Catechol-Based Functionalizable Ligands for Gallium-68 Positron Emission Tomography Imaging

Emission Tomography Imaging

Article

Catechol-Based Functionalizable Ligands for Gallium-68 Positron

M. Andrey Joaqui-Joaqui, Mukesh K. Pandey,* Aditya Bansal, Mandapati V. Ramakrishnam Raju,

Fiona Armstrong-Pavlik, Ayca Dundar, Henry L. Wong, Timothy R. DeGrado,* and Valerie C. Pierre*

Cite This: https://dx.doi.org/10.1021/acs.inorgchem.0c00975

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sı Supporting Information

ABSTRACT: Four tris-bidentate catecholamide (CAM) ligands

were synthesized, characterized, and evaluated as ligands for

radiolabeling of gallium-68 for positron emission tomography

(PET). Three of those ligands, 2,2-Glu-CAM, 3,3-Glu-CAM, and

TREN-bisGlyGlu-CAM, incorporate ligand caps that contain a

pendant carboxylic group for further conjugation to targeting

moieties. The acyclic ligands all exhibited high (>80%) radio-

labeling yields after short reaction times (<10 min) at room

temperature, a distinct advantage over macrocyclic analogues that

display slower kinetics. The stabilities of the four Ga^I^I^Icomplexes

are comparable to or higher than those of other acyclic ligands used

for gallium-68 PET imaging, such as desferrioxamine, with pGa

values ranging from 21 to >24, although the functionalizable

III

ligands are less stable than the parent Ga -TREN-CAM. In vivo imaging studies and ex vivo pharmacokinetic and biodistribution

studies indicate that the parent [ Ga]Ga-TREN-CAM is stable in vivo but is rapidly cleared in <15 min, probably via a renal

pathway. The rapid and mild radiolabeling conditions, high radiolabeling yields, and high stability in human serum (>95%) render

TREN-bisGlyGlu-CAM a promising candidate for gallium-68 chelation.

INTRODUCTION

ligands yield kinetically inert complexes while providing

functional groups that are suitable for further conjugation to

■

Positron emission tomography (PET) is a standard and

powerful imaging modality favored in the medical ﬁeld for

diagnosis and for evaluation of therapeutic methods due to its

high sensitivity and its non-invasive nature. Tracers used in

PET imaging employ positron (β )-emitting radionuclides.

Detection of the γ-rays emitted upon collision of the positrons

β ) with nearby electrons (β ) enables the spatial

reconstruction of the tracer’s distribution in vivo. Two

diﬀerent approaches are pursued in the design of radio-

pharmaceutical imaging agents. The ﬁrst involves organic

molecules, often based on drugs, which incorporate a nonmetal

antibodies or other targeting moieties. Examples used

clinically include [ Ga]Ga-DOTA -1NaI octreotide (DOTA-

NOC), [ Ga]Ga-DOTA -Tyr octreotide (DOTA-TOC), and

[ Ga]Ga-DOTA -Tyr octreotate (DOTA-TATE), three trac-

ers that target somatostatin receptors that are overexpressed in

1−14

−

neuroendocrine tumors (Figure 1).

Unfortunately, the

(

high kinetic inertness of DOTA-based gallium complexes that

advantageously slows dechelation and transmetalation to a

time many-fold greater than the half-life of gallium-68 also

imposes harsh reaction conditions for the formation of the

complex. With such macrocyclic ligands, high temperatures,

low pHs, and long reaction times (e.g., 90 °C, pH 3−5, and 30

min, respectively) are typically required to reach adequate

18 3−7

PET radionuclide such as C, N, O, or F. The second

approach employs PET radioactive metals tightly coordinated

by a chelate and often conjugated to a targeting moiety such as

15−19

radiolabeling yields.

Although such harsh conditions can

an antibody. Among the metallic radionuclides employed for

PET imaging, gallium-68 stands out as an attractive choice due

be used with many peptide conjugates, they are incompatible

with many targeting antibodies. Moreover, much of the

to its convenient decay proﬁle (mean β energy of 0.89 MeV;

t₁_/₂= 68 min) and high positron yield (89%), which allow for

suﬃcient levels of radioactivity for the production of high-

quality images while minimizing the exposure of patients to a

Received: April 1, 2020

prolonged large and damaging dose of radiation.

Past gallium-68 radiopharmaceuticals often employed

chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-

acetic acid [DOTA (Figure 1)] because such macrocyclic

XXXX American Chemical Society

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Figure 1. Structures of some common cyclic and acyclic chelators used for gallium-68 PET.

radioactivity of gallium-68 decays during the long reaction

times needed for DOTA complexation. Such severe limitations

led to the development of new chelators such as NOTA and

NODAGA (Figure 1), whose smaller cages enable complex-

ation of gallium-68 at room temperature in less time, typically

namic stability such as HBED-CC (Figure 1) have more

28,29

potential.

Its prostate-speciﬁc membrane antigen con-

jugate, PSMA-HBED-CC, is currently one of the most widely

used PET imaging agents. Other ligands such as THP and

H DEDPA, AAZTA, DATA, PIDAZTA, and the

recently reported H hox are also promising chelating

10 min. However, complexation with these ligands still

requires the use of acidic conditions that can be problematic

with some targeting biomolecules. Similar drawbacks aﬀect

other macrocyclic ligands that have been reported (Figure 1),

moieties for gallium-68 PET imaging.

Despite the potential of these recent gallium-68 chelators,

there remains a need for new compounds that enable

radiolabeling to be performed with high yields, rapidly, at

room temperature and near-physiological pH. The further

development of such chelators into radiopharmaceuticals

requires that the chelate contain a pendant functional group

for further conjugation to targeting biomolecules. High

thermodynamic stability, which is necessary for kinetically

labile radiolabels to be successful in vivo, can be achieved by

matching the coordination number, geometry, cavity size, and

chelating functionality to those favored by gallium(III). Given

including TRAP, NOPO, PCTA, and porphyrin scaﬀolds

such as TFPP.

Ultimately, for gallium-68 complexation to be performed

eﬃciently and rapidly at room temperature and near-neutral

pH, kinetically labile ligands must be used. Such ligands

include acyclic or linear scaﬀolds such as DTPA [diethylene-

triaminepentaacetatic acid (Figure 1)]. Unfortunately, the

gallium complex of DTPA is not suﬃciently thermodynami-

cally stable to prevent dechelation and/or transmetalation in

5−27

vivo.

Recent linear chelators with increased thermody-

the similarity in coordination chemistry between Ga and Fe

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

members of the Enterobacteriaceae family, is one of the most

Article

ions, those requirements have led researchers to explore the

potential of chelators historically developed and optimized for

iron as templates for ⁶⁸Ga-labeled radiopharmaceuticals. In

particular, siderophores, which are low-molecular weight

natural product iron chelators secreted by bacteria and fungi

is one of the highest known for iron(III) complexes (the higher

III

the pM value, the more stable the complex). The Ga

45,46

complex of enterobactin has also been reported.

Enter-

obactin has, however, not been evaluated for gallium-68

complexation and further application in PET imaging. There

are a number of reasons why it would be a poor choice as a

gallium-68 chelator for in vivo imaging applications. First, it

lacks a chemical handle for further conjugation of a targeting

moiety. Second, the trilactone backbone is prone to hydrolysis

speciﬁcally for Fe³chelation, are promising scaﬀolds. The

gallium-68 and gallium-67 complexes of desferrioxamine [DFO

(

Figure 1)], a hydroxamate-based bacterial siderophore, for

instance, have been successfully used for PET imaging.

Advantageously, targeting moieties can be conjugated to DFO

under physiological conditions, which would render the Ga

via its N-terminus, as in the tumor-targeting [ Ga]Ga-DFO-

complex unstable in vivo.

succinyl-D-Phe-octreotide. Unfortunately, because they are

With this in mind, and to expand the toolkit of molecular

radiotracers, we aim to apply the literature on enterobactin

mimics for iron(III) coordination to the development of novel

not suﬃciently strong chelators, DFO conjugates have been

0−42

reported to leach the radionuclide in vivo.

Similarly, the

45,47,48

Ga complex of pyoverdine, a mixed type siderophore from

ligands for Ga.

Speciﬁcally, we sought to design,

Pseudomonas species with both catecholate and hydroxamate

synthesize, and evaluate new ligands that could chelate

coordinating moieties, has also shown potential for imaging

[ Ga]Ga rapidly under mild conditions at physiological

pH and room temperature with high radiochemical yields and

would be thermodynamically stable as needed for in vivo

applications. To avoid the problem of the propensity of

lactones toward hydrolysis, the backbone of enterobactin was

replaced in our four ligands with more stable amines and

amide caps (Figure 2). As with enterobactin, all four ligands

are tripodal and contain three catecholate coordinating

moieties (CAMs) as needed for strong coordination of the

some bacterial infections by PET. However, pyoverdine lacks

a pendant functional group for further bioconjugation, which

limits its use for targeted imaging.

Enterobactin (Figure 2), a siderophore produced by most

metal ion. Three of the ligands, 2,2-Glu-CAM (2), 3,3-Glu-

CAM (3), and TREN-bisGlyGlu-CAM (4), also contain

pendant carboxylic acid functional groups that enable further

bioconjugation. Those four ligands diﬀer in the size of their

chelating cavity, which is expected to inﬂuence both radio-

labeling yield and thermodynamic stability.

RESULTS AND DISCUSSION

■

Synthesis. The synthesis of the tripodal enterobactin

analogue TREN-CAM (1) was previously reported via a route

that uses methyl-protected catechols and thus necessitates

subsequent harsh deprotection conditions. To use milder

conditions, we have instead employed benzyl protecting

groups and synthesized 1 according to Scheme 1. Benzyl-

Scheme 1. Synthesis of TREN-CAM (1)

Figure 2. Chemical structures of functionalizable enterobactin-based

PET ligands.

powerful siderophores due to its three preorganized catechol

moieties. Accordingly, its pFe value, 35.5, where pFe is

deﬁned as

−

pFe = −log[Fe] when [enterobactin]_t_o_t_a_l= 10 M and

free

Reagents and conditions: (a) TBTU, DIPEA, CH Cl , rt, 24 h; (b)

−

[

^F^e^]total = 10 M

(1)

HCl/AcOH, rt, 24 h.

Inorg. Chem. XXXX, XXX, XXX−XXX

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Article

protected 2,3-dihydroxybenzoic acid (5) was synthesized in

Scheme 3. Synthesis of TREN-bisGlyGlu-CAM (4)

two steps from commercial sources as reported by Gardner et

al. The TREN-capped benzyl-protected tripodal ligand (6)

was subsequently formed under standard coupling conditions

with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tet-

raﬂuoroborate (TBTU). Deprotection under acidic conditions

yielded the ﬁnal ligand TREN-CAM (1) that was stored under

N (g) in the dark at 4 °C due to the susceptibility of

52,53

catecholamides to oxidation.

The ligands 2,2-Glu-CAM (2) and 3,3-Glu-CAM (3) were

synthesized according to Scheme 2 starting from the

Scheme 2. Synthesis of 2,2-Glu-CAM (2) and 3,3-Glu-CAM

(3)

Reagents and conditions: (a) NHS, EDC·HCl, 1,4-dioxane, 0 °C, 24

h; (b) DIPEA, 1,4-dioxane, rt, 30 h; (c) NHS, EDC·HCl, CH Cl , 0

C, 24 h; (d) DIPEA, CH Cl , rt, 30 h; (e) HCl/AcOH, rt, 20 h.

2 2

manner similar to that of Glu( Bu)CAM(Bn) (10) according

to the procedure of Dertz et al. In situ N-hydroxysuccinimide

(

NHS) activation of the arm Glu( Bu)CAM(Bn) (10) with N-

ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride

EDC·HCl) enabled further conjugation to the tris(2-

(

aminoethyl)amine backbone to yield the monosubstituted

TREN intermediate 13. In situ NHS activation of the second

arm, GlyCAM(Bn) (14), enabled conjugation to the two

remaining primary amines of 13 and completion of the fully

protected ligand (15). Simultaneous deprotection of the tert-

butyl and benzyl groups under acidic conditions yielded the

Reagents and conditions: (a) CH Cl , rt, 48 h; (b) TBTU, DIPEA,

CH Cl , rt, 24 h; (c) HCl/AcOH, rt, 30 h.

thiazolidine-activated intermediate CAM(Bn)thiaz (7) that

was previously synthesized according to the procedure

reported by Allred et al. Advantageously, the thiazolidine

activating group enables chemoselective coupling of the CAM

moiety to the primary amines of the diethylenetriamine and

dipropylenetriamine backbones to yield bis-amides 8 and 9,

respectively. Separately, Glu( Bu)-CAM(Bn) (10) was synthe-

sized as reported by Dertz et al. Conjugation of the arm 10

to intermediate 8 or 9 under standard coupling conditions with

TBTU yielded the fully protected ligand 2,2-Glu( Bu)-

CAM(Bn) (11) or 3,3-Glu( Bu)-CAM(Bn) (12), respectively.

ﬁnal ligand 4 that was kept under N (g) in the dark at 4 °C.

III

Stability of Ga(III) Complexes. For labile Ga

complexes to be viable candidates for PET imaging, the

complexes must be suﬃciently stable so that the radioactive

metal does not leach out of the agent and into undesired sites.

Because it is the radionuclide metal ion that is imaged, any

decomplexation would prevent accurate imaging of the desired

targets. Achieving high stability is particularly important given

the low concentration of PET agents used in vivo,

concentrations that favor decomplexation.

The stability of metal complexes with diﬀering numbers of

protonation steps is best compared not by their formation

The tert-butyl and benzyl protecting groups were subsequently

removed simultaneously under acidic conditions to yield the

ﬁnal ligands 2,2-Glu-CAM (2) and 3,3-Glu-CAM (3), both of

constants but by their pM values, where pM = −log[M] (eq

free

1). For Ga complexes, the relevant pM value is pGa. This

thermodynamic parameter is typically reported under physio-

logical pH (7.4) at [L] = 10 μM and [M] = 1 μM.

which were kept under N (g) in the dark at 4 °C.

The ﬁnal ligand, TREN-bisGlyGlu-CAM (4), was synthe-

sized according to Scheme 3. In a ﬁrst step, the benzyl-

protected podand GlyCAM(Bn) (14) was synthesized in a

Advantageously, unlike formation constants (K ), pM values

enable direct comparison of the complexation behavior of

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Article

ligands that diﬀer in basicity, protonation state, or even metal

ion:ligand stoichiometry. The larger the pGa value, the less

free (uncomplexed) Ga is present in solution, and the more

stable the Ga complex.

pM values can be calculated from the association constants

of the metal complex and the protonation steps of both the

free ligand and the metal complex. They can also be directly

evaluated at a given pH by competition with a well-

characterized ligand, without prior determination of the

6,57

protonation constants.

DFO is a ligand whose Ga

complex is well characterized, which undergoes ligand

exchange and reaches thermodynamic equilibrium rapidly,

and which has previously been investigated for gallium-68 PET

imaging; it is thus ideal for determining the stability of the

catechol-based ligands 1−4. Competition titrations were

56,57

performed as previously reported.

Ligand exchange was

monitored by ultraviolet−visible spectroscopy after equilibra-

tion at 25 °C for 24 h. The results are shown in Figure 3. The

concentration of competing DFO necessary to generate an

equal partition of Ga³between the two ligands [log([DFO]/

[

L]) when log([Ga-DFO]/[Ga-L]) = 0] directly gives the

diﬀerence in the pM value between the ligand of interest and

DFO. Because the pGa for Ga-DFO is known (21.2 at pH 7.4

and 25 °C), the pGa values of ligands 1−4 at the same pH

and temperature can be directly calculated. It is noteworthy

that because pM values are independent of the isotope of the

metal ion, these experiments were performed with the naturally

occurring and nonradioactive Ga isotope.

The results of the competition experiments are listed in

Table 1. Notably, TREN-CAM forms such a stable Ga

complex that even a 1000-fold excess of DFO did not result

in enough ligand exchange to be accurately measured. As such,

a precise pGa value for Ga-TREN-CAM could not be

determined, although a lower limit of 24, 3 units higher that

of Ga-DFO (pGa = 21.2), can be conservatively estimated. Ga-

TREN-CAM is thus one of the most stable Ga^I^I^Icomplexes

evaluated for PET imaging. It is at least 1 and 6 orders of

magnitude more stable than TRAP (pGa = 23.1) and DOTA

(

pGa = 18.5), respectively.

The carboxylate-bearing ligands 2,2-Glu-CAM (2), 3,3-Glu-

CAM (3), and TREN-bisGlyGlu-CAM (4) all form less stable

III

Ga complexes than the parent TREN-CAM ligand. The

stabilities of these three functionalizable complexes span nearly

orders of magnitude, with 2,2-Glu-CAM (2), which has the

smallest ligand cap, being the most stable and TREN-

bisGlyGlu-CAM (4), which has the largest ligand cap, being

the least stable. A smaller cap is therefore preferred for the

Ga ion. The decrease in stability compared to that of the

parent Ga-TREN-CAM could be due to a number of

parameters. The tight hydrogen-bonding network of the

ligand’s TREN backbone that predisposes the ligand TREN-

CAM and increases the stability of its metal complex is

signiﬁcantly altered in the other three ligand caps. Previous

work by our group and others on lanthanide and iron

complexes previously reported such a decrease in the pM value

5,59−61

upon modiﬁcation of the TREN backbone.

Complex

III

Figure 3. Competition of Ga complexes with DFO: (a) spectra of

charge is also known to aﬀect the stability of d metal ions that

III

III 57,62

Ga -TREN-CAM upon addition of increasing concentrations of

lack ligand ﬁeld stabilization energy such as Ga .

The

III

DFO, (b) spectra of Ga -2,2-Glu-CAM upon addition of increasing

more negative the charge, the less stable the coordination

complex. The pendant carboxylates of ligands 2−4, which are

deprotonated at neutral pH, thus likely also contribute to the

decrease in the pGa value. Nonetheless, these ligands all form

complexes that are more stable than many other ligands used

concentrations of DFO, and (c) competition titrations of

functionalizable ligands 2−4 against DFO. The x intercepts indicate

the diﬀerence in pGa between each catechol ligand and the competing

DFO. Experimental conditions: 0.01 M TRIS buﬀer, pH 7.4, 0.1 M

KCl, 25 °C.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

[ Ga]GaCl used. As shown in Figure 4 for the best chelator,

Article

Table 1. pGa Values of Tris-catecholate Ga^I^I^IComplexes

Table 2. Radiolabeling Conditions and Yields of Ligands

ligand

pGa − pGa

pGa

complex

yield (%)

DFO

TREN-CAM (1)

,2-Glu-CAM (2)

,3-Glu-CAM (3)

>24

22.8

22.2

21.1

21.2

TREN-CAM (1, n = 10)

2,2-Glu-CAM (2, n = 2)

3,3-Glu-CAM (3, n = 3)

94 ± 6

91 ± 4

83 ± 6

90 ± 1

96 ± 1

92 ± 1

1.60

1.03

−0.14

TREN-bisGlyGlu-CAM (4)

TREN-bisGlyGlu-CAM (4, n = 2)

DFO

THP

Experimental conditions: 0.01 M TRIS(aq), pH 7.4, 0.1 M KCl, 25

C ([Ga ] = 1 μM, [DFO] = 10 μM, pH 7.4, and 25 °C). From ref

HBED

Labeling conditions for this work: T = 25 °C, pH 8.0−9.0, t = 10

min. Reaction volumes were 142 μL. The ligand concentrations used

for radiolabeling were TRENCAM (213 μM), 2,2-Glu-CAM (185

μM), 3,3-Glu-CAM (177 μM), and TREN-bisGlyGlu-CAM (185

clinically for gallium-68 PET imaging, including DOTA.

Additionally, ligands 1−4 have a higher aﬃnity for Ga than

μM). Labeling conditions from ref 58: 500 μM, T = 25 °C, pH 6.5, t

transferrin (pGa = 19.7), a serum protein that is well-known

for its capability to sequester Ga³ions, thereby leading to

= 10 min.

dechelation of weak gallium-68 agents in vivo. While this is

promising for in vivo use of ligands 1−4, it is important to note

that blood is a complex matrix that also contains other

endogenous chelating species that could also lead to

decomplexation of the gallium-68 tracers.

Gallium-68 Radiolabeling Yields. Though the thermo-

dynamic stability of the Ga complexes is important, it is only

one of the parameters that determines the viability of a

chelator for PET imaging. Practically, a chelate must also be

able to complex the radionuclide rapidly. Gallium-68, like

t₁_/₂= 110 min), has a limited half-life (t₁_/₂= 68 min), and

thus, radiolabeling must be achieved eﬃciently and quickly,

ideally in <10 min. This short reaction time does not

necessarily enable the system to reach thermodynamic

equilibrium. Although thermodynamic values such as pGa

give a good indication of the stability of a complex in complex

biological media, they do not necessarily guarantee a high

labeling yield if kinetics of complexation are slower than the

reaction time.

high temperatures (90 °C) and acidic pH to reach such

radiolabeling levels. The catechol ligands 1−4 also compare

positively to other acyclic chelators that are also labile and thus

chelate gallium rapidly. TREN-CAM (1), 2,2-Glu-CAM (2),

and TREN-bisGlyGlu-CAM (4) all exhibit radiolabeling yields

≥90%, values that are typical of the more favored rapid

III

chelators HBED, DFO, and THP (all ≥92%).

From Le Chat

III

elier’s principle, the formation of the Ga

complex is favored under concentrated conditions and

disfavored under diluted ones. The radiolabeling yield is thus

highly dependent on the concentration of ligand and

(

Radiochemical labeling yields were determined for the four

catechol-based ligands as depicted in Scheme 4. All experi-

Scheme 4. Radiolabeling of TREN-CAM with [ Ga]GaCl

Figure 4. Eﬀect of TREN-CAM concentration on radiolabeling yield

ments were performed under the same short reaction time, 10

min, at room temperature under slightly basic conditions.

with [ Ga]GaCl .

[

Ga]GaCl₃was produced via zinc-68 (p,n) gallium-68

nuclear reaction using a cyclotron in a liquid target and

TREN-CAM (1), the radiolabeling yield remains high but

decreases sharply if the ligand concentration is <0.5 mM. This

is a minimum ligand concentration that is slightly higher than

those observed for DOTA, NOTA, and HBED, although

gallium labeling with those ligands is typically performed under

66,67

puriﬁed using hydroxamate resin as reported previously.

The results, given in Table 2, indicate that the ligand that

III

forms the most stable Ga complex, TREN-CAM (1), also

exhibits the highest radiolabeling yield, coordinating [ Ga]-

Ga almost quantitatively in 10 min at room temperature. The

three functionalizable catechol-based ligands 2−4 also enable

high radiolabeling yields of >80% under short reaction times at

room temperature. Though not quantitative, the measured

radiolabeling levels are superior to the radiochemical yields

reported for some DOTA bioconjugates such as DOTA-TOC

and DOTA-NOC. However, the macrocyclic DOTA-based

ligands, unlike the catecholates 1−4, need to be subjected to

acidic conditions.

Stability in Human Serum. The in vitro stability of

radiolabeled ligands 1−4 after incubation for ≤2 h in freshly

harvested human serum at 37 °C was evaluated by iTLC. The

results, shown in Figure 5, indicate that although the gallium-

68 complexes of TREN-CAM ([ Ga]Ga-1) and TREN-

bisGlyGlu-CAM ([ Ga]Ga-4) were remarkably stable in

human serum, almost no decomplexation was observed over

Inorg. Chem. XXXX, XXX, XXX−XXX

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Article

Figure 5. Stability of [ Ga]Ga-TREN-CAM (1), [ Ga]Ga-2,2-

GluCAM (2), and [ Ga]Ga-TREN-bisGlyGlu-CAM (4) in human

serum at 37 °C. Error bars represent ±1 standard deviation (n = 3).

Figure 6. Representative PET images showing the distribution of (a)

[

Ga]GaCl and (b) [ Ga]Ga-TREN-CAM (1) 15, 30, 60, and 120

[

h (approximately twice the half-life of Ga). However, both

min postadministration with [ Ga]Ga-TREN-CAM activity primarily

in the bladder, gut, and kidneys. PET images were normalized to units

of SUV and presented as maximum intensity projection scan (MIPS)

images. The anatomic reference skeleton images are formed by using

the mouse atlas registration system algorithm with information

obtained from the stationary top-view planar X-ray projector and side-

view optical camera. Abbreviations: GU, gut; KI, kidney; BL, bladder.

Ga]Ga-2,2-Glu-CAM ([ Ga]Ga-2) and [ Ga]Ga-3,3-Glu-

CAM ([ Ga]Ga-3) decomplexed almost immediately in

serum. Representative iTLC traces of the stability measure-

ments are shown in Figure S54 . Interestingly, there does not

appear to be a straightforward relationship between the

III

thermodynamic stability of the Ga complex in aqueous

buﬀer, as determined by pGa, and the stability of the complex

in serum. Indeed, Ga-2,2-GluCAM and Ga-3,3-GluCAM are

both more stable than Ga-DFO; yet, both of those former

complexes rapidly leach out Ga³in serum, whereas Ga-DFO

does not. On the other hand, Ga-TREN-bisGlyGlu-CAM,

whose stability is comparable to that of Ga-DFO, remains

remarkably stable in serum. The source of this discrepancy has

not been determined but could potentially be due to the faster

kinetics of transmetalation with the less rigid 2,2-GluCAM and

the in vivo biodistribution proﬁle observed depicts mainly what

can be considered as short-term high stability. This does not

necessarily guarantee that (bio)macromolecular derivatives of

those complexes, which would stay in circulation for longer

periods of time, would remain intact in vivo.

The ex vivo biodistribution of [ Ga]Ga-TREN-CAM was

evaluated after PET imaging on organ tissues harvested 2 h

postinjection. The results, shown in Figure 7, demonstrate that

,3-GluCAM ligands. The TREN ligand cap of TREN-CAM

and TREN-bisGlyGluCAM appears to be necessary for

maintaining the stability of the gallium-68 complex in serum.

Gallium-68 PET Imaging Studies. Given its high

thermodynamic stability (high pGa values), its quantitative

radiochemical yield, and its excellent stability in human serum,

the Ga radiotracer is not exclusively cleared via renal routes.

Indeed, there is a signiﬁcant presence of the radiotracer in the

upper gut (6.3 ± 0.8% ID/g of tissue), liver (2.0 ± 0.3% ID/g

of tissue), and especially cecum feces (108.7 ± 8.9% ID/g of

tissue). These results suggest that at least some [ Ga]Ga-

TREN-CAM also clears through the gastrointestinal tract via

[

Ga]Ga-TREN-CAM was selected for further PET imaging

studies in vivo. Dynamic PET/X-ray imaging studies were

carried out in mice (n = 3 female) to evaluate the

pharmacokinetics and biodistribution of the radiotracer. The

hepatobiliary excretion routes. Importantly, though, and as

needed for further targeted imaging, the ex vivo biodistribution

study indicates that after 120 min, the radiotracer does not

accumulate signiﬁcantly in the organs or tissues not involved in

clearance.

data shown in Figure 6b strongly suggest that Ga-radio-

labeled TREN-CAM undergoes rapid renal clearance. Fifteen

minutes postinjection, the radionuclide was distributed

primarily to the bladder, with some gallium-68 localized in

the gut and kidneys, in accordance with high urinary excretion.

Importantly, the PET images show a discrete distribution of

Biodistribution and Pharmacokinetics of Ga-TREN-

CAM. Biodistribution and pharmacokinetic analysis of Ga-

TREN-CAM was also carried out at higher concentrations with

the nonradioactive natural isotope of gallium. This study was

performed to determine if the injected dose of the metal

complex and the use of a native versus a radioactive isotope

aﬀects the biodistribution and pharmacokinetic behavior of the

[

Ga]Ga-TREN-CAM into speciﬁc soft tissues, with little to

no retention in other organs such as bones. Unchelated

[

Ga]GaCl has a very diﬀerent biodistribution proﬁle (Figure

III

a), with accumulation in heart tissue. The vastly diﬀerent

probe. Ga -TREN-CAM was injected intravenously in the tail

biodistributions of [ Ga]Ga-TREN-CAM and [ Ga]GaCl

vein of mice (n = 3 female mice). In this case, the distribution

III

suggest that TREN-CAM does not release its gallium-68

of the Ga complex was evaluated ex vivo in blood and organ

radionuclide in vivo, in agreement with the high stability in

by inductively coupled plasma mass spectrometry (ICP-MS).

As one can see from Figure 8, the biodistribution of Ga -

TREN-CAM injected at large doses and monitored by

10,35

III

serum determined above.

It is important to highlight that

given the rapid clearance exhibited by [ Ga]Ga-TREN-CAM,

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

except for cecum feces and bladder where n = 2). % ID/g of organ is the percentage of injected dose per gram of organ weight.

Article

Figure 7. Ex vivo biodistribution pattern of [ Ga]Ga-TREN-CAM 120 min postinjection. Error bars represent ±1 standard deviation (n = 3)

Figure 8. Ex vivo biodistribution of Ga-TREN-CAM in mice. Error

bars represent ±1 standard deviation (n = 3).

elemental analysis follows closely that of the prior experiment

by measuring the radioactivity levels of organs. In particular,

the large dose of Ga observed in the kidney 2 h postinjection

conﬁrms that Ga-TREN-CAM is primarily cleared renally.

The curve of the postinjection concentration of Ga in blood

versus time shown in Figure 9 is typical of the pharmacoki-

netics of most xenobiotic agents. Two phases are readily

distinguishable, an early rapid declining phase followed by a

moderately declining period. During the ﬁrst phase (between

time of dosing and 2 h), also known as the distribution phase, a

rapid decrease in Ga concentration in plasma occurs as it is

being distributed from the central compartment (circulation)

into the peripheral compartments (body tissues). This phase

ends when an equilibrium of Ga concentration is established

between the central and peripheral compartments. The second

phase is a distinctive elimination period (from 2 h onward) in

which there is a more gradual decrease in plasma concentration

_F_i_g_u_r_e₉_._B_l_o_o_d_c_o_n_c_e_n_t_r_a_t_i_o_n_o_fnat_G_a_a_f_t_e_r_i_n_j_e_c_t_i_o_n_w_i_t_hnatGa-

TREN-CAM in mice. Error bars represent ±1 standard deviation (n =

3).

temperature and non-acidic pH. These rapid kinetics, which

are attributed to the acyclic nature of the ligands, give them a

distinct advantage over macrocyclic ligands such as DOTA that

require high temperatures, acidic conditions, and long reaction

times to achieve suﬃcient radiolabeling. Addition of a

peripheral carboxylate group for further conjugation to a

targeting moiety can be achieved via modiﬁcation of the ligand

cap and incorporation of a glutamic acid residue. It is

noteworthy that the modular nature of the synthesis enables

facile incorporation of amine or thiol peripheral groups in lieu

of the carboxylate one if so desired for further bioconjugation.

Unfortunately, addition of the amino acid in the ligand

backbone aﬀects the tight hydrogen-bonding network of the

ligand cap. As a result, even though 2,2-Glu-CAM, 3,3-Glu-

as the Ga chelator is excreted from the organism.

CONCLUSION

Tripodal tris-catecholate ligands enable rapid radiolabeling of

gallium-68 in short periods of time (<10 min) at room

III

■

CAM, and TREN-bisGlyGlu-CAM still bind Ga rapidly at

III

room temperature, their corresponding Ga complex is less

stable than Ga-TREN-CAM. Nonetheless, these complexes

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

remain at least as stable as those of common acyclic gallium

chelators evaluated for PET, such as Ga-DFO. Interestingly,

trends of stability in buﬀer do not correspond to those

observed in human serum. Of the four gallium-68 complexes

by ﬂash chromatography over silica eluting with 5% CH

OH/95%

CH Cl to yield TREN-CAM(Bn) (6) as a light-yellow oil (0.57 g,

0%). H NMR (400 MHz, CDCl ): δ 7.80 (t, J = 6 Hz, 3H), 7.62−

.61 (m, 3H), 7.42−7.40 (m, 6H), 7.37−7.32 (m, 9H), 7.27−7.21

(m, 15H), 7.07−7.06 (m, 6H), 5.07 (s, 6H), 5.02 (s, 6H), 3.15 (q, J =

evaluated, two, [ Ga]Ga-TREN-CAM and [ Ga]Ga-TREN-

bisGlyGluCAM, were highly stable in human serum, with

nearly no dechelation observed over 2 h. Importantly, PET

imaging analysis in healthy mice showed that Ga-TREN-CAM

displays high stability in vivo, in agreement with the relative

conditional stability observed in vitro. In vivo PET studies in

Hz, 6H), 2.32 (t, J = 7 Hz, 6H). C NMR (100 MHz, CDCl ): δ

65.2, 151.7, 146.7, 136.6, 136.5, 128.6 (×3), 128.2, 127.8, 127.6,

24.3, 123.1, 116.8, 76.2, 71.2, 52.5, 37.3. ESI-HR-MS. Calcd for

C H N O [M + H] : m/z 1095.4908. Found: m/z 1095.4971.

TREN-CAM (1). TREN-CAM(Bn) (6, 54 mg, 49 μmol) was

dissolved in a mixture of glacial acetic acid and 12 N HCl [1:1 (v/v),

mL] and stirred at room temperature under N (g) for 24 h. The

mice and ex vivo biodistribution analyses of [ Ga]Ga-TREN-

CAM suggest that, after tail vein injection, the radiotracer is

cleared rapidly from mice (<15 min) primarily via renal routes.

The high stability of Ga-TREN-bisGlyGlu-CAM in serum for

extended periods of time combined with the high radiolabeling

yields obtained after short reaction times at room temperature

and the presence of a functional group for further

bioconjugation to targeting group renders TREN-bisGlyGlu-

CAM a promising target for gallium radiopharmaceuticals.

volatiles were then removed under reduced pressure to yield the HCl

salt of the free ligand TREN-CAM as an oﬀ-white solid. (1, 32 mg,

quant. yield). H NMR (400 MHz, CD OD): δ 7.13 (dd, J = 8 Hz, J

2 Hz, 3H), 6.90 (dd, J = 8 Hz, J = 2 Hz, 3H), 6.63 (t, J = 8 Hz, 3H),

3.85 (t, J = 6 Hz, 6H), 3.67 (t, J = 6 Hz, 6H). C NMR (100 MHz,

CD OD): δ 172.7, 149.7, 147.1, 120.0, 119.9, 119.6, 116.4, 55.4, 36.0.

ESI-HR-MS. Calcd for C H N O [M + H] : m/z 555.2091.

Found: m/z 555.2062.

Ga-TREN-CAM (Ga-1). The deprotected ligand 1 (125 mg, 225

μmol) and KOH (37.8 mg, 674 μmol) were ﬁrst mixed in methanol

(

4 mL). Next, Ga(acac) (82.5 mg, 225 μmol) was added, and the

EXPERIMENTAL SECTION

■

resulting crude stirred overnight at room temperature. The product

was precipitated with diethyl ether (10 mL), separated oﬀ, and dried

under reduced pressure to yield Ga-TREN-CAM as a beige powder

(Ga-1, 158 mg, 95%). H NMR (400 MHz, DMSO-d

3H), 6.78 (d, J = 8.1 Hz, 3H), 6.44 (d, J = 7.2 Hz, 3H), 6.10 (t, J = 7.7

Hz, 3H), 3.37 (s, 6H), 2.33 (s, 6H). C NMR (100 MHz, DMSO-

d ): δ 168.2, 158.7, 155.9, 114.6, 114.1, 112.0, 111.7, 54.8, 34.3. ESI-

HR-MS. Calcd for C27H26GaN O [M + 2H] : m/z 619.0956.

4 9

General Considerations. Unless otherwise stated, all reagents

were purchased from commercial sources and used without further

puriﬁcation. Flash chromatography was performed on silica gel

SiliFlash F60 40−63 μm. Distilled water was further puriﬁed by a

): δ 11.12 (s,

Millipore Simplicity UV system (resistivity of 18 × 10 Ω).

Deuterated solvents were obtained from Cambridge Isotope

−

Laboratories (Tewskbury, MA). H and C NMR spectra were

recorded at room temperature on a Bruker Advance III 400

spectrometer at 400 and 100 MHz, respectively, at the LeClaire-

Dow Characterization Facility of the Department of Chemistry at the

University of Minnesota. Residual solvent peaks were used as the

Found: m/z 619.0972.

2,2-CAM(Bn) (8). CAM(Bn)thiaz (7, 1.32 g, 3.02 mmol) and

diethylenetriamine (122 μL, 1.13 mmol) were dissolved in anhydrous

dichloromethane (75 mL). The resulting mixture was stirred at room

temperature under N

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash chromatography over silica eluting with 5% CH OH/95%

CH Cl to yield intermediate 2,2-CAM(Bn) (8, 0.48 g, 58%) as a

colorless oil. H NMR (400 MHz, CDCl ): δ 8.05 (t, J = 6 Hz, 2H),

7.66 (t, J = 4 Hz, 2H), 7.40−7.39 (m, 4H), 7.35−7.25 (m, 16H), 7.06

(d, J = 5 Hz, 4H), 5.06 (s, 4H), 5.03 (s, 4H), 3.26 (q, J = 6 Hz, 4H),

internal reference. H NMR data are reported as follows: chemical

(g) for 48 h. The reaction mixture was then

shifts (δ, ppm), multiplicity (s, singlet; d, doublet; t, triplet; m,

multiplet; br, broad), coupling constants (Hz), and integration.

NMR data are reported as chemical shifts (δ, ppm). Mass spectra

were recorded on a Bruker BioTOF II ESI/TOF-MS instrument at

the Waters Center for Innovation in Mass Spectrometry of the

Department of Chemistry at the University of Minnesota. Ultra-

violet−visible spectra were recorded on a Varian Cary 100 Bio

Spectrometer with 1 cm quartz cuvettes in TRIS(aq) buﬀer (0.01 M)

and KCl (0.1 M) (pH 7.4). Semipreparative high-performance liquid

chromatography (HPLC) was performed with a Varian Prostar 210

HPLC instrument (Agilent, Santa Clara, CA) equipped with a Varian

ProStar 335 diode array detector and an Agilent Zorbax Eclipse XDB-

C18 column (5 μm pore size, 9.4 mm × 250 mm). Unless speciﬁed

otherwise, HPLC measurements were performed at a ﬂow rate of 1.0

2.50 (t, J = 6 Hz, 4H). C NMR (100 MHz, CDCl ): δ 165.5, 151.8,

146.8, 136.6, 136.5, 128.7 (×2), 128.6 (×2), 128.2, 127.7, 127.6,

124.4, 123.1, 116.9, 76.3, 71.2, 53.6, 48.1, 39.3. ESI-LR-MS. Calcd for

C H N O [M + H] : m/z 736.34. Found: m/z 736.44.

2,2-Glu( Bu)-CAM(Bn) (11). DIPEA (198 μL, 0.114 mmol)

followed by TBTU (0.28 g, 0.87 mmol) were added to an ice-cooled

solution of 2,2-CAM(Bn) (8, 0.49 g 0.67 mmol) and Glu( Bu)CAM-

(Bn) (10, 0.43 g, 0.84 mmol) in anhydrous dichloromethane (15 mL)

under N (g). The resulting mixture was stirred at room temperature

−

mL min with the following elution condition: 15% CH CN/85%

water from 0 to 2 min, followed by a linear gradient to 100% CH CN

for 24 h and then concentrated under reduced pressure. The resulting

oil was then suspended in ethyl acetate (100 mL) and washed

from 2 to 23 min, and 15% CH CN/85% water from 30 to 32 min.

All pH measurements were performed using a Thermo Scientiﬁc Ag/

AgCl reﬁllable probe and a Thermo Orion 3 Benchtop pH meter.

successively with saturated NaHCO (aq) (5 × 50 mL) and saturated

NaCl(aq) (2 × 50 mL). The organic layer was dried over Na SO (s),

Synthesis and Characterization. CAM(Bn) (5), CAM(Bn)-

ﬁltered, and concentrated under reduced pressure. The crude product

was puriﬁed by ﬂash chromatography over silica, eluting with 40%

thiaz (7), Glu( Bu)CAM(Bn) (10), and GlyCAM(Bn) (14)

were prepared according to literature procedures with successful

ethyl acetate/60% hexanes to yield 2,2-Glu( Bu)-CAM(Bn) as a

synthesis established by LR MS and H NMR spectroscopy.

colorless oil (11, 0.58 g, 71%). H NMR (400 MHz, CDCl ): δ 8.57

TREN-CAM(Bn) (6). To an ice-cooled solution of CAM(Bn) acid

5, 1.04 g, 3.10 mmol) and tris(2-aminoethyl)amine (TREN, 151 mg,

(d, J = 8 Hz, 1H), 8.13 (s, 1H), 8.02 (t, J = 6 Hz, 1H), 7.65−7.57 (m,

3H), 7.45−7.17 (m, 31H), 7.07−7.01 (m, 6H), 5.18−4.98 (m, 13H),

3.66−3.21 (m, 8H), 2.24−2.08 (m, 2H), 1.95−1.90 (m, 1H), 1.62−

(

.03 mmol) in anhydrous dichloromethane (30 mL) under N (g) was

added diisopropylethylamine (DIPEA, 632 μL, 3.63 mmol) followed

by TBTU (1.09 g, 3.40 mmol). The resulting mixture was stirred at

room temperature for 24 h. The reaction mixture was concentrated

under reduced pressure. The resulting oil was resuspended in ethyl

acetate (100 mL) and washed successively with saturated

1.52 (m, 1H), 1.37 (s, 9H). C NMR (100 MHz, CDCl ): δ 172.6,

172.0, 166.0, 165.7, 164.9, 151.9, 151.8, 151.7, 147.0, 146.8 (×2),

136.6 (×3), 136.4 (×2), 129.3, 129.0, 128.8, 128.7 (×2), 128.6, 128.4

(×2), 128.3 (×2), 128.0, 127.9, 127.8, 127.7 (×2), 127.0, 124.3 (×2),

124.2, 123.3, 123.1 (×2), 117.2, 117.1, 117.0, 80.4, 76.4, 76.2, 76.1,

71.4, 71.2 (×2), 49.0, 47.0, 45.8, 38.5, 37.8, 31.2, 28.2, 28.0. ESI-LR-

NaHCO (aq) (5 × 50 mL) and saturated NaCl(aq) (2 × 50 mL).

The organic layer was dried over Na SO (s), ﬁltered, and

MS. Calcd for C H N O [M + H] : m/z 1237.55. Found: m/z

76 77

concentrated under reduced pressure. The crude product was puriﬁed

1237.57.

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

,2-Glu-CAM (2). 2,2-Glu( Bu)-CAM(Bn) (11, 240 mg, 194

μmol) was dissolved in a mixture of glacial acetic acid and 12 N HCl

1:1 (v/v), 12 mL] and stirred at room temperature under N (g) for

(t, J = 7 Hz, 4H), 2.46 (t, J = 7 Hz, 2H), 2.30−2.28 (m, 1H), 2.11−

2.00 (m, 5H). C NMR (100 MHz, CD OD): δ 173.6, 173.4, 172.1,

[

171.2, 150.1, 149.9, 147.3, 147.2, 119.9, 119.8 (×2), 119.7, 119.3,

0 h. The volatiles were removed under reduced pressure to yield the

free ligand (2) as an oﬀ-white solid (98.0 mg, 80%). H NMR (400

118.9, 116.6, 116.5, 53.2, 46.7, 37.1, 31.1, 27.6, 27.4. ESI-HR-MS.

−

Calcd for C H N O [M − H] : m/z 667.2251. Found: m/z

MHz, DMF-d ): δ 9.15 (s, 1H), 8.93 (s, 1H), 7.50−7.24 (m, 4H),

667.2248.

.02−6.95 (m, 3H), 6.76−6.67 (m, 3H), 4.62 (dd, J = 9 Hz, J = 6

Ga-3,3-Glu-CAM (Ga-3). The deprotected ligand Ga-3,3-Glu-

CAM (3, 59.4 mg, 88.8 μmol) was ﬁrst dissolved in methanol (3 mL),

followed by the addition of KOH (15.0 mg, 267 μmol). Next,

Hz, 1H), 3.66 (br s, 4H), 3.16 (br s, 4H), 2.52 (t, J = 8 Hz, 2H),

.31−2.25 (m, 1H), 2.19−2.15 (m, 1H). C NMR (100 MHz,

CD OD): δ 173.6, 173.4, 172.8, 171.2, 150.2, 149.9, 147.3, 147.2,

Ga(acac) (32.6 mg, 88.8 μmol) was added, and the resulting mixture

20.0, 119.9 (×2), 119.8, 119.3, 119.1, 116.6, 116.3, 53.2, 49.6, 37.4,

stirred at room temperature for 18 h. The desired product was

precipitated oﬀ by addition of diethyl ether (9 mL). The white solid

−

1.1, 27.4. ESI-HR-MS. Calcd for C H N O [M − H] : m/z

39.1938. Found: m/z 639.1963.

Ga-2,2-Glu-CAM (Ga-2). 2,2-Glu-CAM (2, 114 mg, 0.179 mmol)

was then separated oﬀ and dried under reduced pressure (Ga-3, 58.2

mg, 78%). H NMR (400 MHz, DMF-d ): δ 10.28−10.55 (m, 2H),

and KOH (30 mg, 0.53 mmol) were mixed in methanol (5 mL). Once

everything was dissolved, Ga(acac ) (65.4 mg, 0.178 mmol) was

added. The resulting mixture was stirred at room temperature

overnight. After this time, the compound was precipitated oﬀ by

addition of diethyl ether (9 mL), separated oﬀ, and dried under

7.28−6.99 (m, 4H), 6.79−6.63 (m, 3H), 6.25−6.29 (m, 3H), 4.60−

4.70 (m, 1H), 3.78−3.78 (m, 8H + ether), 2.44 (br, 2H), 2.16−1.98

(m, 6H + acetic acid/acetate). ESI-HR-MS. Calcd for

−

C H GaN O [M + 2H] : m/z 733.1274. Found: m/z 733.1224.

TREN-Glu( Bu)-CAM(Bn) (13). N-Hydroxysuccinimide (98 mg,

reduced pressure to yield Ga-2,2-Glu-CAM as an oﬀ-white solid (Ga-

0.85 mmol) was added to a stirred solution of Glu( Bu)CAM(Bn)

(10, 0.37 g, 0.71 mmol) in anhydrous 1,4-dioxane (10 mL), and the

resulting reaction mixture was cooled to 0 °C. N-Ethyl-N′-(3-

dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 163

mg, 850 μmol) was added to the reaction mixture that was then

further stirred at room temperature for 24 h. The resulting active ester

was further added to a prestirred solution of tris(2-aminoethyl)amine

(TREN, 603 mg, 4.13 mmol) and DIPEA (2.3 mL, 13 mmol) in

anhydrous 1,4-dioxane (5 mL). The reaction mixture was stirred at

room temperature for 30 h and then concentrated under reduced

pressure. The crude product was dissolved in dichloromethane (50

mL) and washed with water (2 × 20 mL). The organic layer was dried

, 116.4 mg, 80%). H NMR (400 MHz, DMSO-d ): δ 10.56 (s, 1H),

.31−7.13 (m, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.49 (d, J = 7.4 Hz,

H), 6.26 (t, J = 7.7 Hz, 1H), 4.34 (s, 1H), ∼3.44 (4H) signal

overlapped by a water peak, 2.71 (s, 4H), 2.36−2.19 (m, 2H), 2.00−

−

.89 (m, 2H). ESI-LR-MS. Calcd for C H GaN O [M + 2H] : m/

30 28 4 12

z 705.10. Found: m/z 705.20.

,3-CAM(Bn) (9). The bis-amide 3,3-CAM(Bn) (9) was prepared

according to the literature procedure reported by Allred et al. and

puriﬁed by ﬂash chromatography over silica eluting with 10%

CH OH/90% CH Cl to yield 9 as a white powder (0.49 g, 82%).

H NMR (400 MHz, CDCl ): δ 8.10 (m, 2H), 7.63 (dd, J = 7 Hz, J

3 Hz, 2H), 7.43−7.41 (m, 4H), 7.36−7.29 (m, 16H), 7.08−7.06

with MgSO (s). The volatiles were then removed under reduced

(m, 4H), 5.08 (s, 4H), 5.05 (s, 4H), 4.74 (br s, 1H), 3.32−3.30 (m,

pressure to yield TREN-Glu( Bu)-CAM(Bn) (13) as a yellow paste

H), 2.50−2.46 (m, 4H), 1.63−1.60 (m, 4H). C NMR (100 MHz,

(0.42 g, 91%) that was used immediately in the next step without

CDCl ): δ 165.8, 151.7, 146.7, 136.5, 136.4, 128.8, 128.7 (×2), 128.3,

further puriﬁcation. H NMR (400 MHz, CDCl

): δ 8.46 (d, J = 8

27.7, 127.5, 124.4, 123.0, 117.0, 76.4, 71.2, 50.2, 46.6, 37.2, 28.5.

Hz, 1H), 8.07 (br, 1H), 7.57 (dd, J

= 7 Hz, J

= 3 Hz, 1H), 7.43−

Calcd for C H N O [M + H] : m/z 764.37. Found: m/z 764.38.

7.26 (m, 7H), 7.24−7.16 (m, 3H), 7.11−6.97 (m, 2H), 5.17−4.91

(m, 4H), 4.77−4.39 (m, 1H), 2.75−2.38 (m, 12H), 2.20−2.09 (m,

2H), 2.03−1.90 (m, 1H), 1.72−1.51 (m, 1H), 1.33 (m, 9H). ESI-LR-

,3-Glu( Bu)-CAM(Bn) (12). DIPEA (192 μL, 1.10 mmol)

followed by TBTU (0.271 g, 844 μmol) were added to a solution

of 3,3-CAM(Bn) (9, 0.49 g, 0.65 mmol) and Glu( Bu)CAM(Bn) (10,

−

MS. Calcd for C36

646.29.

[M − H] : m/z 646.36. Found: m/z

.46 g, 0.88 mmol) in anhydrous dichloromethane (16 mL) cooled to

°C under N (g). The resulting mixture was stirred at room

TREN-bisGlyGlu( Bu)-CAM(Bn) (15). To a stirred solution of

GlyCAM(Bn) (14, 0.27 g, 0.69 mmol) in anhydrous dichloromethane

(10 mL) was added N-hydroxysuccinimide (NHS, 0.106 g, 0.921

mmol), and the resulting reaction mixture was cooled to 0 °C. EDC

(0.173 g, 0.902 mmol) was added, and the reaction mixture was

stirred at room temperature for 24 h. The resulting active ester was

temperature for 24 h. The reaction mixture was concentrated under

reduced pressure. The resulting oil was then resuspended in ethyl

acetate (100 mL) and washed successively with saturated

NaHCO (aq) (5 × 50 mL) and saturated NaCl(aq) (2 × 50 mL).

The organic layer was dried over Na SO (s), ﬁltered, and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash chromatography over silica, eluting with a gradient of 100%

hexanes to 100% ethyl acetate to yield the protected ligand 3,3-

then added to a prestirred solution of TREN-Glu( Bu)-CAM(Bn)

(13, 0.15 g, 0.23 mmol) and DIPEA (620 μL, 3.47 mmol) in

dichloromethane (5 mL). The reaction mixture was stirred at room

temperature for 30 h and then concentrated under reduced pressure.

The crude product was dissolved in dichloromethane (60 mL) and

washed with water (2 × 30 mL). The organic layer was dried with

Glu( Bu)-CAM(Bn) as a colorless oil (12, 0.55 g, 67%). H NMR

(

400 MHz, CDCl ): δ 8.54 (d, J = 8 Hz, 1H), 8.04−8.00 (m, 2H),

.69−7.61 (m, 3H), 7.41−7.28 (m, 24H), 7.22−7.16 (m, 6H), 7.08−

.02 (m, 6H), 5.19 (m, 1H), 5.09−4.99 (m, 12H), 3.47−2.98 (m,

H), 2.27−2.10 (m, 2H), 1.97−1.89 (m, 1H), 1.77−1.68 (m, 2H),

MgSO (s), and the solvent removed under reduced pressure. The

crude product was puriﬁed by ﬂash chromatography over silica eluting

.61−1.50 (m, 3H), 1.40 (s, 9H). C NMR (100 MHz, CDCl ): δ

with 5% CH OH/95% CH Cl to yield TREN-bisGlyGlu( Bu)-

72.0, 171.8, 165.6, 165.4, 165.0, 151.9, 151.8 (×2), 147.0, 146.8,

46.7, 136.7, 136.6 (×3), 136.4, 136.3, 129.2, 128.9, 128.8 (×2),

28.7 (×4), 128.6, 128.5, 128.4, 128.3 (×2), 128.2, 127.9 (×2), 127.7,

27.6, 127.1, 124.4, 124.3, 124.2 (×2), 123.0 (×2), 117.3, 117.0,

16.8, 80.5, 76.4, 76.2, 76.1, 71.3 (×2), 71.2, 53.7, 48.7, 45.3, 43.5,

CAM(Bn) as a yellow oil (13, 0.32 g, 40%). H NMR (400 MHz,

CDCl ): δ 8.64−8.56 (m, 3H), 7.75−7.60 (m, 6H), 7.48−7.31 (m,

30H), 7.10 (b, 6H), 5.19−5.09 (m, 12H), 4.59−4.52 (m, 1H), 4.02−

3.88 (m, 4H), 3.50−3.01 (m, 6H), 2.67−2.47 (m, 6H), 2.25−2.18

(m, 2H), 2.03−1.98 (m, 1H), 1.77−1.72 (m, 1H), 1.41 (s, 9H).

7.2, 36.9, 31.1, 29.1, 28.4, 28.2, 27.7. ESI-LR-MS. Calcd for

NMR (100 MHz, CDCl ): δ 172.0, 171.9, 171.8, 171.7, 169.6, 169.5,

C H N NaO [M + Na] : m/z 1287.57. Found: m/z 1287.58.

166.0, 165.5, 151.9, 151.8, 147.0 (×2), 136.4, 136.3 (×2), 136.2

136.1, 129.4, 129.3 (×2), 129.2, 128.7 (×2), 128.6 (×2), 128.5, 128.3,

128.2, 128.0, 127.9, 127.8, 127.7, 127.0, 126.5, 124.4, 123.1, 122.9,

117.3, 80.3, 76.3, 76.2, 71.3 (×2), 54.4, 54.0, 53.5, 43.8, 38.6, 38.3,

,3-Glu-CAM (3). 3,3-Glu( Bu)-CAM(Bn) (12, 0.10 g, 81 μmol)

was dissolved in glacial acetic acid and 12 M HCl [1:1 (v/v), 3 mL]

and stirred at room temperature under N (g) for 30 h. The volatiles

were then removed under reduced pressure to yield 3,3-Glu-CAM as

an oﬀ-white solid (3, 58 mg, quant. yield). H NMR (400 MHz,

CD OD): δ 7.32−7.22 (m, 4H), 6.95−6.93 (m, 3H), 6.75−6.69 (m,

31.9, 28.1, 27.3. ESI-LR-MS. Calcd for C H N O [M + H] : m/z

1394.64. Found: m/z 1394.67.

TREN-bisGlyGlu-CAM (4). TREN-bisGlyGlu( Bu)-CAM(Bn)

(15, 29 mg, 21 μmol) was dissolved in glacial acetic acid and 12 M

H), 4.68 (dd, J = 9 Hz, J = 5 Hz, 1H), 3.51 (t, J = 7 Hz, 4H), 3.08

1 2

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

The Supporting Information is available free of charge at

Article

HCl [1:1 (v/v), 2 mL] and stirred at room temperature under N (g)

for 20 h. The volatiles were removed under reduced pressure to yield

100[(radioactivity at solvent front)/(radioactivity at origin +

radioactivity at solvent front)]. If the radiochemical purities of the

the HCl salt of the free ligand TREN-bisGlyGlu-CAM as an oﬀ-white

Ga-enterobactin-based analogues (2−4) were found to be <95%,

the complexes were puriﬁed by passing the solution through a CM

solid. (4, 17 mg, 96%). H NMR (400 MHz, CD OD): δ 7.32−7.25

(

m, 3H), 6.93 (d, J = 8 Hz, 3H), 6.74−6.69 (m, 3H), 4.58−4.54 (m,

Sep-Pak Light cartridge, thereby stripping the free [ Ga]Ga ions

from the solution. The CM Sep-Pak cartridge was preactivated with 2

mL of water followed by 10 mL of air before use.

H), 4.04 (br, s, 4H), 3.61−3.46 (m, 12H), 2.47−2.43 (m, 2H),

.24−2.21 (m, 1H), 2.09−2.02 (m, 1H). C NMR (100 MHz,

CD OD): δ 173.5, 171.8, 149.8, 147.0, 129.6, 129.5, 129.4, 120.0,

Dilute hydrochloric acid and sterile water were added to formulate

19.9, 119.7, 119.5, 116.7, 116.5, 55.6, 54.8, 43.9, 36.1, 36.0, 31.3,

−

Ga-labeled TREN-CAM analogue solutions to contain approx-

imately 0.9% NaCl at pH ∼7.2. Final solutions were ﬁltered through a

.2 μM sterile ﬁlter and rechecked for radiochemical purity using

7.5, 20.8. ESI-HR-MS. Calcd for C H N O [M − H] : m/z

96.2790. Found: m/z 796.2858.

Ga-TREN-bisGlyGlu-CAM (Ga-4). TREN-bisGlyGlu-CAM (4,

9.8 mg, 75.0 μmol) and KOH (12.6 mg, 225 μmol) were ﬁrst

iTLC before use.

dissolved in methanol (2 mL), followed by the addition of Ga(acac)₃

27.8 mg, 75.7 μmol). The resulting product was precipitated out of

solution by addition of diethyl ether. The solid was separated oﬀ and

Evaluation of In Vitro Serum Stability. The stabilities of

radiolabeled ligands were determined by measuring the radiochemical

purity of each complex after incubation in human serum at 37 °C for

(

dried under reduced pressure to yield Ga-TREN-bisGlyGlu-CAM as a

and 2 h. In each case, the pH was maintained between 8 and 8.5. At

white powder (Ga-4, 33.3 mg, 54%). For H NMR (400 MHz,

each incubation interval, the relative amount of radioactivity

DMSO-d ), the existence of multiple isomers in solution as well as the

associated with free [ Ga]Ga (origin) and radiolabeled complex

reduced solubility of this coordination compound that seems to

facilitate the formation of aggregates results in a complex NMR

(solvent front) was determined using iTLC as described above.

Gallium-68 PET Imaging and Biodistribution Studies. To

spectrum. Assignment of signals is challenging, and thus, the H NMR

determine the biodistribution and pharmacokinetics of the imaging

agent, three female BalbC mice (Envigo, Indianapolis, IN) were

spectrum is instead shown in the Supporting Information . ESI-HRMS.

−

Calcd for C H GaN O [M + 2H] : m/z 862.1811. Found: m/z

injected with [ Ga]Ga-TREN-CAM [1, 8.8−33.3 μg total of injected

862.1763.

labeled (0.3−1.2 MBq) and unlabeled TREN-CAM per animal] via

tail vein injection. At 15, 30, 60, and 120 min postinjection,

anesthetized animals underwent 10 min PET scans using a small

animal PET/X-ray system (Soﬁe BioSystems Genesys4, Culver City,

CA). The anatomic reference skeleton images were formed by using

the mouse atlas registration system algorithm with information

obtained from the stationary top-view planar X-ray projector and side-

view optical camera. PET images were normalized to units of

standardized uptake value {SUV = (activity concentration in tissue)/

Solution Thermodynamics: Competition Titrations. Samples

containing known concentrations of ligand, GaCl (0.01−1 equiv),

and DFO mesylate salt (0.01−100 equiv) were prepared in Tris buﬀer

(

10 mM, pH 7.4) with KCl (0.1 M). The pH of all solutions was

adjusted to 7.40 with HCl(aq) or KOH(aq) as necessary and diluted

to identical volumes. All samples were equilibrated at 25 °C for 24 h,

after which UV−vis spectra were recorded. The concentrations of the

free and complexed ligand in each solution were averaged over 10

wavelengths by using solutions of L and GaL at the same

concentration and under the conditions as the references. Using the

[(injected dose)/(g of whole body weight)]} and presented as

known pGa value for DFO (21.2), the concentration of the

maximum intensity projection scan (MIPS) images. To determine the

competing ligand necessary to generate an equal partition of Ga

biodistribution of [ Ga]Ga-TREN-CAM (1), the mice were

between the ligand of interest and the competitor occurs at the x-

intercept of the log−log plot (Figure 3), which gives the diﬀerence in

pGa values between DFO and the ligand of interest (Table 1).

sacriﬁced after the ﬁnal imaging time point (120 min) and organ

tissues were harvested. The radioactivity in organ tissues was counted

using a gamma counter; the measurements were decay-corrected to

Method of Cyclotron Production and Puriﬁcation of

[⁶⁸

Ga]GaCl . The Ga isotope was produced with a GE PETtrace

the time of [ Ga]Ga-TREN-CAM injection, and the SUV was

(

16.3 MeV energy) cyclotron using a liquid target via the Zn-

p,n) Ga nuclear reaction as described previously. In brief, a 1 M

calculated.

Biodistribution Experiments. Thirty female mice (CD-1, 6−8

weeks of age, Envigo) were injected intravenously with Ga-TREN-

CAM (1 mg/kg in PBS), and groups of three animals were euthanized

at speciﬁc time points (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h,

and 24 h) over a 24 h period. The blood, brain, heart, lung, liver,

spleen, stomach, cecum, muscle (diaphragm), and kidney were

collected from each animal and stored at −20 °C. Tissues were boiled

solution of isotopically enriched (>99%) [ Zn]Zn(NO ) in 1.1 N

HNO (aq) was irradiated with a proton beam at 40 μA for 60 min.

After irradiation, the solution was transferred to a hot cell equipped

with an automated radiochemistry synthesis module (Trasis All-in-

One), puriﬁed with a hydroxamate resin cartridge (100 mg), and

concentrated on an anion exchange resin (Ag-1X-8, 400 mg) as

7,69

described previously.

The ﬁnal [ Ga]GaCl was eluted with 1.0

in HNO (∼10%) until no cloudiness remained in the sample (∼8 h).

mL of water from anion exchange resin. The puriﬁed [ Ga]GaCl

Samples were diluted with mQ water to equivalent volumes and

analyzed for Ga by ICP-MS (ALS Environmental, Salt Lake City,

UT). Data obtained from blood were subjected to pharmacokinetic

analysis (WinNonlin software, Certara USA, Inc., Princeton, NJ)

using a noncompartmental model.

solution was manually transferred to another hot cell to perform

manual radiolabeling of diﬀerent enterobactin-based analogues.

Radiolabeling of Enterobactin Analogues with [ Ga]GaCl .

Radiolabeling of each ligand was performed between pH 8.5 and 9.0

at room temperature (25 °C). In brief, the ligands (600 μg) were

separately suspended in 1.5 mL of water and the pH of the resulting

mixture were adjusted to ∼7.5−8.0 using 1 M NaOH(aq) until the

ASSOCIATED CONTENT

■

solution became clear. Separately, the solution of [ Ga]GaCl was

sı Supporting Information

adjusted to 8.0−9.0 with 1 M NaOH. One milliliter of the pH-

adjusted solution of cyclotron-produced [ Ga]GaCl was subse-

https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c00975.

quently added to the solution of the ligand, and the reaction mixture

was stirred at room temperature for 10 min. Reaction progress was

measured by instant thin-layer chromatography (i-TLC) using a 1:1

mixture of methanol and 1 M ammonium acetate as the mobile phase

Characterization data, including H and ¹³C NMR data

of intermediates and ﬁnal ligands, ESI-MS data of the

intermediates and ligands, HPLC chromatograms of the

ligands, and UV−visible spectra of the ligands and

(

for Ga complexes, f > 0.9; free [ Ga]Ga R_f= 0). The

radiochemical purity was assessed using a rad-TLC scanner (BioScan,

Eckert Ziegler, Berlin, Germany). The percentage of intact radio-

labeled complex was calculated as follows: radiochemical purity =

III

respective Ga complexes (PDF )

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Animals: Evaluation Using a 3D-Mode MicroPET Scanner and

Article

Impact of Reconstruction Algorithms. EJNMMI Res. 2017, 7 (1), 91.

AUTHOR INFORMATION

Corresponding Authors

■

(7) Cai, Z.; Li, S.; Pracitto, R.; Navarro, A.; Shirali, A.; Ropchan, J.;

Valerie C. Pierre − Department of Chemistry, University of

Huang, Y. Fluorine-18-Labeled Antagonist for PET Imaging of Kappa

Opioid Receptors. ACS Chem. Neurosci. 2017 , 8 (1), 12 −16.

(8) Zeglis, B. M.; Lewis, J. S. A Practical Guide to the Construction

of Radiometallated Bioconjugates for Positron Emission Tomography.

Dalt. Trans. 2011, 40 (23), 6168−6195.

Minnesota, Minneapolis, Minnesota 55455, United States;

orcid.org/0000-0002-0907-8395; Email: pierre@umn.edu

Mukesh K. Pandey − Department of Radiology, Mayo Clinic,

Rochester, Minnesota 55905, United States; orcid.org/0000-

(9) Macpherson, D. S.; Fung, K.; Cook, B. E.; Francesconi, L. C.;

002-2332-5305; Email: Pandey.Mukesh@mayo.edu

Zeglis, B. M. A Brief Overview of Metal Complexes as Nuclear

Timothy R. DeGrado − Department of Radiology, Mayo Clinic,

Rochester, Minnesota 55905, United States; orcid.org/0000-

Imaging Agents. Dalt. Trans. 2019, 48 (39), 14547−14565.

(10) Berry, D. J.; Ma, Y.; Ballinger, J. R.; Tavare, R.; Koers, A.;

002-0511-5398; Email: DeGrado.Timothy@mayo.edu

Sunassee, K.; Zhou, T.; Nawaz, S.; Mullen, G. E. D.; Hider, R. C.;

Blower, P. J. Efficient Bifunctional Gallium-68 Chelators for Positron

Emission Tomography: Tris(Hydroxypyridinone) Ligands. Chem.

Commun. 2011, 47 (25), 7068−7070.

Authors

M. Andrey Joaqui-Joaqui − Department of Chemistry,

University of Minnesota, Minneapolis, Minnesota 55455,

United States; orcid.org/0000-0001-7446-6004

Aditya Bansal − Department of Radiology, Mayo Clinic,

Rochester, Minnesota 55905, United States; orcid.org/0000-

(11) Koukouraki, S.; Strauss, L. G.; Georgoulias, V.; Eisenhut, M.;

Haberkorn, U.; Dimitrakopoulou-Strauss, A. Comparison of the

Pharmacokinetics of Ga-DOTATOC and [ F]FDG in Patients

with Metastatic Neuroendocrine Tumours Scheduled for Y-

DOTATOC Therapy. Eur. J. Nucl. Med. Mol. Imaging 2006, 33

(10), 1115−1122.

(12) Haug, A.; Auernhammer, C. J.; Wan

G.; Goke, B.; Bartenstein, P.; Po

001-7657-4981

Mandapati V. Ramakrishnam Raju − Department of

Chemistry, University of Minnesota, Minneapolis, Minnesota

gler, B.; Tiling, R.; Schmidt,

pperl, G. Intraindividual Comparison

5455, United States; orcid.org/0000-0002-8045-5827

of Ga-DOTA-TATE and F-DOPA PET in Patients with Well-

Differentiated Metastatic Neuroendocrine Tumours. Eur. J. Nucl. Med.

Mol. Imaging 2009, 36 (5), 765−770.

Fiona Armstrong-Pavlik − Department of Chemistry,

University of Minnesota, Minneapolis, Minnesota 55455,

United States; orcid.org/0000-0002-9922-5079

Ayca Dundar − Department of Radiology, Mayo Clinic,

Rochester, Minnesota 55905, United States; orcid.org/0000-

(

13) Virgolini, I.; Ambrosini, V.; Bomanji, J. B.; Baum, R. P.; Fanti,

S.; Gabriel, M.; Papathanasiou, N. D.; Pepe, G.; Oyen, W.; De

Cristoforo, C.; Chiti, A. Procedure Guidelines for PET/CT Tumour

002-3128-6801

Imaging with Ga-DOTA-Conjugated Peptides: Ga-DOTA-TOC,

Henry L. Wong − Department of Medicinal Chemistry and

Institute for Therapeutics Discovery & Development, University

of Minnesota, Minneapolis, Minnesota 55414, United States;

orcid.org/0000-0003-1755-1485

Ga-DOTA-NOC, Ga-DOTA-TATE. Eur. J. Nucl. Med. Mol.

Imaging 2010, 37 (10), 2004−2010.

(14) Prasad, V.; Ambrosini, V.; Hommann, M.; Hoersch, D.; Fanti,

S.; Baum, R. P. Detection of Unknown Primary Neuroendocrine

Tumours (CUP-NET) Using Ga-DOTA-NOC Receptor PET/CT.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.inorgchem.0c00975

Eur. J. Nucl. Med. Mol. Imaging 2010, 37 (1), 67−77.

(15) Huo, Y.; Kang, L.; Pang, X.; Shen, H.; Yan, P.; Zhang, C.; Liao,

X.; Chen, X.; Wang, R. Noninvasive PET Imaging of a Ga-68-

Radiolabeled RRL-Derived Peptide in Hepatocarcinoma Murine

Models. Mol. Imaging Biol. 2019, 21 (2), 286−296.

Notes

The authors declare no competing ﬁnancial interest.

(16) Velikyan, I.; Beyer, G. J.; Långstrom, B. Microwave-Supported

ACKNOWLEDGMENTS

The authors acknowledge support from the Minnesota

Partnership for Biotechnology and Genomics (Grant 17.38).

Preparation of Ga Bioconjugates with High Specific Radioactivity.

■

Bioconjugate Chem. 2004, 15 (3), 554−560.

(

17) Velikyan, I.; Sundberg, Å. L.; Lindhe, O; Ho

glund, A. U.;

Eriksson, O.; Werner, E.; Carlsson, J.; Bergstro

Tolmachev, V. Preparation and Evaluation of Ga-DOTA-hEGF for

m, M.; Långstrom, B.;

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