Bifunctional chelates optimized for molecular MRI

Article abstract of DOI:10.1021/ic500085g

Important requirements for exogenous dyes or contrast agents in magnetic resonance imaging (MRI) include an effective concentration of paramagnetic or superparamagnetic ions at the target to be imaged. We report the concise synthesis and characterization of several new enantiopure bifunctional derivatives of (α¹R,α⁴R,α⁷R, α¹⁰R)-α¹,α⁴, α⁷,α¹⁰-tetramethyl-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTMA) (and their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) analogues as controls) that can be covalently attached to a contrast agent delivery system using either click or peptide coupling chemistry. Gd complexes of these derivatives can be attached to delivery systems while maintaining optimal water residence time for increased molecular imaging sensitivity. Long chain biotin (LC-biotin) derivatives of the Eu(III) and Gd(III) chelates associated with avidin are used to demonstrate higher efficiencies. Variable-temperature relaxometry, ¹⁷O NMR, and nuclear magnetic resonance dispersion (NMRD) spectroscopy used on the complexes and biotin-avidin adducts measure the influence of water residence time and rotational correlation time on constrained and unconstrained systems. The Gd(III)-DOTMA derivative has a shorter water residence time than the Gd(III)-DOTA derivative. Compared to the constrained Gd(III)-DOTA derivatives, the rotationally constrained Gd(III)-DOTMA derivative has ～40% higher relaxivity at 37 °C, which could increase its sensitivity as an MRI agent as well as reduce the dose of the targeting agent.

Full text of DOI:10.1021/ic500085g

Article
pubs.acs.org/IC
Open Access on 06/16/2015
Bifunctional Chelates Optimized for Molecular MRI
Erik C. Wiener,† Marie-Caline Abadjian,‡ Raghvendra Sengar,† Luce Vander Elst,§
Christoffel Van Niekerk,‡ Douglas B. Grotjahn,*,‡ Po Yee Leung,‡ Christie Schulte,‡ Curtis E. Moore,⊥
and Arnold L. Rheingold⊥
†Hillman Cancer Center, University of Pittsburgh Medical Center, 5117 Centre Avenue, Pittsburgh, Pennsylvania 15213, United
States
‡Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, California 92182-1030,
United States
§Department of General, Organic and Biomedical Chemistry, University of Mons, 7000 Mons, Hainaut, Belgium
⊥Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0385, United States
S
* Supporting Information
ABSTRACT: Important requirements for exogenous dyes or
contrast agents in magnetic resonance imaging (MRI) include
an effective concentration of paramagnetic or superparamag-
netic ions at the target to be imaged. We report the concise
synthesis and characterization of several new enantiopure
bifunctional derivatives of (α1R,α4R,α7R,α10R)-α1,α4,α7,α10-
tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTMA) (and their 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid (DOTA) analogues as controls) that
can be covalently attached to a contrast agent delivery system using either click or peptide coupling chemistry. Gd complexes of
these derivatives can be attached to delivery systems while maintaining optimal water residence time for increased molecular
imaging sensitivity. Long chain biotin (LC-biotin) derivatives of the Eu(III) and Gd(III) chelates associated with avidin are used
to demonstrate higher efficiencies. Variable-temperature relaxometry, 17O NMR, and nuclear magnetic resonance dispersion
(NMRD) spectroscopy used on the complexes and biotin−avidin adducts measure the influence of water residence time and
rotational correlation time on constrained and unconstrained systems. The Gd(III)-DOTMA derivative has a shorter water
residence time than the Gd(III)-DOTA derivative. Compared to the constrained Gd(III)-DOTA derivatives, the rotationally
constrained Gd(III)-DOTMA derivative has ∼40% higher relaxivity at 37 °C, which could increase its sensitivity as an MRI agent
as well as reduce the dose of the targeting agent.
he enormous versatility of magnetic resonance imaging
T(MRI) offers the promise of combining anatomical images
with noninvasive in vivo histopathology, molecular and cellular
imaging, imaging metabolic pathways, and functional imaging.
Over the years MRI has achieved a number of successes,
including dynamic contrast enhancement (DCE) MRI1,2 as a
marker of changing vascular endothelial growth factor3,4 in
cancer, imaging receptor expression,5−11 stem cell track-
ing,12−16 transporter systems,17−20 using choline21−23 as a
marker for the PI3K pathway,24−26 the use of intracellular
sodium27,28 for imaging cell division,29 and blood oxygen level
monitoring.30−33 No matter the MR technique, one either
needs to use exogenous markers or endogenous markers.
The use of exogenous dyes or contrast agents requires a
suitable concentration of the agent at the desired target. To
achieve detection, a number of approaches have been used
including delivery of relatively large numbers of paramagnetic
or superparamagnetic ions (such as Gd, Mn, and Fe) in the
form of polymeric drug delivery systems34,35 and/or nano-
particles,6,8−10 or a smaller number of more highly efficient
agents, or a combination of the two.7,36,37
Free, unchelated gadolinium is generally toxic; hence,
sequestering it as a kinetically stable chelate is vital to reduce
toxicity to acceptable levels. A synthetic challenge addressed in
this work is the creation of a bifunctional chelating system that
has all of the following favorable properties: (1) rapid water
exchange that allows highly efficient relaxation and increased
sensitivity, (2) tight Gd3+ binding, and (3) easy covalent
attachment to a desired structure while maintaining the
properties that allow highly efficient relaxation and increased
sensitivity.
The sensitivity of any method described above depends on
the efficiency of the agent (relaxivity) either at the level of the
individual ion or the entire molecule. Relaxivity is calculated as
the change in relaxation rate (1/T1 or 1/T2) per unit
concentration of ion. Higher relaxivities result in more efficient
contrast agents and lower doses or an increased ability to detect
low-expression targets. The most common form of magnetic
Received: January 14, 2014
Published: June 16, 2014
© 2014 American Chemical Society
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Figure 1. Known (1−3) chelators for Gd and novel bifunctional compounds (4−7) made in this study.
resonance imaging (MRI) contrast agent works through
dipole−dipole interactions of the paramagnetic ion with
water protons. The chemist can alter variables in three
categories (inner-,38−41 second-,42 and outer-sphere43,44 con-
tributions) to optimize efficiency of a contrast agent. The inner-
sphere contribution depends on the number of water molecules
directly coordinated to the metal ion (q), the time that the
water molecules remain coordinated to the ion (the water
residence time, τM), the number of unpaired electrons (or spin
quantum number, S), the distance between the metal ion dipole
and dipole of the interest (r), the electronic relaxation time
(τS), and a measure of rotational diffusion or the rotational
correlation time (τR). The second-sphere contribution depends
on similar variables, which are associated with the second
coordination sphere. The outer-sphere contribution depends
on the number of unpaired electrons or spin quantum number
(S), the electronic relaxation time (τS), the distance between
the metal ion dipole and dipole of the interest (d), and the
relative diffusion coefficient D such that d2/D is the
translational correlation time (τD). For T1-based agents with
long electronic relaxation times, such as gadolinium [Gd(III)]
in the 0.2 to 3.0 T range commonly used in the clinic, the best
method to increase the efficiency of an agent is to increase the
rotational correlation time (τR).
Attaching ion-chelate complexes to macromolecules in-
creases the rotational correlation time and the relaxiv-
ity.36,37,41,45 Initial studies conjugated monofunctional chelates
to macromolecules by linking one of the chelate carboxylic
acids to an amine on the macromolecule to form an amide
bond. This resulted in an increase in relaxivity that was
significantly less than expected.41 Using variable-temperature
relaxivity studies Lauffer et al.41 predicted that the water
residence time was so long that it limited the relaxivity gains
associated with increasing the rotational correlation time. Using
the same method, others46 confirmed in general that Gd(III)
chelates containing amide ligands had water residence times
long enough to negate relaxivity gains associated with
increasing the rotational correlation time. At about the same
time by direct measurement of the water−oxygen exchange
rate, Gonzalez et al.47 confirmed a long water residence time for
the bismethyl−amide derivative of diethylenetriamine penta-
acetic acid (DTPA) (1, Figure 1) complexed to Gd(III) (2325
ns at 25 °C). They also reported that the water residence times
of both Gd(III)-DTPA and Gd(III)-DOTA are long, 244 and
208 ns, respectively (DOTA = 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid).
Theoretical studies predict that the optimum water residence
time for maximizing the relaxivity by increasing the rotational
correlation time of Gd(III)-based agents is between 10 to 60
ns.41,48 The direct measurements of the water residence time of
Gd complexes with either DTPA49 or DOTA49 (1 or 2, Figure
1) or their amide47 derivatives mentioned above confirmed that
the water residence times are greater than 200 ns. When
coupled with variable-temperature relaxometry studies, direct
measurements confirm that the water residence time of
Gd(III)-DOTA and Gd(III)-DTPA limits the gains in relaxivity
associated with increasing the rotational correlation
time.48,50−53
For the DOTA chelate two conformational isomers are
present, each with different water residence times and relaxivity
properties. It is well-known that modifications in the structure
of DOTA or its analogues such as adding a methyl group to
each acetate arm (to yield (α1R,α4R,α7R,α10R)-α1,α4,α7,α10-
tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTMA)) so that the complex is homochiral (Gd-
R,R,R,R-DOTMA, 3) or fusing cyclohexyl rings to the
backbone results in faster water exchange rates or shorter
water residence times and higher relaxivities of rotationally
constrained systems. The higher relaxivities result from shifting
the relative population of isomers to the one(s) with faster
water exchange.54−57
The discussion above indicates that contrast agents prepared
from Gd(III)-DOTMA should be more efficient and have
higher relaxivities than those prepared from Gd(III)-DOTA,
but the problem until the work reported here is that the existing
strategy for covalent attachment of the macrocycle−use of one
carboxylate for amide formation−leads to longer water
residence times and reduced relaxivities. Therefore, to leave
the tetracarboxylate ligand system intact, we designed and
prepared new bifunctional derivatives of DOTA and DOTMA
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Scheme 1. Synthesis of 4b-NaOTf and Amine Analogue 5b-NaOTf
(Figure 1), either bearing an azide-bearing side chain (4a and
4b, respectively) that can be linked to alkynes using click
chemistry58 (not shown in this work) or bearing an amine (5a
and 5b, respectively) for linking to carboxylic acids using
standard peptide ligation methods, as shown in Scheme 1.
Once coordinated to Gd or Eu, the resulting bifunctional agents
would be novel, versatile complexes that could selectively react
through click chemistry or peptide coupling, depending on the
need, to conjugate the chelate to a molecule of interest (see
Scheme 2). In contrast, commercially available bifunctional
imaging agents typically use carboxylate-active ester or
isothiocyanate groups for covalent attachment, unfortunately
compromising one or more of the chelating carboxylate arm(s)
and disrupting the optimal coordination geometry favored by
homochiral DOTMA; thus, they are not as effective. In this
report we describe the synthesis of 4a,b and 5a,b and the
derivatization of 5a,b with either a Cbz group or biotinylated
chain. Chelates for the low molecular weight rapidly rotating
models were derivatized at the amino group to demonstrate
bifunctionality and to prevent the free amine from wrapping
around and coordinating to the Gd(III), Scheme 3.
Scheme 2. Model Reactions Showing Feasibility of
Complexation of the various new bifunctional DOTA and
DOTMA species to Gd(III) allowed us to determine water
residence time and relaxivity measurements of low molecular
weight and macromolecular complexes of the amine derivatives.
The optimal water residence time varies as a function of field
strength. The complexes based on the DOTMA framework of 5
have optimal water residence times and significantly higher
relaxivities (more than 50%) when rotationally constrained by
biotin−avidin binding than the corresponding Gd-DOTA
derivative. These higher relaxivities are expected to translate
to lower doses or to the ability to detect targets at lower
expression levels.
Deprotection of N-Formyl Group by 9 and Water
Scheme 3. Synthesis of N3 and Cbz Model Complexes M-4a, M-4b, M-6a, and M-6b (M = Eu, Gd)
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MHz) showing peaks for 9: δ 5.25 (q, J = 7.0, 1H), 3.86 (s, 3H), 1.72
(d, J = 7.0, 3H). 19F NMR (CDCl3, 376.1 MHz): δ −75.2 (s)
trifluoromethanesulfonate (triflate, TfO) on 9 and −78.4 (s) free
triflate ion.
EXPERIMENTAL SECTION
■
General. Unless specified, all the reactions were done in oven-dried
or flame-dried glassware in an atmosphere of dry argon gas using
standard Schlenk techniques. The anhydrous solvents were purchased
from Acros and Aldrich chemical companies and used in the reactions
without further purification. All workup and chromatographic
purifications of compounds were done in air using Optima grade
solvents obtained from Fisher Scientific. Column chromatography was
done using a Teledyne ISCO CombiFlash Rf automated system.
Commercial grade preloaded Redi Sep Rf columns were used for silica
chromatography, and HiTrap SP HP columns purchased from GE
Healthcare were used for ion-exchange chromatography. The stains
used to visualize the thin-layer chromatography (TLC) plates
(aluminum backed 200 μm silica) were Hanessian’s Stain [CeSO4
(5 g) and (NH4)Mo7O24.4H2O (25 g) dissolved in water (450 mL)
and concentrated sulfuric acid (50 mL)], and potassium permanganate
stain [KMnO4 (1.5 g) and K2CO3 (10 g) dissolved in 10% NaOH
(1.25 mL) in water (200 mL)].
Synthesis of 11. A sample of 860,61 (3.44 g, 15.68 mmol) was taken
up in CH2Cl2 (40 mL), and the resulting solution was dried over
molecular sieves (beads, grade 512 type 4 Å, 4−8 mesh) overnight.
The flask was then chilled in an ice bath under nitrogen. Dry (distilled
from CaH2) N,N-diisopropylethylamine (12.47 g, 96.48 mmol) was
added dropwise, followed by the reaction mixture described above,
containing 9 (47.10 mmol assuming 100% yield) via cannula, and the
reaction flask was rinsed with CH2Cl2 (3 mL) to complete the transfer
of alkylating agent. After 3 h, an aliquot was removed from the
supernatant, solvent was removed using a nitrogen stream, and the
1
residue was analyzed in CD3OD by H NMR spectroscopy, showing
complete disappearance of the formylcyclen signal at 8.12 ppm and the
appearance of one major (>92%) singlet at 8.02 ppm. After a total of 4
h of reaction time, the mixture was filtered through a Buchner funnel
̈
containing Whatman filter paper (No. 40) to remove molecular sieves,
and the solids were rinsed with cold CH2Cl2 (50 mL). The combined
CH2Cl2 filtrates were washed with ice water (100 mL). The CH2Cl2
phase was washed with 3% NaOH (3 × 100 mL). The aqueous layers
were combined and back-extracted with CH2Cl2 (100 mL). All organic
layers were combined and washed with brine (100 mL) and dried over
Na2SO4. The mixture was filtered, and the filtrate was concentrated by
rotary evaporation, leaving an amber oil (8.21 g) containing
intermediate 10 and some 9. For 10 in the mixture: 1H NMR
(CD3OD, 399.8 MHz) δ 8.02 (s, 1H), 4.17 (ddd, J = 5.3, 8.4, 14.0,
1H), 3.89 (ddd, J = 4.2, 9.3, 13.7 1H), 3.678 (s, 3H), 3.671 (s, 6H) [In
addition, correlation spectroscopy data suggests additional peaks
overlap peaks in this region], 3.585 and 3.583 (two q, each with J =
7.0, total 2H), 3.47 (td, J = 5.2, 14.3 1H), 2.95−2.81 (m, 7H), 2.75
(td, J = 5.1, 14.2, 2H), 2.69−2.62 (m, 3H), 2.50−2.38 (m, 2H), 1.258
(d, J = 7.0, 3H), 1.250 (d, J = 7.2, 3H), 1.215 (d, J = 7.2, 3H). 13C
NMR (CD3OD, 100.5 MHz): δ 175.7, 175.6, 175.5, 165.5, 61.2, 60.0,
57.2, 53.8, 51.9, 51.8, 51.7, 51.6, 51.3, 51.1, 51.0, 50.2, 44.0, 15.7, 15.6,
15.2. 19F NMR (CD3OD, 376.1 MHz): δ −80.1 (s). ESI-MS m/z
459.2 (M + H), 481.3 (M + Na), (calculated for M = C21H38O7N4,
458.27). In addition, minor peaks in the 1H and 13C NMR spectra are
present for DOTMA or its Na+ OTf− adduct, an undesired side-
product. There was also evidence of a minor presence of DOTMA by
ESI-MS m/z 539.3 (M + Na), (calculated for M = C24H44O8N4,
516.32). The undesired DOTMA-NaTfO was not removed at this
point to minimize the number of purification steps to preserve yield
and reduce possible racemization of product
The crude product was dissolved in methanol (100 mL), and the
resulting solution was stirred as the flask was chilled in ice. Triflic acid
(5.09 g, 33.9 mmol, 2.0 equiv relative to the amount of 8 used; the
yield of intermediate 10 was assumed to be 100%) was added over 5
min. After an additional 5 min, the mixture was allowed to warm to
room temperature; then it was heated in a 60 °C oil bath for 7 h before
being cooled to room temperature and concentrated by rotary
evaporation. The residue was stored under vacuum for 10 h, leaving 11
(26.26 g, 33.9 mmol, 100%) as crispy beige foam, which quickly turns
sticky on exposure to moisture in air. 1H NMR (CD3OD, 399.8 MHz,
digital resolution = 0.20 Hz) δ 5.48 (s, 1H), 4.66 (q, J = 7.2, 1H), 4.37
(q, J = 7.0, 1H), 3.85 (s, 3H), 3.73 (s, 3H), 3.66 (s, 3H), 3.95−3.56
(m, 6H, partially overlapping with other signals and partially hidden
quartet at 3.72 ppm), 3.29−3.22 (m, 4H), 3.15−2.95 (m, 6H), 2.84−
2.60 (m, 3H), 1.64 (d, J = 7.24, 3H), 1.37 (d, J = 7.1, 3H), 1.36 (d, J =
6.8, 3H). 13C NMR (CD3OD, 100.5 MHz) δ 176.7, 176.5, 171.4,
121.9 (q, J = 318.6), 60.8, 56.5, 56.0, 55.7, 54.1, 53.2, 53.2, 48.8, 48.4,
46.3, 44.6, 44.3, 44.1, 44.0, 42.6. 19F NMR (CD3OD, 376.1 MHz): δ
−79.9 (s). 23Na NMR (CD3OD, 376.1 MHz): δ −3.4 (s).
Caution! Although we did not observe any unusual decompositions of
the azides reported, organic azides can be explosive materials and should
be handled with care.
1H, 13C, 23Na, and 19F NMR spectra were recorded using Bruker
1
ACP-500 or Varian spectrometers at 30 °C or room temperature. H,
13C, and 19F NMR chemical shifts are reported in ppm referenced to
residual solvent resonances (1H NMR: δ 7.27 for CHCl3 in CDCl3,
and 3.31 for CHD2OD in CD3OD. 13C NMR: δ 77.23 for CDCl3 and
49.15 for CD3OD. 19F NMR: −76.55 for CF3COOH). Deuterated
solvents for NMR were obtained from Cambridge Isotope
Laboratories and were used without purification. Electrospray
ionization mass spectra (ESI MS) were collected on a Micromass
Quattro II, triple quadrupole mass spectrometer using both negative
and positive ionization modes. Elemental analyses were performed at
NuMega Resonance Laboratories, San Diego, California. Liquid
chromatography−mass spectrometry (LCMS) spectra were recorded
on Agilent Technologies 6330 Ion Trap instrument. High-resolution
electrospray ionization time-of-flight (HR-ESI-TOF) analyses were
performed at Scripps Center for Metabolomics and Mass Spectrom-
etry, La Jolla, California. IR spectra were obtained on a Nicolet Nexus
670 FT-IR Instrument using KBr pellets.
Gadolinium concentration was measured by inductively coupled
argon plasma (ICP) mass spectrometry (University of Illinois at
Urbana−Champaign {UIUC}, Illinois Sustainability Technology
Center, Division of the Institute of Natural Resource Sustainability).
The sample (0.0500 mL) was added to Optima grade nitric acid
(0.500 mL) and heated to 80 °C in a closed vial overnight. The
solution was left for 7 weeks before being shipped to UIUC. Samples
were also measured by inductively coupled plasma optical emission
spectrometry (ICP-OES) (San Diego State University, Ecology
Analytical Facility).
X-ray Crystallography. After purification of 4a-NaOTf and 4b-
NaOTf by chromatography, the compounds were crystallized, and
data were acquired and analyzed by the Small Molecule X-ray
Crystallography facility at the University of California, San Diego.
Compound 4a-NaOTf was crystallized by slow evaporation of
CH2Cl2, whereas 4b-NaOTf was crystallized by vapor diffusion of
diethyl ether into a CH2Cl2 solution at −10 °C.
Synthesis of 9.59 To a dry 3-neck flask was added triflic anhydride
(13.7469 g, 48.7237 mmol, 1.03 equiv relative to (S)-methyl lactate)
and dry CH2Cl2 (15 mL). An addition funnel was placed on the 3-
neck flask and filled with (S)-methyl lactate (4.9040 g, 47.1040 mmol),
dry N,N-diisopropylethylamine (6.3931 g, 49.4631 mmol), and dry
CH2Cl2 (5 mL). The contents of the addition funnel were added
dropwise under nitrogen to the reaction mixture as the flask was
cooled in an ice bath over a period of 10 min before the bath was
allowed to warm to room temperature. Reaction completion was
monitored by analysis of aliquots by proton and fluorine NMR. The
proton NMR showed 2.1% of unreacted alcohol in the reaction
mixture. The crude reaction mixture was used directly in the next step
Synthesis of 4b-NaOTf. Triflic anhydride (4.8468 g, 17.2 mmol)
was added to a dry 3-neck flask containing dry CH2Cl2 (15 mL).
Ester−azide alcohol 12 (2.497 g, 15.7 mmol) and dry iPr2NEt (2.2375
g, 17.3 mmol) were added dropwise over 10 min using an addition
funnel. The mixture was transparent pale brown. Using a gastight
syringe, 90 min after the first Tf2O had been added, a 50 μL aliquot
1
without further purification. H NMR of an aliquot (CD3OD, 399.8
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was removed and quickly added to dry CDCl3 (0.6 mL) in an NMR
tube. Analysis by 19F NMR spectroscopy showed singlets at −74.6 and
−78.4 ppm for 13 and triflate ion, respectively, and almost no
detectable peak at −79.2 (Tf2O). Analysis by 1H spectroscopy showed
a singlet at 3.89 ppm (CH3O2C of product, 3.0 units) and a small
singlet at 3.83 (CH3O2C of reactant 12, 0.10 units), indicating that
approximately 10% of alcohol 12 remained. An additional portion of
Tf2O (0.5985 g, 2.12 mmol) was added 120 min after the beginning
the first Tf2O addition. Another 6.5 h elapsed before the solution of
triflate 13, which was kept in the freezer, was added to the other
reactant solution, prepared as follows: in a separate flask under
nitrogen, to salt 11 (26.26 g, derived from 15.68 mmol of formylcyclen
8) was added CH2Cl2 (80 mL) and dry iPr2NEt (10.52 g, 81.4 mmol).
The flask was chilled in ice, and the solution of triflate ester 13 was
added via cannula over 7 min. After an additional 8 min the ice bath
was removed, and the mixture was allowed to warm to ambient
temperature. After an additional 6 h, using a gastight syringe, a 50 μL
aliquot was removed and quickly added to dry CDCl3 (0.6 mL) in an
NMR tube. Analysis by 19F NMR spectroscopy showed one major
singlet at −80.1 ppm (triflate ion) and a very minor peak at −76.8
(13). The flask was chilled in ice, and an ice-cold solution of NaOH (6
g) in water (50 mL) was added. The aqueous phase was extracted with
CH2Cl2 (2 × 50 mL). The combined CH2Cl2 phases were
concentrated by rotary evaporation, and the residue was stored
under vacuum for 5 h, leaving a brownish solid (10.98 g). Analysis by
19F NMR spectroscopy showed singlets at −76.8 and −80.1 ppm for
13 and product, respectively. The crude product was purified by silica
gel flash chromatography (gradient from CH2Cl2 to acetonitrile;
product elutes with acetonitrile−CH2Cl2 0.4:1), affording a beige
foamlike solid (total 6.076 g, 8.170 mmol, 52% overall yield from
formylcyclen 8, with 5% DOTMA tetramethyl ester−NaOTf
calculated from NMR spectra (not shown)). 1H NMR (CD3OD,
399.8 MHz, digital resolution = 0.20 Hz): δ 3.84−3.80 (m, 4H), 3.78
(s, 3H), 3.76 (s, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.73−3.67 (m, 2H),
3.65−3.63 (m, 1H), 3.57−3.50 (m, 1H), 3.11−2.07 (m, 4H), 2.75−
2.65 (m, 4H), 2.43−2.38 (m, 4H), 2.00−1.91 (m, 2H), 1.26 (d, J =
7.0, 3H), 1.25 (d, J = 7.1, 6H). 13C NMR (CD3OD, 599.8 MHz): δ
178.4, 178.2, 178.2, 177.4, 59.6, 57.8, 57.8, 57.8, 53.2, 53.1, 53.0, 51.6,
48.5, 46.0, 45.9, 45.9, 45.9, 24.6, 7.83, 7.80. 23Na NMR (CD3OD,
399.8 MHz): δ 6.6 (br s) and −3.4 (sharper s) in a ratio of 98 to 2,
respectively, based on integration of NMR peaks. 19F NMR (CD3OD,
399.8 MHz): δ −79.9 (s). ESI-LCMS m/z 572.3 (M + H) (calculated
29.6, 26.9, 23.2, 19.8. 23Na NMR (CD3OD, 105.8 MHz): δ 6.5 (br s),
−3.4 (s). 19F NMR (CD3OD, 376.1 MHz): δ −79.7 (s).
Synthesis of 5b (isolated product to be formulated as
monohydrate). To a round-bottom flask containing 4b-NaOTf (7.53
g, contained 7% DOTMA-NaOTf, 10.12 mmol if 100% pure) was
added 5% palladium on carbon (0.7530 g) along with MeOH (150
mL) under nitrogen. The mixture was put in an ice bath, and TfOH
(1.5781 g, 10.515 mmol) was added over 10 min. Hydrogen gas was
bubbled gently through the mixture for 6 h. The vent needle was then
removed, and the reaction was kept under hydrogen gas overnight.
The mixture was then filtered through a Celite pad into a tared flask.
The Celite pad was rinsed with MeOH (2 × 50 mL). The filtrate was
concentrated by rotary evaporation and then put under vacuum for 4
d, affording a pale orange syrup (8.85 g, 10.20 mmol, quantitative
1
yield). H NMR (CD3OD, 599.6 MHz): (δ 3.8−3.6, includes signal
from DOTMA) δ 3.80 (s, 3H), 3.75 (s, 3H), 3.74 (s, 6H), 3.67−3.65
(m, 1H), 3.34−3.31 (m, 4H) likely 2H, but obscured by solvent signal,
3.14−3.06 (m, 1H), 3.02 (t of narrow multiplet, J ≈ 14.0, 5H), 2.77
(tdd, J = 13.7, 5.0, 2.7, 2H), 2.69 (tdd, J = 13.7, 6.5, 2.6, 3H), 2.44−
2.39 (m, 5H), 2.29−2.20, (m, 4H), 2.09−1.98 (m. 2H), 1.27 (d, J =
7.3, 3H), 1.25 (d, J = 7.0, 8H, 6H from 5b as well as signal from
DOTMA). 13C NMR (CD3OD, 599.8 MHz): δ 178.4, 178.2, 178.2,
178.1, 176.7, 60.6, 57.7(t), 53.4, 53.0, 48.8, 48.5, 45.9, 40.2, 23.3, 7.8.
23Na NMR (CD3OD, 105.7 MHz): δ 6.6 (br s), −3.4 (s). 19F NMR
(CD3OD, 376.1 MHz): δ −79.9 (s). Anal. Calcd for C27H48F6-
N5NaO14S2 (867.81): C, 37.37; H, 5.58; N, 8.07. Found: C, 36.71; H,
5.78; N, 7.57%. Calcd for C27H48F6N5NaO14S2 + H2O (885.82): C,
36.61; H, 5.69; N, 7.91.
Model Reaction of DMF with 9. To a dry J. Young resealable
NMR tube was added 9 (0.0184 g, 0.07791 mmol, 1.0 equiv) and dry
dimethylformamide (DMF) (0.0107 g, 0.14639 mmol, 1.88 equiv),
and CD2Cl2 was added until the total volume was ∼0.55 mL. The
NMR tube was left at room temperature. Proton and fluorine NMR
spectra were acquired at different time points (11 min, 1.2 h, 2.2 h, 3.2
h, 6.5 h, 19.7 h, and 7 d) to monitor the evolution and stability of
product(s) formed. 19F NMR (CD2Cl2, 376.1 MHz): δ −79.1 (s) for
the free triflate of the product and −75.7 (s) for the covalently bound
triflate of the starting material. As time elapsed the peak at δ −75.7
decreased as a peak at δ −79.1 increased. After 1.3 h, integration of the
19F NMR peaks showed that 77% of the fluorine was in the free triflate
ion. After 19.7 h had passed the only 19F NMR peak was the one at δ
−79.1 ppm, showing complete consumption of 9. For the intermediate
compound A in the reaction mixture: 1H NMR (CD2Cl2, 399.8 MHz):
δ 8.93 (s, 1H), 7.96 (s, 1H), 5.55 (q, J = 7.0, 1H), 3.82 (s, 3H), 3.50 (s,
3H), 3.29 (s, 3H), 1.73 (d, J = 7.0, 3H). 13C NMR (CD2Cl2, 399.8
MHz): δ 167.9, 160.5, 121.2 (q, J = 320.1, 1C) 81.7, 54.0, 42.6, 37.2,
17.3. 19F NMR (CD2Cl2, 399.8 MHz): δ −79.2 (s).
for M+ = C25H45O8N7 = 571.33). Anal. Calcd for C25H45N7O8
+
CF3NaO3S (743.73): C, 41.99; H, 6.10; N, 13.18. Found: C, 41.70; H,
5.93; N, 13.58%. IR (KBr): 3442.9 br, 2956.4 s, 2845.7 s, 2099.2 s,
1728.2 s, minor peaks at 2360.4, 1637.5. For examples of Na+
complexes like 4b-NaOTf, see refs 62 and 63.
After 22 d, the product formed appears to be stable as seen by the
proton NMR spectrum. To test our proposed hydrolysis hypothesis, in
the glovebox deoxygenated water (5.5 μL, 0.30 mmol, 1.2 equiv) was
added to the reaction mixture in the NMR tube. The reaction was
monitored by proton NMR spectroscopy, showing that after 25 min
the proton peaks corresponding to A were not detectable, and only the
peaks for hydrolysis products were seen, indicating that the hydrolysis
occurred relatively quickly, which supports the notion that trialkylation
of compound 8 to form 10 is accompanied by some overalkylation and
loss of the formyl group, leading to the formation of small amounts of
DOTMA-NaOTf. For the hydrolysis mixture: 1H NMR (CD2Cl2,
399.8 MHz): δ 8.07 (s, 1H), 5.19 (q, J = 7.0, 1H), 3.72 (s, 3H), 1.49
(d, J = 7.0, 3H); in addition, peaks for residual excess DMF were seen
at 7.96 (s, 1H), 2.92 (s, 3H), 2.82 (s, 3H), and a broad peak for water
at 4.60 ppm. 13C NMR (CD2Cl2, 399.8 MHz): δ 171.1, (163.1, DMF
carbonyl carbon), 160.6, 68.6, 52.8, 36.9, 36.0, 31.7, 17.3. 19F NMR
(CD2Cl2, 399.8 MHz): δ −79.1 (s).
Synthesis of 5a. Methanol (250 mL) was added to a mixture of 4a-
NaOTf (12.96 g, 18.5 mmol) and Pd on carbon (5%, 1.29 g) in a 1 L
flask so as to wet the catalyst as quickly as possible. The mixture was
stirred as the flask was placed in an ice bath. After 10 min, TfOH
(2.868 g, 19.1 mmol) was added over 1 min. The ice bath was
removed, and hydrogen was bubbled slowly through the mixture for 9
h. The mixture was stirred for an additional 38 h under static hydrogen
atmosphere, hydrogen gas being bubbled through the mixture once for
5 min in the middle of this time period. The mixture was filtered
through Celite, and the filter cake was rinsed with methanol (4 × 100
mL). The combined filtrates were concentrated by rotary evaporation,
and the syrupy residue was stored under vacuum for 2 weeks, leaving
5a (15.17 g) as crispy foam, which quickly turns sticky on exposure to
moisture in air. The NMR data suggest the presence of two species in
CD3OD solution, likely a mixture of the structure shown (with Na+
associated with the macrocycle, δ
= 6.5 ppm) along with free
Na
1
solvated Na+ (δ = −3.4 ppm) and the sodium-free macrocycle. H
General Method for the Synthesis of Gadolinium and
Europium Complexes of N3-DOTA and N3-DOTMA Chelates.
Complexes Gd-4a, Gd-4b, Eu-4a, and Eu-4b. In an open flask, N3-
ester−chelate (4a or 4b, 1.00 mmol, 1.0 equiv) was dissolved in
tetrahydrofuran (THF) (15 mL) followed by the addition of water (4
mL). To the above mixture was added LiOH·H2O (6.0 molar equiv)
Na
NMR (CD3OD, 399.8 MHz): δ 3.81 (s, 3H), 3.79−3.74 (m, 9H),
3.69−3.63 (m, 1H), 3.63−3.40 (m, 4H), 3.27−3.08 (m, 5H), 3.08−
2.90 (m, 5H), 2.84−2.57 (m, 4H), 2.48−2.19 (m, 5H), 2.19−1.30 (m,
6H). 13C NMR (CD3OD, 599.8 MHz): δ 176.6, 176.1, 175.9, 175.1,
174.0, 173.4, 60.5, 56.0, 54.2, 53.9, 52.8, 50.6, 47.0, 45.7, 40.1, 39.3,
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while being stirred at 0 °C. The reaction was brought to room
temperature, and stirring was continued overnight (16 h). The
reaction could be monitored by IR [before addition of LiOH: 2100.5
cm−1 (N3), 1733.4 cm−1 (CO); after LiOH: 2092.2 cm−1 (N3),
1595.3 cm−1 (carboxylate)]. Once complete, the reaction mixture was
concentrated by rotary evaporation and stored under oil pump
vacuum. The crude hydrolysis product was further used without
purification, and the yield was assumed to be 100%; the solid (1.0
equiv from 4a or 4b starting material) was dissolved in water (40 mL),
and GdCl3·6H2O or EuCl3·6H2O (1.1 molar equiv) was added at
room temperature. The pH was adjusted to 7 using 1 M HCl, and the
mixture was stirred overnight at 80 °C, under nitrogen. Completion
was determined by ESI-MS (direct injection). The solution was then
concentrated by freeze-drying, and the residue was dried over
phosphorus pentoxide in a desiccator for at least 2 d. Yields could
not be calculated because of the uncertain composition of the
complexes’ counterions (X) as well as other ions accumulated during
the synthesis. Even after attempts at purification using ion-exchange
columns, elemental analyses tended to give low values, which others
have noted for lanthanide-DOTA complexes.64,65 Since the
purification of complexes Gd-4a, Gd-4b, Eu-4a, and Eu-4b was not
trivial, analogous complexes (Gd-6a, Gd-6b, Eu-6a, and Eu-6b) were
purified after functionalization with Cbz group.
1H), 1.27−1.08 (m, 4H). ESI-MS (+): 638.59 (M + H, 100%) [M =
C30H47N5O10, 637.72].
DOTMA-Cbz-ester 6b: (1.23 g, 74%) was obtained from the
starting ester−chelate 5b (1.34 g, 2.45 mmol). 1H NMR (CDCl3, 500
MHz): δ 7.42−7.23 (m, 5H), 6.50 (s, 1H), 5.09 (s, 2H), 3.77−3.59
(m, 12H), 3.58−3.23 (m, 6H), 3.19−3.01 (m, 2H), 2.99−2.32 (m,
14H), 2.11−1.68 (m, 2H), 1.32−1.11 (m, 9H). ESI-MS (+): 680.31
(M + H, 100%), 546.28 (M−Cbz + H, 55%) [M = C33H53N5O10,
679.4].
General Method for the Synthesis of Gadolinium and
Europium Complexes of Cbz-DOTA and Cbz-DOTMA Chelates.
Complexes Gd-6a, Gd-6b, Eu-6a, and Eu-6b. In an open flask, Cbz-
ester−chelate (6a or 6b, 1.00 mmol) was dissolved in a mixture of
THF (6 mL) and MeOH (3 mL) followed by the addition of water (2
mL). An aqueous solution of LiOH·H2O (230 mg in 1 mL of water,
5.45 mmol) was added to the above mixture while being stirred at 0
°C. The reaction was brought to room temperature, and stirring was
continued overnight. The mixture was concentrated and redissolved in
water (4 mL) followed by adjusting the pH to ∼7 using 0.5 M HCl.
Water was removed by freeze-drying, and the remaining solid was
1
checked by H NMR spectroscopy to confirm the absence of OMe
signals. The crude hydrolysis product was further used without
purification; the solid was dissolved in water (20 mL), and a 1 M
aqueous solution of GdCl3·6H2O or EuCl3·6H2O (1 mL, 1.00 mmol)
was added at room temperature. The pH was adjusted to 7 and
maintained by adding a 1.0 M solution of LiOH during the course of
the reaction. The mixture was stirred overnight at 65 °C. Solvent was
removed by freeze-drying, and the remaining solid was purified over
reverse-phase C18 silica using methanol and water (0% to 100%) as
eluents. Fractions containing desired compounds were identified by
TLC and mass spectral analysis. An aqueous solution of Gd
compounds was further passed through a HiTrap SP HP column,
which removed lithium ions.
Li[Gd(DOTA-Cbz)] (Gd-6a) was obtained (255 mg, 38%) from
the starting ester−chelate 6a (630 mg, 0.91 mmol). ESI-MS(−):
735.26 (M − Li, 100%) (calculated m/z for M = C26H35GdLiN5O10,
742.18].
Li[Gd(DOTMA-Cbz)] (Gd-6b) was obtained (313 mg, 47%) from
the starting ester−chelate 6b (580 mg, 0.85 mmol). ESI-MS(−):
777.20 (M − Li, 100%). ESI-MS(+): 791.24 (M + 2Li, 100%)
(calculated m/z for M = C29H41GdLiN5O10, 784.23).
[Gd(DOTA-N3)]X (Gd-4a) was obtained (0.6601 g) from the
starting ester−chelate 4a (0.4516 g, 0.6436 mmol). 23Na NMR
(CD3OD, 105.7 MHz): δ −2.10 (br s). 19F NMR (CD3OD, 376.1
MHz): δ −80.11 (s). 7Li NMR (CD3OD, 155.4 MHz): δ −0.39 (br s).
ICP-OES (Gd at 342.247 and 335.047 nm); 143.5−145.2 ppm,
expected 215.6 ppm (if all material was only Gd-DOTA-N3 anion).
HRMS (ESI-TOF) of Gd-DOTA-N3: m/z 651.1130 (M + Na + H),
(calculated m/z for (M + Na + H) = (12C)18(1H)27(158Gd)-
(14N)7(16O)8, 651.1138).
[Gd(DOTMA-N3)]X (Gd-4b) was obtained (0.7033 g) from the
starting ester−chelate 4b (0.4628 g, 0.6223 mmol). 23Na NMR
(CD3OD, 105.7 MHz): δ 0.08 (s). 19F NMR (CD3OD, 376.1 MHz): δ
−78.80 (s). 7Li NMR (CD3OD, 155.4 MHz): δ 0.12 (s). ICP-
OES(Gd at 342.247 and 335.047 nm); 204.1−207.7 ppm, expected
284.0 ppm (if all material was only Gd-DOTMA-N3 anion. HRMS
(ESI-TOF) of Gd-DOTMA-N3: m/z 671.1777 (M + 2H), (calculated
m/z for (M + 2H) = (12C)21(1H)33(158Gd)(14N)7(16O)8, 671.1788).
[Eu(DOTA-N3)]X (Eu-4a) was obtained (1.7075 g) was obtained
from the starting ester−chelate 4a (1.0020 g, 1.4281 mmol). 23Na
NMR (CD3OD, 105.7 MHz): δ −0.42 (br s). 19F NMR (CD3OD,
Li[Eu(DOTA-Cbz)] (Eu-6a) (68 mg, 32% yield) was obtained
from the starting ester−chelate 6a (185 mg, 0.27 mmol). ESI-MS(+):
738.21 (M + H, 100%) (calculated m/z for M = C26H35EuLiN5O10,
737.18).
Li[Eu(DOTMA-Cbz)] (Eu-6b) (125 mg, 68% yield) was obtained
from the starting ester−chelate 6b (160 mg, 0.24 mmol). ESI-MS(+):
780.24 (M + H, 74%), 786.15 (M + Li, 100%) (calculated m/z for M
= C29H42EuLiN5O10, 779.22).
7
376.1 MHz): δ −79.99 (s). Li NMR (CD3OD, 155.4 MHz): δ 0.00
(br s). ESI-MS(+): m/2z 318.5 (M + 2Li) (calculated m/z for M =
C18H27EuN7O8, 622.11).
[Eu(DOTMA-N3)]X (Eu-4b) (0.5507 g) was obtained from the
starting ester−chelate 4b (0.3478 g, 0.4676 mmol). 23Na NMR
(CD3OD, 105.7 MHz): δ 0.15 (s). 19F NMR (CD3OD, 376.1 MHz): δ
7
To create rotationally constrained chelates with increased rotational
correlation times, a biotin moiety for avidin binding was added as
outlined in Scheme 4.
−78.76 (s). Li NMR (CD3OD, 155.4 MHz): δ 0.12 (s). ESI-MS(+):
m/z 666.4 (M + 2H); ESI-MS(−): m/z 664.1 (M); (calculated m/z
for M = C21H33GdN7O8, 664.16).
General Method for the Synthesis of Biotinylated Chelate
Esters 7a and 7b. A round-bottom flask was charged with ester−
chelate (5a or 5b, 0.15 mmol) and biotinamidohexanoic (LC-biotin)
acid NHS-ester (60 mg, 0.13 mmol) followed by the addition of THF
(5 mL). The flask was kept on an ice bath, and the mixture was stirred
for 15 min before the addition of triethylamine (150 μL, 1.1 mmol).
The ice bath was removed, and the reaction mixture was stirred at
room temperature for 16 h. The solvent was removed in a rotary
evaporator, and the remaining residue was dissolved in CH2Cl2 (20
mL) and washed with water (3 × 5 mL). The organic layer was
separated, dried over anhydrous sodium sulfate, filtered, and
evaporated to give an off-white solid. The compound was purified
using SiO2 and a gradient of CH2Cl2 in methanol (from 0 to 80%
methanol).
General Method for the Synthesis of Cbz-Protected Esters
6a and 6b. A round-bottom flask was charged with tetraester amine
(5a or 5b, 2.4 mmol) and N-(benzyloxycarbonyloxy)succinimide (910
mg, 3.66 mmol), and CH2Cl2 (80 mL) was added. The flask was kept
on an ice bath, and the mixture was stirred for 15 min before the
addition of triethylamine (1.3 mL, 9.7 mmol). The ice bath was
removed, and the reaction mixture was stirred at room temperature
overnight. Solvent was removed by rotary-evaporation, and the
remaining residue was dissolved in methylene chloride (50 mL) and
washed with water (3 × 20 mL). The organic layer was separated,
dried over anhydrous sodium sulfate, filtered, and evaporated to give a
light yellow gummy mass. The compound was purified using normal
phase silica chromatography and a solvent gradient of CH2Cl2 in
methanol (from 0 to 80% methanol).
1
DOTA-Cbz-ester 6a: (1.24 g, 81%) was obtained from the starting
ester−chelate 5a (1.21 g, 2.4 mmol). 1H NMR (DMSO-d6, 500 MHz):
δ 7.44−7.20 (m, 5H), 5.01 (s, 2H), 3.87−3.44 (m, 16H), 3.37−2.77
(m, 16H), 2.76−2.56 (m, 2H), 2.31−1.82 (m, 3H), 1.80−1.62 (m,
7a (86 mg, 74%) was obtained from 5a (78 mg, 0.155 mmol). H
NMR (CDCl3, 500 MHz): δ 7.29 (t, 1H, J = 5.2 Hz), 6.88 (t, 1H, J =
5.3 Hz), 6.01 (s, 1H), 5.69 (s, 1H), 4.54−4.48 (m, 1H), 4.36−4.29 (m,
1H), 3.81−3.69 (m, 12H), 3.58−3.52 (m, 1H), 3.51−3.34 (m, 3H),
6559
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MS(+): 942.10 (M − Li + 2H, 100%) [M = C34H54Gd1Li1N8O11S1,
946.30]. ESI-HRMS(−): 942.4705 (M − Li + 2H, 96%), 964.4579 (M
− Li + H + Na, 100%) (calculated m/z for M = C34H54Gd1-
Li1N8O11S1, 947.3034).
Scheme 4. Synthesis of Biotin−Chelate Conjugates Gd-7a
and Gd-7b
Gd-7b (37 mg, 53% yield) was obtained from the starting ester−
chelate 7b (62 mg, 0.07 mmol). ESI-MS(−): 982.3 (M − Li, 100%).
ESI-MS(+): 984.3 (M − Li + 2H, 100%) [calculated m/z for M =
C37H60Gd1Li1N8O11S1, 989.35). ESI-HRMS(+): 990.3564 (M + H,
100%) (calculated m/z for M = C37H60Gd1Li1N8O11S1, 989.3503).
Oxygen-17 NMR Measurements. NMR measurements of 17O
were performed as described by Laurent et al.66 Solutions of Gd-6a
and Gd-6b were prepared in distilled water (pH 6.5−7.0) at
concentrations of 23 mM. Solutions (0.35 mL) were transferred to
5 mm o.d. NMR tubes, and 17O NMR measurements were made at
11.7 T in a Bruker AVANCE-500 spectrometer (Bruker, Karsruhe,
Germany). The temperature was regulated by air or nitrogen flow
controlled by a Bruker BVT 3200 unit. 17O transverse relaxation times
of distilled water (pH = 6.5−7) were measured using a Carr−Purcell
Meiboom−Gill (CPMG) sequence and a subsequent two-parameter
fit of the data points. The 90° and 180° pulse lengths were 27.5 and 55
μs, respectively. The 17O T2 values of water in the solutions of
complexes were obtained from line width measurements. All spectra
were proton decoupled. Data are presented as the reduced transverse
R
p
relaxation rate (1/T2 = 55.55/(q × [complex] × 1/T2 ), where
[complex] is the molar concentration of the complex, q is the number
of inner sphere coordinated water molecules, and 1/T2 is the
p
paramagnetic transverse relaxation rate. Data analysis and treatment
was performed as described by Vander Elst et al.,67 Muller et al.,68 and
Laurent et al.69
Proton Nuclear Magnetic Relaxation Dispersion (NMRD)
Measurements. Proton NMRD measurements were made over a
magnetic field range of 0.47 mT to 1.0 T on a Stelar Spin fast field
cycling (FFC) NMR relaxometer (Stelar, Mede (PV), Italy). Three
different solutions of each complex were prepared in 50 mM N-(2-
hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer, pH =
7.35, with 150 mM NaCl. Measurements were performed on 0.800 mL
samples in 10 mm o.d. NMR tubes. Additional relaxation rates were
measured at 20 and 60 MHz on a Bruker Minispec mq-20 and mq-60
at temperatures specified in Figure 4 and matching temperatures used
for all samples.
Biotin−Avidin Complexation. Three separate sample concen-
trations of 7a and 7b were prepared with avidin. In separated tubes a
solution of 50 mM HEPES, pH = 7.35, with 150 mM NaCl was added
to six tubes containing avidin for final concentrations of 0.22 mM.
Chelates were added such that [chelate]/[avidin] never exceeded 3.2
(i.e., the ratio was always less than 4 to prevent saturation of avidin) at
concentrations of 0.553, 0.692, and 0.700 mM Gd(III) for 7a and
0.267, 0.315, and 0.448 mM Gd(III) for 7b.
3.24−3.12 (m, 5H), 3.11−2.81 (m, 8H), 2.79−2.70 (m, 1H), 2.65−
2.01 (m, 16H), 1.96−1.71 (m, 3H), 1.70−1.28 (m, 11H). ESI-MS(+):
843.60 (M + H, 10%), 865.64 (M + Na, 100%) (calculated m/z for M
= C38H66N8O11S, 842.46).
1
7b (62 mg, 53%) was obtained from 5b (86 mg, 0.157 mmol). H
NMR (CDCl3, 500 MHz): δ 7.31 (m, 1H), 6.82 (m, 1H), 5.92 (s,
1H), 5.47 (s, 1H), 4.51 (m, 1H), 4.34 (m, 1H), 3.85−3.61 (m, 15H),
3.55−3.41 (m, 2H), 3.40−3.32 (m, 1H), 3.28−3.11 (m, 4H), 3.01−
2.82 (m, 5H), 2.79−2.69 (m, 1H), 2.61−2.44 (m, 4H), 2.44−2.13 (m,
11H), 2.02−1.29 (m, 15H), 1.29−1.13 (m, 9H). ESI-MS(+): 907.68
(M + Na, 100%) (calculated m/z for M = C41H72N8O11S, 884.50).
General Method for the Synthesis of Gadolinium Com-
plexes of Biotin-Chelate Gd-7a and Gd-7b. In an open flask,
biotin−tetraester derivative 7a or 7b (0.1 mmol) was dissolved in a
mixture of THF (2 mL) and MeOH (1 mL) followed by the addition
of water (0.5 mL). An aqueous solution of LiOH·H2O (21.5 mg in 0.5
mL water, 0.5 mmol) was added to the above mixture while being
stirred at 0 °C. The reaction was brought to room temperature, and
stirring was continued overnight. The mixture was concentrated and
redissolved in water (4 mL) followed by adjusting the pH to ∼7 using
0.5 M HCl. Water was completely removed by freeze-drying, and the
remaining solid was checked by 1H NMR spectroscopy to confirm the
absence of OMe signals. The crude hydrolysis product was further
used without purification; the solid was dissolved in water (5 mL), and
a 1 M aqueous solution of GdCl3 (115 μL, 0.12 mmol) was added at
room temperature. The pH was adjusted to 7 and maintained by
adding a 1.0 M solution of LiOH during the course of the reaction.
The mixture was stirred for 40 h at 55 °C. The solvent was removed
by freeze-drying, and the remaining solid was purified over reverse-
phase C18 silica using methanol and water (0% to 100%) as eluents.
Fractions containing desired salts Gd-7a or Gd-7b were identified by
TLC and mass spectral analysis. Aqueous solutions of Gd salts were
further passed through a HiTrap SP HP column.
RESULTS
■
The progress of trialkylation of 8 (Scheme 1) was followed by
analysis of aliquots by H NMR spectroscopy, looking for
1
disappearance of the formyl resonance for 8, as well as any
peaks other than those for the intermediate 9. Subsequent
deprotection of the N-formyl group required only 2 equiv of
acid in methanol, giving compound 11 as a triflate salt in
quantitative yield.
Subsequent alkylation of the triflate salt of 11 in situ using
freshly prepared 13 led to isolation of 4b-NaOTf after column
chromatography using CH2Cl2−CH3CN. The structure of 4b-
NaOTf as a NaOTf adduct was strongly suggested by clearly
visible peaks in both 23Na and 19F NMR spectra, and was
ultimately verified by X-ray crystallography (see Discussion
below). Careful analysis of bulk samples of the product revealed
a second, minor (<10%) component, ultimately identified as
the NaOTf adduct of the tetramethyl ester of DOTMA, which
must have arisen at some stage from loss of the N-formyl group
of 8 or 9.
Gd-7a (40 mg, 42% yield) was obtained from the starting ester−
chelate 7a (86 mg, 0.1 mmol). ESI-MS(−): 940.30 (M-Li, 100%). ESI-
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Figure 2. Proton NMR of Eu-Cbz chelates showing the SAP/TSAP
ratio. (top) Eu-6a; (bottom) Eu-6b.
Figure 4. Variable-temperature relaxometry at 0.47 T showing
dependence of relaxivity on temperature. (A) Gd-6a; (B) Gd-6b.
Standard errors were determined using three samples.
peak for free triflate ion appeared at −79.1 ppm. Upon addition
of water, A quickly hydrolyzed to B, as shown by proton and
fluorine NMR data. The proton on the chiral carbon shifted
upfield to 5.19 ppm and the formyl proton shifted upfield to
8.07 ppm, whereas in 19F NMR spectra the free triflate ion
signal remained at −79.1 ppm.
Below we discuss the X-ray crystal structure of 4b-NaOTf.
Samples of 4b-NaOTf containing 5 to 10% of DOTMA
impurity were used in subsequent experiments, where the
presence of the impurity did not interfere with conjugation
because it lacks a reactive side chain group.
Subsequent hydrogenation of the azide (Scheme 1)
proceeded smoothly in the presence of one added equivalent
of TfOH, without which the amine formed during hydro-
genation attacked the ester of the unique side chain, resulting in
an unwanted lactam. The resulting triflate salts of the amine
derivative 5b-NaOTf was neutralized by Et3N and acylated by
an external reagent (e.g., in Schemes 3 and 4, biotinamidohex-
anoic acid of Cbz NHS esters, forming 6a, 6b, 7a, or 7b).
Subsequent basic hydrolysis using LiOH and water−THF
mixtures, followed by complexation with Gd(III) or Eu(III) at
slightly acidic pH, delivered chelates for further study.
Figure 3. 17O NMR measurement of the reduced transverse relaxation
●
time as a function of the reciprocal of the temperature: Gd-6a ( ) and
○
Gd-6b ( ).
The point at which the formyl protecting group was lost is
strongly suggested by results of a model reaction using DMF
(Scheme 2). Compound 9 and dry DMF were combined in
CD2Cl2 under dry conditions in a resealable NMR tube,
allowing for in situ monitoring of reaction progress without
having to disturb the reaction by removing aliquots. With a
half-life of about 1 h, a stable intermediate formed (compound
A) as evidenced by proton and fluorine NMR spectral data.
The methine proton on the chiral carbon appeared at 5.55
ppm, the formyl proton appeared at 8.93 ppm, and a fluorine
The difference in the water residence times on Gd(III)
complexes of DOTA and DOTMA result from the differences
in the relative populations of conformational isomers, denoted
square antiprismatic (SAP) and twisted square antiprismatic
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Table 1. Parameters Obtained by the Theoretical Analysis of the 17O NMR Experimental Data
ΔH‡ (kJ mol−1
)
B × 1020 (s−2
)
EV (kJ mol−1
)
A/ℏ × 106 (rad s−1
)
ΔS‡ (J mol−1 K−1
)
310
298
τM (ns)
τV (ps)
Gd-6a
Gd-6b
100.8 4.7
41.6 5.2
4.86 0.56
5.23 0.40
54.1 0.52
1.99 0.229
6.37 0.493
0.102 19
8.12 3.98
−2.95 0.203
−3.0 0.146
63.3 2.2
63.1 0.198
99.7 0.407
(TSAP). The TSAP isomer exchanges more quickly. Alpha
substitution of the acetate arms attached to the four nitrogens
on DOTA creates DOTMA, featuring four chiral centers, and
favors the TSAP isomer with the homochiral diastereomer
(either the R,R,R,R or the S,S,S,S enantiomer) having the
time and that rotationally constrained compounds made from
the DOTMA analogue 6b should have higher relaxivities. To
test this hypothesis, we prepared the biotinylated version of the
two chelates (Gd-7a and Gd-7b) and used the avidin−biotin
complexation (ABC) reaction to increase the rotational
correlation time or rotationally constrain the two compounds.
The small-molecule biotin analogues Gd-7a and Gd-7b are
rapidly rotating and have the same relaxivities and similar
NMRD profiles, Figure 5. Adding avidin results in the binding
highest TSAP/SAP ratio.54,56,57 Woods et al.57 used H NMR
1
spectroscopy of Eu(III) complexes to differentiate the various
diastereomers and determine the relative ratio of TSAP/SAP in
each case. We therefore used proton NMR of Eu(III)
complexes of Cbz derivatives Eu-6a and Eu-6b, Figure 2.
The Eu-6b has a much higher TSAP/SAP ratio (Figure 2B)
than that of Eu-6a (Figure 2A). Moreover, the fact that only
two sets of peaks are seen demonstrates that the use of base to
deprotect the methyl esters did not significantly epimerize the
chiral centers, which would have resulted in up to 16
stereoisomers and a large number of peaks in the axial region
(downfield of 15 ppm). Moreover, the high TSAP/SAP ratio
for Eu-6b demonstrates that the conformational properties of
the macrocycle or its complexes are not detectably altered by
inclusion of unique side chain of novel derivative 6b. Woods et
al.57 assumed that the conformational behavior of Eu(III) and
Gd(III) complexes would be the same in their analysis, and we
use the same reasonable assumption. This implies that the
bifunctional Gd(III)-DOTMA derivatives should have shorter
water residence times than their Gd(III)-DOTA analogues.
To confirm the proton NMR data and calculate the actual
water residence time, we used 17O NMR to measure the
reduced transverse relaxation rate of the water molecules in the
inner coordination sphere of the complexes as a function of
temperature (see Figure 3).
Analysis of the results at 37 °C showed that the new DOTA
analogue Gd-6a had a longer water residence time than the new
DOTMA analogue Gd-6b, 100.8 versus 41.6 ns, respectively
(Table 1). Decreasing the temperature increases the rotational
correlation time and the water residence time, which have
opposite effects on the relaxivity. For rapidly rotating
complexes, whose relaxivity is not limited by long water
residence times, decreasing the temperature with the
subsequent increase in rotational correlation time will increase
the relaxivity. If the water residence time limits the relaxivity,
decreasing the temperature will decrease the relaxivity. We used
variable-temperature relaxometry to analyze the temperature
dependence of the relaxivity of Gd-6a and Gd-6b, Figure 4. At
37 °C the relaxivities of the two chelates are approximately
equal, which is expected for similar Gd(III) systems with one
inner sphere water molecule whose rotational correlation time
dominates the relaxivity. Decreasing the temperature results in
an increase in relaxivity for both chelates until about 10 °C.
When the relaxivity for the Gd-6a levels off (Figure 4A), the
relaxivity of Gd-6b continues to increase (Figure 4B), which is
expected on the basis of the τM data in Table 1. In the case of
Gd-6b, where the data benefit from smaller error bars, the value
at 2.5 °C is clearly greater than the value at 10 °C and at all
other higher temperatures.
Figure 5. NMRD profiles of Gd-7a (circles) and Gd-7b (squares)
with (filled) and without (open) avidin at 37 °C, showing the effects of
biotin−avidin binding on relaxivity.
of biotin and increases the rotational correlation time and
relaxivity. The Cbz derivatives 6a and 6b were studied to
provide preliminary data for the further investigation of
derivatives 7a and 7b.
NMRD profiles of the long chain (LC) biotinylated Gd
chelates in the presence of avidin show a peak in the high-field
region characteristic of slowly rotating complexes.41 In the
presence of avidin, Gd-7b has higher relaxivities than Gd-7a at
all corresponding magnetic field strengths, consistent with a
shorter water residence time on the new DOTMA derivative
Gd-7b.
The variable-temperature relaxometry results, coupled with
the calculated water residence times of the Cbz derivatives Gd-
6a and Gd-6b, and the NMRD profiles of biotinylated
derivatives Gd-7a and Gd-7b predict that the relaxivity of the
rotationally constrained Gd-7a + avidin might be limited by the
water residence time, while that of the Gd-7b + avidin might
be dominated by the rotational correlation time. To test this
hypothesis, we performed variable-temperature relaxometry on
the avidin complexed derivatives (Figure 6). Decreasing the
temperature has no effect on the relaxivity of the Gd-7a +
avidin, whereas the relaxivity of Gd-7b + avidin monotonically
increases with decreasing temperature.
DISCUSSION
■
The results for the Cbz derivatives Gd-6a and Gd-6b suggest
that the relaxivity of rotationally constrained analogues of 6a
with an ethylamino linker are limited by the water residence
Synthesis. As evidenced mainly by NMR, X-ray crystallog-
raphy, and mass spectroscopy, we prepared four new
bifunctional chelates (4a-, 4b-, 5a-, and 5b-NaOTf) and
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side product to levels below 10%, though we could not avoid its
formation entirely. Formamide alkylations are known (for
examples, see refs 72 and 73), and subsequent hydrolyses are
also known.74−76
Turning to the deprotection of 10, though HCl could be
used, the presence of chloride counterion turned out to
interfere with the subsequent alkylation by 13, because of
chloride displacement of the triflate. In theory, one could
neutralize the HCl salt and isolate the free amine for alkylation,
but in practice, we found that higher overall yields could be
obtained by the procedure shown, with triflic acid.
Using NMR, elemental analysis, and X-ray crystallography, it
was found that the azide analogues had a sodium cation
coordinated to macrocycle as well as a triflate counterion.
These ions were present even after purification by liquid
chromatography and crystallization. There are similar macro-
cycles62,63,77−84 that contain a sodium ion bound in place by
ring nitrogens and carbonyl-containing arms. The average bond
distances between Na−O and Na−N of 4a-NaOTf were
2.540(8) Å and 2.556(7) Å, respectively. And the average bond
distances between Na−O and Na−N of 4b-NaOTf (Figure 7)
were 2.502(7) Å and 2.562(7) Å, respectively.
Figure 6. Variable-temperature relaxometry of Gd-7a (filled circles)
and Gd-7b (filled squares) with avidin at 0.47 T. Note that for a given
temperature, each Gd-7b value is significantly different from the Gd-
7a value at P < 0.001. Among Gd-7a values, the 50 °C value is
significantly different (P ≤ 0.001) from all others, and the 3 °C value is
significantly different (P ≤ 0.001) than all others except the 10 °C
value. Other groups of values significantly different (P ≤ 0.001): 37 °C
and 3, 10, 20 °C; 30 °C and 3 and 10 °C; 25 °C and 3 and 10 °C.
successfully complexed them to Gd to make novel contrast
agents. Two different functional groups (N3, 4 and NH2, 5) of
each chelate (DOTA, a; DOTMA, b) are designed for coupling
to either alkynes using click chemistry or carboxylic acids using
standard peptide coupling chemistry.
The syntheses of 4a- and 4b-NaOTf were designed to
minimize the role of chromatographic purifications, and they
make exclusive use of triflate as a counterion for several reasons.
Previous work had shown that the leaving group from 9 could
be displaced in an SN2 fashion with high fidelity, and because of
relatively rapid reaction rates with amine nucleophiles,
subsequent epimerization of the newly formed chiral center
was minimized.59 None of the other leaving groups used (other
sulfonates, halides) gave satisfactory results. The work also
showed that although 9 could be isolated and even distilled,
such treatment tended to lower the enantiomeric purity of both
9 and its alkylation products.59 Thus, here both triflate
electrophiles 9 and 13 were made fresh and used as mixtures,
rather than being isolated or stored.
Figure 7. Thermal ellipsoid plot of 4b-NaOTf drawn at 35%
probability level. Hydrogen atoms and triflate counterion were
omitted for clarity.
These bond distances fall reasonably within the average range
of distances found for similar crystal structures (CCDC search
results: Na−O: 2.513(30) Å and Na−N: 2.622(7) Å). An
interesting trend found in the crystal structure of 4b-NaOTf,
among the four molecules that form the unit cell, was the Na−
N bond was longest for the nitrogen with the azide moiety; this
could be due to steric effects. In contrast, only one of the two
molecules in the unit cell of 4a-NaOTf had a significantly
longer Na−O and Na−N distance (2.823(11) Å and 2.642(11)
Å, respectively) on the nitrogen and oxygen that are part of the
azide moiety. The crystallographic data obtained of 4a-NaOTf
and 4b-NaOTf show that the azide moiety does not
significantly alter the conformation of macrocycle, for example
by disrupting chelation by the ester oxygens or ring nitrogens.
The proton NMR spectrum of Eu-6b (Figure 2B) is
consistent with an R,R,R,R configuration for the compound,
given the broken symmetry caused by the unique amino butyric
acid arm, which causes four unique max peaks to be seen
between δ 18 and 20 ppm. Similarly, four max peaks were seen
for the R,R,R,S isomer of Eu(III)-DOTMA where the R-
N-formylcyclen (8) was an attractive monoprotected cyclen
derivative,70 readily made according to literature procedures,
which are reported to give the monohydrate.60,61 Previously, in
our hands the trialkylation of 8 with the primary halide
t
BrCH2CO2 Bu and subsequent N-deprotection proceeded
without incident,71 whereas here, after similar treatment of 8
with 9, because some tetraalkylated side product was formed,
evidence was gathered that the N-formyl group was being lost
during alkylation. The control experiment shown in Scheme 2
shows how incomplete drying of the alkylation reaction might
lead to formyl group loss: DMF was quickly alkylated, and the
resulting iminium ion suffered rapid hydrolysis. We hypothe-
sized that the water in the hydrated form of 8 and/or wet
methanol helped facilitate partial premature loss of the N-
formyl group and allowed a fourth alkylation event, leading to
formation of DOTMA tetramethyl ester−NaOTf adduct.
Indeed, drying the formylcyclen−CH2Cl2 solution with
molecular sieves prior to alkylation reduced the amount of
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configured center breaks the symmetry.57 Our NMR data for
Eu-6b convincingly show the presence of either the R,R,R,R or
S,S,S,S stereoisomer, but by themselves do not discriminate
between these two possibilities; what does discriminate
between the possible stereoisomers is the synthetic starting
materials and known SN2 inversion. Our synthesis of 5b and
derivatives was designed to produce the R,R,R,R enantiomer.
The crystal structure of intermediate 4b-NaOTf verifies its
R,R,R,R configuration, and for 4a-NaOTf, the single stereo-
center was in the R configuration. However, the one caveat is
that the assignments are rigorously proven only for the single
crystals analyzed. In the case of 4b-NaOTf, analysis of bulk
sample (50−100 mg) was accomplished by 13C NMR
spectroscopy, with particular attention paid to the carbonyl
region (178.5 to 178.0 ppm). Other than a minor peak for
DOTMA-NaOTf at 178.1 ppm, only four singlets were seen, at
178.3, 178.2, 178.2, and 177.4 ppm. We conclude that no other
diastereomers were present in greater than 3% concentration. If
another diastereomer were present, unless all of its carbonyl
peaks were overlapping with those seen, we estimate that the
limit of detection was 3%.
were limited by the water residence time, then decreasing the
temperature would decrease the relaxivity. Figure 4 shows a
variable-temperature relaxometry experiment on the Gd-6a and
Gd-6b. Both chelates exhibit increasing relaxivities as the
temperature is decreased from 37 to 15 °C. However, from 15
to 3 °C the relaxivity of the Gd-6a levels off, while that of Gd-
6b continues to increase. The relaxivity of Gd-6a levels off
because the increase in relaxivity associated with increasing the
rotational correlation time is now offset by the decrease in
relaxivity associated with increasing the water residence time. In
contrast, the water residence time of Gd-6b at 37 °C is lower
than that of Gd-6a, but even by lowering the temperature to 3
°C, it has not yet reached a value that offsets changes in the
relaxivity associated with increasing the rotational correlation
time.
The shorter water residence time and the variable-temper-
ature relaxometry indicate that the rotationally constrained
Gd(III)-DOTMA derivative should have a higher relaxivity
than a similarly constrained Gd(III)-DOTA derivative. Rota-
tionally constraining low molecular weight Gd(III) chelates
increases the rotational correlation time. One can rotationally
constrain the chelates by increasing the viscosity55 or attaching
the Gd(III) chelate to a macromolecule either covalently or
noncovalently.40,41,45 We elected to increase the rotational
correlation time of the two types of chelates by noncovalently
attaching them to avidin through the ABC reaction. ABC was
first done for a derivative of Gd(III)-diethylenetriaminepentaa-
cetate (Gd(III)-DTPA) by Langereis et al.87 and later by
Geninatti Crich et al.88 Biotin binds to avidin and streptavidin
with an extremely high noncovalent binding constant. Each
protein has four biotin binding sites. Prior to the addition of
avidin to the biotinylated chelates, the NMRD profiles are
found to be the same with the characteristic features of rapidly
rotating molecules. This is expected as the rotational
correlation time dominates the relaxivity for small Gd(III)
chelates with these water residence times. Adding avidin
increases the rotational correlation time and significantly
increases the relaxivity of both biotinylated chelates, with the
relaxivity of Gd-7b increasing more than that of Gd-7a. The
NMRD profiles of Gd-7a + avidin and Gd-7b + avidin also
exhibit the characteristic peak of the relaxivity in the high-field
region. The higher relaxivity across all magnetic field strengths
for Gd-7b relative to Gd-7a are consistent with the well-
accepted view that the water residence time limits the relaxivity
of constrained Gd(III)-DTPA and Gd(III)-DOTA chelate
systems.
We note the landmark 1992 paper85 of Renn and Meares
with title “Large Scale Synthesis” of a bifunctional DOTA
analogue in quantities of “up to 10 g” in a linear nine-step
synthesis with overall yield 5.6%. Several steps required reverse-
phase HPLC purifications. We thank a reviewer for pointing
out that the Meares work accomplished synthetically
challenging cyclen ring substitution in the form of a para-
nitrobenzyl group. Subsequent insightful papers from the
groups of Sherry and Woods86 report synthesis of the
corresponding para-nitrobenzyl DOTMA analogue and a series
of significant studies of the structure and dynamics of their
lanthanide complexes, but somewhat surprisingly, our search of
the literature found no reported use of the bifunctional nature
of these DOTMA derivatives to date. In contrast, here we
report synthesis of arm-substituted bifunctional DOTMA
analogues on similar ca. 10 g scale but in only five steps from
cyclen to the azide derivative 4b-NaOTf in about 40% overall
yield, with only one chromatography step. Moreover, of equal
or greater significance, as highlighted below, we show that the
proposal that a macromolecular DOTMA agent should have
higher relaxivity than its DOTA counterpart is correct, thanks
to our ability to conjugate the new DOTMA derivative in such
a way as to leave the coordination sphere intact.
Relaxivity Properties. Dunand et al.56 demonstrated that
“the water-exchange rate is definitely independent of the
solution structure for both the SAP and TSAP isomers, and,
hence, the overall water exchange only depends on the SAP/
TSAP isomeric ratio.” The proton NMR of Eu-6a and Eu-6b
clearly shows the SAP/TSAP ratio is much greater for Eu-6a
relative to Eu-6b. The water residence time for the Gd-6a at 37
°C is approximately 2.5 times longer than that of Gd-6b (101
vs 42 ns), which is consistent with the larger SAP/TSAP ratio.
The difference in water residence times of the Gd(III)-Cbz
chelate complexes coupled with the small molecular size imply
that the relaxivities at 25 and 37 °C should be similar, but
following an increase in the rotational correlation times, the
values of relaxivity should differ. Therefore, we increased the
rotational correlation time by decreasing the temperature. If the
relaxivity of a chelate was limited by the rotational correlation
time, then decreasing the temperature will increase the
relaxivity. Decreasing the temperature also has the effect of
increasing the water residence time. If the relaxivity of a chelate
We used the long-chain (LC) derivative of biotin that
consists of biotin plus an aminohexanoic acid chain. This longer
chain helps guarantee unimpeded access of the biotin conjugate
to the avidin binding site, meaning the affinity of the biotin
conjugate would be equal to that of biotin itself, but also has the
potential for additional rotational freedom about the long chain
linking the chelate to the protein. There is the possibility for a
shorter overall rotational correlation time resulting from
segmental motions. We therefore used variable-temperature
relaxometry to determine if further increases in the rotational
correlation time increased the relaxivity. The relaxivity at 20
MHz of the Gd-7a with avidin (Gd-7a + avidin) was constant
while decreasing the temperature from 50 to 3 °C, whereas the
relaxivity of Gd-7b (Gd-7b + avidin) almost doubled (see
Figure 6). A one-way repeated-measures analysis of variance
gave P ≤ 0.001 and a power of the test with α equal to
0.050:1.000. We used the Holm−Sidak method for a pairwise
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multiple comparison to isolate the groups that differ from one
another. In the presence of avidin the relaxivities of Gd-7b at all
temperatures, except 50 °C, significantly differed from the
relaxivities at all temperatures of Gd-7a with P ≤ 0.001. In the
presence of avidin the relaxivity of Gd-7b at 50 °C was not
significantly different from the relaxivities of Gd-7a at 3, 10, 20,
25, 30, 37, and 50 °C. In the presence of avidin none of the
relaxivities of the Gd-7a were significantly different at any
temperature from itself in the presence of avidin. The
relaxivities of Gd-7b in the presence of avidin at all
temperatures differed significantly, P ≤ 0.001, except the
relaxivities at 3 and 10; 10 and 20; 20 and 25; 20 and 30; 25
and 30; 25 and 37; 30 and 37 °C.
Table 2. Comparison of Relaxivities (r1) of Selected
Compounds Similar to Those in This Work
compound
Gd(III)-DTPA
r1 (mM−1 s−1
)
tesla temp (°C)
ref.
4.2
3.4
4.2
30
0.47
0.47
0.47
0.59
35
37
40
35
46
91
92
48
Gd(III)-DOTA
Gd(III)-DOTMA
G = 5 PAMAM (Gd-
DOTA)
G = 6 PAMAM (Gd-
31
34
36
0.59
0.59
0.47
35
25
23
48
48
48
DOTA)
G = 7 PAMAM (Gd-
DOTA)
G =10 PAMAM
(Gd-DOTA)
The implication from the foregoing data that the water
residence time limits the relaxivity of rotationally constrained
DOTA complexes (e.g., Gd-7a + avidin) is consistent with
other reports. We have shown that the water residence time of
a generation 6 ammonia core polyamidoamine dendrimer with
an isothiocyanatobenzyl-DOTA derivative, having the linker
attached to a ring carbon, limits the relaxivity.48 Increasing the
temperature from 5 to 35 °C results in an increase in the
relaxivity from 25 to 32 (s × mM)−1. The increase in relaxivity
associated with decreasing the water residence time (that
results from increasing the temperature) offsets and exceeds the
decrease in relaxivity associated with decreasing the rotational
correlation time (that results from increasing the temperature).
The water residence time dominates when the relaxivity
increases with increasing temperature. Using the same chelate
attached to ethylenediamine core polyamidoamine dendrimers
Bryant et al.89 reported that the relaxivity levels off at around
generation 7. The relaxivities at 23 °C and 20 MHz for the
generations 5, 7, 9, and 10 are 30, 35, 36, and 36 (s × mM)−1,
respectively. These values are consistent with the 32 (s ×
mM)−1 obtained for the generation 6 ammonia core, reported
by us.48 Tweedle’s results are also consistent with the
relaxivities reported for dendrimers.55 His group used viscous
solutions to reduce the rotational correlation time and obtained
maximum relaxivity values of 35 (s × mM)−1 for rotationally
constrained Gd(III)-DOTA systems. The experimentally
obtained values match closely to the theoretical values
calculated with Solomon−Bloembergen−Morgan theory using
experimental values for the water residence time, which
predicted the water residence time limits the relaxivity of
rotationally constrained Gd(III)-DOTA systems. While our
results demonstrate that the relaxivity of the Gd(III)-DOTA
derivative Gd-7a is limited by the water residence time, the
maximum relaxivity observed for Gd-7a + avidin is
substantially lower 22 (s × mM)−1 (see Table 2) than that
reported for other systems89 [30 to 36 (s × mM)−1]. It is
possible that the lower relaxivity results from a longer water
residence time caused by the association of the chelate with
avidin. However, the Gd-7a + avidin relaxivity is similar to that
of other biotin−avidin systems (see Table 2).
biotinylated Gd-DTPA
6.1 0.2
8.7
1.5
20
37
46
(biotinylated) Gd-7a
0.47
this
work
(biotinylated) Gd-7b
8.7
0.47
0.47
0.47
1.5
37
25
25
20
20
20
this
work
avidin−biotinylated
18.1
17.4
17.5 0.3
22
93
GdL1/Av (DOTA)
avidin−biotinylated
93
GdL2/Av (DOTA)
avidin−biotinylated
46
Gd-DTPA
avidin−biotinylated Gd-7a
avidin−biotinylated Gd-7b
0.47
0.47
this
work
45
this
work
are reasonable, and therefore we cannot disregard the results of
using either model: letting the water residence time float or
fixing it to values measured by 17O NMR.
While letting the water residence time float gives similar
rotational correlation times, using fixed water residence times
gives us different rotational correlation times. We cannot
eliminate the second possibility, because such a change in
rotational correlation time could result from protein−chelate
associations.94,95 The constant relaxivity observed for Gd-7a
with avidin in the variable-temperature NMRD data of Figure 5
supports an increase in the water residence time to longer
values on binding to avidin. One would expect a decrease in the
relaxivity associated with a decrease in temperature as was
observed with a generation 6 ammonia core PAMAM
dendrimer coated with a Gd(III)-DOTA surface.48 The
inability to differentiate between changes in rotational
correlation time or changes in the water residence time
associated with the binding of the chelate to avidin highlight the
main problem with multiparameter NMRD profile fits: there is
no unique solution, and often multiple solutions have similar
quality of fits. At best the parameter values obtained are
estimates and suffer from a lack of accuracy. Large errors can
result in the values of the parameters because the fits start with
assigning a value for the distance between the water proton and
metal ion. Usually values for proton−metal distances are
obtained from X-ray crystallographic data,96 and it is well-
known from protein studies that solution structures and crystal
structures can differ significantly. In the case of the water
proton−metal distances a small error in the proton−metal
distance can cause significantly larger errors in the five fitted
parameters because of the inverse sixth power relationship 1/r6
of the water proton−metal distance. In short, while the
quantitative accuracy of the fits are always subject to question,
qualitative conclusions can be made from NMRD profile fits
especially when comparing similar chelates.
Avidin is a highly glycosylated protein, and the −OH groups
may act like those on poly(ethylene glycol) to reduce the
relaxivity.90 Note that fitting the NMRD profile while letting
the water residence time float, instead of fixing it at the value
for the Cbz-Gd(III)-DOTA measured by 17O NMR, yields a
value for the water residence time that is quite long relative to
that measured for our DOTA derivative Gd-6a (408 vs 101 ns,
respectively). This is about 10 times the value calculated for
Gd-7b + avidin (45 ns) by letting the water residence time
float during the NMRD fitting process. Both fitting methods
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The relaxivity of Gd-7b in the presence of avidin [32 2 (s
× mM)−1 at 37 °C] is lower than that reported for highly
constrained derivatives, such as the 58 (s × mM)−1 value
obtained by Tweedle55 for DOTMA in viscous solution.
However, our own results at a lower temperature of 3 °C [52
5 (s × mM)−1] show the effects of increased rotational
correlation time. The lower relaxivity of Gd-7b + avidin at 37
°C may be the result of segmental motions associated with
using the long-chain version of biotin. Using biotin itself,
without the hexanoic acid spacer, should result in higher
relaxivities. The fact that observed relaxivity for the rotationally
constrained Gd-7b is consistent with those reported in the
literature, whereas that of the Gd(III)-LC-DOTA, Gd-7a,
derivative is much lower may result from steric effects of the
CH3 groups on the ligand arms. The CH3 groups on the ligand
arms of Gd-7b may interfere with any interactions that were
hypothesized, to prolong the water residence time or reduce
the number of inner-sphere water molecules, to occur between
Gd-7a and avidin.
The four novel bifunctional chelates (4a-, 4b-, 5a-, and 5b-
NaOTf) are readily accessible for further studies of their
lanthanide complexes. The bifunctional octadentate chelate
design enables Gd(III) to be coordinated while maintaining
respectrable contrast properties and accessibility for more
chemistry at the azide/amine moiety. Measurements show that
the constrained derivative of Gd-7b has a higher relaxivity than
that of constrained derivative of Gd-7a due to a larger TSAP/
SAP ratio; moreover, the relaxivity of Gd-7b is dominated by
rotational correlation time, unlike Gd-7a, which is influenced
more by water residence time.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: dbgrotjahn@mail.sdsu.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support from the U.S. National Institutes of Health (R01
DK078500-01) is acknowledged. At SDSU, we thank Dr.
LeRoy Lafferty for his assistance with NMR experiments and
Lisa Thurn (Department of Biology) for assistance with ICP-
OES experiments. L.V.E. thanks the ARC Programs of the
French Community of Belgium, the FNRS (Fonds National de
la Recherche Scientifique), the support and sponsorship
concerted by COST Actions (D38 and TD1004).
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CONCLUSIONS
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We reported a concise and efficient synthesis of new
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ASSOCIATED CONTENT
■
S
* Supporting Information
CIF files for 4a- and 4b-NaOTf, an ORTEP diagram for 4a-
NaOTf, ESI-TOF of Gd-4a and Gd-4b, and the NMRD fitting
data. This material is available free of charge via the Internet at
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