A submicrogram-scale protocol for biomolecule-based PET imaging by rapid 6π-azaelectrocyclization: Visualization of sialic acid dependent circulatory residence of glycoproteins

Article abstract of DOI:10.1002/anie.200702989

On target: Lysine residues in picomole samples of a peptide, proteins, and a monoclonal antibody were rapidly and selectively labeled by a dota-based probe (1, see scheme). Positron emission tomography imaging of [⁶⁸Ca]dota- labeled orosomucoid

Full text of DOI:10.1002/anie.200702989

Communications
DOI: 10.1002/anie.200702989
Molecular Imaging
A Submicrogram-Scale Protocol for Biomolecule-Based PET Imaging
by Rapid 6p-Azaelectrocyclization: Visualization of Sialic Acid
Dependent Circulatory Residence of Glycoproteins**
Katsunori Tanaka, Tatsuro Masuyama, Koki Hasegawa, Tsuyoshi Tahara, Hiroshi Mizuma,
Yasuhiro Wada, Yasuyoshi Watanabe, and Koichi Fukase*
In memory of Yoshihiko Ito
Molecular imaging is an important topic that has gained
significant attention in the fields of chemical biology, drug
discovery, and diagnosis. Positron emission tomography
(PET) is a widely used, noninvasive method that quantita-
tively visualizes the locations and levels of radiotracer
accumulation with high imaging contrast.[1] 2-deoxy-2-
[18F]fluoro-d-glucose ([18F]FDG) has become an important
small-molecule-based PET tracer used especially in the
detection of primary and metastatic cancers. Current efforts
focus on the development of biomolecule-based tracers,
which are composed of peptides, monoclonal antibodies
(mAbs), and oligonucleotides.[1] Generally, PET imaging of
these biomolecules is achieved by conjugation to metal-
chelating agents, such as dota (1,4,7,10-tetraazacyclodecane-
1,4,7,10-tetraacetic acid) or dtpa (diethylenetriaminepenta-
acetic acid), prior to radiolabeling with a suitable b+-emitting
metal. The chelating agents can be introduced either during
the solid-phase synthesis of peptides or more directly by the
reaction of lysine residues or N-terminal amino groups with
dota-O-sulfosuccinimidyl ester.[2] However, the latter
method, using the activated dota ester, usually proceeds
slowly and requires as much as a few milligrams of sample to
keep the reaction concentrations high. Because PET experi-
ments require only small amounts of the tracers, and because
important biomolecules are sometimes obtained only in small
amounts, a submicrogram-scale conjugation methodology
would greatly expand the applicability of PET imaging.
Herein, we describe the submicrogram-scale labeling of lysine
residues by a rapid 6p-azaelectrocyclization, which led to an
efficient PET protocol; a first visualization of sialic acid
dependent circulatory residence of glycoproteins was real-
ized.
Although various labeling methods for biomolecules have
been reported,[3] an efficient, rapid, and mild protocol that can
be applied in various buffer solutions remains to be found.
Recently, Katsumura and co-workers reported that unsatu-
rated (E)-ester aldehydes quantitatively react with primary
amines, including lysine, within five minutes in water, thus
providing 1,2-dihydropyridines in irreversible reactions
(Scheme 1).[4] These reactions proceed by smooth azaelec-
trocyclization of the intermediary Schiff bases (1-azatrienes),
which is strongly accelerated owing to the efficient interac-
tions between the highest occupied and lowest unoccupied
molecular orbitals (HOMO and LUMO) within the 1-
azatriene systems; these interactions are favored by the
electron-withdrawing substituent at C4 and the conjugation at
C6.[4d] Inspired by Katsumuraꢀs findings, we designed and
synthesized a new dota labeling probe, dota-(E)-ester alde-
hyde 4a (Scheme 2). (Z)-Vinyl bromide 2 and glycine-linked
(E)-stannane 1 were heated to 1108C in the presence of
[Pd2(dba)3] (dba = trans,trans-dibenzylideneacetone), P(2-
furyl)3, and LiCl in DMF to provide the Stille coupling
product,[4d] which was subsequently treated with 3m hydro-
chloric acid to give aminoalcohol 3 in 73% yield for two steps.
[*] Dr. K. Tanaka, T. Masuyama, Prof. Dr. K. Fukase
Department of Chemistry, Graduate School of Science
Osaka University
1-1 Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043 (Japan)
Fax: (+81)6-6850-5419
E-mail: koichi@chem.sci.osaka-u.ac.jp
Homepage: http://www.chem.sci.osaka-u.ac.jp/lab/fukase/
Dr. K. Hasegawa, Dr. T. Tahara, Dr. H. Mizuma, Y. Wada,
Prof. Dr. Y. Watanabe
Molecular Imaging Program, RIKEN
6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe-shi
Hyogo 650-0047 (Japan)
Prof. Dr. Y. Watanabe
Department of Physiology, Graduate School of Medicine
Osaka City University
1-4-3, Asahimachi, Abeno-ku, Osaka 545-8585 (Japan)
[**] We acknowledge Prof. Shigeo Katsumura in Kwansei Gakuin
University for helpful discussion and HRMS measurements. The
present work was financially supported by Grant-in-Aid for Scientific
Research No. 18850014 and 19681024 to K.T. from the Japan Society
for the Promotion of Science. PET=positron emission tomography.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Scheme 1. Rapid reaction with lysine through 6p-azaelectrocyclization.
102
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 102 –105

Angewandte
Chemie
Table 1: Labeling of biomolecules by probes 4a–c.[a]
Entry Probe
Biomolecule
(m [mg])
Biomolecule Equiv
conc [m]
t
Labeled
probe [min] Lys[c]
1[b] 4a
somatostatin (170) 5.5”10À4
100
25
10
500
10
10
30
10
10
10
30
30
1
5
2
20
2
3
2
3
4
5
6
4a
4a
4a
4a
4a
albumin (100)
albumin (100)
albumin (100)
5.0”10À5
5.0”10À5
5.0”10À5
orosomucoid (62) 4.5”10À6
asialoorosomucoid 4.5”10À6
(62)
Scheme 2. Synthesis of dota and fluorescent labeling probes: a) [Pd2-
(dba)3], P(2-furyl)3, LiCl, DMF, 1108C, 73%; b) 3m HCl, MeOH/H2O
(1:1), RT, quantitative yield; c) dota-OSu, Et3N, DMF, RT, 58% (for
4a); coumarin-OSu, CH2Cl2, RT, 68% (for 4b); TAMRA-OSu, DMF/
CH2Cl2 (1:1), RT, 74% (for 4c); d) Dess–Martin periodinane, DMF/
CH2Cl2 (3:10), RT for 4a; polystyrene-supported o-iodoxybenzoic acid
(IBX), DMF/CH2Cl2 (1:1), RT for 4b and 4c.
7[b] 4a(Gd)[d] somatostatin (32) 3.6”10À4
31
7
7
30
30
30
30
1
8
9
10
4b
4c
4c
albumin (120)
albumin (12)
anti-GFP mAb
(2.0)
1.7”10À5
2.1”10À5
1.1”10À6
1[e]
1[e]
2[e]
20
[a] Unless otherwise noted, reactions were performed in 0.1m phosphate
buffer (pH 7.4) at 248C. [b] in H2O. [c]The number of labeled lysine residues
was calculated by g counting of 57Co introduced to dota (see the Supporting
Information) [d] Gd was introduced by the reaction of 4a with 0.1m GdCl3 in
H2O. [e] Estimated by emission spectra at 470 nm (coumarin) and 555 nm
(TAMRA).
Selective acylation of the amino group in 3 was achieved in
58% yield by the reaction with dota-O-succinimidyl ester
(dota-OSu)[5] in the presence of Et3N in DMF. Finally, the
allylic alcohol was oxidized by Dess–Martin periodinane in
DMF/CH2Cl2 (3:10) to afford the desired aldehyde 4a, which
was used for labeling as its DMF solution after size-
partitioning filtration and removal of CH2Cl2. By applying
the established route in Scheme 2, various functionalities can
be introduced at the amino group in 3, thereby providing
access to general probes; the coumarin (4b) and the carboxy-
tetramethylrhodamine (TAMRA, 4c) derivatives have been
prepared for fluorescent labeling (Scheme 2).
With probes 4a–c in hand, we tested the reactivity toward
lysine residues of various biomolecules, namely, somatostatin
as a peptide as well as albumin, orosomucoid, and anti-GFP
antibody (GFP = green fluorescent protein) as proteins
(Table 1). To our satisfaction, the reaction of somatostatin
(170 mg) with 100 equivalents of dota probe 4a at room
temperature for 30 min provided mono-dota labeled soma-
tostatin in 96% yield based on the HPLC analysis (Table 1,
entry 1). Interestingly, under these labeling conditions, only
one of the two lysine residues in somatostatin, namely that not
involved in receptor binding, was modified (see the
Supporting Information).[6] In contrast, under conventional
labeling conditions, using the dota-activated ester required a
much longer reaction time (over 24 h), which resulted in
indiscriminant modification at both lysine residues and the
N terminus as well as the recovery of the intact peptide. Thus,
the high reactivity of 4a enabled the lysine azaelectrocycliza-
tion modification to be completed in a very short time, which
resulted in selective labeling of the more accessible lysine
residue. Similarly, the incubation of human serum albumin
(100 mg) with 25 equivalents of probe 4a for just 10 min and
subsequent quick size-partitioning gel filtration successfully
provided the modified protein with five incorporated dota
units (Table 1, entry 2). Noteworthy is that labeling by
azaelectrocyclization proceeds in nearly quantitative yields
based on the lysine residues, both at high (greater than 10À3 m)
and at low (less than 10À5–10À6 m) concentrations of the probe
4a. As a result, the number of dota units introduced to a
protein can be precisely controlled by adjusting the number of
equivalents of 4a used. For example, with a reaction time of
ten minutes, ten equivalents of 4a provided the labeled
protein with two molecules of dota, while use of 500 equiv-
alents resulted in the introduction of 20 dota molecules
(Table 1, entries 3 and 4).
To further demonstrate the performance of this labeling
method, proteins that are available in only small amounts
(62 mg of glycoproteins, orosomucoid, and asialoorosomu-
coid), were labeled successfully with the incorporation of two
or three units of dota by incubating the respective protein
with ten equivalents of 4a for 30 min (Table 1, entries 5 and
6). Furthermore, the paramagnetic metal Gd3+ can be
chelated by the dota unit of 4a prior to peptide labeling
(Table 1, entry 7). This new process could allow for the
labeling of valuable or unstable materials.
The present electrocyclization protocol is also applicable
to rapid fluorescent labeling; 10–100 mg albumin was labeled
Angew. Chem. Int. Ed. 2008, 47, 102 –105
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
103

Communications
with probes 4b and 4c(Table 1, entries 8 and 9). As little as
2 mg of anti-GFP mAb (ca. 10 pmol) was successfully labeled
by 4c within 30 min (Table 1, entry 10); 20 equivalents of 4c
resulted in preferential labeling at the more accessible Fc
fragment with two molecules of fluorophore, thereby retain-
ing its GFP recognition activity with 90% of the intact mAb
(see the Supporting Information).
To verify the applicability of the new labeling method to
PET imaging, the dota–somatostatin adduct was treated with
68Ga3+ as a b+ emitter.[7] The resulting 68Ga-labeled adduct
was investigated in rabbits (Figure 1). Somatostatin receptors
are expressed on pancreas, kidney, gastrointestinal tract, and
endocrine tumors. Somatostatin receptor imaging has been
used for the diagnosis of endocrine tumors by using radio-
labeled octreotide, a metabolically stable analog of somatos-
tatin. In our case, the microPET imaging of [68Ga]dota–
somatostatin successfully visualized accumulation of the
tracer in the pancreas two to four hours after the injection,
thus establishing this method as a powerful PET labeling
protocol.[7,8]
Figure 1. MicroPET images of [68Ga]dota–somatostatin in rabbits (axial
view, overlaid with computed tomography (CT) images). The images
represent time-dependent changes of the accumulation in the liver
(large arrow), the kidney (small arrow), and the pancreas (arrowhead)
after injection of [68Ga]dota–somatostatin. SUV=standardized uptake
value.
Furthermore, microPET images of [68Ga]dota–orosomu-
coid and asialoorosomucoid adducts in rabbits (Figure 2)
successfully visualized the asialo glycoprotein being cleared
through the kidneys faster than orosomucoid,[9] thus achieving
the first visualization of sialic acid dependent circulatory
residence of glycoproteins.[8]
peptides, proteins, and other amine-containing molecules.
In vivo kinetic studies of proteins such as the cytokines is
currently in progress.
In summary, we have developed a new lysine-based
labeling method for biomolecules on
the basis of rapid 6p-azaelectrocycli-
zation. Both dota as a metal-chelating
agent (either for MRI, PET, or other
radiopharmaceutical purposes, such
as single photon emission computed
tomography (SPECT) with g emit-
ters) and fluorescent groups were
efficiently and selectively introduced
to lysine residues in either phosphate
or Tris–HCl buffer solutions (Tris =
tris(hydroxymethyl)aminometha-
ne)[4b] within 30 min at room temper-
ature. The dota-labeled somatostatin
and glycoproteins were subsequently
radiometallated with 68Ga, and the
receptor-mediated accumulation of
somatostatin in the pancreas was
observed. Furthermore, oligosacchar-
ide-dependent circulatory residence
of glycoproteins was visualized for
the first time by microPET. There are
two distinct features of this labeling
method: 1) Labeling requires samples
of only a few micrograms, or even less,
Figure 2. Dynamic microPET images and time–activity curves of [68Ga]dota glycoproteins in
rabbits. a) Time course of accumulation of [68Ga]dota–orosomucoid (top) and [68Ga]dota–asialoor-
osomucoid (bottom) in some peripheral organs (axial views). These PET images were overlaid on
anatomical images obtained by CT. b,c) Changes in the standardized uptake value (SUV) of
if desired. 2) Near-quantitative reac-
tion at the lysine residues of biomol-
ecules enables the use of a simple
isolation procedure (i.e. quick gel
filtration or centrifugal separation).
The protocol developed herein will
68
68
&
*
[ Ga]dota–orosomucoid (b) and [ Ga]dota–asialoorosomucoid (c) in the liver ( ), the spleen ( ),
~
and the kidney ( ). SUV was used as a quantitative evaluation of PET data and normalized by
radioactivity in the tissue region, the injected dose, and the weight of the subjects. These values
expand the applicability of PET on represent the mean and standard deviation.
104
www.angewandte.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 102 –105

Angewandte
Chemie
Experimental Section
[1] S. S. Gambhir, Nat. Rev. Cancer 2002, 2, 683 – 693.
Representative procedure for labeling with probe 4a (Table 1,
entry 5): Probe 4a (14 nmol, 1.5 mL) in DMF was added to
orosomucoid (62 mg, 1.4 nmol, 295 mL, pH 7.4) in phosphate-buffered
saline (PBS) at room temperature (concentration: 4.5 ” 10À6 m for
orosomucoid, 4.5 ” 10À5 m for 4a). After the mixture had stood at
room temperature for 30 min, the dota-labeled protein was purified
by ultrafiltration (30000 cut). Solutions of ammonium acetate (0.25m,
20 mL, pH 7.0) and CoCl2 (4.0 nmol, 400 mm, 5 mL, containing more
than 350000 cpmmLÀ1 57CoCl2) were added to a PBS solution of the
labeled protein (6.67 ” 10À10 mol, 5.74 mgmLÀ1, 5 mL, pH 7.4). After
the mixture was incubated at 408C for 3 h, a solution of dota (40 mg,
100 nmol, 10 mm, 10 mL) was added, and the mixture was incubated at
408C for 30 min. A 1.0-mL aliquot of the resulting solution was
spotted on a TLC plate and developed until the solvent (MeOH/
H2O = 1:1) reached 7 cm from the origin. The plate was cut into seven
fractions, for each of which 57Co was counted by a gamma counter.
The number of the 57Co chelates per orosomucoid was calculated by
the ratio of 57Co at the bottom of TLC plate (Rf = 0–0.14, corre-
sponding to 57Co–dota–protein) to the rest of the plate, which
contained 57Co chelated by free dota.
[2] M. R. Lewis, J. Y. Kao, A.-L. J. Anderson, J. E. Shively, A.
Raubitschek, Bioconjugate Chem. 2001, 12, 320 – 324, and refer-
ences therein.
[3] a) I. Chen, A. Y. Ting, Curr. Opin. Biotechnol. 2005, 16, 35 – 40;
b) L. W. Miller, V. W. Cornish, Curr. Opin. Chem. Biol. 2005, 9,
56 – 61; c) Y. Takaoka, H. Tsutsumi, N. Kasagi, E. Nakata, I.
Hamachi, J. Am. Chem. Soc. 2006, 128, 3273 – 3280, and refer-
ences therein.
[4] a) K. Tanaka, M. Kamatani, H. Mori, S. Fujii, K. Ikeda, M.
Hisada, Y. Itagaki, S. Katsumura, Tetrahedron Lett. 1998, 39,
1185 – 1188; b) K. Tanaka, M. Kamatani, H. Mori, S. Fujii, K.
Ikeda, M. Hisada, Y. Itagaki, S. Katsumura, Tetrahedron 1999, 55,
1657 – 1686; c) K. Tanaka, S. Katsumura, Org. Lett. 2000, 2, 373 –
375; d) K. Tanaka, H. Mori, M. Yamamoto, S. Katsumura, J. Org.
Chem. 2001, 66, 3099 – 3110; e) K. Tanaka, S. Katsumura, J. Am.
Chem. Soc. 2002, 124, 9660 – 9661; f) K. Tanaka, T. Kobayashi, H.
Mori, S. Katsumura, J. Org. Chem. 2004, 69, 5906 – 5925.
[5] Available from Macrocyclics; http://www.macrocyclics.com/.
[6] These probes only react with the e-amino group of lysine, and
other amino acids or N termini were not labeled at RT; see
reference [4b].
Received: July 5, 2007
Revised: September 13, 2007
Published online: November 16, 2007
[7] H. R. Maecke, M. Hofmann, U. Haberkorn, J. Nucl. Med. 2005,
46, 172S – 178S.
[8] Detailed PETanalysis of [68Ga]dota-somatostatin and [68Ga]dota-
glycoproteins will be reported in full accounts of this work.
[9] Representative example: A. G. Morell, R. A. Irvine, I. Sternlieb,
I. H. Scheinberg, G. Ashwell, J. Biol. Chem. 1968, 243, 155 – 159.
Keywords: fluorescent probes · glycoproteins · imaging agents ·
.
lysine · positron emission tomography
Angew. Chem. Int. Ed. 2008, 47, 102 –105
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
105