Improved syntheses and applicability of different DOTA building blocks for multiply derivatized scaffolds

Article abstract of DOI:10.1016/j.bmc.2007.11.044

DOTA (1,4,7,10-tetraazacyclodocecane-N,N′,N″,N?-tetraacetic acid), which forms extremely stable complexes with a large number of metal ions, is one of the most important and most commonly used chelators for in vivo applications such as cancer diagnosis an

Full text of DOI:10.1016/j.bmc.2007.11.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2606–2616
Improved syntheses and applicability of different DOTA
building blocks for multiply derivatized scaffolds
C. Wa¨ngler,a B. Wa¨ngler,a M. Eisenhut,a U. Haberkornb and W. Mierb,*
aGerman Cancer Research Center, Radiopharmaceutical Chemistry, INF 280, 69120 Heidelberg, Germany
bUniverstiy Clinics of Heidelberg, Radiologische Klinik INF 400, Heidelberg, Germany
Received 7 September 2007; revised 12 November 2007; accepted 16 November 2007
Available online 22 November 2007
Abstract—DOTA (1,4,7,10-tetraazacyclodocecane-N,N0,N00,N000-tetraacetic acid), which forms extremely stable complexes with a
large number of metal ions, is one of the most important and most commonly used chelators for in vivo applications such as cancer
diagnosis and therapy. However, many of the published synthesis protocols for DOTA derivatives are complicated and give the
products in low yields. Here we report improved synthesis routes for tris-tBu-DOTA, tris-benzyl-DOTA, and thiol-DOTA, and also
describe the synthesis of the novel compound tris-4-nitro-benzyl-DOTA. In addition, we determined the applicability of the DOTA
derivatives tris-tBu-DOTA, thiol-DOTA, tris-benzyl-DOTA, tris-4-nitrobenzyl-DOTA, tris-allyl-DOTA, DOTA-PFP-ester, and
DOTA-PNP-ester for multimerization reactions using amino functionalized PAMAM dendrimers of different sizes. Thiol-DOTA
was found to be the best compound for efficient reactions with dendritic scaffolds generating highly homogeneous DOTA-multimers.
This DOTA derivative could be quantitatively conjugated to a 128-mer dendrimer.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
thesecompounds,amuchhigherspecificactivitycanbeob-
tainedbymodifyingonly somesitesofthe carrier molecule.
DOTA (1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetra-
acetic acid) is one of the most important chelators in
radiochemistry as it forms complexes with extremely
high stability constants with a large number of main
group and transition metal ions. Due to this, DOTA is
the most commonly used chelator for in vivo applica-
tions such as cancer therapy and clinical diagnosis.
For this purpose, it is introduced into biomolecules such
as peptides and antibodies and subsequently labeled
with radionuclides.1–6 Furthermore, some of the cur-
rently used MRI contrast agents such as GadovistÒ,
ProHanceÒ, and DotaremÒ are based on DOTA-gado-
linium complexes. Gadomer-17Ò, a recently developed
MRI contrast agent, uses a multiply DOTA-derivatized
dendrimer which shows much longer retention times and
improved visualization properties due to higher signals
compared to low molecular weight gadolinium chelates.7
As DOTA is the chelator of choice, protocols are re-
quired that allow the facile synthesis of derivatives that
are applicable for multimeric derivatizations of biomol-
ecules. However, many of the published synthesis proce-
dures are complex or give the products in low yields.
In the current work, some synthesis protocols are im-
proved to enable faster and reliable DOTA-synthon syn-
theses. Further on, several DOTA derivatives were
studied with regard to their suitability for multimeriza-
tion reactions. PAMAM dendrimers were used as model
compounds for these reactions and were reacted with
the different DOTA derivatives to define the most suit-
able synthon for quantitative derivatizations of multi-
meric scaffold molecules.
This kind of multimerization of DOTA is also advanta-
geous for the synthesis of protein conjugates as it enables
an enhanced specific activity of biomolecules.8–10 With
2. Results and discussion
2.1. Improved synthesis of tris-tBu-DOTA, tris-benzyl-
DOTA and thiol-DOTA and synthesis of the novel
compound tris-4-nitro-benzyl-DOTA
Keywords: DOTA; Chelators; Synthesis; Multivalency; Dendrimers.
*
Conjugation reactions with sensitive biomolecules, such
as antibodies or cytokines, have to be conducted under
Corresponding author. Tel.: +49 6221 567720; fax: +49 6221
5633629; e-mail: walter.mier@med.uni-heidelberg.de
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.044

C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
2607
mild conditions. Therefore, the high number of synthe-
ses that could be used for conjugation reactions is lim-
ited to biocompatible reactions.11 For the multiple
conjugation to dendritic structures the spectrum of
applicable reactions is broader and a large spectrum of
activated chelators can be used. DOTA derivatives have
gained high importance in radiochemistry and therefore
protocols are required that allow the facile and uncom-
plicated synthesis of these compounds.
the critical points of the synthesis: the low temperature
required during the reaction and purification of the first
intermediate product (1,4,7,10-tetraaza-cyclododec-1-
yl)-acetic acid benzyl ester (1) and a rapid purification
in all steps of the synthesis (Scheme 1).
Higher reaction temperatures and long incubation with
the silica chromatography material lead to intramolecu-
lar ring formations or fragmentations of the products
requiring complex purification steps and reduced syn-
thesis yields.
Besides the in situ activation,12 two different kinds of
DOTA-derivatives are known: active esters which do
not need to be deprotected after coupling13 and pro-
tected derivatives that are reacted using additional cou-
pling reagents and deprotected after the coupling.14,15
Many of the published synthesis protocols for DOTA-
derivatives are complicated with difficult purification
steps or give the products in low yields. Especially for
tris-tBu-DOTA (3), which is the derivative of choice
for the introduction of DOTA in solid phase synthesis,
several synthesis protocols have been described in the lit-
erature.16,17 However, these protocols do not emphasize
The synthesis protocol described for thiol-DOTA (5)18
starts from tris-tBu-DOTA which is either expensive
or has to be synthesized according to the multi-step pro-
tocol described above. Furthermore, the slow deprotec-
tion of the tBu-groups and fragmentation products
produced during the long incubation time in TFA result
in low yields (Fig. 1).
The synthesis can be simplified by starting from DOTA
that is directly reacted with S-trityl-protected mercap-
O
O
O
O
OH
O
O
H
H
H
H
H
H
N
N
N
N
N
N
N
N
N
N
N
b
N
N
N
a
c
O
O
O
O
O
N
N
H
O
O
O
O
O
O
1
3
2
Scheme 1. Synthesis of tris-tBu-DOTA. Reagents and conditions: (a) benzyl-bromoacetate, CHCl3, rt, 3 h, 86%; (b) tert-butyl bromoacetate, K2CO3,
acetonitrile, rt, 2–5 h, 67%; (c) Pd/C, H2, THF/MeOH 1:1, rt, 2–8 h, 71%.
HO
O
HO
O
H
N
H
N
O
O
S H
S H
250
200
150
100
50
N
N
N
N
N
N
O
O
N
N
O
HO
O
O
OH
O
O
H
N
O
S
N
N
N
O
O
S i
N
O
O
O
HO
O
H
N
O
S H
N
N
O
N
N
HO
O
OH
0
0
1
2
3
4
5
time
Figure 1. Chromatograms of the deprotection reaction of S-trityl-mercapto-tris-tBu-DOTA with concentrated TFA after 16 h (compact line) and the
educt (dashed line). Elution gradient: 0–100% MeCN + 0.1% TFA in 5 min.

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C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
HO
HO
HO
O
O
O
H
N
H
N
S
SH
OH
N
N
N
N
N
N
N
N
N
a
b
O
O
O
O
O
O
N
N
N
HO
HO
HO
O
O
O
OH
OH
OH
4
5
Scheme 2. Synthesis of thiol-DOTA starting from DOTA. Reagents and conditions: (a) S-trityl-mercapto-ethanolamine trifluoroacetate, DCC,
water/MeCN 1:1, pyridine, rt, 10 h, 23%; (b) TIS, TFA, rt, 5 min, 56%.
toethanol19 and subsequently deprotected with concen-
trated TFA (Scheme 2) leading to much less side prod-
ucts in the final deprotection step (Fig. 2).
However, using the synthesis approach described above,
much less side products can be observed in the final
deprotection step (Fig. 4).
Tris-benzyl-DOTA (8) is a valuable DOTA synthon
especially if a mild deprotection step of the coupled
DOTA is required. The synthesis of this com-
pound20 can be facilitated by using an allyl-pro-
tected acetic acid derivative instead of a tert-butyl-
protected one in the first step of the synthesis.
The allyl protecting group can easily be removed
with tetrakis-(triphenylphosphine)-palladium as the
catalyst (Scheme 3).
Moreover, a new DOTA-derivative, tris-4-nitro-benzyl-
DOTA (10), was synthesized. As the 4-nitro-benzyl-
group can readily be cleaved with TBAF (tetrabutylam-
monium fluoride) in organic solvents,21 this compound
should be deprotectable under very mild conditions
and should therefore be suitable for the derivatization
of sensitive molecules with DOTA. The compound
could be obtained in a three-step synthesis in moderate
yields. However, it was found to be highly sensitive,
and therefore it could not successfully be used for cou-
pling experiments.
This is advantageous because the benzyl protecting
groups are not inert against the harsh deprotection con-
ditions required for the tert-butyl-groups with 12 N
HCl/dioxane 1:1 in the last step of the synthesis leading
to fragmentations of the product and low reaction yields
(Fig. 3).
2.2. Applicability of different DOTA derivatives for
multimerization reactions
For some applications it is necessary to introduce a high
number of chelators into one molecule. This is, for
TFA
250
S i
200
HO
H
N
O
S H
150
100
50
N
N
N
O
O
N
HO
O
OH
0
0
1
2
3
4
5
time [min]
Figure 2. Chromatogram of the deprotection reaction of S-trityl-mercaptoethylamino-DOTA with concentrated TFA after 5 min. Elution gradient:
0–100% MeCN + 0.1% TFA in 5 min (note that the product does not contain a strong chromophore absorbing at 214 nm).

C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
2609
O
O
O
O
O
OH
N
N
N
N
N
N
N
c
O
O
O
O
N
O
O
O
O
8
O
7
O
b
d
O
H
H
H
H
H
H
N
N
N
N
N
N
N
N
a
O
NO2
H
NO2
O
O
6
O
O
O
OH
O2N
O2N
N
N
N
N
N
e
O
O
O
O
N
N
N
O
O
O
O
10
9
O
O
O2N
O2N
Scheme 3. Synthesis of tris-benzyl-DOTA and tris-4-nitro-benzyl-DOTA. Reagents and conditions: (a) allyl-chloroacetate, CHCl3, 1 h 4 °C, 6 h, rt,
66%; (b) benzyl-bromoacetate, K2CO3, MeCN, rt, 10 h, 44%; (c) tetrakis-(triphenylphosphine)-palladium, morpholine, CH2Cl2, rt, 3 h, 71%; (d) 4-
nitrobenzyl-bromoacetate, K2CO3, MeCN, rt, 2 h, 78%; (e) tetrakis-(triphenylphosphine)-palladium, morpholine, CH2Cl2, rt, 1 h, 27%.
600
tris-benzyl-DOTA
400
200
0
0
1
2
3
4
5
time [min]
Figure 3. Decomposition of tris-benzyl-DOTA with 12 N HCl/dioxane 1:1 after 10 min (dashed line) and 12 h (compact line). Elution gradient: 0–
100% MeCN + 0.1% TFA in 5 min.
example, favorable for the synthesis of MRI contrast
agents or radiotracers which require high amounts of
metal ions, for example, gadolinium, per molecule to
accomplish high contrasts or for achieving high specific
activities of radiolabeled compounds.
reactions. For the multimerization reactions PAMAM
dendrimers containing a polyethyleneglycol linker with
a terminal protected thiol group and one to 128 amine
functions were used.8 These dendrimers were reacted
with tris-tBu-DOTA (3), thiol-DOTA (5), tris-benzyl-
DOTA (8), tris-4-nitrobenzyl-DOTA (10), tris-allyl-
DOTA (11),22 DOTA-PFP-ester (12)13, and DOTA-
PNP-ester (13)13 (Scheme 4).
Therefore, several DOTA-derivatives were investigated
with regard to their coupling yields in multimerization

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C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
1400
1200
1000
800
600
400
200
0
O
O
OH
N
N
N
N
O
O
O
O
O
catalyst
0
1
2
3
4
5
time [min]
Figure 4. Chromatogram of the final deprotection step of 7 to tris-benzyl-DOTA with tetrakis-(triphenylphosphine)-palladium as catalyst after 3 h.
Elution gradient: 0–100% MeCN + 0.1% TFA in 5 min.
These experiments showed that tris-tBu-DOTA and
tris-allyl-DOTA could be coupled to the dendrimers
containing one to eight amino functions giving homo-
geneous products in good yields (Table 1 and Fig. 5).
However, the subsequent deprotection steps of the
DOTA-multimers were problematic. In the case of
the tris-tBu-DOTA-multimers, the deprotection with
concentrated TFA gave rise to fragmentations of the
polyethyleneglycol linker of the dendrimer molecules
within 10 min. Furthermore, the deprotection reaction
was very slow and led to heterogeneous products
due to incomplete removal of the protecting groups.
In the case of the tris-allyl-DOTA-multimers, the
deprotection reaction was quantitative within short
reaction times but gave reaction mixtures that were
difficult to purify as the catalyst tetrakis-(triphenyl-
phosphine)-palladium gave very high UV-signals
which prevented the HPLC purification of the
products.
The active esters of DOTA should be advantageous
building blocks for multimerization reactions, as they
can be coupled directly without the use of additional
coupling reagents and furthermore, subsequent depro-
tection steps are dispensable. DOTA-PFP-ester and
DOTA-PNP-ester were reacted with dendrimers con-
taining one to eight amino functions. It could be ob-
served that the active esters reacted inefficiently with
the amino groups of the dendrimers producing inhomo-
geneous products even when applied in high excess of up
to five equivalents of active ester per amino function
(Table 1). However, it could be seen that the DOTA-
PFP-ester showed a much higher reactivity than the
DOTA-PNP-ester (Table 1).
Table 1. Coupling yields determined by HPLC of the different DOTA derivatives with dendrimers containing a different number of amino functions
DOTA-derivative
Coupling yields in % with n amino functions per dendrimer
n = 1
n = 2
n = 4
n = 8
n =16
n = 32
n = 64
n = 128
Tris-tBu-DOTA (3)
Thiol-DOTA (5)
100
(83)a
100
(91)a
60
100
(89)a
100
(79)a
4
100
(73)a
100
(83)a
0
95
n.d.
n.d.
n.d.
n.d.
(70)a
100
(89)a
n.d.
93
100
100
100
100
(73)a
n.d.
n.d.
(85)a
n.d.
n.d.
(70)a
n.d.
n.d.
(65)a
n.d.
n.d.
Tris-benzyl-DOTA (8)
Tris-allyl-DOTA (11)
100
(79)a
56
100
(76)a
43
94
(84)a
28
DOTA-PFP-ester (12)
DOTA-PNP-ester (13)
10
0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
60
40
6
n.d., not determined.
a Yields of isolated products.

C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
2611
A
B
C
DMSO / PyBOP
DMSO / PyBOP
Phosphate
1500
1250
1000
750
500
250
0
1000
750
500
250
0
heterogeneously
derivatized
600
400
200
0
product
dendrimer
product
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
time [min]
time [min]
time [min]
Figure 5. Chromatograms of the derivatization reactions of an 8 amino functions containing dendrimer with tris-tBu-DOTA (A), tris-benzyl-DOTA
(B), and thiol-DOTA (C). Elution gradient: 0–100% MeCN + 0.1% TFA in 5 min.
Tris-benzyl-DOTA showed coupling properties similar
to the active esters of DOTA in the multimerization
reactions and also gave inhomogeneous products due
to insufficient coupling yields (Table 1 and Fig. 5).
tris-allyl-DOTA could be multimerized and gave homo-
geneous coupling products. However, the deprotection
of the products was problematic showing that the appli-
cation of tris-tBu-DOTA and tris-allyl-DOTA is re-
stricted to non-sensitive molecules and solid phase
syntheses, respectively. Tris-benzyl-DOTA, DOTA-
PNP-ester, and DOTA-PFP-ester showed low reactivi-
ties toward the dendritic core molecules producing het-
erogeneous products and thus were not suitable for
multimerization reactions.
In contrast to this, thiol-DOTA could be introduced
quantitatively after previous derivatization of the den-
drimer amino groups with maleimido caproic acid8 (Ta-
ble 1). This approach produces DOTA-multimers that
do not require an additional deprotection step and is
therefore also highly suited for the derivatization of bio-
molecules. Furthermore, the coupling reaction was com-
plete within a few minutes and the products could be
obtained in high homogeneity (Fig. 5).
However, it was found that thiol-DOTA has superior
coupling properties regarding its applicability for a mul-
tiple introduction into dendritic molecules: it could be
introduced quantitatively generating highly homoge-
neous DOTA-multimers which do not require deprotec-
tion after coupling. Thus, thiol-DOTA is suitable for
the derivatization of multimeric scaffolds and the ob-
tained DOTA-multimers could, when introduced into
carrier molecules, generate high contrasts in molecular
imaging.
In summary, tris-tBu-DOTA and tris-allyl-DOTA can
be coupled quantitatively even to high numbers of ami-
no functions but show unfavorable deprotection proper-
ties. Due to the high UV-signals produced by the
deprotection catalyst, tris-allyl-DOTA is especially sui-
ted for solid phase chemistry. Tris-benzyl-DOTA,
DOTA-PFP-ester, and DOTA-PNP-ester show insuffi-
cient reactivities when coupled to a high number of ami-
no functions and are therefore not suited for
multimerization reactions, although the active esters
give good results when conjugated to biomolecules.
Thiol-DOTA quantitatively reacts with maleimide-
derivatized dendrimers and the products can be ob-
tained in good yields and high homogeneity. Due to this,
thiol-DOTA is a suitable compound for generating
DOTA-multimers applicable in molecular imaging pro-
viding high contrasts.
4. Experimental
4.1. General
All commercially available chemicals were of analytical
grade and used without further purification prior to use.
An Agilent 1100Ò system with a ChromolithÒ Perfor-
mance (RP-18e, 100–4.6 mm, Merck, Germany) column
was used for analytical purposes. Semi-preparative
HPLC purifications were performed using a Gyncotech
P-580 system (Germering, Germany) equipped with a
variable SPD 6-A UV detector and a C-R5A integrator
(both Shimadzu, Duisburg, Germany). The column ap-
plied was a ChromolithÒ (RP-18e, 100–10 mm, Merck,
Germany).
3. Conclusion
In summary, the synthesis routes for tris-tBu-DOTA,
tris-benzyl-DOTA, and thiol-DOTA could be improved
and the synthesis of the novel compound tris-4-nitro-
benzyl-DOTA could be established.
The MALDI-TOF spectra were obtained on a Kratos
Analytical Compact Maldi III system. ESI spectra were
obtained using a Triple–Quadrupole-mass spectrom-
eter TSQ 7000 (Thermo Fisher Scientific, Bremen).
NMR spectra were taken using a Varian Mercury Plus
300 MHz and a Varian NMR System 500 MHz,
respectively.
Further on, we determined the applicability of the
DOTA derivatives tris-tBu-DOTA, thiol-DOTA, tris-
benzyl-DOTA, tris-4-nitrobenzyl-DOTA, tris-allyl-
DOTA, DOTA-PFP-ester, and DOTA-PNP-ester for
multimerization reactions of PAMAM dendrimers of
different sizes. It was found that tris-tBu-DOTA and

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C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
O
O
a
O
H
N
N
N
NH2
N
n
N
O
O
O
O
n = 1 - 8
(14a - d)
n
O
O
N
b
N
O
H
N
N
O
O
N
O
O
n
n = 1 - 8
(15a - d)
HO
N
O
c
O
H
N
N
N
O
OH
N
O
OH
n
n = 1 - 8
(16a - d + 17a - d)
O
d
O
O
H
N
N
N
N
O
N
O
O
O
n
n = 1 - 4
(18a - c)
e, f
HO
NH
O
O
OH
N
N
N
N
O
O
N
O
O
N
S
H
O
OH
n
n = 1 - 128
(19a - h)
Scheme 4. Reaction of the PAMAM dendrimers containing 1–128 amine functions with the different DOTA derivatives. Reagents and conditions:
(a) tris-tBu-DOTA, PyBOP, DIPEA, DMF, rt, 3 h, 70–83%; (b) tris-allyl-DOTA, PyBOP, DIPEA, DMF, rt, 2.5 h, 76–84%; (c) tris-benzyl-DOTA,
PyBOP, DIPEA, DMF, rt, 5 h, products not isolated; (d) DOTA-PFP-ester or DOTA-PNP-ester, DIPEA, DMF, rt, 1 h or 10 h, products not
isolated; (e) maleimido caproic acid, PyBOP, DIPEA, DMF, rt, 40 min, 29–84%; (f) thiol-DOTA, PBS (0.05 M P, 0.15 M NaCl, pH 7.2), rt, 10 min,
65–91%.
Tris-allyl-DOTA, DOTA-PFP-ester, and DOTA-PNP-
ester were synthesized according to the literature
methods.13,22
8.7 mmol) in 5 mL CHCl3 was added dropwise over a
period of one hour at room temperature. After two
hours, the solvent was evaporated at room temperature
and the product was purified via column chromatogra-
phy on silica with CHCl3/EtOH/NH4OH(conc.) 8:9:4 as
the eluent (Rf = 0.6).
4.2. (1,4,7,10-Tetraaza-cyclododec-1-yl)-acetic acid ben-
zyl ester (1)
To a solution of cyclene (3 g, 17.4 mmol) in 20 mL
a
Special care has to be taken that the temperature of the
soluted product never rises above 25 °C as this leads to
CHCl3,
solution of benzyl-bromoacetate (2 g,

C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
2613
an unwanted internal ring formation. The product
was obtained as a colorless oil (2.38 g, 7.44 mmol,
86%). H NMR (DMSO-d6) (d, ppm; J, Hz): 7.41 (s,
4.5. S-Trityl-mercaptoethylamino-DOTA (S-trityl-thiol-
DOTA) (4)
1
5H); 5.21 (s, 2H); 3.51–3.43 (m, 2H); 2.64–2.38 (m,
16H). 13C NMR (DMSO-d6) (d, ppm): 175.8; 145.1;
130.1; 129.3; 127.5; 75.6; 57.8; 54.9; 53.1; 53.0; 49.2.
MALDI-MS (m/z) for [M+H]+(calculated): 320.6
(321.4).
To a solution of S-trityl-mercaptoethanolamine trifluo-
roacetate (2.54 g; 5.9 mmol) and DOTA (3 g;
5.9 mmol) in 40 mL water/MeCN 1:1, a solution of
DCC (1.21 g; 5.86 mmol) in 5 mL pyridine was added
dropwise at room temperature. After reaction over-
night, the solvents were evaporated and the product
was purified via column chromatography on silica with
water/MeCN 1:1 + 0.1% TFA as the eluent (Rf = 0.8).
The product was obtained as a light yellow solid
(952 mg, 1.4 mmol, 23%) after lyophilization. 1H
NMR (DMSO-d6) (d, ppm; J, Hz): 7.35–7.21 (m,
15H); 3.96–3.04 (m, 18H); 3.02–2.97 (m, 8H); 2.24 (t,
2H, J3 = 6.7). 13C NMR (DMSO-d6) (d, ppm):
158.87; 158.45; 145.00; 129.71; 128.74; 127.45; 66.73;
46.54; 46.48; 26.62. MALDI-MS (m/z) for [M+H]+(cal-
culated): 706.1 (706.3).
4.3. (4,7,10-Tris-tert-butoxycarbonylmethyl-1,4,7,10-tet-
raaza-cyclododec-1-yl)-acetic acid benzyl ester (2)
To a suspension of (1,4,7,10-tetraaza-cyclododec-1-yl)-
acetic acid benzyl ester (2.38 g, 7.44 mmol) and pow-
dered K2CO3 (4.27 g, 30.5 mmol) in 40 mL acetonitrile,
a
solution of tert-butyl bromoacetate (5.8 g,
29.76 mmol) in 10 mL acetonitrile was added dropwise
over a period of one hour at room temperature. The
reaction was monitored by TLC with CHCl3/EtOH
9:1 as mobile phase. When the reaction was complete,
the solids were removed and the volatile components
were evaporated. As the product showed a consider-
able decomposition when incubated for longer than
two hours with silica, it was rapidly purified via col-
umn chromatography on silica with first CHCl3 and
subsequently CHCl3/EtOH 9:1 as the eluent
(Rf = 0.5). The product was obtained as a colorless
4.6. Mercaptoethylamino-DOTA (thiol-DOTA) (5)
S-Trityl-thiol-DOTA (952 mg, 1.4 mmol) was dissolved
in a mixture of 200 lL TIS and 5 mL TFA and reacted
for 5 min at room temperature. The volatile components
were evaporated and the product was purified using
semi-preparative HPLC with 0–20% MeCN + 0.1%
TFA in 3 min as the gradient (Rt = 1.1 min). The prod-
uct was isolated as a yellow oil (363 mg, 0.78 lmol, 56%)
1
foam (3.29 g, 4.97 mmol, 67%). H NMR (DMSO-d6)
(d, ppm; J, Hz): 7.35 (s, 5H); 5.13 (s, 2H); 3.46–3.39
(m, 2H); 3.31 (s, 2H); 3.29–2.71 (bs, 7H); 2.43–1.94
(bs, 6H); 1.44 (s, 9H); 1.40 (bs, 18H). 13C NMR
(DMSO-d6) (d, ppm): 174.3; 172.9; 144.7; 129.8;
128.7; 127.3; 85.7; 75.3; 58.3; 57.1; 53.8; 52.6; 52.3;
50.5; 31.2. MALDI-MS (m/z) for [M+H]+(calculated):
662.8 (663.4).
1
after lyophilization. H NMR (DMSO-d6) (d, ppm; J,
Hz): 3.98 (bs, 2H); 3.84 (bs, 2H); 3.59 (bs, 6H); 3.37–
3.09 (m, 16H); 2.54 (q, 2H, J3 = 7.5); 2.41 (t, 1H,
J3 = 7.6). 13C NMR (DMSO-d6) (d, ppm): 158.93;
158.51; 54.61; 53.47; 51.22; 51.07; 49.29; 48.99; 42.96;
23.97. MALDI-MS (m/z) for [M+H]+(calculated):
463.9 (464.1). ESI-MS (m/z) for [M+H]+ (calculated):
464.2 (464.1).
4.4. (4,7,10-Tris-tert-butoxycarbonylmethyl-1,4,7,10-tet-
raaza-cyclododec-1-yl)-acetic acid (tris-tBu-DOTA) (3)
4.7. (1,4,7,10-Tetraaza-cyclododec-1-yl)-acetic acid allyl
ester (6)
To a solution of (4,7,10-tris-tert-butoxycarbonylmeth-
yl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid benzyl
ester (830 mg, 1.25 mmol) in 40 mL THF/MeOH 1:1,
palladium on carbon (10% Pd, 114 mg) was added
and the reaction mixture was stirred in a hydrogen
atmosphere at room temperature. The reaction was
monitored by HPLC. After completion of the reaction,
the catalyst was removed by filtration, the solvents
were evaporated, and as the product showed a consid-
erable decomposition when incubated for longer than
three hours with silica it was rapidly purified via col-
umn chromatography on silica with a gradient of
water/MeCN + 0.1% TFA starting with MeCN
(Rf = 0.5 with water/MeCN 1:1 + 0.1% TFA). The
product was obtained as a colorless solid (507 mg,
0.89 mmol, 71%). 1H NMR (DMSO-d6) (d, ppm; J,
Hz): 3.46 (m, 2H); 3.36 (s, 6H); 3.33 (bs, 4H); 2.87
(bs, 4H); 2.72 (bs, 4H); 2.64 (bs, 4H); 1.41 (s, 18H);
1.40 (s, 9H). 13C NMR (DMSO-d6) (d, ppm): 179.63;
179.30; 89.53; 65.63; 64.61; 61.88; 60.78; 58.59; 56.09;
36.88; 36.83. MALDI-MS (m/z) for [M+H]+(calcu-
lated): 572.6 (573.4). ESI-MS (m/z) for [M+H]+ (calcu-
lated): 573.4 (573.4).
To a solution of cyclene (560 mg, 3.26 mmol) in 40 mL
CHCl3,
a solution of allyl-chloroacetate (190 lL,
1.63 mmol) in 10 mL CHCl3 was added dropwise over
one hour at 4 °C. After six hours at room temperature,
the solvent was evaporated and the product was purified
via column chromatography on silica with CHCl3/
EtOH/NH4OH(conc.) 8:9:4 as the eluent (Rf = 0.5). The
product was obtained as a colorless oil (290 mg,
1.07 mmol, 66%). 1H NMR (DMSO-d6) (d, ppm; J,
Hz): 5.97–5.84 (m, 1H); 5.33–5.17 (m, 2H); 4.55–4.53
(m, 2H); 3.38 (s, 2H); 2.64–2.38 (m, 16H). 13C NMR
(DMSO-d6) (d, ppm): 171.46; 133.30; 118.47; 64.95;
55.93; 51.83; 47.42; 46.45; 45.51. MALDI-MS (m/z)
for [M+H]+(calculated): 270.0 (271.2). ESI-MS (m/z)
for [M+H]+ (calculated): 271.2 (271.2).
4.8. (4-Allyloxycarbonylmethyl-7,10-bis-benzyloxycarbon-
ylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid
benzyl ester (7)
To a solution of (1,4,7,10-tetraaza-cyclododec-1-yl)-
acetic acid allyl ester (290 mg, 1.07 mmol) in 40 mL

2614
C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
MeCN, powdered K2CO3 (600 mg, 4.28 mmol) and a
solution of benzyl-bromoacetate (671 lL, 4.28 mmol)
in 10 mL MeCN were added at room temperature.
After reaction overnight, the suspension was filtered,
the solvent evaporated and the product purified via
column chromatography on silica with CHCl3/EtOH
9:1 as the eluent (Rf = 0.2). The product was obtained
as a colorless foam (340 mg, 0.48 mmol, 44%). 1H
NMR (DMSO-d6) (d, ppm; J, Hz): 7.41–7.29 (m,
15H); 6.00–5.85 (m, 1H); 5.38–5.22 (m, 2H); 5.17–
5.07 (m, 6H); 4.62–4.57 (m, 2H); 3.97–3.94 (m, 5H);
3.49–2.54 (m, 16H). 13C NMR (DMSO-d6) (d, ppm):
174.04; 173.81; 136.45; 132.97; 129.09; 128.85; 128.66;
118.76; 66.67; 66.57; 65.78; 65.44; 55.51; 55.40; 53.21;
49.12. MALDI-MS (m/z) for [M+H]+(calculated):
714.6 (715.4). ESI-MS (m/z) for [M+H]+ (calculated):
715.3 (715.4).
850.2 (850.3). ESI-MS (m/z) for [M+H]+ (calculated):
850.4 (850.3).
4.11. (4,7,10-Tris-4-nitrobenzyloxycarbonylmethyl-1,4,7,
10-tetraaza-cyclododec-1-yl)-acetic acid (tris-4-nitroben-
zyl-DOTA) (10)
To a solution of (4-allyloxycarbonylmethyl-7,10-bis-4-
nitro-benzyloxycarbonylmethyl-1,4,7,10-tetraaza-cyclo-
dodec-1-yl)-acetic acid 4-nitrobenzyl ester (720 mg,
0.87 mmol) in 40 mL CH2Cl2, morpholine (1.48 mL;
1.47 g, 17 mmol) and tetrakis-(triphenylphosphine)-pal-
ladium (294 mg, 0.25 mmol) were added at room tem-
perature. After one hour, the solvent was evaporated
and the product was purified via column chromatogra-
phy on silica with water/MeCN 1:1 + 0.1% TFA as the
eluent (Rf = 0.5). The product was obtained as a color-
less foam (190 mg, 0.23 mmol, 27%). 1H NMR
(DMSO-d6) (d, ppm; J, Hz): 8.23 (d, 6H, J3 = 8.8);
7.69 (d, 6H, J3 = 8.8); 5.37–5.24 (m, 6H); 4.12–4.04
(m, 2H); 4.03 (s, 6H); 3.21 (s, 16H). 13C NMR
(DMSO-d6) (d, ppm): 168.96; 168.21; 147.63; 144.01;
129.28; 124.18; 66.84; 65.49; 65.30; 53.69; 53.19; 49.11.
MALDI-MS (m/z) for [M+H]+(calculated): 809.7
(810.3).
4.9. (4,7,10-Tris-benzyloxycarbonylmethyl-1,4,7,10-tetra-
aza-cyclododec-1-yl)-acetic acid (tris-benzyl-DOTA) (8)
To a solution of (4-allyloxycarbonylmethyl-7,10-bis-
benzyloxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-
1-yl)-acetic acid benzyl ester (340 mg, 0.48 mmol) in
40 mL CH2Cl2, morpholine (830 lL; 827 mg,
9.5 mmol) and tetrakis-(triphenylphosphine)-palladium
(165 mg, 0.14 mmol) were added at room tempera-
ture. After three hours, the solvent was evaporated
and the product was purified via column chromatog-
raphy on silica with water/MeCN 45:55 + 0.1% TFA
as the eluent (Rf = 0.5). The product was obtained
as a colorless foam (230 mg, 0.34 mmol, 71%). 1H
NMR (DMSO-d6) (d, ppm; J, Hz): 7.37–7.30 (m,
15H); 5.14–5.09 (m, 6H); 4.11–4.02 (m, 2H); 3.80–
3.74 (m, 6H); 3.42–3.07 (m, 16H). 13C NMR
(DMSO-d6) (d, ppm): 170.80; 169.72; 136.22;
129.12; 128.84; 128.75; 66.70; 63.92; 54.29; 53.65;
51.48; 49.17. MALDI-MS (m/z) for [M+H]+(calcu-
lated): 673.9 (675.3). ESI-MS (m/z) for [M+H]+ (cal-
culated): 675.3 (675.3).
4.12. Reaction of PAMAM dendrimers with tris-tBu-
DOTA (14a–d)
A solution of PyBOP (2.9 equiv per amine function of
the respective dendrimer) and DIPEA (3 equiv per
amine function) in 300 lL DMF was added to tris-
tBu-DOTA (3) (3 equiv per amine function) and reacted
for two minutes. Subsequently, this mixture was added
to a solution of the respective full generation dendrimer
containing 1–8 amine functions (50 lmol) in 200 lL
DMF. After 3 h, the products were purified using
semi-preparative HPLC using a gradient of 10–100%
MeCN + 0.1% TFA over 12 min. The products were iso-
lated as pale yellow solids in yields of 70–83% after
lyophilization.
4.10. (4-Allyloxycarbonylmethyl-7,10-bis-4-nitrobenzyl-
oxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-
acetic acid 4-nitrobenzyl ester (9)
G0—1 DOTA: MALDI-MS (m/z) for [M+H]+ (calcu-
lated): 1137.5 (1138.6). ESI-MS (m/z) for [M+H]+ (cal-
culated): 1138.8 (1138.6).
To a solution of (1,4,7,10-tetraaza-cyclododec-1-yl)-
acetic acid allyl ester (291 mg, 1.08 mmol) in 40 mL
MeCN, powdered K2CO3 (604 mg, 4.31 mmol) and a
G1—2 DOTA: MALDI-MS (m/z) for [M+H]+ (calcu-
lated): 1921.5 (1921.2). ESI-MS (m/z) for [M+H]+ (cal-
culated): 1921.4 (1921.2).
solution
of
4-nitrobenzyl-bromoacetate
(1.18 g,
G2—4 DOTA: MALDI-MS (m/z) for [M+H]+ (calcu-
lated): 3489.2 (3487.2). ESI-MS (m/z) for [M+H]+ (cal-
culated): 3486.8 (3487.2).
4.31 mmol) in 10 mL MeCN were added at room tem-
perature. After two hours, the suspension was filtered,
the solvent evaporated, and the product purified via
column chromatography on silica with CHCl3/EtOH
9:1 as the eluent (Rf = 0.1). The product was obtained
as a colorless foam (720 mg, 0.87 mmol, 78%). 1H
NMR (DMSO-d6) (d, ppm; J, Hz): 8.23 (d, 6H,
J3 = 8.8); 7.69 (d, 6H, J3 = 8.8); 5.98–5.85 (m, 1H);
5.39–5.21 (m, 8H); 4.61–4.58 (m, 2H); 4.05 (s, 6H);
3.96 (s, 2H); 3.24 (s, 16H). 13C NMR (DMSO-d6)
(d, ppm): 169.06; 168.41; 147.85; 144.07; 132.85;
129.39; 124.23; 118.99; 65.51; 65.34; 53.74; 53.21;
49.15. MALDI-MS (m/z) for [M+H]+(calculated):
G3—8 DOTA: ESI-MS (m/z) for [M+H]+ (calculated):
6618.4 (6619.4).
4.13. Reaction of PAMAM dendrimers with tris-allyl-
DOTA (15a–d)
A solution of PyBOP (2.4 equiv per amine function of
the respective dendrimer) and DIPEA (5 equiv per
amine function) in 300 lL DMF was added to tris-al-
lyl-DOTA (2.5 equiv per amine function) and reacted

C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
2615
for two minutes. Subsequently, this mixture was added
to a solution of the respective full generation dendri-
mer containing 1–8 amine functions (50 lmol) in
200 lL DMF. After 2.5 h, the products were purified
using semi-preparative HPLC using a gradient of
15–100% MeCN + 0.1% TFA over 12 min. The prod-
ucts containing 1–4 DOTA moieties were isolated as
yellow solids in yields of 76–84% after lyophilization.
MeCN + 0.1% TFA over 8 min. The products were
isolated as white to ocher solids in yields of 65–91%
after lyophilization.
Acknowledgment
The authors thank T. Timmermann from the Faculty of
Pharmacy at the University of Heidelberg for recording
NMR spectra.
G0—1 DOTA: MALDI-MS (m/z) for [M+H]+ (calcu-
lated): 1089.9 (1090.6).
G1—2 DOTA: MALDI-MS (m/z) for [M+H]+ (calcu-
lated): 1825.2 (1825.0).
Supplementary data
G2—4 DOTA: MALDI-MS (m/z) for [M+H]+ (calcu-
lated): 3298.0 (3295.9). ESI-MS (m/z) for [M+H]+ (cal-
culated): 3295.2 (3295.9).
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.11.044.
G3—8 DOTA: ESI-MS (m/z) for [M+H]+ (calculated):
6232.7 (6233.6).
4.14. Reaction of PAMAM dendrimers with DOTA-
PNP-ester (16a–d)
References and notes
1. de Jong, M.; Breeman, W. A. P.; Valkema, R.; Bernard, B.
F.; Krenning, E. P. J. Nucl. Med. 2005, 46, 13S.
2. Smith, C. J.; Volkert, W. A.; Hoffman, T. J. Nucl. Med.
Biol. 2005, 32, 733.
3. Reubi, J. C.; Waser, B. Eur. J. Nucl. Med. Mol. Imaging
2003, 30, 781.
A solution of DOTA-PNP-ester (2.5 equiv per amine
function of the respective dendrimer) and DIPEA (6 equiv
per amine function) in 300 lL DMF was added to a solu-
tionoftherespective full generation dendrimercontaining
1–8 amine functions (50 lmol) in 200 lL DMF and re-
acted overnight. The products were not isolated.
4. Meyer, A.; Auernheimer, J.; Modlinger, A.; Kessler, H.
Curr. Pharm. Des. 2006, 12, 2723.
5. Witzig, T. E.; Molina, A.; Gordon, L. I.; Emmanouilides,
C.; Schilder, R. J.; Flinn, I. W.; Darif, M.; Macklis, R.;
Vo, K.; Wiseman, G. A. Cancer 2007, 109, 1804.
6. Ringhoffer, M.; Blumstein, N.; Neumaier, B.; Glatting,
G.; von Harsdorf, S.; Buchmann, I.; Wiesneth, M.;
Kotzerke, J.; Zenz, T.; Buck, A. K.; Schauwecker, P.;
Stilgenbauer, S.; Do¨hner, H.; Reske, S. N.; Bunjes, D. Br.
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7. Kobayashi, H.; Kawamoto, S.; Choyke, P. L.; Sato, N.;
Knopp, M. V.; Star, R. A.; Waldmann, T. A.; Tagaya, Y.;
Brechbiel, M. W. Mag. Reson. Med. 2003, 50, 758.
8. Wa¨ngler, C.; Moldenhauer, G.; Eisenhut, M.; Haberkorn,
U.; Mier, W. Unpublished results.
4.15. Reaction of PAMAM dendrimers with DOTA-
PFP-ester (17a–d)
A solution of DOTA-PFP-ester (2.5 equiv per amine
function of the respective dendrimer) and DIPEA (6
equiv per amine function) in 300 lL DMF was added
to a solution of the respective full generation dendrimer
containing 1–8 amine functions (50 lmol) in 200 lL
DMF and reacted for one hour. The products were
not isolated.
4.16. Reaction of PAMAM dendrimers with tris-benzyl-
DOTA (18a–c)
9. Kobayashi, H.; Sato, N.; Saga, T.; Nakamoto, Y.;
Ishimori, T.; Toyama, S.; Togashi, K.; Konishi, J.;
Brechbiel, M. W. Eur. J. Nucl. Med. 2000, 27,
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Cancer Res. 2003, 9, 3756.
A solution of PyBOP (2.9 equiv per amine function of
the respective dendrimer) and DIPEA (5 equiv per
amine function) in 300 lL DMF was added to tris-ben-
zyl-DOTA (8) (3 equiv per amine function) and reacted
for two minutes. Subsequently, this mixture was added
to a solution of the respective full generation dendrimer
containing 1–4 amine functions (50 lmol) in 200 lL
DMF. The products were not isolated.
11. Hermanson, G. T. Bioconjugate Techniques; Academic
Press, 1996.
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4.17. Reaction of PAMAM dendrimers with thiol-DOTA
(19a–h)
To a solution of the respective maleimide-derivatized
dendrimer containing 1–128 maleimide functions
(10 lmol) in 200 lL MeCN was added a solution of
thiol-DOTA (4 equiv per maleimide) in 250 lL PBS
(0.05 M, 0.15 M NaCl, pH 7.2) and the mixture was
reacted for 10 min. After acidification with 1 M HCL
(200 lL) the products were purified using semi-pre-
16. Heppeler, A.; Froidevaux, S.; Ma¨cke, H. R.; Jermann, E.;
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Behe, M.; Powell, P.; Hennig, M. Chem. Eur. J. 1999, 5,
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parative HPLC using
a
gradient of 20–100%

2616
C. Wa¨ngler et al. / Bioorg. Med. Chem. 16 (2008) 2606–2616
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