Reassessment of diethylenetriaminepentaacetic acid (DTPA) as a chelating agent for indium-111 labeling of polypeptides using a newly synthesized monoreactive DTPA derivative

Article abstract of DOI:10.1021/jm950949+

Previous studies on indium-111 (¹¹¹In) labeling of polypeptides and peptides using cyclic diethylenetriaminepentaacetic dianhydride (cDTPA) as a bifunctional chelating agent (BCA) have indicated that DTPA might be a useful BCA for ¹¹¹

Full text of DOI:10.1021/jm950949+

J . Med. Chem. 1996, 39, 3451-3460
3451
Rea ssessm en t of Dieth ylen etr ia m in ep en ta a cetic Acid (DTP A) a s a Ch ela tin g
Agen t for In d iu m -111 La belin g of P olyp ep tid es Usin g a New ly Syn th esized
Mon or ea ctive DTP A Der iva tive
Yasushi Arano,*,† Takashi Uezono,† Hiromichi Akizawa,† Masahiro Ono,† Kouji Wakisaka,† Morio Nakayama,‡
Harumi Sakahara,§ J unji Konishi,§ and Akira Yokoyama†
Department of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, and Department of Nuclear Medicine,
Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-01, J apan, and Department of Radiopharmaceutical Chemistry,
Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862, J apan
Received December 27, 1995X
Previous studies on indium-111 (111In) labeling of polypeptides and peptides using cyclic
diethylenetriaminepentaacetic dianhydride (cDTPA) as a bifunctional chelating agent (BCA)
have indicated that DTPA might be a useful BCA for 111In labeling of polypeptides at high
specific activities when DTPA can be incorporated without inducing intra- or intermolecular
cross-linking. To investigate this hypothesis, a monoreactive DTPA derivative with a maleimide
group as the peptide binding site (MDTPA) was designed and synthesized. A monoclonal
antibody (OST7, IgG1) was used as a model polypeptide, and conjugation of MDTPA with OST7,
111In radiolabeling of MDTPA-OST7, and the stability of 111In-MDTPA-OST7 were investi-
gated using cDTPA and benzyl-EDTA derivatives as references. SDS-PAGE analysis
demonstrated that while cDTPA induced intramolecular cross-linking, no such undesirable
side reactions were observed with MDTPA. MDTPA generated 111In-labeled OST7 with high
radiochemical yields at higher specific activities than those produced using cDTPA and benzyl-
EDTA derivatives as the BCAs. Incubation of each 111In-labeled OST7 in human serum
indicated that MDTPA generated 111In-labeled OST7 of much higher and a little lower stability
than those derived from cDTPA and benzyl-EDTA derivatives, respectively. These findings
indicated that the low in vivo stability of cDTPA-conjugated antibody reported previously is
not attributable to low stability of 111In-DTPA but to formation of intramolecular cross-linking
during cDTPA conjugation reactions. The present study also indicated that MDTPA and its
precursor, the tetra-tert-butyl derivative of DTPA, would be useful BCAs for 111In radiolabeling
of polypeptides that have rapid blood clearance with high specific activities.
In tr od u ction
ization of somatostatin-positive tumors has been reported
in numerous clinical studies.5 These results suggest
that cDTPA would be a suitable BCA for 111In radiola-
beling of other oligopeptides and polypeptides such as
vasoactive intestinal peptide for receptor scintigraphy.6,7
cDTPA has long been used as a BCA for 111In labeling
of various polypeptides, especially monoclonal antibod-
ies (mAbs), due to its simple conjugation reaction with
polypeptides and its ready availability from commercial
sources. Extensive studies of 111In-labeled mAbs using
cDTPA as the BCA, however, have demonstrated low
in vivo stability of the radiolabel. In animal studies,
use of cDTPA caused higher hepatic radioactivity levels
of 111In-labeled mAbs when compared to 111In-labeled
mAbs using benzyl-DTPA and benzyl-EDTA derivatives
as the BCAs.8 The lower kinetic stability of 111In-
cDTPA-mAbs in plasma was also demonstrated.9
In contrast to these observations, recent metabolic
studies of 111In-labeled neoglycoalbumins have indicated
that 111In remains stable as intact DTPA chelates in
vivo.10,11 Further analyses of radiometabolites after
administration of 111In-cDTPA-neoglycoalbumins in
mice have confirmed that 111In is present as 111In-
DTPA-lysine and 111In-DTPA in vivo.12 Since neogly-
coalbumins are prepared by modifying large amounts
of accessible amine residues in the albumin backbone
with sugar derivatives,11,13,14 only limited numbers of
amine residues are available for cDTPA conjugation.
Occupation of amine residues in the albumin molecule
with sugar derivatives may also induce steric interfer-
Low molecular weight oligopeptides such as soma-
tostatin analogs and chemotactic peptides have at-
tracted attention as new vehicles for delivery of radio-
activity to target tissues in diagnostic nuclear
medicine.1-3 Since the mechanisms of localization of
many peptides to the target tissues involve specific
binding to receptors expressed on the target cells,
radiolabeled peptides of high specific activities are
required for diagnostic application.3 The somatostatin
analog, octreotide, was successfully radiolabeled with
indium-111 (111In) at high specific activity and high
radiochemical yields with cyclic diethylenetriaminepen-
taacetic dianhydride (cDTPA; Figure 1, compound 3)
used as a bifunctional chelating agent (BCA). In
addition, attachment of an 111In-DTPA moiety to the
octreotide molecule altered the excretion pathway from
hepatobiliary to urinary excretion while maintaining the
target localization levels, which reduced upper abdomi-
nal radioactivity levels as was observed with a radio-
iodinated octreotide analog.4 As a result, clear visual-
* Address all correspondence to Yasushi Arano, Ph.D., Department
of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sci-
ences, Kyoto University, Sakyo-ku, Yoshida-Shimoadachi-cho, 606-01
Kyoto, J apan. TEL: 81-75-753-4567. FAX: 81-75-753-4568. E-
mail: arano@pharm.kyoto-u.ac.jp.
† Department of Radiopharmaceutical Chemistry, Kyoto University.
‡ Department of Radiopharmaceutical Chemistry, Kumamoto Uni-
versity.
§ Department of Nuclear Medicine, Kyoto University.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(95)00949-6 CCC: $12.00 © 1996 American Chemical Society

3452 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Arano et al.
F igu r e 1. Chemical structures of monoreactive DTPA derivative, 3,6-bis(carboxymethyl)-9-(((2-maleimidoethyl)carbamoyl)methyl)-
3,6,9-triazaundecanedioic acid (MDTPA, 1) and of diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic dianhydride (cDTPA, 3), 1-(4-
isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA, 4), and 1-[4-[(5-maleimidopentyl)amino]benzyl]eth-
ylenediaminetetraacetic acid (EMCS-Bz-EDTA, 5).
ence against the formation of intramolecular cross-
linking during the conjugation reaction with cDTPA. On
the other hand, formation of intra- and intermolecular
cross-linking has been observed in the conjugation
reaction of cDTPA with mAbs.15 Studies of the reaction
of cDTPA with insulin have also demonstrated the
unavoidable formation of such unfavorable side prod-
ucts.16 Thus, it was thought that the difference in
stability of 111In between cDTPA-conjugated mAbs and
neoglycoalbumins might be attributable to the presence
or absence of intramolecular cross-linking. In other
words, DTPA might provide 111In-labeled peptides and
polypeptides of sufficient stability for diagnostic ap-
plications in nuclear medicine if it can be incorporated
into polypeptides without inducing unfavorable side
reactions, although X-ray structural characterization
indicated that the octadentate ligand structure of DTPA
is superior to the heptadentate structure of an amide
bound DTPA for the thermodynamic and kinetic stabi-
lization of indium.17 If DTPA actually forms a 111In
chelate with sufficient in vivo stability for application
in nuclear medicine, DTPA would become an attractive
BCA, due to its ability to produce 111In-labeled polypep-
tides of high specific activities with high radiochemical
yields. This was supported by much faster 111In com-
plexation with DTPA-conjugated mAb than benzyl-
DTPA-conjugated mAb.15 However, this hypothesis can
be confirmed only when DTPA-conjugated polypeptides
can be prepared free from unfavorable side products.
In the present study, a monoreactive DTPA derivative
was designed and synthesized (MDTPA; Figure 1,
compound 1) to investigate this possibility. A mAb
against osteogenic sarcoma (IgG1, OST7) was used as a
model polypeptide because there have been numerous
studies of 111In radiolabeling of mAbs using cDTPA and
because of the ease of detection of interchain cross-
linking of antibodies by SDS-PAGE under reducing
conditions. The conjugation reaction of MDTPA with
OST7, radiochemical yields of the conjugate with 111In,
and the stability of the resulting 111In-labeled OST7 in
serum were investigated using cDTPA as a reference.
and EMCS-Bz-EDTA include an EDTA chelating group
and no DTPA group, comparable stability of 111In-Bz-
EDTA to 111In-Bz-DTPA has been documented when
conjugated to mAbs.9 Thus, both Bz-EDTA chelating
agents were used as representative BCAs to generate
111In-labeled mAbs with in vivo stability sufficient for
diagnostic applications. Reassessment of DTPA as BCA
for 111In labeling of proteins and peptides is discussed.
Ch em istr y
Syn th esis of Mon or ea ctive DTP A Der iva tive.
The monomaleimide derivative of DTPA (MDTPA,
compound 1 in Figure 1) was synthesized according to
the procedure outlined in Schemes 1 and 2. The first
isolated synthetic intermediate, compound 6 was pre-
pared by trifluoroacetylation of one terminal amine of
diethylenetriamine using ethyl trifluoroacetate, followed
by alkylation of unprotected amines with tert-butyl
bromoacetate with a yield of 66%. The amide nitrogen
was then alkylated with tert-butyl bromoacetate in the
presence of sodium hydride to produce compound 7 in
relatively high yield (71%). The trifluoroacetyl protect-
ing group was removed with anhydrous hydrazine in
dry tert-butyl alcohol, and alkylation of the resulting
secondary amine was followed by treatment with benzyl
bromoacetate to prepare compound 9 at a yield of 52%.
Catalytic hydrogenation with Pd/C in ethyl acetate
provided compound 2 in almost quantitative yield (98%).
The amine derivative of maleimide, compound 12, was
synthesized by the reaction of N-methoxycarbonylma-
leimide with N-(tert-butoxycarbonyl)ethylenediamine in
a saturated aqueous solution of NaHCO3, followed by
deprotection of the Boc group of the intermediate with
trifluoroacetic acid.
The coupling of compound 2 with compound 12 was
achieved by formation of an active ester. The carboxylic
acid of compound 2 was converted to N-hydroxysuccin-
imide ester in the presence of N,N′-dicyclohexylcarbo-
diimide and N-hydroxysuccinimide. The active ester
was then reacted with the amine derivative of maleim-
ide without further isolation. After purification of the
reaction mixture by silica gel chromatography, the
monomaleimide-derived tetra-tert-butyl ester of DTPA
(compound 13) was obtained in a yield of 45%. The
monomaleimide derivative of DTPA (MDTPA; com-
pound 1) was then obtained after deprotection of the
In
addition,
1-(4-isothiocyanatobenzyl)ethylene-
diaminetetraacetic acid (SCN-Bz-EDTA; Figure 1, com-
pound 4) and 1-[4-(5-maleimidopentyl)aminobenzyl]-
ethylenediaminetetraacetic acid (EMCS-Bz-EDTA; Figure
1, compound 5) were used as references to estimate the
applicability of MDTPA. Although both SCN-Bz-EDTA

Monoreactive DTPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3453
Sch em e 1a
a
i
Reagents: (a) CF3COOC2H5; (b) BrCH2COOBut, Pr2NEt; (c) BrCH2COOBut, NaH; (d) NH2NH2, ButOH; (e) BrCH2COOCH2C6H5,
iPr2NEt; (f) Pd/C, H2.
Sch em e 2a
a
i
Reagents: (a) dicyclohexylcarbodiimide, N-hydroxysuccinimide; (b) NaHCO3; (c) CF3COOH; (d) Pr2NEt; (e) CF3COOH.
tert-butyl esters in the presence of trifluoroacetic acid
at a yield of 73%.
and EMCS-Bz-EDTA-conjugated OST7s indicated two
bands at molecular masses of 25 and 50 kDa, corre-
sponding to light and heavy chains, respectively (Figure
2). However, cDTPA-OST7 showed multiple additional
bands at higher molecular weight although cDTPA-
OST7 was purified by size-exclusion HPLC to remove
intermolecular cross-linking.
111In Ra d iola belin g of Con ju ga tes. 111In labeling
of each conjugate was performed by two methods, A and
B (see the Experimental Section for details). In method
A, 111In labeling was performed in the presence of high
acetate concentration to stabilize 111In against hydroly-
sis. When non-chelate-conjugated OST7 (original OST7)
was labeled with 111In by method A, no radioactivity was
detected in the protein fractions by cellulose acetate
electrophoresis (CAE) analysis. Addition of EDTA
Con ju ga tion of MDTP A, cDTP A, SCN-Bz-EDTA,
a n d EMCS-Bz-EDTA w ith OST7. Conjugation of
MDTPA or EMCS-Bz-EDTA with the mAb against
osteogenic sarcoma (OST7, IgG1) was performed by
maleimide-thiol chemistry using the maleimide group
of each reagent and freshly thiolated OST7 by 2-imi-
nothiolane. The number of chelating agents introduced
per molecule of OST7 was estimated to be 2.2 for both
MDTPA- and EMCS-Bz-EDTA-conjugated OST7s by
measuring the thiol groups of OST7 before and after
each conjugation reaction. Conjugation of cDTPA or
SCN-Bz-EDTA with OST7 was achieved by reaction of
acid anhydride or isothiocyanate with primary amine
residues of the proteins. 111In labeling of unpurified
cDTPA- and SCN-Bz-EDTA-OST7 conjugates by method
A (see the Experimental Section) and subsequent analy-
ses by thin layer chromatography estimated the number
of each chelating agent attached per molecule of OST7
to be 2.2 for cDTPA-OST7 and 2.8 for SCN-Bz-EDTA-
OST7, respectively. When analyzed by SDS-PAGE
under reducing conditions, MDTPA-, SCN-Bz-EDTA-,
solution (100 molar excess over OST7) to the OST7
111
solution caused migration of more than 93% of the
-
In activity to the 111In-EDTA fractions (Figure 3).
Figure 4 illustrates the radioactivity profiles of OST7-
cDTPA-111In and OST7-MDTPA-111In by method A
when analyzed by size-exclusion HPLC (A-C) and CAE
(D-F). OST7-cDTPA-111In indicated a small radio-

3454 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Arano et al.
activities (3 and 6 mCi (111 and 222 MBq)/mg). OST7-
MDTPA generated 111In-labeled OST7 in radiochemical
yields of more than 92% from low to high specific
activity. No significant changes in the radiochemical
purities were observed before and after EDTA addition
to OST7-MDTPA-111In. OST7-cDTPA showed lower
radiochemical yields than OST7-MDTPA with similar
radiochemical yields under both radiolabeling condi-
tions. Both OST7-SCN-Bz-EDTA and OST7-EMCS-
Bz-EDTA indicated radiochemical yields similar to
OST7-MDTPA at low specific activity. However, sig-
nificant decreases in the radiochemical yields were
observed with increases in the specific radioactivity.
Although OST7-SCN-Bz-EDTA contained a slightly
more chelating agent per molecule of protein than
OST7-EMCS-Bz-EDTA, the latter provided higher ra-
diochemical yields.
F igu r e 2. SDS-PAGE profiles of the four conjugates under
reducing conditions. Molecular weight standards (lane 6) are
myosin (200 kDa), â-galactosidase (116.25 kDa), phospholi-
pase-b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbu-
min (45 kDa), carbonoic anhydrase (31 kDa), and trypsin
inhibitor (21.5 kDa). OST7-SCN-Bz-EDTA (lane 2), OST7-
EMCS-Bz-EDTA (lane 3), and OST7-MDTPA (lane 4) indi-
cated two bands corresponding to heavy and light chains,
respectively, as also observed with the unmodified OST7 (lane
1). On the other hand, OST7-cDTPA (lane 5) showed multiple
bands with molecular weights higher than the other two
bands, although intermolecular cross-linking was excluded by
size-exclusion HPLC prior to SDS-PAGE analysis. Intramo-
lecular cross-linking was thus demonstrated in the OST7-
cDTPA conjugates.
Sta bility Estim a tion of 111In -La beled OST7s.
OST7s labeled with 111In using MDTPA, cDTPA, and
SCN-Bz-EDTA as the BCAs were purified by size-
exclusion HPLC and incubated at 37 °C in freshly
prepared human sera. Figure 5 illustrates typical
radioactivity profiles of size-exclusion HPLC of OST7-
cDTPA-111In before incubation (Figure 5A) and after
48 h of incubation (Figure 5B). The size-exclusion
HPLC profile of the radioactivity of 111In-labeled apo-
transferrin is also shown in Figure 5C. At 48 h
incubation, approximately 15% of the initial radioactiv-
ity of OST7-cDTPA-111In was present in the same
fractions containing transferrin. These results are
summarized in Figure 6, where the initial radiochemical
purities of each 111In-labeled OST7 are normalized to
100%. OST7-cDTPA-111In released the radioactivity
after incubation, and 26% of the initial radioactivity was
detected at the transferrin fractions at 120 h postincu-
bation. On the other hand, only a slight migration of
the radioactivity (less than 5%) to the transferrin
fractions was observed even at 120 h postincubation of
OST7-SCN-Bz-EDTA-111In. OST7-MDTPA-111In sig-
nificantly reduced the migration of radioactivity from
antibody to transferrin fractions when compared with
OST7-cDTPA-111In. At 48 and 120 h postincubation,
OST7-MDTPA-111In liberated approximately 6 and
10% of the initial radioactivity, respectively.
F igu r e 3. Cellulose acetate electrophoresis profiles of 111In-
labeled unmodified OST7 by method A (see the Experimental
Section) before (left) and after (right) EDTA addition. No
radioactivity was found at the protein fractions, and more than
94% of the radioactivity was detected at the 111In-EDTA
fractions after addition of EDTA. Thus, hydrolysis of 111In was
minimized under these conditions.
Discu ssion
activity peak other than the protein fraction, which was
observed at a longer retention time (ca. 26 min) on
HPLC and ca. 0-1 cm anode on CAE (Figure 4A,D).
While HPLC purification of the OST7-cDTPA-111In
increased the radiochemical purity as determined by
HPLC analysis (Figure 4B), CAE analysis still indicated
the presence of ca. 20% of the non-protein-associated
radioactivity at the 0-1 cm anode (Figure 4E). Addition
of excess EDTA and subsequent purification by centri-
fuged gel-filtration column did not improve the radio-
chemical purities of OST7-cDTPA-111In determined by
CAE analysis (data not shown). On the other hand,
more than 92% of the radioactivity of OST7-MDTPA-
111In was present in the antibody fraction as determined
by both HPLC (Figure 4C) and CAE (Figure 4F)
analyses without any further purification.
A monoreactive DTPA with a coordination geometry
similar to cDTPA can be prepared by protecting one
carboxylate attached to a terminal amine with a reagent
that can be removed under conditions different from the
protecting group of the other four carboxylates in the
DTPA skeleton. We selected the benzyl-protecting
group for one carboxylate and the tert-butyl protecting
group for the rest of the four carboxylates (compound
9). Since compound 9 has an asymmetrical structure,
the symmetrical diethylenetriamine was first modified
to an asymmetrical structure by acylation of one of the
two terminal amines with ethyl trifluoroacetate, fol-
lowed by alkylation of the unprotected primary and
secondary amines with tert-butyl bromoacetate to gen-
erate compound 6. Alkylation of the amide nitrogen of
this molecule was performed with another tert-butyl
bromoacetate in the presence of sodium hydride to
produce compound 7. In the deprotection reaction of
the trifluoroacetyl group of compound 7, the standard
procedure using aqueous NaHCO3 solution failed to
Radiochemical yields of the four 111In-labeled OST7s
are summarized in Table 1. Method A was used for
preparation of 111In-labeled OST7s at the specific activ-
ity of 0.25 mCi (9.25 MBq)/mg, whereas method B was
employed for 111In labeling of OST7s at higher specific

Monoreactive DTPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3455
F igu r e 4. Size-exclusion HPLC (A-C) and cellulose acetate electrophoresis (D-F) profiles of OST7-cDTPA-111In and OST7-
MDTPA-111In by method A. Panels A and D are of OST7-cDTPA-111In; panels B and E are of OST7-cDTPA-111In after
purification by size-exclusion HPLC; panels C and F are of OST7-MDTPA-111In. OST7-cDTPA-111In showed a radioactivity
peak in fractions other than those of protein (A and D). Although size-exclusion HPLC purification was shown to increase
radiochemical purity of OST7-cDTPA-111In by repeated size-exclusion HPLC analysis (B), CAE analysis indicated the presence
of such radioactive species (E). OST7-MDTPA-111In showed much higher radiochemical yields by both analytical methods (C
and F).
Ta ble 1. Radiochemical Yieldsa of 111In-Labeled OST7s
specific
activity
OST7-
OST7-
OST7-
OST7-
(mCi/mg) MDTPA-111In (+)-EDTAb cDTPA-111In (+)-EDTAb SCN-Bz-EDTA-111In (+)-EDTAb EMCS-Bz-EDTA-111In (+)-EDTAb
0.25
3.0
6.0
92.5 (1.0)
94.5 (1.5)
93.3 (1.6)
93.3 (1.1)
93.4 (0.5)
92.4 (1.6)
70.2 (3.5)
ndc
64.5 (5.4)
65.5 (5.8)
nd
64.2 (8.2)
92.9 (1.5)
24.5 (8.3)
5.3 (2.6)
93.7 (0.7)
12.8 (2.6)
3.8 (1.8)
92.3 (1.4)
76.1 (9.0)
30.4 (5.3)
91.5 (2.1)
72.1 (13.1)
22.7 (1.3)
a
b
Radiochemical yields determined by CAE. Each value represents the mean (1 sd) for three experiments. To estimate nonspecific
binding of 111In in each preparation, EDTA was added to each 111In-labeled OST7 (EDTA to OST7 molar ratio of 100:1). After incubation
for 1 h at 23 °C, the antibody-associated radioactivity was reanalyzed by CAE. c nd ) not determined.
cleave the protecting group. Deprotection under stron-
ger alkaline conditions accompanied solvolysis reactions
of the tert-butyl ester groups. Among the various
reagents tested, anhydrous hydrazine in dry tert-butyl
alcohol provided a high degree of selective deprotection.
The resulting secondary amine of this key intermediate
was then alkylated with benzyl bromoacetate to produce
compound 9. The precursor of the monoreactive DTPA,
compound 2, was obtained in high yield by the catalytic
hydrogenation of the benzyl ester.
This compound is a monocarboxylic acid derivative
of DTPA with the rest of the four carboxylates being
protected with acid-removable tert-butyl esters, which
are soluble in organic solvents such as acetonitrile and
dimethylformamide (DMF). Since low reaction yields
of cDTPA with oligopeptides were caused by intermo-
lecular cross-linking, even when the reactions were
performed with oligopeptides possessing only one amine
residue per molecule or under solid-phase synthesis
conditions,1,2,18 compound 2 would serve as a good
reagent to introduce a DTPA skeleton to oligopeptides
with high reaction yields. The high solubility of com-
pound 2 in organic solvent would render the reagent
versatile for incorporating DTPA groups into oligopep-
tides by either liquid- or solid-phase synthesis.
carboxylic acid of compound 2 to N-hydroxysuccinimide
ester and subsequently deprotected the tert-butyl esters.
However, compound 10 underwent decomposition dur-
ing purification by silica gel column chromatography.
Use of other alcohols such as tetrafluorophenol in place
of N-hydroxysuccinimide did not allow isolation of the
active ester precursor. Therefore, we selected a male-
imide group as the protein binding site, and freshly
prepared unpurified compound 10 was reacted with an
amine derivative of maleimide, compound 12. After
column chromatography on silica gel, compound 13 was
isolated in pure form. The final product, MDTPA
(compound 1), was then obtained by cleavage of the tert-
butyl ester in the presence of trifluoroacetic acid.
MDTPA was conjugated to OST7 by maleimide-thiol
chemistry using 2-IT.19,20 A similar method was em-
ployed for EMCS-Bz-EDTA conjugation of OST7 while
cDTPA and SCN-Bz-EDTA were conjugated by reaction
of amine residues of the mAb.8,9,11,21-23 When analyzed
by SDS-PAGE under reducing conditions, MDTPA-,
EMCS-Bz-EDTA-, and SCN-Bz-EDTA-conjugated OST7s
demonstrated two bands, corresponding to the light and
heavy chains (Figure 2), which indicated the absence
of intermolecular and intramolecular (interchain) cross-
linking of the three conjugates. However, although
OST7-cDTPA was purified from intermolecularly cross-
linked forms by size-exclusion HPLC prior to analysis,
SDS-PAGE of cDTPA-OST7 showed multiple bands
at a molecular mass higher than 66 kDa as well as the
The monoreactive DTPA derivative was prepared as
illustrated in Scheme 2. When considering the struc-
tural similarity to cDTPA, a monoactive ester of DTPA
would be desirable. We attempted to convert the free

3456 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Arano et al.
hydrolysis kinetics of 111In under these reaction condi-
tions was confirmed by the high-yield production (>94%)
of 111In-EDTA chelate after addition of EDTA to a
mixed solution of 111In acetate and unmodified OST7
(Figure 3). Under these conditions, MDTPA-, SCN-Bz-
EDTA-, and EMCS-Bz-EDTA-conjugated OST7s showed
radiochemical yields of more than 92% as determined
by CAE analyses. However, OST7-cDTPA-111In indi-
cated significantly lower radiochemical yields (Table 1).
Furthermore, although size-exclusion HPLC purification
of OST7-cDTPA-111In increased the radiochemical
purity (>95%) when reanalyzed by HPLC, CAE analysis
demonstrated that ca. 20% of the radioactivity was still
present in the non-protein fractions (Figure 4E). The
addition of a large amount of EDTA to the OST7-
cDTPA-111In did not alter the antibody-associated
radioactivity when analyzed by CAE as shown in Table
1. These results indicated that OST7-cDTPA-111In
contained 111In species that could not be removed by
size-exclusion HPLC purification or EDTA chase but
were dissociated from the conjugate during CAE analy-
sis. Similar results were observed with 111In radiola-
beling of OST7-MDTPA and OST7-cDTPA at higher
specific activities by method B. While OST7-MDTPA
afforded radiochemical yields of over 92%, significantly
lower yields were observed with OST7-cDTPA (ca. 65%)
(Table 1). Since MDTPA possesses coordination geom-
etry similar to cDTPA when cDTPA is introduced
without any side reactions, the differences in radio-
chemical yield between MDTPA- and cDTPA-conjugated
OST7 would be attributable to the presence of intramo-
lecular (interchain) cross-linking in OST7-cDTPA, as
demonstrated by SDS-PAGE (Figure 2). DTPA mol-
ecules that formed intramolecular cross-links may have
acted as another chelating site for 111In and hindered
111In complexation with the DTPA moiety in OST7-
cDTPA. Since such 111In species were not removed by
size-exclusion HPLC, which is commonly used for the
purification of 111In-cDTPA-labeled polypeptides, con-
tamination by such 111In species in 111In-cDTPA-labeled
polypeptides would constitute one reason for the ob-
served instability of the radiolabel.
To further characterize the 111In complexation reac-
tion with respect to ligand structure, radiochemical
yields of MDTPA-OST7 were compared with the two
Bz-EDTA-conjugated OST7s. While MDTPA-OST7
provided radiochemical yields of over 92% under the
three conditions tested, both Bz-EDTA-conjugated OST7s
showed decreased radiochemical yields with increases
in their specific activities. These results may have been
due to the rigid ligand structure of Bz-EDTA chelating
agents, which confers high kinetic stability on the
resulting 111In chelate but which may also hinder the
complexation reaction. These findings indicated an
important advantage of monoreactive DTPA for the
preparation of 111In-labeled polypeptides at high specific
activities. The higher radiochemical yields of EMCS-
Bz-EDTA-OST7 than SCN-Bz-EDTA-OST7 suggested
the importance of the linkage structure between protein
binding site and chelating site to increase the number
of chelating groups available for complexation with
metal ions.24,25 Although a similar explanation may
account for the radiochemical yields of MDTPA- and
cDTPA-OST7s, the similar radiochemical yields of
OST7-cDTPA-111In under the two radiolabeling condi-
F igu r e 5. Size-exclusion HPLC profile of OST7-cDTPA-111
-
In before (A) and after 48 h (B) incubation in human serum
at 37 °C and size-exclusion HPLC profile of 111In-labeled
apotransferrin (C). Approximately 15% of the radioactivity
was transferred from the antibody to the fractions containing
transferrin during 48 h incubation.
F igu r e 6. Percent radioactivity in the antibody fractions after
incubation of 111In-MDTPA-OST7, 111In-cDTPA-OST7, and
111In-SCN-Bz-EDTA-OST7 in freshly prepared human serum
at 37 °C. Each value was calculated by dividing the antibody-
bound radioactivity at various incubation times by the radio-
chemical purities of respective 111In-labeled antibody imme-
diate after size-exclusion HPLC purification. OST7-MDTPA-
111In liberated radioactivity slightly higher and much lower
than OST7-SCN-Bz-EDTA-111In and OST7-cDTPA-111In,
respectively. The radioactivity was transferred from the
antibody to serum components such as transferrin.
two bands corresponding to heavy and light chains
(Figure 2). Although cDTPA conjugation reactions were
performed at various cDTPA-to-OST7 molar ratios,
similar SDS-PAGE profiles were observed. Thus,
intramolecular (interchain) cross-linking was unavoid-
able when cDTPA was used as a BCA with polypeptides,
as has been documented previously.15,16 The effects of
such side reactions on complexation with 111In and on
the stability of the resulting 111In-labeled protein were
then examined.
Since the radiolabeling of 111In with chelate-conju-
gated proteins is a competitive reaction between com-
plexation and hydrolysis of 111In, radiolabeling of each
conjugate was performed at high acetate concentration
to minimize the hydrolysis of 111In (method A). Slow

Monoreactive DTPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3457
vacuo, and the oily residue was dissolved in dry acetonitrile
(40 mL). The solution was cooled below 0 °C, and N,N-
diisopropylethylamine (4.65 g, 36 mmol) was added to the
solution, followed by tert-butyl bromoacetate (7.02 g, 36 mmol)
while the reaction temperature was kept below 0 °C. After
the reaction mixture was stirred at room temperature over-
night, the solution was concentrated in vacuo. Ethyl acetate
(50 mL) was added to the residue, and the organic phase was
washed three times with a saturated aqueous solution of
sodium bicarbonate. After the organic layer was dried over
anhydrous calcium sulfate, ethyl acetate was removed in
vacuo. The oily residue was chromatographed on silica gel
using dichloromethane-methanol (70:1) as an eluent to pro-
duce compound 6 (3.68 g, 66%) as a pale yellow oil. 1H-NMR
(CDCl3): δ 1.45 (27H, s, t-Bu), 2.75-2.79, 2.83-2.88 (6H, m,
NCH2CH2NCH2), 3.29 (2H, s, NCH2COO-t-Bu), 3.33-3.38 (2H,
m, CH2NHCO), 3.44 (4H, s, NCH2COO-t-Bu), 8.59 (1H, br,
amide). FABMAS calcd for C24H43F3N3O7 (MH+): m/z 542,
found 542. Anal. Calcd for (C24H42F3N3O7) C, H, N.
Syn th esis of Di-ter t-bu tyl 3,6-Bis((ter t-bu toxyca r bon -
yl)m et h yl)-9-(t r iflu or oa cet yl)-3,6,9-t r ia za u n d eca n ed io-
a te (7). To a suspension of 60% sodium hydride (0.122 g, 3.05
mmol) in dry DMF (13 mL) was added compound 6 (1.38 g,
2.54 mmol) in dry DMF (2 mL) to maintain the reaction
temperature below -15 °C. The reaction temperature was
then brought to room temperature, and stirring was continued
until evolution of hydrogen ceased. The reaction solution was
chilled to below 0 °C again, and tert-butyl bromoacetate (0.74
g, 3.81 mmol) was added dropwise while the reaction temper-
ature was kept below 0 °C. The solution was warmed to room
temperature again, and the stirring was continued overnight.
Ethyl acetate (50 mL) was added to the reaction solution, and
the organic phase was washed three times with a saturated
aqueous solution of sodium bicarbonate. The organic phase
was dried over anhydrous calcium sulfate, and the solvent was
removed in vacuo. The oily residue was chromatographed on
silica gel using ethyl acetate-hexane (1:6) as an eluent to
produce compound 7 as a pale yellow oil (1.18 g, 71%). 1H-
NMR (CDCl3): δ 1.45-1.47 (36H, s, t-Bu), 2.76-2.79, 2.85-
2.92 (6H, m, NCH2CH2NCH2), 3.31 (2H, s, NCH2COO-t-Bu),
3.42 (4H, s, NCH2COO-t-Bu), 3.55 (2H, m, CH2NCOCF3), 4.16
(1H, s, NCH2COO-t-Bu), 4.29 (1H, d, NCH2COO-t-Bu). FAB-
MAS calcd for C30H53F3N3O9 (MH+): m/z 656, found 656. Anal.
Calcd for (C30H52F3N3O9) C, H, N.
tions (methods A and B) suggested that this hypothesis
is unlikely.
Finally, stability of the label, 111In, derived from
cDTPA-, MDTPA-, and Bz-EDTA-conjugated antibodies
was estimated in freshly prepared human serum.
OST7-cDTPA-111In released significantly more radio-
activity than OST7-MDTPA-111In and OST7-SCN-Bz-
EDTA-111In (Figure 6). Most of the radioactivity
released from OST7-cDTPA-111In was observed in the
same fractions containing transferrin (Figure 5), rein-
forcing the involvement of transchelation of 111In from
cDTPA-conjugated antibodies to transferrin in
plasma.15,26-29 Since OST7-cDTPA-111In contained ca.
20% of the non-chelate-associated radioactivity even
after size-exclusion HPLC purification, and the differ-
ence in radioactivity release between OST7-cDTPA-
111In and OST7-MDTPA-111In was close to the value
(ca. 15%) at 120 h postincubation (Figure 6), the
presence of nonchelated 111In species in OST7-cDTPA-
111In appears to account for the higher radioactivity
release from the conjugate. These studies also indicated
that Bz-EDTA forms an 111In chelate with a little higher
kinetic stability than DTPA even when DTPA was
conjugated to mAb without inducing intra- or intermo-
lecular cross-linking.
In conclusion, these findings suggested that low in
vivo stability of 111In-labeled polypeptides derived from
cDTPA may be caused primarily by a formation of
intramolecular cross-linking during conjugation reac-
111
tions and may not be due to inherent stability of
-
In-DTPA chelate. Use of monoreactive DTPA in place
of cDTPA as the BCA increased the radiochemical yield
and the stability in serum of 111In-labeled polypeptide.
Furthermore, MDTPA facilitated generation of 111In-
labeled OST7 in high radiochemical yields even when
radiolabeling was performed at high specific activities.
Thus, MDTPA and its precursor appear to be appropri-
ate BCAs for 111In radiolabeling of polypeptides at high
specific activities. However, their applications would
be limited to polypeptides with rapid blood clearance
due to the slightly lower stability of 111In-MDTPA than
Bz-EDTA-111In. The discrepancies in the stability
between 111In-cDTPA-labeled mAbs and neoglycoalbu-
mins could be attributed to the higher level of intramo-
lecular cross-linking in the former and much faster
blood clearance rate of the latter.
Syn th esis of 1-Ben zyl ter t-Bu tyl 3,6,9-Tr is((ter t-bu -
t oxyca r b on yl)m et h yl)-3,6,9-t r ia za u n d eca n ed ioa t e (9).
Compound 7 (2.81 g, 4.28 mmol) was dissolved in tert-butyl
alcohol (28 mL), and anhydrous hydrazine (13.5 mL, 428
mmol) was added below 0 °C. After 3.5 h of stirring at the
same temperature, dichloromethane (150 mL) was added to
the reaction mixture, and the organic phase was washed with
a saturated aqueous solution of sodium bicarbonate. The
organic layer was separated and dried over anhydrous calcium
sulfate, and the solvent was removed in vacuo. The residue
was then dissolved in dry DMF (22 mL), and N,N-diisopropy-
lethylamine (0.78 g, 6.03 mmol) was added. After the reaction
solution was chilled to below 0 °C, benzyl bromoacetate (1.38
g, 6.03 mmol) was added dropwise while the temperature was
maintained below 0 °C. The solution was warmed to room
temperature, and stirring was continued overnight. Ethyl
acetate (150 mL) was added to the solution, and the organic
phase was washed with a saturated aqueous solution of sodium
bicarbonate. After the organic layer was dried over anhydrous
calcium sulfate, the solvent was removed in vacuo. The
residue was chromatographed on silica gel using ethyl acetate-
hexane (1:3) as an eluent to produce compound 9 (1.59 g, 52%)
as a pale yellow oil.
Exp er im en ta l Section
Proton nuclear magnetic resonance (1H-NMR) spectra were
recorded on an AC 300 (300 MHz) spectrometer (Bruker), and
the chemical shifts are reported in ppm downfield from an
internal tetramethylsilane standard. Fast atom bombardment
mass spectra (FABMS) were obtained with a J MS-HX/HX110
A model (J EOL Ltd., Tokyo, J apan). Size-exclusion HPLC was
performed with Cosmosil 5Diol-300 (7.5 × 600 mm) eluted with
0.1 M phosphate buffer (pH 6.8) at a flow rate of 1 mL/min.
Mon oclon a l An tibod y. The monoclonal antibody against
osteogenic sarcoma (OST7, IgG1) was produced by standard
hybridoma technology, as described previously.30
1H-NMR (CDCl3): δ 1.44 (36H, s, t-Bu),
2.74-2.86 (8H, m, NCH2CH2NCH2CH2N), 3.33 (2H, s, NCH2-
COO-t-Bu), 3.43 (4H, s, NCH2COO-t-Bu), 3.46 (2H, s, NCH2-
COO-t-Bu), 3.64 (2H, s, NCH2COOBz), 5.14 (2H, s, CH2Ph),
7.33-7.36 (5H, m, aromatic). FABMAS calcd for C37H62N3O10
Syn th esis of Di-ter t-bu tyl 3-((ter t-Bu toxyca r bon yl)-
m eth yl)-6-(2-((tr iflu or oa cetyl)a m in o)eth yl)-3,6-d ia za oc-
ta n ed ioa te (6). Diethylenetriamine (1.06 g, 10.3 mmol) was
dissolved in dry dichloromethane (8 mL), and ethyl trifluoro-
acetate (1.46 g, 10.3 mmol) in dry dichloromethane (12 mL)
was added dropwise to keep the reaction temperature below
0 °C. After 2 h of stirring below 0 °C, the reaction mixture
was stirred for an additional 1.5 h at room temperature. The
solvent and unreacted ethyl trifluoroacetate were removed in
(MH+): m/z 708, found 708. Anal. Calcd for (C37H61N3O10
)
C, H, N.
Syn th esis of 1-ter t-Bu tyl Hyd r ogen 3,6,9-Tr is((ter t-
bu toxyca r bon yl)m eth yl)-3,6,9-tr ia za u n d eca n ed ioic Acid
(2). A mixture of compound 9 (1.57 g, 2.22 mmol) and 10%

3458 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Arano et al.
palladium on carbon (0.1 g) in ethyl acetate (18 mL) was
hydrogenated at room temperature for 6 h. The catalyst was
removed by filtration, and the filtrate was evaporated in vacuo
to provide compound 2 as a pale yellow oil (1.36 g, 98%). 1H-
NMR (CDCl3): δ 1.45-1.47 (36H, s, t-Bu), 2.85-3.00 (8H, m,
NCH2CH2NCH2CH2N), 3.44 (4H, s, NCH2COO-t-Bu), 3.47 (2H,
s, NCH2COO-t-Bu), 3.48 (2H, s, NCH2COO-t-Bu), 3.49 (2H, s,
NCH2COOH), 5.16 (1H, br, COOH). FABMAS calcd for
C30H56N3O10 (MH+): m/z 618, found 618. Anal. Calcd for
(C30H55N3O10‚1/2H2O) C, H, N.
Syn th esis of N-(2-((ter t-Bu toxyca r bon yl)a m in o)eth yl)-
m a leim id e (11). To a solution of N-(tert-butoxycarbonyl)-
ethylenediamine31 (1.28 g, 8 mmol) in a saturated aqueous
bicarbonate (50 mL) was added N-(methoxycarbonyl)maleim-
ide32 (1.55 g, 10 mmol) at 0 °C. After 30 min of stirring at the
same temperature, the mixture was stirred for an additional
1 h at room temperature. The aqueous phase was extracted
with chloroform (50 mL) three times. The combined organic
phase was dried over anhydrous calcium sulfate, concentrated
in vacuo, and chromatographed on silica gel using a mixture
of chloroform-acetonitrile (20:1) as an eluent to yield com-
pound 11 (0.9 g, 46.8%). 1H-NMR (CDCl3): δ 1.41 (9H, s, t-Bu),
3.30-3.36 (2H, m, CH2NHBoc), 3.64-3.68 (2H, m, CH2N-
maleimide), 4.76 (1H, br, NH-Boc), 6.71 (2H, s, maleimide).
FABMAS calcd for C11H17N2O4 (MH+): m/z 241, found 241.
Anal. Calcd for (C11H16N2O4) C, H, N.
Syn th esis of N-(2-Am in oeth yl)m a leim id e Sa lt of Tr i-
flu or oa cetic Acid (12). Compound 11 (0.4 g, 1.66 mmol) was
stirred in a mixed solution of trifluoroacetic acid (5 mL) and
anisole (200 µL, 1.84 mmol) for 1 h at room temperature. After
trifluoroacetic acid was removed in vacuo, the residue was
treated with dry ether to produce compound 12 as a white
crystal (0.33 g, 78%). 1H-NMR (DMSO-d6): δ 3.00 (2H, t,
NH2CH2), 3.66 (2H, t, CH2N-maleimide), 7.06 (2H, s, maleim-
ide), 7.96 (2H, br, NH2). FABMAS calcd for C6H9N2O2
(MH+): m/z 141, found 141. Anal. Calcd for (C6H8N2O2‚CF3-
COOH) C, H, N.
Syn th esis of Di-ter t-bu tyl 3,6-Bis((ter t-bu toxyca r bon -
yl)m eth yl)-9-(((2-m aleim idoeth yl)car bam oyl)m eth yl)-3,6,9-
tr ia za u n d eca n ed ioa te (13). To a solution of compound 2
(0.5 g, 0.798 mmol) in dry acetonitrile (5 mL) was added
dropwise a solution of N,N′-dicyclohexylcarbodiimide (175 mg,
0.85 mmol) in dry acetonitrile (1.5 mL) while the reaction
temperature was maintained below 0 °C. After 15 min of
stirring, N-hydroxysuccinimide (98 mg, 0.85 mmol) in dry
acetonitrile (0.4 mL) was added. After 30 min of stirring below
0 °C, the reaction solution was stirred at room temperature
for 5.5 h. A mixture of compound 12 (205 mg, 0.809 mmol)
and N,N-diisopropylethylamine (148 µL, 0.85 mmol) in dry
acetonitrile (3 mL) was then added to the solution, and the
reaction mixture was stirred at room temperature overnight.
After filtration of the organic salt, the filtrate was evaporated
in vacuo, and the residue was chromatographed on silica gel
using ethyl acetate as an eluent to produce compound 13 as a
pale yellow oil (267 mg, 45%). 1H-NMR (CDCl3): δ 1.45 (36H,
s, t-Bu), 2.69-2.83 (8H, m, NCH2CH2NCH2CH2N), 3.21 (2H,
s, CH2CONH), 3.30 (2H, s, NCH2COO-t-Bu), 3.34 (2H, s, NCH2-
COO-t-Bu), 3.44 (4H, s, NCH2COO-t-Bu), 3.46-3.50 (2H, m,
maleimide-NCH2CH2), 3.65-3.69 (2H, m, maleimide-NCH2-
CH2), 6.69 (2H, s, maleimide), 8.31 (1H, t, amide). FABMAS
calcd for C36H62N5O11 (MH+): m/z 740, found 740. Anal. Calcd
for (C36H61N5O11‚1/2H2O) C, H, N.
P r ep a r a tion of Con ju ga tes: MDTP A-OST7. Conjuga-
tion of MDTPA to OST7 was performed by maleimide-thiol
chemistry using 2-iminothiolane (2-IT), as described previ-
ously.19 To a solution of OST7 (10 mg/mL) in well-degassed
0.16 M borate buffer (pH 8.0) containing 2 mM ethylenedi-
aminetetraacetic acid (EDTA) was added 45 µL of 2-IT (2 mg/
mL) in the same buffer. The reaction mixture was gently
agitated at room temperature for 1 h. After the unreacted
reagent was removed by centrifuged gel-filtration column22
using Sephadex G-50 (Pharmacia Biotech, Tokyo, J apan)
equilibrated with 0.1 M phosphate-buffered saline (PBS, pH
6.0) containing 2 mM of EDTA, the protein concentration was
adjusted to 5 mg/mL with the same buffer. MDTPA solution
(92.5 µL; 8.4 mg/mL) in the same buffer was then added to
the freshly thiolated solution of OST7 (1.85 mL), and the
reaction mixture was stirred gently at room temperature for
2 h. The solution was further agitated gently for 30 min after
addition of iodoacetamide (190 µL; 10 mg/mL) in the same
buffer. The number of MDTPA groups introduced per mol-
ecule of OST7 was estimated by measuring the number of thiol
groups before and after MDTPA addition using 2,2′-dithiopy-
ridine,33 as previously described.19 The OST7-MDTPA con-
jugate was separated from unreacted small molecules using
Sephadex G-50 column chromatography (1.8 × 40 cm) equili-
brated and eluted with 20 mM of 2-(N-morpholino)ethane-
sulfonic acid (MES)-buffered saline (pH 6.0). The conjugate
fractions collected were subsequently adjusted to 1 mg/mL
with the same buffer.
EMCS-Bz-EDTA-OST7. EMCS-Bz-EDTA-OST7 conju-
gate was prepared according to the procedure described above
using EMCS-Bz-EDTA (Dojindo Labs., Kumamoto, J apan) in
place of MDTPA. The number of EMCS-Bz-EDTA groups
attached per molecule of OST7 was also estimated using 2,2′-
dithiopyridine, as described above.
cDTP A-OST7. Conjugation of cDTPA to OST7 was per-
formed according to the procedure of Paik et al.23 with slight
modifications. To a solution of OST7 (400 µL; 6 mg/mL) in
0.05 M borate-buffered saline (pH 8.5) was added 20 µL of
cDTPA (Dojindo Labs.) in dry dimethyl sulfoxide (5.7 mg/mL).
After 30 min of gentle stirring at room temperature, a small
aliquot (10 µl) was removed for 111In radiolabeling to estimate
the number of DTPA molecules conjugated to each OST7
molecule, according to the procedure described previously34,35
with slight modifications. Briefly, 10 µL of acetate buffer (0.1
M, pH 4.5) was added to the reaction mixture, and 5 µL of
111In acetate, prepared by method A (see 111In radiolabeling
section), was then added. After being left to stand at room
temperature for 1 h, protein-bound radioactivity was deter-
mined by thin layer chromatography (Merck Art 5553) devel-
oped with a mixture of 10% aqueous ammonium formate-
methanol-0.2 M aqueous citric acid (2:2:1). The remainder
of the reaction mixture was purified by size-exclusion HPLC,
followed by Sephadex G-50 column chromatography (1.8 × 40
cm) equilibrated with 20 mM of MES-buffered saline (pH 6.0).
The protein concentration was adjusted to 1 mg/mL with the
same buffer.
SCN-Bz-EDTA-OST7. 1-(4-Isothiocyanatobenzyl)ethyl-
enediaminetetraacetic acid (SCN-Bz-EDTA; Dojindo Labs.)
was conjugated to OST7 according to the previously described
procedure.13 Briefly, a 20 µL solution of SCN-Bz-EDTA in dry
DMF (15 mg/mL) was added to a 1 mL solution of OST7 (10
mg/mL) in borate-buffered saline (0.05 M, pH 8.5) at 37 °C for
20 h. OST7-SCN-Bz-EDTA was then purified by gel perme-
ation chromatography using Sephadex G-50 (1.8 × 40 cm), as
described above. The number of SCN-Bz-EDTA molecules
attached per molecule of OST7 was measured by a procedure
similar to that used for OST7-cDTPA.
Syn th esis of 3,6-Bis(ca r boxym eth yl)-9-(((2-m a leim i-
d oet h yl)ca r b a m oyl)m et h yl)-3,6,9-t r ia za u n d eca n ed ioic
Acid (MDTP A, 1). Compound 13 (120 mg, 0.16 mmol) was
stirred in a mixed solution of trifluoroacetic acid (2 mL) and
anisole (80 µL, 0.736 mmol) at room temperature for 3 h. After
trifluoroacetic acid was removed in vacuo, dry tetrahydrofuran
was added to the residue. Compound 1 was obtained as a
white crystal after addition of hexane to the solution (72.8 mg,
73%). 1H-NMR (DMSO-d6): δ 2.93-3.00 (4H, m, CH2CH2),
3.18-3.24 (8H, m, NCH2CH2NCH2CH2N), 3.40 (2H, s, NCH2-
CONH), 3.47-3.49 (6H, m, NCH2COOH), 3.98 (2H, s, NCH2-
COOH), 6.99 (2H, s, maleimide), 8.14 (1H, t, amide). FABMAS
calcd for C20H30N5O11 (MH+): m/z 516, found 516. Anal. Calcd
for (C20H29N5O11‚1/2CF3COOH‚1/2C4H8O‚H2O) C, H, N.
SDS-P AGE. Each conjugate was characterized by SDS-
PAGE (Mini PROTEAN II, BioRad Co. Ltd., Richmond, CA)
under reducing conditions using 4-20% gradient gels (BioRad
Co. Ltd.). Each conjugate (approximately 1 µg) was denatured
by dilution in an equal volume of sample buffer containing 65
mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophe-
nol blue, and 5% 2-mercaptoethanol. All the samples were
heated in boiling water for 3 min before being loaded onto the
gel. Molecular weight marker proteins (BioRad Co. Ltd.),

Monoreactive DTPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3459
which included myosin (200 kDa), â-galactosidase (116 kDa),
phosphorylase-b (97 kDa), bovine serum albumin (66 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybeen
trypsin inhibitor (22 kDa), were also treated in the same way.
111In Ra d iola belin g of Con ju ga tes. Meth od A. 111In
radiolabeling of each conjugate was performed according to
the procedure of Brechbiel et al.8 with slight modifications as
described previously.13 Briefly, 5 µL of 111InCl3 (2 mCi (74
MBq)/mL) in 0.02 M HCl was added to a mixed solution (5
µL) of 1 M sodium acetate and 1.75 M hydrochloric acid (4:1).
After 3 min, a 40 µL aliquot of conjugate or unmodified OST7
(1 mg/mL) was added, and the reaction mixture was kept at
23 °C for 1 h.
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Meth od B. Each OST7 conjugate was also labeled with
111In at higher specific activity. A solution of sodium acetate
(1M, 2.25 µL) was mixed with a 30 µL solution of 111InCl3 (2
mCi/mL) in 0.02 N HCl. After the mixture was allowed to
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for 30 min.
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0.8 mA/cm for 45 min in a veronal buffer (I ) 0.06, pH 8.6).
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111In radiolabeling of bovine apotransferrin (Nacalai Tesque,
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in 0.02 M HCl was mixed with a 40 µL solution of sodium
acetate (1 M), and the resulting 111In acetate solution (50 µL)
was added to a protein solution (150 µL; 5 mg/mL) in PBS (0.1
M, pH 7.4). After a 10 min incubation at 37 °C, a 20 µL
solution of EDTA (2 mM in PBS) was added. After a 30 min
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centrifuged gel-filtration column.
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with 111In by method B and purified by size-exclusion HPLC.
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reach a final OST7 concentration of 20 µg/mL, and the
solutions were incubated at 37 °C. After 1, 2, 3, and 5 days of
incubation, a 5-10 µL aliquot of each sample was drawn, and
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tions were subsequently collected, and the radioactivity was
determined using a well counter (ARC 2000, Aloka, Tokyo,
J apan). Each value was calculated by dividing the antibody-
bound radioactivity at various intervals by the radiochemical
purities of freshly purified respective preparations. Data are
expressed as means and sd of three experiments.
Sta tistica l An a lysis. Data are expressed as means (
standard deviation where appropriate. Results were analyzed
for statistical significant using Student’s unpaired t-test.
Differences were considered statistically significant when the
p value was less than 0.05.
Ack n ow led gm en t. The authors are grateful to
Nihon Medi-Physics Co. Ltd. (Takarazuka, J apan) for
their kind gift of 111InCl3. The authors are also grateful
to Drs. Naoshige Akimoto and Masako Akaji (Faculty
of Pharmaceutical Sciences, Kyoto University) for taking
FAB mass and NMR spectra, respectively. This work
was supported in part by a Grant-in-Aid for Developing
Scientific Research (No. 07274108 for A.Y. and No.
07672419 for Y.A.) from the Ministry of Education,
Science and Culture of J apan.

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