Amino acids labeled with [99mTc(CO)3]+ and recognized by the L-type amino acid transporter LAT1

Article abstract of DOI:10.1021/ja066002m

We have synthesized amino acids conjugated at the α-carbon through an alkyl spacer to a small tripod ligand. The tripod coordinates to the fac-[M(CO)₃]⁺ moiety (M = Re, ^99mTc). Depending on the lengths of the spacers, these metal complexes with pendent α-amino acids are recognized and transported by the L-type amino acid transporter LAT1. The best result was achieved with a butyl spacer. The K_i value of the corresponding complex is comparable to that of the artificial amino acid BCH. Efflux of [³H]-L-phenylalanine shows that the labeled amino acids do not only bind to the transporter but are transported into the cells. These are the first metal-labeled small molecules which are actively internalized to the intracellular space. Copyright

Full text of DOI:10.1021/ja066002m

Published on Web 12/01/2006
Amino Acids Labeled with [99mTc(CO)3]+ and Recognized by the L-type Amino
Acid Transporter LAT1
Yu Liu,† Jae Kyoung Pak,† Paul Schmutz,† Matthias Bauwens,‡ John Mertens,‡ Hector Knight,§ and
Roger Alberto*,†
Institute of Inorganic Chemistry, UniVersity of Zu¨rich, Winterthurerstr. 190, 8057 Zu¨rich, Switzerland,
Radiopharmaceutical Chemistry, Vrije UniVersiteit Brussels, Laarbeeklaan103, 1090 Brussels, Belgium, and
Mallinckrodt Medical B.V., NL-1755 Petten, The Netherlands
Received August 18, 2006; E-mail: ariel@aci.unizh.ch
Scheme 1. Synthesis of 8 (PG ) Protecting Group, Cbz) [(i)
NaOEt, EtOH; (ii) NaBH4, NiCl2, Boc2O, MeOH; (iii) NaOH, then
HCl; (iv) [ReBr3(CO)3]2-, MeOH, rt]
The labeling of small biomolecules for PET (positron emission
tomography) and SPECT (single photon emission tomography) imag-
ing represents the incentive for radiopharmaceutical chemistry. The
success of 18FDG (18F-deoxyglucose) in molecular PET imaging in
oncology, neurology, and cardiology emphasizes the importance of
radioactive metabolic tracers.1 Since 99mTc is cheap, permanently
available in many clinics, and burdens a low dose to patients, it would
be desirable to have comparable molecular imaging agents with
99mTc instead of cyclotron-produced 18F or 11C. The preparation of
99mTc-labeled glucose as 18FDG analogue has been attempted,
though without breakthrough so far.2-4 The failure of the metal-
labeled small biomolecules is not surprising since a complex will
affect the molecular recognition by transporters, receptors, or enzymes.
No small biomolecules such as glucose or amino acids with pendent
metal complexes retaining recognition and specific transportation
through the cell membrane have been reported so far.
Lipophilic and neutral amino acids are taken up by the L-type
amino acid transporter LAT1 (a Na+ independent antiport trans-
porter) which is highly overexpressed in some tumor cell lines.5-7
Yanagida et al. stated that the LAT systems show a relatively broad
and symmetrical selectivity but strongly asymmetrical substrate
affinity. Intracellular amino acids control their antiport activity. A
variety of natural and artificial amino acids such as iodinated
phenylalanine and tyrosine analogues, the anticancer drug mel-
phalan, and even BCH (Scheme 1) are efficiently transported by
the LAT system.8-12 Kersemans et al. showed that the D isomer of
2-I-phenylalanine was taken up in different types of tumor cells
expressing the LAT1.13 Insights into substrate recognition reveal
binding sites for the zwitterionic amino acid group, while a pocket
interacts with the neutral side chains.11 We present in this study
the first example of a metal-labeled amino acid which is accepted
by the LAT1 transmembrane transporter.
alkyl side chain. One of the pathways to amino acid-tripod
compounds is exemplified in Scheme 1 for a butyl linker (5). A
similar approach yields the corresponding conjugates with pentyl
(6) and hexyl (7) spacers (Supporting Information).
The R-C-H in 4 is deprotonated with NaOEt. Reaction with
protected ꢀ-mes-lysine (i) followed by reduction and deprotection
(ii and iii, respectively) gave the desired amino acid-tripod
conjugate 5. The R-C in the tripod is chiral and present in its D-
and L-configuration after the synthesis. Coupling with an enantio-
merically pure amino acid results in two diastereomers (e.g., 5 from
L-lysine). Starting from D,L-amino acids, racemic mixtures of two
diastereomers are received (6 and 7). Due to the low specificity of
the pocket interacting with the side chain, the selection of one single
diastereomer is not yet decisive for the proof of concept.
Compounds 5-7 react in water with 1 or 1a to form the
complexes [Re(5)(CO)3] (8), [Re(6)(CO)3] (9), [Re(7)(CO)3] (10),
and the corresponding 99mTc analogues. The amino acid part and
the tripod provide similar sets of donors, but binding to the tripod
was found preferentially (>95%). The complexes 8-10 are
hydrophilic and the 99mTc complexes serum stable for at least 24 h.
Within a structure-activity relationship (sar) study comprising
a series of natural and custom synthesized amino acid analogues
(S-alkylated derivatives of cysteine), the rhenium complexes 8-10
The complexes [M(OH2)3(CO)3]+ (M ) Re (1), 99mTc (1a)) bind
to a variety of ligands.14 Active transport of labeled small molecules
through protein channels requires complexes of low molecular
weight and “slim” shape. Compound 2 (Scheme 1) has rarely been
used as a tripod ligand. It has a low molecular weight (103.1 vs 65
for [C5H5]- or 129 for 9-ane-N3) and forms strong complexes with
1 or 1a. Reaction of 2 with 1 gives [Re(2)(CO)3] (3) and with 1a
at high dilution [99mTc(2)(CO)3] in quantitative yield. An X-ray
structure of 3 is shown in Figure 1 together with a sketched van
der Waals radii based size comparison with [CpRe(CO)3] from the
top view, along a hypothetical amino acid side chain.
3
have been subjected to competitive uptake studies with H-L-Phe
in R1M rhabdomyosarcoma cells. These cells overexpress LAT1
and allow one to calculate Ki (affinity constant) and to measure
stimulated efflux (Figure 2). The bioassays followed literature
procedures and are described in the Supporting Information.15
The LAT transport system works by a 1:1 exchange of amino
acids.12 This allows one to investigate if nonradioactive compounds
enter the cell by measuring the efflux of an already taken up
radiolabeled amino acid such as 3H-L-Phe.5 Hence, it is possible to
To afford a symmetric amino acid-metal complex conjugate, 3
must be attached at its R-carbon to the terminus of an amino acid
† University of Zu¨rich.
‡ Vrije Universiteit Brussels.
§ Mallinckrodt Medical B.V.
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15996
J. AM. CHEM. SOC. 2006, 128, 15996-15997
10.1021/ja066002m CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
an R-amino acid, can be combined with a complex while retaining
affinity and transport.
We emphasize that conjugation of many other ligands such as
histidine or methionine to an R-amino acid, regardless of spacer
type, resulted in complete loss of affinity. This implied that those
conjugates were too bulky. However, the spatial orientation of the
complex relative to the receptor binding part is at least as important.
Changing the anchoring atom in 8 from the R-C to the 3-NH2 group
yields 21 (Scheme 2). The number of atoms in 21 is not changed
with respect to 8, but affinity for LAT1 is lost. Consequently, for
the successful labeling of small molecules, the ligand sizes have
to be considered but the orientation of the complex relative to the
receptor binding site is equally important.
Figure 1. ORTEP presentation of 3 and size comparison with [CpRe-
(CO)3] based on van der Waals radii.
In conclusion, we have synthesized new R-amino acid derivatives
conjugated to small tripod ligands and their fac-[Re(CO)3]+ based
complexes. They represent the first examples of small molecule-
metal complex conjugates that are actively internalized into cells
by a transporter (LAT1). In vivo studies will show if the 99mTc
analogues are sufficiently accumulated for imaging of cancer tissue.
Figure 2. Ki values and stimulated efflux of the rhenium amino acids and
reference compounds; Ki values of inhibitor with 3H-L-Phe/L-Phe as
substrate; efflux of H-L-Phe out of R1M cells after 1 min.
Acknowledgment. This research was in part supported by an
Eureka project and Mallinckrodt Medical B.V.
3
Scheme 2. Structure-Activity Relationships between 10 and 20
and 8 and 21 (8 and 21 have the same stoichiometric
composition)
Supporting Information Available: Synthetic details of Ki’s, efflux
determination, and crystallographic data for 4 in CIF format. This
material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 2 shows the standard BCH substrate (17) (Ki ) 153 µM
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The data for 8-10 show that they all exhibit affinity for LAT1
and cause efflux. Complex 8 has the highest (Ki ) 308 µM) and 9
the lowest affinity (Ki ) 5300 µM). Compound 10 is in between
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3
antiport LAT1 system, all compounds causing efflux of H-L-Phe
are also taken up into these cells. Competitive inhibition of the
uptake together with the coupled stimulated efflux proves that 8-10
are transported by the same LAT system as L-Phe. The example of
8 shows for the first time that a small biological moiety, such as
JA066002M
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J. AM. CHEM. SOC. VOL. 128, NO. 50, 2006 15997