Enantiomeric Recognition of d - And l -Lactate by CEST with the Aid of a Paramagnetic Shift Reagent

Article abstract of DOI:10.1021/jacs.7b08292

A previous report demonstrated that EuDO3A could be used as an NMR shift reagent for imaging extracellular lactate produced by cancer cells using CEST imaging. In this work, a series of heptadentate macrocyclic YbDO3A-trisamide complexes with δ-chiral car

Full text of DOI:10.1021/jacs.7b08292

Subscriber access provided by READING UNIV
Article
Enantiomeric recognition of D- and L-lactate by
CEST with the aid of a paramagnetic shift reagent
Lei Zhang, Andre F Martins, Piyu Zhao, Michael Tieu, David Esteban-
Gomez, Gregory T. McCandless, Carlos Platas-Iglesias, and A. Dean Sherry
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08292 • Publication Date (Web): 30 Oct 2017
Downloaded from http://pubs.acs.org on October 31, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Enantiomeric recognition of D- and L-lactate by CEST with the aid of
a paramagnetic shift reagent
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†,
†,‡,
†
†
§
∥
Lei Zhang, André F. Martins, ∥ Piyu Zhao, Michael Tieu, David Esteban-Gómez, Gregory T.
McCandless,† Carlos Platas-Iglesias§ and A. Dean Sherry*, †,‡
† Department of Chemistry, University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, United
States, ‡ Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390,
United States, § Departamento de Química, Facultade de Ciencias & Centro de Investigacións Científicas Avanzadas
(CICA), Universidade da Coruña, 15071 A Coruña, Spain
KEYWORDS: MRI contrast agent, Lactate shift reagent, Stereochemistry, Chemical exchange saturation transfer (CEST).
ABSTRACT: A previous report demonstrated that EuDO3A can be used as a NMR shift reagent for imaging extracellular lactate
produced by cancer cells using CEST imaging. In this work, a series of heptadentate macrocyclic YbDO3A-trisamide complexes
with δ-chiral carbons in the three pendant side-arms were examined as shift reagents for lactate detection. High resolution 1H NMR
spectra and DFT calculations provided evidence for formation of stereoselective lactate•YbDO3A-trisamide complexes each with a
different CEST signature. This stereoselectivity allowed discrimination of D- versus L-lactate by both high-resolution NMR and
CEST. This work demonstrates that lanthanide-based paramagnetic shift reagents can be designed to selectively detect important
metabolites by CEST MRI.
1. INTRODUCTION.
has even been reported for octadentate systems such as Eu-
DOTP even on the basis of forming strong ion-pair com-
plexes.11,12
The binding of biologically important molecules to paramag-
netic lanthanide DO3A-type complexes offers new avenues for
molecular imaging of such species.1–4 Lactate, for example, is
overproduced in most cancers yet methods to quantify lactate
production in vivo are lacking, especially methods that can dis-
tinguish intracellular lactate from extracellular lactate.5,6 Re-
cently, Zhang et al. reported a strategy to image extracellular
lactate produced by cancer cells by using the shift reagent (SR),
EuDO3A.7 This SR forms a bidentate complex with lactate even
in the presence of variety of competing biological anions. Once
bound to EuDO3A, lactate was easily detected via CEST-MRI
(chemical exchange saturation transfer magnetic resonance im-
aging) by selective activation of the highly shifted, exchanging
lactate –OH proton. Two major lactate-EuDO3A species of
equal population as detected by CEST suggested that the metal
ion-bound L-lactate lifts the magnetic degeneracy of the two di-
astereomers, Λ(δδδδ) and Δ(λλλλ), known to be present in so-
lution. Evidence supporting this hypothesis was reported by
Dickins et al. in a study of a chiral heptadentate lanthanide com-
plex which also forms a stable ternary complex with L-lactate.8,9
In that case, the two diasteriomeric ternary complexes were re-
solved by 1H NMR yet only a single Λ(δδδδ) crystal was iso-
lated for crystallographic studies. Somewhat later, Terreno et
al.10 also found that a variety of -hydroxy-carboxylate mole-
cules form ternary complexes with heptadentate YbDO3AM-
type complexes and some result in rather specific stereochemi-
cal discrimination of complex isomers. This suggests that bi-
dentate coordination of a -hydroxy-carboxylate ligand such as
lactate to a LnDO3A-type complex can result in isomer discrim-
ination likely on the basis of differences in affinity. This is not
terribly surprising given that complex isomer discrimination
The current study was initiated to investigate this phenome-
non in more detail by using various YbDO3A-derivatives as
shift reagents for L-lactate with the overarching goal of improv-
ing the sensitivity of CEST for detection of lactate. Previous
studies of various YbDOTA-tetraamide complexes with chiral
centers on the pendent arms often result in a single species in
solution due to the steric and torsional strain of placing larger
groups in pseudo-axial positions.13–15 One exception reported
by Mani et al.16 was that the chirality introduced by sec-butyl
amide substituents resulted in complexes where up to four dia-
1
stereoisomeric coordination isomers could be observed by H
NMR. Given our previous observation that addition of lactate
to EuDO3A results in discrimination of lactate•EuDO3A stere-
oisomers by CEST but not high resolution H NMR,7 we were
1
interested in knowing 1) whether introduction of chiral centers
onto the shift reagent side-arms results in improved stereoiso-
mer discrimination to yield a single CEST-active species and 2)
whether the proton exchange rate between the bound lactate –
OH proton and solvent water is sensitive to such structural
changes in the SR. One of our overarching goals is to optimize
the lactate –OH proton exchange rate to achieve maximum
CEST sensitivity.
2. RESULTS AND DISCUSSION
The ligand designs shown in Figure 1 were chosen to vary in
1) overall charge and affinity for lactate, 2) position and stere-
ochemistry of the δ chiral carbons on the side-arms, and 3) ca-
pability to form stable heptadentate LnDO3A-type complexes.
Carboxylates and carboxylate esters were used as bulky groups
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Structures of the YbDO3A derivatives used in this study. All chiral molecules are drawn as the (S)-configuration.
shifted 1H resonances, commonly referred to as the axial H4 eth-
ylene protons, were made by comparison with analogous
YbDOTMA, YbDOTTA, YbDTMA complexes.20–23 The H4
resonances in the more compact square antiprism isomers often
appear around 150 ppm for YbDOTA-type complexes (carbox-
ylate donors) and around 100 ppm or less for LnDOTAM-type
complexes (amide donors). In comparison, the H4 proton reso-
nances in the less-compacted twisted square antiprism isomers
are less highly shifted, typically <40 ppm. For axially symmet-
ric LnDOTAM-tetraamide complexes, the H4 protons are mag-
netically equivalent and appear as a single resonance24 while the
four H4 resonances in asymmetric YbDO3A-type complexes
shown in Fig. 3 show four magnetically non-equivalent reso-
nances appearing in the chemical shift range of ~50-100 ppm.
Thus, one can conclude that these complexes exist in solution
largely as SAP coordination isomers.
on the δ chiral carbon centers and Yb(III) was selected among
the lanthanide series for its greater charge density, small ionic
radius, and ability to induce large hyperfine NMR shifts without
extensive line-broadening. Heptadentate macrocyclic com-
plexes with Yb3+ are known to increase the binding affinity to
lactate by one order of magnitude over the corresponding Eu3+
complexes.10 The synthetic details are either given in Support-
ing Information or described elsewhere.17
Octadentate LnDOTA-type chelates are known to provide
four isomeric structures determined by the orientation of the
ethylene groups in the macrocycle (positive = δδδδ, negative =
λλλλ) or arm rotation (positive = Δ, negative = Λ) relative to the
four nitrogen atoms, see Fig. 2). In the absence of chiral centers,
Δ(δδδδ)/Λ(λλλλ) and Λ(δδδδ)/Δ(λλλλ) form enantiomeric pairs
so only two isomers are typically detected by 1H NMR.16,18 Hep-
tadentate Ln-complexes also exist in solution as multiple spe-
cies,10 but a report have shown that positively charged Yb3+-
complexes favor a single SAP isomer.8 The presence of chiral
centers in the ligand framework increases the number of iso-
mers that can be potentially detected by NMR. With chiral lac-
tate bound as a bidentate ligand, many more structures are pos-
sible since lactate can bind Yb3+ with the either the hydroxyl or
carboxylate group at the capping position of the square an-
tiprism (SAP) or twisted square antiprism (TSAP) coordination
polyhedron.7 The 1H NMR shifts observed for Yb3+ complexes
are largely pseudocontact in origin (훿ipse) and produce large lan-
thanide induced shifts (LIS) with negligible contact contribu-
tions. These properties also make Yb3+ a preferred lanthanide
for structural elucidations of metal-complexes by NMR. The
hyperfine NMR shifts induced by the paramagnetic Yb3+ in
these complexes are somewhat smaller compared to those ob-
served for the analogous octadentate YbDOTA-type com-
plexes.19
The high resolution 1H spectra of Yb1, Yb3, Yb4 show multi-
ple, broad overlapping proton resonances in the absence of lac-
tate (Fig. S2) but become much sharper and well-resolved after
addition of one equivalent of lactate (Fig. 3). The one exception
is Yb2 which shows moderately sharp 1H resonances even in the
absence of lactate (Fig. S2). This indicates that the interconver-
sion rate between the multiple isomers present in solution is
moderately fast in the free complexes but much slower once
lactate is bound. The assignment of most highly downfield
Figure 2: Schematic representation of the isomeric forms
of heptadentate Yb3+ complexes with one equivalent of
lactate. Arm rotation results in interconversion between
enantiomers while ring flips results in interconversion be-
tween diastereoisomers. The arrangement is described
through (λλλλ)/(δδδδ) and the side arm orientation, leading
to Λ/∆ metal coordination.
ACS Paragon Plus Environment

Page 3 of 8
Journal of the American Chemical Society
Table 1. Chemical shifts of the lactate–OH proton resonances and methyl for each of the D- or L-lactate•Ybx complexes,
1
2
proton exchange rate of Bound L and D lactate, 298 and 310 K and CEST amplitude at 298 K and 310 K with B1 of 600 Hz.
3
4
Yb0
Yb1
Yb2
Yb3
Yb4
5
6
Lactate type
L-lac
D-lac
L-lac
D-lac
L-lac
D-lac
L-lac
D-lac
L-lac
D-lac
7
8
9
δ-OH at 298 and
164/149
152/140
164/149
152/140
136
125
145
132
129
120
145
133
157
143
168
154
155
142
166
153
310 K
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH-lac (ppm)
CH3-lac (ppm)[a]
kex 298 K (s-1)[b]
kex 310 K (s-1)
54.9/36.5
29.4/19.2
54.9/36.5
29.4/19.2
31.1
22.1
1800
2800
8.5
42.0
14.9
1700
2800
12
31.2
24.1
2600
4100
20
44.8
15.7
2600
4200
20
38.5
30.1
2000
2600
14
56.4
20.6
2000
2300
12
36.7
29.8
1500
1800
6.6
54.9
19.3
1400
1800
4.4
1900/1800 2200/2000
3000/2900 2800/2700
CESTeff% (298 K)[c]
CESTeff% (310 K)
7.0/7.8
19/18
8.2/9.0
19/18
19
22
35
34
30
27
16
16
[a]. 1H NMR spectra were collected in D2O. [b]. Omega plot was performed with 50 mM complex plus 50 mM lactate. Pre-saturation pulse of 3s with B1
from 100 Hz to 1000 Hz was applied. [c]. CEST experiment was performed with 50 mM complex plus 50 mM lactate. A pre-saturation pulse of 3s with B1
of 600 Hz was applied at 298 K and 310 K, respectively. Proton exchange rate and CEST amplitude are in 10 percent error range.
Among these five complexes, a striking difference was ob-
served in the 1H NMR spectra of Yb0, the only complex lacking
a chiral carbon. Here, each H4 proton resonance appears as two
equally intense peaks with very similar chemical shifts while
the lactate CH and CH3 resonances also appear as two reso-
nances of equal intensity separated by ~15 and ~10 ppm, re-
spectively. This suggests that lactate binds to both SAP isomers,
Λ(δδδδ) and Δ(λλλλ), of Yb0 and that the protons in these two
diastereoisomeric L-lactate•Yb0 complexes are not magneti-
cally equivalent. The 1H spectrum of Yb0 in the presence of one
equivalent of D-lactate was identical (Fig. 3). In comparison,
the 1H NMR spectra of D- or L-lactate with the four complexes
containing chiral carbons in their side-arms (Yb1-4) each dis-
played four sharp ethylene proton resonances (Fig. 3) and single
lactate CH and CH3 resonances. This suggests that the lactate
Yb1-4 complexes either exist in solution as a single species or
inversion/rotation between these diastereomers is much faster
than that seen for the lactate Yb0 complexes. Close inspection
of these spectra show that the chemical shifts of the ethylene
protons and lactate protons differed in all four L-lactate•Yb1-4
complexes. Furthermore, the chemical shifts of the protons in
the D-lactate•Yb1-4 complexes differed from those seen in spec-
tra of the L-lactate•Yb1-4 complexes (see Table 1). This sug-
gests that a unique single stereoisomer, either Λ(δδδδ) or
Δ(λλλλ), is formed in each of these complexes.
To examine these effects further, 2D EXSY NMR (Exchange
Spectroscopy) spectra were collected on samples of L-lac-
tate•Yb0 and L-lactate•Yb4 (Fig. 4). The EXSY mixing time
was varied and optimized to obtain optimal exchange peaks for
L-lactate•Yb0 and this same value was then used for the spec-
trum of L-lactate•Yb4. As seen in Fig. 4, the EXSY spectrum
of L-lactate•Yb0 shows exchange cross-peaks between the axial
and equatorial proton resonances and between the non-equiva-
lent CH and CH3 lactate resonances characteristic of intercon-
Figure 3. 1H NMR spectra of Ybx complexes containing 1:1 L-lactate (left) or 1:1 D-lactate (right). All NMR spectra were recorded at 25oC
in D2O at 9.4 T. pD = 6.5. Arrows point to the H4 proton resonances.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 8
protons of the cyclen unit separated by three bonds; iii) the gem-
version between the two diastereotopic L-lactate•Yb0 com-
plexes while the EXSY spectrum of L-lactate•Yb4 showed no
cross peaks under identical experimental conditions. This sup-
ports our earlier conclusion that that the later complex exists as
a single species in solution. An alternative explanation could
be that interconversion rate between two diastereomers in L-
lactate•Yb4 is much faster that that seen in L-lactate•Yb0. How-
ever, this is highly unlikely because interconversion between
diastereomers requires side-arm rotation and his would not be
expected to occur more rapidly in the bulkier L-lactate•Yb4
complex. Thus, the only reasonable conclusion is that L-lac-
tate•Yb4 exists as a single species in solution and, by compari-
son, the remaining L-lactate•Yb1-4 likely do as well. Unfortu-
nately, none of the lactate-Yb1-4 complexes formed crystals
likely due to their high solubility in polar protic solvents. One
of the complexes, Yb3, crystalized as a dimer where one of the
carboxylate groups of the ligand acted as a bridge between the
two metal ions (Fig. S3). Lactate was not present in this struc-
ture. This behavior has been reported previously in another sim-
ilar lanthanide complex.25 In the structure shown in Fig. S3, the
Yb3 complex was present as the S-Δ(λλλλ) isomer in both metal
centers.
1
2
3
4
5
6
7
8
inal protons of the methylenic groups of the pendant arms; iv)
the CH and methyl groups of the pendant arms; and v) the CH
and methyl groups of lactate (Table S2, Supporting Infor-
mation).
A detailed analysis of the pseudocontact shifts induced by Yb
was performed using the following expressions: 29
 y2
r5
 4xy 
2
2








1
3
3z  r2
x
ipse ( zz

Tr )
 xy






 ( xx   yy )
r5
r5








9
 4xz 
 4yz 
 xz

   yz





10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

r5
r5




(1)
(2)
r 
x
2  y2  z2
where
In these equations x, y and z are the Cartesian coordinates of
nuclei i relative to the location of the paramagnetic ion, and pk
are the components of the magnetic susceptibility tensor (p, k =
x, y or z). In a principal magnetic axis system, xy = xz = yz =
0, so that only the first two terms of Equation 1 remain, repre-
senting the axial and rhombic contributions to the pseudocon-
tact shifts. Equation 1 was used to analyze the pseudocontact
shifts by following the methodology of Forsberg,30 which in-
The chemical shifts of the bound lactate protons in these
complexes are also informative (Table 1). Most notable is that
the chemical shift differences between the D- and L-lactate
complexes are much larger (~10 ppm) in comparison to the eth-
ylene proton resonances. These large chemical shift differences
between D- and L-lactate observed here by 1H NMR are much
larger than those observed using non-aqueous chiral agents.26–
volves a least-squares fit procedure including the axial [zz
–
1/3(xx + yy + zz)] and rhombic (xx − yy) anisotropies of the
magnetic susceptibility tensor  as fitting parameters, as well as
three Euler angles that relate the orientation of the Cartesian
axes of the input structure and the principal magnetic axis sys-
tem.31 As structural models we used molecular geometries ob-
tained with DFT calculations following standard procedures
(see computational details, Supporting Information).32,33 These
calculations provided four local energy minima corresponding
to the S-Λ(δδδδ), S-Δ(λλλλ), S-Δ(δδδδ) and S-Λ(λλλλ) diaster-
eoisomers. According to DFT, the minimum energy confor-
mation corresponds to the S-Λ(δδδδ) isomer for both L-lac-
tate•Yb3 and D-lactate•Yb3. A similar structure was observed
in the solid state for a lactate-YbDO3A complex with chiral D-
phenyl groups.8 The orientation of the lactate anion is such that
28
This indicates that the geometrical positions of the CH and
CH3 protons in D-lactate•Ybx versus L-lactate•Ybx are quite
different with respect to the magnetic axes governing the Yb-in-
duced pseudocontact shifts. To investigate the origin of these
differences further, a complete NMR assignment of all proton
resonances in spectra of the Yb3 complexes with D- and L-lac-
1
tate was undertaken by analysis of their H,1H-COSY spectra
(Fig. S4). Cross-peaks in these spectra were observed for i) the
axial and equatorial protons of the cyclen structure; ii) the axial
Figure 4. (Left). 2D EXSY NMR spectrum of Yb0•L-lactate (1:1) in D2O using a mixing time of 15 ms. (Right). 2D EXSY NMR spectrum
of Yb4•L-lactate (1:1) in D2O using a mixing time of 15 ms. The red arrows reflect exchange of the lactate CH and CH3 resonances while
the blue arrows reflect exchange of the axial proton resonances between the two SAP diasterisomers.
ACS Paragon Plus Environment

Page 5 of 8
Journal of the American Chemical Society
the carboxylate group occupies one of the coordination posi- magnetic axis (the z axis) is not placed perpendicular to the ideal
1
2
3
4
5
6
7
8
tions in the upper plane of the SAP coordination polyhedron,
while the hydroxyl group of lactate coordinates at the apical po-
sition. DFT calculations indicated that this coordination mode
was far more favorable (> 9.0 kcal mol-1) than the reverse situ-
ation in which the apical position is occupied by the carboxylate
group. This likely reflects the preference of the weaker hy-
droxyl donor atom to occupy the more hindered capping posi-
tion.34 The analysis of the pseudocontact shifts confirmed the
predictions of DFT, as the chemical shifts calculated with Eq
(1) showed an excellent agreement with the experimental ones,
with deviations < 5.5 ppm for L-lactate•Yb3 and < 4.9 ppm for
D-lactate•Yb3 (Table S2, Supporting Information). Much larger
deviations were observed by using the geometry of the
S-Δ(λλλλ) isomer, particularly for the CH protons of the side
arms (up to 15-17 ppm, Supporting Information).
plane defined by the four nitrogen atoms of the macrocycle, but
parallel to that plane. This surprising result is however in agree-
ment with recent studies reported for DyDOTA.35 Furthermore,
the analysis provides virtually identical orientations of the mag-
netic axes in L-lactate•Yb3 and D-lactate•Yb3 so the slightly
different chemical shifts observed for the lactate resonances in
L-lactate•Yb3 and D-lactate•Yb3 cannot be attributed to the for-
mation of a different isomer, or to a different magnetic anisot-
ropy of the complex (the axial and rhombic anisotropies are
very similar, Table S2, Supporting Information). Instead, the
origin of the different chemical shifts is related to the different
position of lactate proton nuclei with respect to the magnetic
axes (Figure 5). Indeed, the CH proton of lactate presents virtu-
ally identical distances to the paramagnetic center in both L-
lactate•Yb3 and D-lactate•Yb3 (r = 3.860 and 3.820 Å, respec-
tively), but different z coordinates (1.160 and 1.755 Å), which
leads to somewhat different axial pseudocontact shifts (-14.3
and -8.4 ppm). However, the different position of the lactate
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5 shows the orientation of the molecular geometries
obtained for L-lactate•Yb3 and D-lactate•Yb3 in the principal
magnetic axis system. The analysis shows that the principal
L-lactate•Yb3
D-lactate•Yb3
Figure 5. Top: Molecular geometries of the L-lactate•Yb3 and D-lactate•Yb3 systems obtained with DFT showing the orientation of the
magnetic axes. Circles highlight the position of lactate CH protons. Middle: Contour plots showing the rhombic contribution of the pseu-
docontact shifts (ppm) in the plane parallel to the x and y axis containing the lactate CH proton. Bottom: Axial contribution to the pseu-
docontact shifts (ppm) in the same plane. White circles indicate the position of lactate CH protons. Negative pseudocontact shifts corre-
spond to shifts to lower fields.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 8
protons in the xy plane is the main reason for the different chem-
ical shifts observed for L-lactate•Yb3 and D-lactate•Yb3, lead-
ing to rhombic contributions of -15.3 and -45.3 ppm (Table S2,
Supporting Information).
from 9-16 ppm. This observation is again consistent with enan-
tiomeric discrimination of D– or L–lactate by the Yb1-4 com-
plexes (Table 1).
1
2
3
4
5
6
7
8
The amplitude of the CEST exchange peaks for the D- versus
L-lactate in each complex varied to some extent. For example,
the CEST intensities of the D-lactate –OH exchange peaks were
larger than the corresponding L-lactate –OH exchange peaks in
the Yb1 complexes, about equal in the Yb3 complexes, and
CEST spectra. To investigate the utility of these Ybx com-
plexes for imaging lactate by CEST,7 we collected CEST spec-
tra of samples containing each complex (Yb0-4) with one equiv-
alent of L-lactate or D-lactate. As shown in Fig. 6, the chemical
shifts of the exchanging lactate –OH proton in the Ybx com-
plexes are substantially larger (~130-170 ppm) compared to the
shifts observed when using EuDO3A as a shift reagent.7 For the
achiral complex, Yb0, two sharp CEST peaks at 149 and 164
ppm were detected that are likely attributable to two the equally
favored diasteroisomeric complexes, Λ(δδδδ) and Δ(λλλλ).
This is supported by DFT calculations, which provide two vir-
tually isoenergetic minima for the Λ(δδδδ) and Δ(λλλλ) isomers
of the L-lactate•Yb0 and D-lactate•Yb0 adducts. Identical
CEST exchange peaks were observed when either D-lactate or
L-lactate was added to a sample of Yb0 similar to that seen in
the 1H NMR spectra. Upon addition of either D- or L-lactate to
any one of the chiral complexes, Yb1-4, only a single CEST peak
was observed, (Table 1) but again, the chemical shifts of the
CEST exchange peaks for D- versus L-lactate complexes varied
1
smaller in the Yb2 and Yb4 complexes. The corresponding H
9
spectra show that the concentrations of the D- versus L-lactate
species are identical so these intensity differences must reflect
differences in proton exchange rates. With a temperature in-
crease to 310 K, larger CEST amplitudes were observed for all
Yb1-4-lactate complexes. The proton exchange rates were eval-
uated using the Omega plot method at both temperatures and,
indeed, faster proton exchange rates were observed at 310 K
than at 298 K, as would be expected. The proton exchange rates
at 310 K were close to optimal for CEST and hence should ben-
efit experiments performed at physiological temperatures. In
each complex, the D-lactate –OH exchange peak was shifted
further downfield compared to the analogous L-lactate –OH ex-
change peak. On the basis of the results described above, this
can be attributed to slightly different positions of the OH pro-
tons with respect to the orientation of the magnetic axes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. CONCLUSIONS
In this study, we investigated whether the substitution of a 
chiral carbon in the pendant arms of YbDO3A-amide type com-
plexes can limit the number of stereoisomers present in solution
and thereby enhance detection of lactate by CEST NMR. Our
results show that with different substitutions at the -position
on the pendant arms, a single energetically favorable isomer is
1
formed. This was supported by both high resolution H NMR
and CEST data. In the presence of (D or L) lactate, the achiral
Yb0 complex forms two equally populated Λ(δδδδ) and Δ(λλλλ)
isomers with lactate whereas the Ybx complexes having  chiral
centers yield a single isomer (shown to be the S-Λ(δδδδ) isomer
for the Yb3 complexes by analysis of the pseudocontact shifts).
A second finding of this study was the observation of enantio-
meric discrimination of D- and L-lactate by the chiral DO3A-
derived Ybx complexes. In summary, our study shows that hep-
tadentate macrocyclic Yb-complexes with  chiral centers serve
as key predictors of conformational orientation of lanthanide
complexes with macrocyclic ligands and powerful discrimina-
tors of enantiomeric substrates such as D- and L-lactate. Ter-
nary complexes are formed with the lactate OH group bound to
the Yb3+ ion in an apical position as reported previously for
lactate•EuDO3A.7 The lactate OH proton is highly shifted
downfield in the presence of the Yb-complexes as seen by
CEST NMR. The approach used magnifies the chemical shift
of lactate OH CEST which may ultimately prove usefull in the
design of metabolite-specific shift reagents for functional MRI.
L-lactate is generally considered the only form produced by
cancer cells while D-lactate, although rarely produced by hu-
man cells, is a metabolic product of some essential bacteria that
exist in the human microbiota. Consequently, D-lactate acidosis
has been associated with several diseases including bowel syn-
drome and D-lactate encephalopathy.36,37 Given that the devel-
opment of enantiopure agents is of interest in molecular imag-
ing, this work is a positive step forward towards optimizing in-
organic responsive agents (shift reagents) for selective identifi-
cation of important biomarkers and metabolic profiles.
Figure 6. CEST spectra of Yb(III)-complexes with either L-
lactate or D-lactate in H2O. (50 mM complex with 50 mM D-
lactate or L-lactate). Pre-saturation pulse of 4 s with B1 of 14.1
T was applied at 298 K using a 9.4 T NMR spectrometer. The
pH was adjusted to 7.4 in all samples just prior to data collec-
tion. CEST spectra were only shown the downfield part for
clarification.
ACS Paragon Plus Environment

Page 7 of 8
Journal of the American Chemical Society
(13)
(14)
(15)
Howard, J. A. K.; Kenwright, A. M.; Moloney, J. M.; Parker,
D.; Woods, M.; Howard, J. A. K.; Port, M.; Navet, M.; Rous-
seau, O. Chem. Commun. 1998, 1381.
Vipond, J.; Woods, M.; Zhao, P.; Tircsó, G.; Ren, J.; Bott, S.
G.; Ogrin, D.; Kiefer, G. E.; Kovacs, Z.; Sherry, A. D. Inorg.
Chem. 2007, 46, 2584.
Mani, T.; Opina, A. C. L.; Zhao, P.; Evbuomwan, O. M.; Mil-
burn, N.; Tircso, G.; Kumas, C.; Sherry, A. D. JBIC J. Biol.
Inorg. Chem. 2013, 19, 161.
Mani, T.; Tircsó, G.; Zhao, P.; Sherry, A. D.; Woods, M. In-
org. Chem. 2009, 48, 10338.
Zhang, S.; Winter, P.; Wu, K.; Sherry, A. D. J. Am. Chem.
Soc. 2001, 123, 1517.
Delli Castelli, D.; Terreno, E.; Aime, S. Angew. Chem. Int.
Ed. 2011, 50, 1798.
Aime, S.; Botta, M.; Ermondi, G. Inorg. Chem. 1992, 31,
4291.
Brittain, H. G.; Desreux, J. F. Inorg. Chem. 1984, 23, 4459.
Aime, S.; Barge, A.; Bruce, J. I.; Botta, M.; Howard, J. A. K.;
Moloney, J. M.; Parker, D.; de Sousa, A. S.; Woods, M. J. Am.
Chem. Soc. 1999, 121, 5762.
Rohovec, J.; Kývala, M.; Vojtíšek, P.; Hermann, P.; Lukeš, I.
Eur. J. Inorg. Chem. 2000, 2000, 195.
Aime, S.; Botta, M.; Garda, Z.; Kucera, B. E.; Tircso, G.;
Young, V. G.; Woods, M. Inorg. Chem. 2011, 50, 7955.
Dickins, R. S.; Parker, D.; Bruce, J. I.; Tozer, D. J. Dalton
Trans. 2003, 1264.
Kang, S. I.; Ranganathan, R. S.; Emswiler, J. E.; Kumar, K.;
Gougoutas, J. Z.; Malley, M. F.; Tweedle, M. F. Inorg. Chem.
1993, 32, 2912.
1
2
3
4
5
6
7
8
ASSOCIATED CONTENT
Supporting Information
The SI contains the synthetic details for preparing the Yb(III) com-
plexes, the experimental details of the CEST experiments, experi-
1
mental and calculated H NMR shifts, computational details and
high-resolution NMR of the Yb complexes.
(16)
(17)
(18)
(19)
9
AUTHOR INFORMATION
Corresponding Author
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*E-mail: dean.sherry@utsouthwestern.edu; sherry@utdallas.
edu.
(20)
(21)
Author Contributions
∥These authors contributed equally.
(22)
(23)
(24)
(25)
ACKNOWLEDGMENT
The authors acknowledge partial financial support for this work
from the National Institutes of Health (CA-115531, EB-01598, EB-
00482), Harold C. Simmons Cancer Center through an NCI Cancer
Center Support Grant, 1P30 CA142543, and the Robert A. Welch
Foundation (AT-584). The authors C. P.-I. and D. E.-G. thank Min-
isterio de Economía y Competitivad (CTQ2016-76756-P) and Cen-
tro de Supercomputación de Galicia (CESGA) for providing the
computer facilities.
(26)
Ema, T.; Tanida, D.; Sakai, T. J. Am. Chem. Soc. 2007, 129,
10591.
(27)
(28)
(29)
Ema, T.; Tanida, D.; Sakai, T. Org. Lett. 2006, 8, 3773.
Iwaniuk, D. P.; Wolf, C. J. Org. Chem. 2010, 75, 6724.
Piguet, C.; Geraldes, C. F. G. C. In Handbook on the Physics
and Chemistry of Rare Earths; K.A. Gschneidner, J.-C. G. B.
and V. K. P., Ed.; Handbook on the Physics and Chemistry of
Rare Earths; Elsevier: United Kingdom, 2003; 353–463.
Forsberg, J. H.; Delaney, R. M.; Zhao, Q.; Harakas, G.; Chan-
dran, R. Inorg. Chem. 1995, 34, 3705.
Regueiro-Figueroa, M.; Bensenane, B.; Ruscsák, E.; Esteban-
Gómez, D.; Charbonnière, L. J.; Tircsó, G.; Tóth, I.; Blas, A.
de; Rodríguez-Blas, T.; Platas-Iglesias, C. Inorg. Chem. 2011,
50, 4125.
(30)
(31)
REFERENCES
(1)
(2)
(3)
(4)
Terreno, E.; Botta, M.; Fedeli, F.; Mondino, B.; Milone, L.;
Aime, S. Inorg. Chem. 2003, 42, 4891.
Aime, S.; Delli Castelli, D.; Fedeli, F.; Terreno, E. J. Am.
Chem. Soc. 2002, 124, 9364.
Botta, M.; Aime, S.; Barge, A.; Bobba, G.; Dickins, R. S.;
Parker, D.; Terreno, E. Chem. – Eur. J. 2003, 9, 2102.
Terreno, E.; Botta, M.; Boniforte, P.; Bracco, C.; Milone, L.;
Mondino, B.; Uggeri, F.; Aime, S. Chem. – Eur. J. 2005, 11,
5531.
(32)
(33)
(34)
Regueiro-Figueroa, M.; Platas-Iglesias, C. J. Phys. Chem. A
2015, 119, 6436.
Esteban-Gómez, D.; de Blas, A.; Rodríguez-Blas, T.; Helm,
L.; Platas-Iglesias, C. ChemPhysChem 2012, 13, 3640.
Rodríguez-Rodríguez, A.; Regueiro-Figueroa, M.; Esteban-
Gómez, D.; Rodríguez-Blas, T.; Patinec, V.; Tripier, R.;
Tircsó, G.; Carniato, F.; Botta, M.; Platas-Iglesias, C. Chem.
– Eur. J. 2017, 23, 1110.
Cucinotta, G.; Perfetti, M.; Luzon, J.; Etienne, M.; Car, P.-E.;
Caneschi, A.; Calvez, G.; Bernot, K.; Sessoli, R. Angew.
Chem. Int. Ed. 2012, 51, 1606.
de Bari, L.; Moro, L.; Passarella, S. FEBS Lett. 2013, 587,
467.
Kowlgi, N. G.; Chhabra, L. Gastroenterol. Res. Pract. 2015,
2015, e476215.
(5)
(6)
(7)
Heiden, M. G. V.; Cantley, L. C.; Thompson, C. B. Science
2009, 324, 1029.
Rato, L.; Alves, M. G.; Socorro, S.; Carvalho, R. A.; Cavaco,
J. E.; Oliveira, P. F. Biosci. Rep. 2012, 32, 61.
Zhang, L.; Martins, A. F.; Mai, Y.; Zhao, P.; Funk, A. M.;
Clavijo Jordan, M. V.; Zhang, S.; Chen, W.; Wu, Y.; Sherry,
A. D. Chem. - Eur. J. 2017, 23, 1752.
(35)
(36)
(37)
(8)
(9)
Dickins, R. S.; Love, C. S.; Puschmann, H. Chem. Commun.
2001, 2308.
Dickins, R. S.; Aime, S.; Batsanov, A. S.; Beeby, A.; Botta,
M.; Bruce, J. I.; Howard, J. A. K.; Love, C. S.; Parker, D.;
Peacock, R. D.; Puschmann, H. J. Am. Chem. Soc. 2002, 124,
12697.
(10)
(11)
(12)
Terreno, E.; Botta, M.; Fedeli, F.; Mondino, B.; Milone, L.;
Aime, S. Inorg. Chem. 2003, 42, 4891.
Wenzel, T. J.; Morin, C. A.; Brechting, A. A. J. Org. Chem.
1992, 57, 3594.
Jiang, J.; Li, A.-H.; Jang, S.-Y.; Chang, L.; Melman, N.;
Moro, S.; Ji, X.; Lobkovsky, E. B.; Clardy, J. C.; Jacobson,
K. A. J. Med. Chem. 1999, 42, 3055.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 8
Table of Content Graphic
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
ACS Paragon Plus Environment