+Na]+), 630 ([M+K]+). Anal. Found: C,
51.7; H, 7.8; N, 19.8. C27H45N9O6ꢃ2H2O requires C, 51.7; H, 7.9; N,
20.1.
3.1.6. 1,4,7,10-Tetraazacyclododecane-1,4,7-tri(methylcarbonyla-
mide) (9)
10% Palladium on carbon (0.2 g) was added to a solution of 8
(0.93 g, 1.5 mmol) in ethanol (30 mL). The reaction mixture was
placed in a hydrogenation vessel for 72 h with the H2 pressure
set to 50 psi. The sample was mixed for 10–15 min twice per day
during this 72 h period. The catalyst was filtered through Cel-
iteÒ545 (Aldrich), the solvents were removed under reduced pres-
sure to afford the title compound colorless oil (0.57 g, 99% yield).
1H NMR (270 MHz, D2O pD 9): d = 2.54 (4H, s br, ring CH2), 2.63

1112
O. Vasalatiy et al. / Bioorg. Med. Chem. 19 (2011) 1106–1114
(4H, s br, ring CH2), 2.69–2.70 (9H, m, NHCH3), 2.77 (4H, s br, ring
CH2), 3.06–3.10 (6H, m, CH2CONH), 3.23–3.24 (4H, s br, ring CH2).
13C NMR (67.5 MHz, D2O pD 9) d = 25.8 (CH3), 26.9 (CH3), 44.9 (CH2
ring), 49.5 (CH2 ring), 51.1 (CH2 ring), 53.4 (CH2 ring), 56.3
(CH2CONH), 58.3 (CH2CONH), 174.1 (CONH), 174.5 (CONH). FTIR
(NaCl pallet, CHCl3): 1111, 1212, 1305, 1417, 1546, 1647, 2842,
2924, 3091, 3317 cmꢀ1. m/z (ESI+) 386 ([M+H]+), 408 ([M+Na]+).
Anal. Found: C, 45.3; H, 9.0; N, 21.5. C17H35N7O3 ꢂ 3.5H2O requires
C, 45.5; H, 9.4; N, 21.8.
(CH2 ring, br), 50.2 (CH2 ring, br), 51.3 (CH2 ring, br), 51.9
(CH2CONH), 54.6 (CH2CONH), 56.1 (CH2CO2), 166.2 (CONH), 169.5
(CONH), 172.0 (CONH), 172.9 (CONH), 172.9 (CONH), 176.0 (CO2).
FTIR (NaCl pallet, CHCl3): 769, 1212, 1642, 1992, 3060,
3414 cmꢀ1. m/z (ESI-) 499 (100% [MꢀH]ꢀ).
3.2. General procedure for preparation of lanthanide chelates
An aqueous solution of the appropriate ligand was adjusted to a
pH of ꢁ5.5 using 1 M NaOH/1 M HCl as necessary. A stoichiometric
quantity of standardized lanthanide chloride aqueous solution was
added and the reaction mixture was stirred at room temperature
with constant adjustment of pH to 5.0–5.5 using 1 M NaOH. The
extent of the chelation reaction was monitored by following
changes in the reaction pH, and were typically observed to be com-
plete ꢁ30 min. The absence of free lanthanide ions was established
with xylenol orange (0.15 M acetate buffer, pH 5.5). The pH was
then adjusted to 7 and the samples were filtered and freeze-dried
to give the chelates. Eu-1: FTIR (KBr): 2116 (N@C@S) cmꢀ1; m/z
(FAB+) 754 and 756 [Eu3+1–2H]+; Eu-2: m/z (FAB+) 649 and 651
[Eu3+2ꢀ–H]+; Eu-6: m/z (FAB+) 710 and 712 [Eu3+6ꢀ–H]+ with an
appropriate isotope pattern was observed.
3.1.7. N-(2-Bromoacetyl) glycine benzyl ester (10)
K2CO3 (13.83 g, 10 mmol) was added to a solution of glycine
benzyl ester, p-toluenesulfonate salt (1.68 g, 5 mmol) in dichloro-
methane (50 mL). The suspension was cooled to 0 °C and, with vig-
orous stirring, bromoacetyl bromide (0.99 g, 5 mmol) was added
drop wise over ꢁ5 min. After the addition was complete, the reac-
tion mixture was stirred at 0 °C for 1 h and at room temperature for
an additional 2 h. Water (10 mL) was then added and the organic
layer was separated and dried over Na2SO4 overnight. The drying
agent was then filtered and the solvent removed under reduced
pressure. The resulting residue was crystallized from hexane to af-
ford the title compound as a colorless solid (1.04 g, 73% yield). 1H
NMR (270 MHz, CDCl3): d = 3.9 (2H, s, CH2CO), 4.11 (2H, d,
CH2CO2), 5.2 (2H, s, CH2Ph), 6.97 (1H, s br, NH), 7.36 (5H, m, Ph).
13C NMR (67.5 MHz, CDCl3): d = 28.7 (CH2CONH), 42.0 (CH2CO2),
67.6 (CH2Ph), 128.6 (Ph), 128.8 (Ph), 134.9 (Ph), 165.8 (CONH),
169.1 (CO2). FTIR (KBr): 940, 1041, 1188, 1262, 1382, 1444, 1538,
1662, 1763, 2877, 2966, 3064, 3282 cmꢀ1. m/z (ESI-) 284 (100%
[MꢀH]ꢀ).
3.2.1. Conjugation of Eu-1 to HSA
HSA (40 mg, 0.6
lmol) was dissolved in 1.5 mL of HEPES/HCl
buffer (5 mM, 140 mM NaCl, pH 8.6). Eu-1 (11.3 mg, 12
lmol)
was dissolved in 0.15 mL of water and added to the protein solu-
tion. The reaction mixture was incubated at room temperature
for 3 h, then purified by dialysis against HEPES/HCl buffer (5 mM,
140 mM NaCl, pH 7.4) for 48 h (4 ꢂ 2 L) at room temperature and
followed by gel permeation chromatography over Sephadex (G-
25) equilibrated with HEPES/HCl buffer (5 mM, 140 mM NaCl, pH
7.4). The purified conjugate (ꢁ2.2 mL) was concentrated to
ꢁ0.2 mL using an Ultrafree-MC Microcentrifuge filter (NMWL
5000 Da). After the first concentration step, 0.3 mL of fresh buffer
was added and the volume was reduced again to ꢁ0.4 mL.
3.1.8. 1-N-Acetyl benzyl glycinate-1,4,7,10-tetraazacyclodode-
cane-4,7,10-tri(methylcarbonyl amide) (11)
K2CO3 (0.21 g, 1.5 mmol) was added to a solution of 9 (0.5 g,
1.3 mmol) in acetonitrile (10 mL), the suspension was heated for
20 min at 70 °C, and N-(2-bromoacetyl) glycine benzyl ester 10
(0.43 g, 1.5 mmol) was added in one portion. The reaction mixture
was stirred at 65–70 °C overnight, then filtered and solvent re-
moved under reduced pressure. The resulting residue was purified
by silica gel column chromatography. The column was first eluted
with CHCl3/MeOH (9.5:0.5) to remove impurities, and the title
compound was then eluted with CHCl3/MeOH/NH4OH (3:2:1) as
the slowest moving fraction. The fractions were concentrated, dis-
solved in 2% methanol in chloroform, and filtered and the solvents
were removed under vacuum to afford title compound (0.61 g, 79.5
% yield). Rf = 0.65 (SiO2, CHCl3/MeOH/NH4OH, 3:2:1). 1H NMR
(270 MHz, CD3CN): d = 2.56–3.19 (33H, ring CH2, CH3, CH2CO),
3.95 (2H, d, NHCH2CO2), 5.13 (2H, s, OCH2Ph), 6.73 (4H, m br,
CONH), 7.38 (5H, m, Ph). 13C NMR (67.5 MHz, CD3CN) d = 25.3
(CH3), 25.5 (CH3), 41.0 (CH2COO), 50.1–50.9 (CH2 ring br), 56.9
(NCH2CO), 57.1 (NCH2CO), 66.5 (CH2Ph), 128.2 (Ph), 128.3 (Ph),
129.7 (Ph), 136.1 (Ph), 169.7 (CONH), 172.0 (CONH), 172.5 (CO2).
FTIR (NaCl pallet, CHCl3): 672, 761, 1216, 1642, 1988, 3080,
3394 cmꢀ1. m/z (ESI+) 591 ([M+H]+, 613 ([M+Na]+).
3.2.2. Conjugation of Eu-1 to 3G4
3G4 (23 mg/mL, 77 nmol) was dialyzed in a Tube-O-Dialyzer™
(2 mL, GBioscences) against HEPES/HCl buffer (5 mM, 140 mM
NaCl, pH 8.6) for 24 h (2 ꢂ 2 L) at 4 °C. The final concentration
was obtained by measuring the absorbance at 280 nm
(
e
= 1.4 mL/(cm ꢂ mg)). Eu-1 (3.6 mg, 3.85
lmol) was dissolved in
0.1 mL of water and added to the 3G4 solution (22 mg/mL). The
mixture was incubated at room temperature for 3 h, followed by
dialysis against HEPES/HCl buffer (5 mM, 140 mM NaCl, pH 7.4)
for 48 h (4 ꢂ 2 L) at 4 °C. The purified conjugate was concentrated
using absorbent (Spectra/Gel Absorbent, SpectrumÒ, Spectrum
Lab) for 6 h at 4 °C.
3.2.3. Conjugation of Eu-2 to HSA
Europium 1-N-hydroxysuccinimidylacetatoamido glycinate-
1,4,7,10-tetraazacyclododecane- 4,7,10-tri(methylcarbonylamide)
chloride was obtained by the method of Lewis et al.29 Eu-2
(0.11 g, 0.12 mmol) was dissolved in water (0.86 mL, 4 °C), and
the pH adjusted to 5.5, followed by addition of N-hydroxy-
succinimide (13.7 mg, 0.12 mmol) in 0.1 mL of water. The reaction
mixture was stirred for 5 min and 0.02 mL 1-ethyl-3-[3-dimethyl-
3.1.9. 1-N-Acetylglycine-1,4,7,10-tetraazacyclododecane-4,7,10-
tri(methylcarbonylamide) (2)
10% Palladium on carbon (0.1 g) was added to a solution of 11
(0.45 g, 0.76 mmol) in ethanol (10 mL). The suspension was placed
on a hydrogenation apparatus operating at 45 psi and room tem-
perature for 96 h. The catalyst was filtered through CeliteÒ545 (Al-
drich) and the solvents removed under reduced pressure to afford
the title compound as a colorless amorphous solid (0.37 g, 97 %
yield). 1H NMR (270 MHz, D2O pD = 8): d = 2.72–2.73 (9H, 2 s,
CH3), 2.9 (4H, s br, ring CH2), 3.1–3.4 (12H, m br, ring CH2), 3.5
(4H, s, CH2CONH), 4.73–4.75 (6H, m, CH2CO). 13C NMR
(67.5 MHz, D2O) d = 25.9 (CH3), 26.0 (CH3), 43.5 (CH2CO2), 49.81
aminopropyl]carbodiimide
hydrochloride
(EDC)
(12.2 mg,
60 mol) aqueous solution was added. The reaction mixture was
l
stirred for 30 min at 0 °C followed by pH adjustment to 7.5 with
0.25 M Na2HPO4. The europium activated ester chelate 12 was
used for conjugation without isolation.
HSA (40.0 mg, 0.6 lmol) was dissolved in 0.78 mL of HEPES/HCl
buffer (5 mM, 140 mM NaCl, pH 7.4) followed by addition of the
europium activated ester chelate 12 (theoretical amount:

O. Vasalatiy et al. / Bioorg. Med. Chem. 19 (2011) 1106–1114
1113
60
l
mol). The reaction mixture was then incubated at room tem-
the way in which various parameters affect the fitting routine.
Each saturation peak must be fitted to an appropriate intensity
and width (shape). The intensity of the peak is predominantly
determined by two factors the concentration of the exchanging
pool and the T1 of protons within that pool. In general it is prefer-
able to experimentally determine both these parameters. However,
the exchangeable protons associated with the protein represent a
diverse range of protons and environments consisting of: amide,
hydroxyl and amine protons of the protein as well as water mole-
cule hydrogen bound to the protein structure. For this reason
determining a precise value of concentration or T1 is not possible.
And yet, it is possible to take advantage of the nature of the fitting
routine’s reliance on these two parameters for determining peak
intensity to produce a valid fitting without knowing either value
exactly. The T1 value of this pool was allowed to float substantially
and even take unrealistic values. In so doing the fitting routine it-
self effectively self corrects for any error in the chosen concentra-
tion value—if the concentration value is too high then the T1 value
used in fitting will be shorter to compensate.
The third exchange pool is inhomogeneous in chemical shift
and will necessarily be broad. The line-width (shape) in the
model is predominantly determined by the exchange rate of each
pool with the bulk (note, there is no exchange in this model
between the bound water pool and the third exchanging pool) and
T2. One may take advantage of this to overcome the uncertainty
in chemical shifts by allowing the center of the peak to shift over
a reasonable diamagnetic range (1–6 ppm) and allowing T2 to
adopt unrealistically short values. In this way the peak is allowed
to become wide enough to encompass spins of all chemical
shifts. Since there is little or no shift difference between the bulk
water pool and this third pool, exchange between these pools
will have little effect upon the shape of either peak and the
shape of the peak of the third pool will be predominantly deter-
mined by T2.
perature for 2 h. The reaction mixture was purified by dialysis
against HEPES/HCl buffer (pH 7.4) for 48 h (4 ꢂ 2 L) at room tem-
perature followed by gel permeation chromatography over Sepha-
dex (G-25) equilibrated with HEPES/HCl buffer (pH 7.4). The
purified conjugate (ꢁ2.2 mL) was concentrated to ꢁ0.2 mL using
Ultrafree-MC Microcentrifuge filters (NMWL 5000 Da). After the
first concentration step, 0.3 mL of fresh buffer was added and the
once again the volume was reduced to ꢁ0.4 mL.
3.3. Determination of Eu-antibody/protein ratio
The number of europium chelates per antibody or HSA was
measured by Inductively Coupled Plasma Mass Spectrometry (ICP
MS). The uncertainty in the measurement reflects the deviation
of three parallel measurements (n = 3). The peak intensities were
fitted to a four point calibration curve to obtain a mean value.
The antibody/protein concentrations were determined by standard
Lowry assay. Three measurements were performed on each conju-
gate preparation.
3.4. ELISA assays
Eu-1–3G4 samples were diluted with 2% fetus bovine serum
(FBS) in PBS buffer to a concentration of 2 mg/mL antibody. As a
control, unmodified 3G4 was used at the same concentration.
Phosphatidylserine (50 lL, 10 lg/mL) in chloroform was added to
each well in the 96-well plate and chloroform was evaporated at
room temperature. 0.2 mL of the blocking buffer (10% FBS in PBS)
was added to each well and this was incubated at 37 °C for 1 h.
The blocking buffer was removed and the plate was washed three
times with PBS buffer. Samples were prepared in duplicate using a
two fold dilution of 2 mg/mL 3G4 with blocking buffer. After incu-
bating the plate at 37 °C for 2 h, it was washed three times with
PBS to remove unbound 3G4. 0.1 mL of 0.8
l
g/mL HRP (Horserad-
Since it is exchange between bound and bulk water pools that is
ish peroxidase) conjugated goat anti-mouse IgG was then added to
each well and the samples were incubated at 37 °C for 1 h. The
plate was then washed three times with PBS buffer followed by
being probed, the values concerning the bulk water and Eu3+
-
bound water pools were then tightly constrained: T1 values were
experimentally determined for the bulk and values previous deter-
mined for the bound pool in a related system employed.33 T2 values
were constrained to be shorter than T1 and fall within the range of
values previously determined for similar systems. In this way the
MATLAB™ fitting routine was constrained to fit the shape of the
bound pool predominantly by altering water proton exchange
alone, and was thus unaffected by the apparent increase in the
width of the direct saturation peak at 0 ppm. This approach to fit-
ting accommodates the signal arising from the protein without
affecting the line-widths and shapes of the two exchange pools
of interest. However, the fitting does as a consequence become
inherently more dependent upon the line-shape of the bound pool;
as a result, as B1 is increased and the spectrum broadens the values
of sHM obtained in the fitting shorten slightly because the shape of
this peak is broader but less well defined and therefore provides
a somewhat less reliable fit.
addition 100
lL of a developing reagent (10 mL of 0.2 M Na2PO4,
10 mL of 0.1 M citric acid, 10 mg of 1,2-diaminobenzene, 10
lL of
H2O2) to detect the second antibody. The samples were incubated
for 10 min, followed by addition of 0.1 mL of 0.18 M H2SO4 to stop
the reaction. The optical density at 490 nm was read using a 96
well plate reader (7520-Microplate reader, Cambridge Technology,
Inc).
3.5. Fitting of CEST spectra (Z-spectra) to a three pool exchange
model
The water exchange kinetics of each chelate-conjugate were
determined by fitting the acquired Z-spectra to the Bloch equations
modified for exchange using a process described previously.34 In
general EuDOTA-tetraamide chelates can only be fitted by using
a 3-pool exchange model that includes bulk water, the Eu3+-bound
water molecule and the ligand amide protons (typically four per
chelate). The amide protons in these Eu3+ chelates experience
rather small paramagnetic shifts and are often not fully resolved
from the bulk water peak itself. Nevertheless, inclusion of these
known exchange sites in the fitting procedure is important in cor-
rectly fitting the shape of the bulk water peak and in obtaining the
Acknowledgements
This work was supported in parts by grants from the National
Institutes of Health (CA-115531 and RR-02584) and the Robert A.
Welch Foundation (AT-584). We also thank Dr. Matthew Ley-
bourne for his assistance with ICP MS measurements.
correct exchange rate constants for the more highly shifted Eu3+
bound water exchange peak.
-
For Eu3+ chelates conjugated to a protein, the CEST fitting proce-
dure is further complicated by a large number of exchangeable
protons and/or water molecules closely associated with the pro-
tein. In order to fit these spectra it is important to be mindful of
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