Maleimido-functionalized NOTA derivatives as bifunctional chelators for site-specific radiolabeling

Article abstract of DOI:10.3390/molecules16065228

Two basic and simple synthetic routes for mono- and bis-maleimide bearing 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) chelators as new bifunctional chelators are described. The syntheses are characterized by their simplicity and short reaction times, as well as practical purification methods and acceptable to very good chemical yields. The usefulness of these two synthetic pathways is demonstrated by the preparation of a set of mono- and bis-maleimide functionalized NOTA derivatives. In conclusion, these two methods can easily be expanded to the syntheses of further tailored maleimide-NOTA chelators for diverse applications.

Full text of DOI:10.3390/molecules16065228

Molecules 2011, 16, 5228-5240; doi:10.3390/molecules16065228
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Maleimido-Functionalized NOTA Derivatives as Bifunctional
Chelators for Site-Specific Radiolabeling
Christian Förster *, Maik Schubert, Hans-Jürgen Pietzsch and Jörg Steinbach
Institute of Radiopharmacy, Helmholtz-Zentrum Dresden-Rossendorf, P.O. Box 510119,
Dresden 01314, Germany
* Author to whom correspondence should be addressed; E-Mail: c.foerster@hzdr.de.
Received: 24 May 2011; in revised from: 10 June 2011 / Accepted: 17 June 2011 /
Published: 22 June 2011
Abstract: Two basic and simple synthetic routes for mono- and bis-maleimide bearing
1,4,7-triazacyclononane-N,N’,N’’-triacetic acid (NOTA) chelators as new bifunctional
chelators are described. The syntheses are characterized by their simplicity and short
reaction times, as well as practical purification methods and acceptable to very good
chemical yields. The usefulness of these two synthetic pathways is demonstrated by the
preparation of a set of mono- and bis-maleimide functionalized NOTA derivatives. In
conclusion, these two methods can easily be expanded to the syntheses of further tailored
maleimide-NOTA chelators for diverse applications.
Keywords: 1,4,7-triazacyclonone-1,4,7-triacetic acid; NOTA; site-specific; maleimide;
ethylene glycol spacer
1. Introduction
1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA) is an intensively investigated macrocyclic,
multidentate chelator used for complexation of a broad variety of bi- and trivalent metal ions [1-3].
The size and geometry of the N3O3 coordination sphere seems to be perfect for Ga3+ ions due to the
formation of a highly stable complex (log K ~ 31) [4]. The favorable complexation kinetics in aqueous
solution, even at room temperature, spotlighted NOTA as chelating agent for gallium radionuclides for
radiopharmaceutical experiments [5]. For covalent binding of NOTA to in vivo targeting entities, several
modified NOTA chelators bearing reactive functional groups were developed. Such NOTA-based

Molecules 2011, 16
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bifunctional chelators (BFC) possess either carboxyl, amino or isothiocyanate groups for conjugation
reactions with various biologically active molecules [6-8].
However, a site-specific introduction of NOTA into proteins of high molecular weight
(e.g., antibodies), which consist of large amounts of amino and carboxyl groups at their side chains, is
very difficult or impossible. Such conjugation reactions afford conjugates with an average number of
randomly located NOTA chelators per protein. Another disadvantage of this unpredictable NOTA
distribution could be a decreased target affinity of the resulting protein-NOTA conjugates, particularly
when NOTA is bound to target recognizing sites. Therefore, the introduction of NOTA via the thiol
groups of cysteine residues could be more favorable. Accessible and reactive thiol groups provided by
cysteine are often very rare compared to amino and carboxyl groups. For example, human serum
albumin (HSA) is a protein consisting of 585 amino acids (Mw ~ 67 kDa) which can be modified via
its amino, carboxyl, or thiol groups. The covalent binding of diethylene triamine pentaacetic acid
(DTPA) chelators via HSA amino groups results in a broad statistical distribution with regard to
binding position and the amount of bound DTPA. For instance, Wagner et al. were able to attach up to
17 DTPA units per HSA molecule [9]. In contrast, a modification via the intrinsic single thiol group
resulted in well-defined conjugates using maleimide-functionalized reagents [10]. Another approach of
site-specific labeling is based on the introduction of a cysteine into a cysteine-free proteinaceous
targeting molecule. Such cysteine-free targeting vectors can easily be screened, identified and selected
by phage-display technology. Tolmachev et al. are working with a recombinant anti-HER2 affibody
for molecular imaging of human epidermal growth factor receptor type 2 (HER2) expressions in malignant
tumors [11]. After introduction of a C-terminal cysteine residue into the cysteine-free anti-HER2
affibody scaffold (58 amino acids; ~7 kDa) the resulting conjugate was modified by a maleimide-bearing
57
DOTA chelator and subsequently radiolabeled with 111In [12] as well as Co [13]. This site-specific
labeling method afforded well-defined products and thus reliable and reproducible biodistribution data.
67
68
64
67
18
NOTA conjugates labeled with Ga, Ga, Cu, Cu, 111In, and potentially F via [18F]AlF [14]
are suitable for diagnostic and therapeutical approaches. To our knowledge, only a few references
referring to maleimide-functionalized NOTA chelating agents are known [15-17]. Cox et al. [15]
described the syntheses of two mono-maleimido- and one bis-maleimido-functionalized NOTA
derivatives. However, the lack of appropriate precursors necessitated a 10 steps synthetic route with an
overall yield of less than 2% for the backbone modified NOTA-maleimide product. Eder et al. [16]
presented a NOTA conjugated derivative of the vascular endothelial growth factor (VEGF). As
spacing unit between the NOTA and VEGF moieties a large poly (ethylene glycol) was introduced.
However, no information about the exact reaction conditions, yields, and analytical characterizations
were given. A recently published work of Tolmachev et al. [17] emphasizes the necessity of maleimide
derivatives of NOTA for site-specific labeling. Therefore, N-(2-aminoethyl)maleimide was coupled to
one of the carboxyl groups of NOTA forming amide bond. The resulting MMA-NOTA product was
bound to an anti-HER2 affibody having a single cysteine residue at the terminal C-position.
In the present work, we describe the convenient syntheses of a set of mono- and bis-maleimido-
functionalized-NOTA chelators bearing different linkers and one or two maleimide entities in high
chemical yields. The aim was to develop simple as well as effective synthetic routes to enable access
to tailored maleimide-functionalized NOTA chelators with high flexibility in terms of molecular size,
shape, and physicochemical properties of the resulting conjugates. The introduction of two maleimide

Molecules 2011, 16
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groups has several advantages as well as further possibilities for applications: first, the binding
probabilities are increased, which can be very helpful, especially when targeting molecules of high
molecular weight. For smaller targeting molecules, bis-maleimide NOTA derivatives can be used to
design radioactive, dimeric targeting structures to enhance their in vivo binding affinity due to
the multivalency effect [18-20]. Second, such derivatives are suitable for grafting two different
thiol-bearing species, e.g., a targeting molecule and a “pharmaceutical modifier”. Such modifiers are
used for discrete manipulation and optimization of in vivo pharmacokinetics. In general, radioactive
substances should be characterized by low blood circulation times to minimize radiation injury of
healthy tissue. However, some radiolabeled compounds such as peptides or proteins of lower
molecular size [21] show too rapid blood clearances. Schlesinger et al. [22,23] investigated radiolabeled,
mirror-image L-oligonucleotides (L-ON) as a complementary system for tumor-pretargeting-
approaches. Biodistribution studies showed that L-ON exhibit very low blood availabilities due to the
polyanionic character and therefore fast renal excretion. To increase short in vivo half-lifes the usage of
pharmaceutical modifiers are often applied [21,24-26]. Further important benefits of this strategy can
be higher targeting rates as well as enhancement of the conjugate’s polarity to reduce liver uptake,
lower immunogenicity, and minimization of unspecific tissue uptake.
2. Results and Discussion
The maleimido-functionalized NOTA chelators were synthesized by using (S)-p-SCN-Bn-NOTA 1
and (S)-p-NH2-Bn-NOTA 2 as starting compounds due to their commercial availability and to keep the
synthetic routes as short and simple as possible. Moreover, these two bifunctional NOTA chelators
already have a modified backbone with functional groups (–NCS, –NH2) for the covalent binding of
different maleimido-based residues. As pointed out in Scheme 1, there are two general possibilities for
modifications of the NOTA to create bifunctional chelators: substitution of the backbone (C-site) or
the methylene group of the acetic acid arm (N-site).
Scheme 1. General possibilities of NOTA modification.
COOH
COOH
COOH
HOOC
HOOC
HOOC
N
N
N
N
N
N
N
N
N
R
R
HOOC
HOOC
HOOC
C-site
N-site
C-site modification
NOTA chelating system
N-site modification
The first synthetic route we were investigating was based on derivatization of starting compound 1.
The isothiocyanate group was used for nucleophilic addition reactions with amino-functionalized
mono- or bis-maleimide compounds to form stable thiourea bonds (Scheme 2).

Molecules 2011, 16
Scheme 2. First reaction pathway for the syntheses of products 3a and 3b.
5231
O
TFA*H2N
HOOC
HOOC
N
O
H
H
N
N
N
N
N
O
NCS
N
N
TEA; anhydr. DMF
N
N
S
HOOC
HOOC
-TEA*TFA
O
HOOC
HOOC
1
3a
O
O
N
O
HOOC
HN
O
O
H
H
N
N
N
N
NH
N
NH
O
N
O
HOOC
S
O
O
NH
TFA*H2N
NH
N
HOOC
O
O
TEA; anhydr. DMF
-TEA*TFA
HN
O
O
1
3b
N
O
The presence of triethylamine (TEA) is necessary to transform the trifluoroacetic ammonium salts
of both maleimide starting materials into reactive primary amino groups. To prevent side-reactions
such as hydrolysis of maleimide or isothiocyanate groups, anhydrous DMF and TEA of the highest
available quality was used. HPLC was utilized for isolation of the products to provide a very high
purity which is required for this kind of application. A HPLC system based on a C-18 reverse-phase
column and water as well as methanol (both containing 0.1 v/v % of TFA) as mobile phases gave
optimal purification results. After isolation of and subsequent freeze-drying of 3a, samples were taken
and analyzed by analytical HPLC to assure its purity. Besides the expected product peak, additional
peaks appeared in all chromatograms, so product samples were taken for mass spectrometric (ESI-MS
and MALDI-TOF) analyses and the by-products were thus identified as oligomeric structures of 3a.
We also found that the amount of by-products was proportional to the time between HPLC isolation
and freeze-drying. This indicates that the formation of by-products was related to the HPLC eluents.
As mentioned above, 0.1 v/v % of TFA in both mobile phases was used to prevent maleimide
hydrolysis during and after HPLC purification. Moreover, acidic conditions provided best purification
and isolation results in terms of sufficient differences in retention times. Unfortunately, TFA
apparently shifts the tautomeric equilibrium of the thiourea bond towards the two isothiourea isomers I
and II (Scheme 3A). The reactive isothiourea-thiol-groups undergo nucleophilic addition reactions
with maleimide groups resulting in the observed oligomeric products (Scheme 3B).
To minimize the mentioned side-reactions, the product fractions were shock-frozen with liquid
nitrogen immediately after collecting from HPLC and subsequently freeze-dried. Unfortunately, this
approach was characterized by a lower reproducibility concerning the amount of by-products, so we
decided to use 0.1 v/v % of acetic acid instead of TFA in both mobile phases. These HPLC conditions
in combination with shock-freezing immediately after HPLC isolation the side-reactions were

Molecules 2011, 16
5232
prevented. Following this procedure, we were able to synthesize and characterize the mono-
maleimido-NOTA chelator 3a as well as the bis-maleimido-NOTA chelator 3b.
Scheme 3. Tautomeric equilibrium (A) and oligomerization of product 3a (B).
(A)
O
O
O
H
N
H
N
H
H
N
N
N
N
N
N
N
SH
S
SH
NOTA
NOTA
NOTA
O
O
O
isothiourea I isomer of 3a
thiourea isomer of 3a
isothiourea II isomer of 3a
O
(B)
HOOC
HOOC
N
H
H
N
O
O
N
N
N
N
O
n
N
N
N
S
N
N
N
N
N
HOOC
HOOC
HS
HS
H
N
O
O
HOOC
HOOC
3a
HOOC
N
N
COOH
N
HOOC
oligomeric by-products
n-1
Only the two amino-group-bearing maleimide compounds presented above were commercially
available. To overcome this pitfall and to prevent additional syntheses of appropriate maleimide
starting compounds, we started to work out another strategy by modifying NOTA compound 2 for the
syntheses of NOTA-maleimides 4a–4d (Scheme 4).
Scheme 4. Second reaction pathway for the syntheses of 4a–4d.
O
O
1
R
O
N
N
HOOC
HOOC
O
O
H
O
O
N
N
N
N
1
N
R
NH
2
N
N
N
HOOC
HOOC
O
O
HOOC
HOOC
1
2
4a
R
= C H
2
4
1
4b
4c
4d
R
= C H
3
6
1
R
= C H
5
10
H
1
R
=
N
O
28
O
Several maleimide derivatives with N-hydroxysuccinimidyl esters and different spacers are
available. We have chosen three aliphatic, heterobifunctional cross-linkers as well as an oligo(ethylene
glycol) (OEG) derivative containing 28 ethylene glycol units. Different spacer lengths between
targeting molecule and labeling site are often necessary to preserve the high target affinity of the native
molecule [19,20]. Moreover, we found that variation of linker length influences radiolabeling

Molecules 2011, 16
5233
efficiency (unpublished results). We also synthesized NOTA-OEG28-Mal 4d, because we purposefully
wanted to influence the biodistribution properties of our targeting vectors (L-ON for pretargeting-
approaches) by using this large spacer. Modifications of biological active molecules with OEG or
poly(ethylene glycol) (PEGylation) is a commonly applied procedure to alter pharmacokinetic
parameters such as the solubility of (radio-)pharmaceuticals [25,26]. This strategy of a simultaneous
modification of targeting molecules within one chemical reaction can be very useful to save time and
costs. Relating to our scientific interests, the OEG size of 28 units seemed to be suitable, but this
concept can easily be adjusted to bigger as well as smaller OEG spacers according to the size and
shape of the targeting molecule. Several Mal-OEGx/PEG-NHS esters are commercially available to gain
tailored NOTA-OEG/PEG-Mal derivatives (e.g., x = 2, 4, 6, 8, 24, 27 as well as up to several kDa sizes).
The product identification and isolation by HPLC was challenging because of nearly equal retention
times of maleimide-OEG28-NHS ester, product 4d, and the by-products formed by hydrolysis reactions
of Mal-OEG28-NHS ester and 4d. Using either TFA or acetic acid conditions no satisfying separations
were possible. Therefore, different buffer solutions with various pH values were tested as mobile
phases. Good results were obtained with triethylammonium acetate at pH = 5.0. The HPLC product
peak was identified by taking reaction samples to which three different chemicals were added. The
product peak shifted after the addition of GaCl3, sodium hydroxide or HS-OEG8-OMe (purchased from
IRIS Biotech). The complexation of Ga3+ ions served as indicator for the presence of NOTA, sodium
hydroxide (inducing ring opening by formation of corresponding maleic acid derivative) and
HS-OEG8-OMe (nucleophilic addition) as indicators for the presence of the maleimido group.
Short reaction times and high chemical yields were obtained when performing the reactions in DMF
in addition to phosphate buffered solution (1:1 v/v). DMF was necessary to dissolve the poorly water-
soluble maleimide starting compounds (except for the synthesis of 4d). The weak acidic conditions
(phosphate buffer, pH = 6.6) very effectively minimized maleimide hydrolysis of the formed products
during the reaction procedures. On the other hand, at this pH value the aromatic amino group of 2 is
not protonated to allow reactions with NHS ester groups of the maleimide derivatives. An excess of
maleimide reagents relative to 2 was necessary, because even at weakly acetic conditions NHS esters
are rapidly hydrolyzed into their corresponding non-reactive carboxyl groups. Unfortunately, we could
not develop an appropriate HPLC method to isolate product 4c without contamination by the starting
maleimide compound. The maleimide-NHS ester was always exhibiting nearly the same retention time
as the product 4c. Therefore, investigations using different HPLC columns for better separations have
to be undertaken. In contrast to all synthesized products, the use of 1H-NMR to characterize compound
4d was not conclusive because signals were strongly overlapped in the NMR spectrum.
Initial reactions using anhydrous DMF as solvent without the addition of phosphate buffer and TEA
as base to convert the aromatic ammonium chloride salt of 2 into a more reactive amino group, gave
unsatisfying chemical yields and very long reaction times (up to two weeks). The poor chemical yields
could be ascribed to formation of hydroxyl anions caused by the presence of water in the NOTA
starting material 2 (≤10 mass% of H2O according to supplier; 2 was delivered as p-NH2-Bn-
NOTA*4HCl), which undergo hydrolysis reactions of NHS ester and maleimide residues.
We also synthesized a bis-maleimide derivative 6 (Scheme 5). Therefore, the bis-maleimide starting
compound was reacting with succinic anhydride to introduce a carboxyl group (compound 5). In the
next step, a reactive ester of 5 was generated in-situ and subsequently coupled to starting compound 2.

Molecules 2011, 16
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Unsuccessfully, we tried to synthesize and to isolate the NHS ester of 5 in different ways.
Alternatively, we were investigating several in situ-coupling reagents such as N-(3-dimethylamino-
propyl)-N-ethylcarbodiimide
hydrochloride
(EDC·HCl),
N,N,N′,N′-tetramethyluronium-O-(N-
succinimidyl)tetrafluoroborate (TSTU), N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexa-
fluorophosphate (HBTU), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluoro-
phosphate (HATU). Best results were found when using (1-cyano-2-ethoxy-2-oxoethylidene-
aminooxy)-dimethylaminomorpholinocarbenium hexafluorophosphate (COMU). Products 4a–4d, 5
and 6 were also purified and isolated by HPLC techniques. As expected, no acid-induced side-reactions
after HPLC separation of these products occurred.
Scheme 5. Synthesis of NOTA-bis-mal product 6.
O
O
O
O
O
N
N
O
O
O
HN
HN
O
TEA; anhydr. DMF
-TEA*TFA
O
O
O
O
O
H
H
HO
C H
5
N
C H
5
N
10
10
N
TFA*H N
N
N
2
H
O
O
O
O
O
5
O
O
N
O
HN
HOOC
O
O
1) COMU, TEA, anhydr. DMF
2) 2
O
H
H
N
N
N
N
C H
5
10
5
N
N
N
H
HOOC
O
O
O
HOOC
6
3. Experimental
3.1. Materials
Chemical reagents and solvents were purchased from commercial sources in the highest available
purity and were used without further purification. (S)-p-SCN-Bn-NOTA (1) as well as (S)-p-NH2-Bn-
NOTA (2) were purchased from Macrocyclics (Dallas, TX, USA). The maleimide starting materials
were purchased from IRIS Biotech (Germany). Reactions were performed in 0.5, 1.5, and 2.0 mL
DNA low-binding tubes (Eppendorf, Germany) using a Thermomixer (Eppendorf, Germany).
1H-NMR (400 MHz) were recorded at room temperature on a Varian Inova 400 spectrometer using the
residual proton resonance in deuterated water (D2O) as reference. MALDI-TOF spectra were obtained
with a Micromass Tandem Quadropole instrument. The MALDI-TOF matrix, 3-hydroxypicolinic acid

Molecules 2011, 16
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(3-HPA), was purchased from Bruker. The matrix was a mixture of 3-HPA (50 mg/mL in ultrapure
water and CH3CN 1:1) and diammonium hydrogen citrate (100 mg/mL) with a ratio of 10:1. For
ESI-MS-spectrometry all products were dissolved in Ultra-pure water.
3.2. Chromatographic Purifications and Analyses
HPLC analyses were performed with following equipment and methods: The HPLC columns used
were: (A) Eurospher 100 C18 column (Knauer, Germany), 5 μm, 4 × 250 mm; (B) Eurospher 100 C18
column (Knauer, Germany), 5 μm, 8 × 250 mm. Method A1: flow 1 mL/min, UV 220 nm, water-methanol
(90/10 v/v %) containing 0.1 v/v % acetic acid (a) and water-methanol (10/90 v/v %) containing
0.1 v/v % acetic acid (b), gradient (a): 0 min 100 %, 0–20 min 0%. Method A2: flow 1 mL/min, UV
220 nm, water-methanol (90/10 v/v %) containing 0.1 vol % trifluoroacetic acid (TFA) (a) and
water-methanol (10/90 v/v %) containing 0.1 v/v % TFA (b), gradient (a): 0 min 100%, 0–20 min 0%.
Method A3: flow 1 mL/min, UV 220 nm, water containing 0.1 v/v % TFA (a) and water-acetonitrile
(30/70 v/v %) containing 0.1 v/v % TFA (b), gradient (a): 0 min 100%, 0–36 min 49%. Method A4:
flow 1 mL/min, UV 220 nm, water-acetonitrile (90/10 v/v %) containing 1 mM triethyl-ammonium
acetate (TEAA; pH = 5.0) (a) and water-acetonitrile (10/90 v/v %) (b), gradient (a): 0 min 100%, 0–25 min
50%. Method B1: flow 3 mL/min, UV 254 nm, water-methanol (90/10 v/v %) containing 0.1 vol %
acetic acid (a) and water-methanol (10/90 v/v %) containing 0.1 v/v % acetic acid (b), gradient (a): 0 min
100%, 0–30 min 0%. Method B2: flow 3 mL/min, UV 220 nm, water-methanol (90/10 v/v %)
containing 0.1 vol % TFA (a) and water-methanol (10/90 v/v %) containing 0.1 v/v % TFA (b),
gradient (a): 0 min 100 %, 0–30 min 0%. Method B3: flow 3 mL/min, UV 220 nm, water containing
0.1 vol % TFA (a) and water-acetonitrile (30/70 v/v %) containing 0.1 vol % TFA (b), gradient (a): 0 min
100%, 0–56 min 49%. Method B4: flow 3 mL/min, UV 220 nm, water-acetonitrile (90/10 v/v %)
containing 1 mM triethyl-ammonium acetate (TEAA; pH = 5.0) (a) and water-acetonitrile (10/90 v/v %)
(b), gradient (a): 0–5 min 100%, 5–40 min 50%.
3.3. General Procedure for the Syntheses of Thiourea Bound NOTA-Maleimides
To a solution of 1 (7.8 mg, 15 μmol) in anhydrous dimethylformamide (DMF, 100 μL)
triethylamine (8.5 μL, 60.8 μmol) was added. The corresponding ammonium trifluoroacetate
maleimide (15.8 μmol) was dissolved in anhydrous DMF (100 μL) and added dropwise to the solution
of 1. The reaction mixture was incubated at 25 °C for 2 h. Acetic acid (21 μL, 369 μmol) was added
and the reaction mixture was fractionized by HPLC. HPLC-fractions containing the desired product
were immediately shock-frozen with liquid nitrogen and lyophilized.
(S)-2-{2-(4-{3-[2-(2,5-dioxo-2,5-dihydro-(1H)-pyrrole-1-yl)ethyl]thiourea}benzyl)-4,7-bis(carboxy-methyl)
-1,4,7-triazacylononane}acetic acid (3a). HPLC method A1: tR = 11.4 min. HPLC method B1:
tR = 14.5 min. Yield: 3.3 mg (38%; white powder). ESI-MS m/z 591 [M+H]+, 613 [M+Na]+. 1H-NMR:
2.92–4.03 (m, 23H), 7.00 (s, 2H, CH=CH), 7.32 (d, J = 7.6 Hz, 2H, aromatic CH), 7.51 (d, J = 7.6 Hz,
2H, aromatic CH).

Molecules 2011, 16
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(S)-2-(2-{4-[3-(6-{2,6-bis[3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamido]-hexanamido}-hexyl)
thiourea]benzyl}-4,7-bis(carboxymethyl)-1,4,7-triazacylononane)acetic acid (3b). HPLC method A1:
tR = 17.3 min. HPLC method B1: tR = 19.9 min. Yield: 4.5 mg (28%; white powder). ESI-MS m/z 510
1
[M+H+Na]2+, 521 [M+2Na]2+, 997 [M+H]+, 1019 [M+Na]+, 1035 [M+K]+. H-NMR: 1.09–1.60
(m, 14H, CH2), 1.91 (s, 3H, CH3COOH), 2.28–2.41 (m, 4H, CH2CONH), 2.60–3.73 (m, 29H), 3.93 (t,
J = 7.2 Hz, 1H, CH), 6.68 (s, 4 H, CH=CH), 7.06 (d, J = 7.6 Hz, 2H, aromatic CH), 7.18 (m, 2H,
aromatic CH).
3.4. General Procedure for the Syntheses of Amide Bound NOTA-Maleimides
To a solution of 2 (2.8 mg, 5 μmol) in phosphate buffer (150 μL, pH = 6.6) the corresponding
N-hydroxysuccinimide ester functionalized maleimide (30 μmol, dissolved in 150 μL DMF) was
added. The slightly yellow dispersion was incubated at 25 °C for 1.5 h. The reaction mixture was
centrifuged and the supernatant was taken for HPLC purification. HPLC-fractions containing the
product were lyophilized.
(S)-2-(2-){4-[3-(2,5-dioxo-2,5-dihydro-(1H)-pyrrole-1-yl)propanamido]benzyl}-4,7-bis(carboxy-methyl)
-1,4,7-triazacylononane)acetic acid (4a). HPLC method A2: tR = 12.7 min. HPLC method B2:
tR = 15.3 min.Yield: 2.4 mg (86%; white powder). ESI-MS m/z 560 [M+H]+, 582 [M+Na]+. 1H-NMR:
2.53 (t, J = 6.2 Hz, 2H, CH2CONH), 2.60–3.70 (m, 19H), 3.74 (t, J = 6.2 Hz, 2H, CH2N), 6.70 (s, 2H,
CH=CH), 7.10–7.30 (m, 4H, aromatic CH).
(S)-2-(2-{4-[4-(2,5-dioxo-2,5-dihydro-(1H)-pyrrole-1-yl)butanamido]benzyl}-4,7-bis-(carboxymethyl)-
1,4,7-triazacylononane)acetic acid (4b). HPLC method A2: tR = 13.6 min. HPLC method B2:
tR = 14.8 min. Yield: 2.4 mg (83%; white powder). ESI-MS m/z 574 [M+H]+, 596 [M+Na]+, 612 [M+K]+.
1H-NMR: 1.86 (q, J = 6.6 Hz, 2H, CH2CH2CH2), 2.29 (t, J = 6.6 Hz, 2H, CH2CO), 2.40–3.80 (m, 21H),
6.62 (s, 2H, CH=CH), 7.10–7.26 (m, 4H, aromatic CH).
(S)-2-(2-{4-[6-(2,5-dioxo-2,5-dihydro-(1H)-pyrrole-1-yl)hexanamido]benzyl}-4,7-bis-(carboxymethyl)-
1,4,7-triazacylononane)acetic acid (4c). HPLC method A2: tR = 14.8 min. HPLC method B2:
tR = 16.3 min. Yield: 2.6 mg (85%; white powder). ESI-MS m/z 602 [M+H]+, 624 [M+Na]+ (the
product contains small amounts of 6-(maleimido)hexanoic acid N-hydroxysuccinimide ester due to
insufficient separation capacity).
(S)-2-[2-(4-{87-[2-(2,5-dioxo-2,5-dihydro-(1H)-pyrrole-1-yl)ethanamido]4,7,10,13,16,19,22,25,28,31,
34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,81,84-octacosaoxa-heptaoctacontan}benzyl)-4,7-bis-
(carboxymethyl)-1,4,7-triazacylononane]acetic acid (4d): To a solution of 2 (2.0 mg, 3.6 μmol) in 200 μL
phosphate buffer (pH = 6.6) Mal-OEG28-NHS ester (10.0 mg, 6.4 μmol) was added. The slightly
yellow solution was incubated at 26 °C for 2.0 h. Product 4d was isolated by HPLC separation.
HPLC-fractions containing product 4d were lyophilized to obtain 4d as a yellow-white powder. HPLC
method A4: tR = 20.4 min. HPLC method B4: tR = 32.4 min. Yield: 4.7 mg (50%; white powder).
ESI-MS m/z 944 [M+H+Na]2+, 955 [M+2Na]2+. MALDI-TOF m/z 933 [M+2H]2+, 1864 [M+H]+, 1886
[M+Na]+, 1902 [M+K]+.

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4-(6-{2,6-bis[3-(2,5-dioxo-2,5-dihydro-(1H)-pyrrole-1-yl)propanamido]hexanamido}hexylamino)-4-
oxobutanoic acid (5). To a solution of the bis-maleimide starting compound Bis-mal-Oc-NH2*TFA
(20 μmol, 13.2 mg) in anhydrous DMF-TEA (100 μL, 20 μmol, 2.0 mg, 2.8 μL) was added. Succinic
anhydride (23 μmol, 2.3 mg) was dissolved in anhydrous DMF (70 μL) and added dropwise. The
reaction mixture was incubated at 26 °C for 30 min. After the addition of TFA (40 μmol, 4.6 mg, 3 μL)
to the colorless solution, the reaction mixture was fractionized by HPLC. HPLC-fractions containing
product 5 were lyophilized to obtain 5 as a white powder. Yield: 8.1 mg (63%; white powder). ESI MS
(m/z): 647 [M+H]+, 669 [M+Na]+, 685 [M+K]+, 645 [M−H]−. HPLC method A2: tR = 13.0 min. HPLC
1
method B2: tR = 17.5 min. H-NMR: 1.32–1.50 (m, 6H, CH2), 1.52–1.66 (m, 6H, CH2CH2NH),
1.72–1.85 (m, 2H, CH2CH), 2.59–2.78 (m, 8H, CH2CO), 3.20–3.35 (m, 6H, CH2NH), 3.88–3.95 (m,
4H, CH2N), 4.21 (t, J = 7.0 Hz, 1H, CH), 6.99 (s, 4H, CH=CH), 8.13 (t, J = 4.0 Hz, 3H, NH) 8.40 (d,
J = 7.2 Hz, 1H, NH).
(S)-2-(2-{4-[4-(6-{2,6-bis[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-propanamido]-hexanamido}-
hexylamino)-4-oxobutanamido]benzyl}-4,7-bis-(carboxymethyl)-1,4,7-triazacylononane)acetic acid (6).
Compound 5 (6.7 μmol) was dissolved in anhydrous DMF and TEA (120 μL, 6.7 μmol, 0.7 mg, 0.9 μL)
was added. An anhydrous DMF solution (35 μL) of COMU (5.4 μmol, 2.3 mg) was added dropwise. After
reaction time for a maximum of 40 min at 26 °C, a solution of 2 (1.9 mg, 3.4 μmol), dissolved in DMF
(55 μL) and TEA (2 μL, 13.5 μmol, 1.4 mg), was added. The reaction mixture was incubated at 26 °C
over night. After addition of TFA (40.4 μmol, 4.6 mg, 3 μL) the reaction mixture was fractionized by
HPLC. HPLC-fractions containing product 6 were lyophilized to obtain 6 as a white powder. Yield:
1.46 mg (42 %; white powder). ESI MS (m/z): 520 [M + 2H]2+, 531 [M + H + Na]2+, 1038 [M + H]+, 1060
1
[M + Na]+. HPLC method A3: tR = 31.8 min. HPLC method B3: tR = 51.8 min. H-NMR: 1.20–1.34
(m, 6H, CH2), 1.37–1.48 (m, 6H, CH2CH2NH), 1.58–1.74 (m, 2H, CH2CHNH), 2.45–2.78 (m, 8H,
CH2CONH), 2.80–4.02 (m, 2H), 4.1 (t, J = 7.0 Hz, 1H, CH), 6.85 (s, 4H, CH=CH), 7.30 (d, J = 8.0 Hz,
2H, aromatic CH), 7.40 (d, J = 8.4 Hz, 2H, aromatic CH).
4. Conclusions
We have developed two basic and simple synthetic pathways for the preparation of a variety of new
mono- and bis-maleimido-NOTA chelators. The first route is based on nucleophilic addition reactions
of p-SCN-Bn-NOTA 1 and maleimide starting materials with primary amino groups under formation
of thiourea bonds. In the second pathway, maleimide derivatives with reactive carboxylic acid ester
residues and p-NH2-Bn-NOTA 2 were coupled to form amide bound products. The practicability of
these two methods is demonstrated by the preparation of a set of different derivatives 3a–3b, 4a–4d
and 6. The isolation of thiourea bond-bearing products 3a and 3b was initially difficult due to
side-reactions after HPLC separation. The side-reactions were suppressed by increasing the pH value
of both mobile phases during HPLC purification.
We also synthesized a derivative 4d, which is characterized by a very long spacer consisting of 28
ethylene glycol units. Such compounds can be used to enable site-specific radiolabeling and optimization
in terms of blood circulation half life, enhanced targeting rates, conjugate polarity, lower
immunogenicity, or minimization of unspecific tissue uptake. Several maleimide compounds with

Molecules 2011, 16
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reactive NHS ester and different OEG- or PEG-spacer lengths are commercially available to allow the
preparation of suitable derivatives according to individual requirements.
The described synthetic methods can be transferred to any other amino group or active ester bearing
maleimide starting material. This enables access to tailored bifunctional NOTA-maleimide chelators
for modification and subsequent radiolabeling of potential targeting molecules.
Prospective investigations include the conjugation of selected NOTA-maleimide products with
target-specific compounds as well as the evaluation of their radiolabeling ability and in vivo behavior.
Similarly, other chelating systems will be modified by utilization of the developed synthetic methods.
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