New synthetic routes for 1-benzyl-1,4,7,10-tetraazacyclododecane and 1,4,7,10-tetraazacyclododecane-1-acetic acid ethyl ester, important starting materials for metal-coded DOTA-based affinity tags

Article abstract of DOI:10.1515/znb-2007-0313

Two improved routes to synthesize 1-benzyl-1,4,7,10-tetraazacyclododecane (6) and 1,4,7,10-tetraazacyclododecane-1-acetic acid ethyl ester (11) are described as well as the synthesis of 1-{2-[4-(maleimido-N-propylacetamidobutyl) amino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (17) and its Y, Ho, Tm, and Lu complexes. The ¹H and ¹³C NMR spectra of the new compounds as well as the single crystal X-ray structure analyses of the intermediates 4-benzyl-1,7-bis(p-toluenesulfonyl) diethylenetriamine (3) and 1,4,7-tris(p-toluenesulfonyl)diethylenetriamine (7) are reported and discussed. The rare earth complexes of 17 have been characterized by ¹H NMR spectroscopy and MALDI-TOF mass spectrometry.

Full text of DOI:10.1515/znb-2007-0313

New Synthetic Routes for 1-Benzyl-1,4,7,10-tetraazacyclododecane and
1,4,7,10-Tetraazacyclododecane-1-acetic Acid Ethyl Ester, Important
Starting Materials for Metal-coded DOTA-Based Affinity Tags
Stephan W. Kohla, Katharina Kusea, Markus Hummerta, Herbert Schumanna,
Clemens Mu¨ggeb, Katharina Janekc, and Hardy Weißhoffb
a Institut fu¨r Chemie, Technische Universita¨t Berlin, Straße des 17. Juni 135, D-10623 Berlin,
Germany
b Institut fu¨r Chemie, Humboldt-Universita¨t zu Berlin, Brook-Taylor-Straße 2, D-12489 Berlin,
Germany
c Universita¨tsklinikum Charite´, Humboldt-Universita¨t zu Berlin, Monbijoustraße 2, D-10098 Berlin,
Germany
Reprint requests to Prof. Dr. H. Schumann. Fax +49 30 31422168.
E-mail: schumann@chem.tu-berlin.de
Z. Naturforsch. 2007, 62b, 397 – 406; received November 7, 2006
Dedicated to Prof. Helgard G. Raubenheimer on the occasion of his 65th birthday
Two improved routes to synthesize 1-benzyl-1,4,7,10-tetraazacyclododecane (6) and 1,4,7,10-
tetraazacyclododecane-1-acetic acid ethyl ester (11) are described as well as the synthe-
sis of 1-{2-[4-(maleimido-N-propylacetamidobutyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclodo-
decane-4,7,10-triacetic acid (17) and its Y, Ho, Tm, and Lu complexes. The 1H and 13C NMR spec-
tra of the new compounds as well as the single crystal X-ray structure analyses of the intermediates
4-benzyl-1,7-bis(p-toluenesulfonyl)diethylenetriamine (3) and 1,4,7-tris(p-toluenesulfonyl)diethyl-
enetriamine (7) are reported and discussed. The rare earth complexes of 17 have been characterized
by 1H NMR spectroscopy and MALDI-TOF mass spectrometry.
Key words: Tetraazacyclododecane, Macrocycle, DOTA, Affinity Tag, Rare Earth Complexes
Introduction
Macrocyclic polyaminopolycarboxylates have
tags for relative and absolute quantitation (ITRAQ)
[9 – 13].
Metal-coded affinity tags are reagents composed
been intensively studied because of their numerous of a chelating ligand, a monoisotopic metal ion, pre-
applications, which often require selective functional- dominantly rare earth cations, and a reactive group
ization [1, 2]. Metal ion-conjugated peptides with 1,4, with specificity towards thiol or amino groups. The
7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid affinity can be achieved for example by an incorpo-
(DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid rated group like biotin [11] or by interaction with
(NOTA) or 1,4,8,11-tetraazacyclotetradecane-1,4,8, antibodies [4, 14]. The principle of MECATs is de-
11-tetraacetic acid (TETA) ligands are ideal agents rived from ICAT, but instead of the stable isotope la-
for a spectrum of applications in biomedicine, beling of proteins or peptides a metal ion labeling
as therapeutic radiopharmaceuticals, luminescent is applied. The protein mixture of two or more sets
probes for biochemical analysis, or MRI contrast of cell states is independently labeled with MECAT
agents [3 – 6]. Recently a new class of DOTA con- reagents containing different metal ions; the sam-
jugates was introduced, the so-called element- or ples are combined, and then conventionally cleaved.
metal-coded affinity tags (MECAT) [7, 8]. These The MECAT labeled peptides are isolated by affin-
reagents can be used in quantitative proteomics, as ity chromatography and analyzed by LC-ESI-MS/MS.
an additional or alternative method to established Peptide sequence information is obtained by tandem
2D-GE and recently developed methods employing mass spectrometry and computer searches of pro-
isotope-coded affinity tags (ICAT) and isobaric tein data banks. Quantitation of proteins in two cell
0932–0776 / 07 / 0300–0397 $ 06.00 © 2007 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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S. W. Kohl et al. · Important Starting Materials for Metal-coded DOTA-Based Affinity Tags
Scheme 1. Route
I for
the synthesis of 1-benzyl-
1,4,7,10-tetraazacyclodo-
decane (6).
states is performed by comparing the intensity of Results and Discussion
the identical peptide peak pair from the samples de-
fined by the mass difference of the complex ions
chosen.
Synthesis of 1-benzyl-1,4,7,10-tetraazacyclododecane
(6) and 1,4,7,10-tetraazacyclododecane-1-acetic acid
ethyl ester (11)
Metal ions, and particularly rare earth cations, are
suitable for ICP-MS and permit low detection limits of
quantitation. Many of the rare earth elements are nat-
urally monoisotopic. Thus, a variety of MECATs with
desired mass differences can be synthesized by pair-
wise integration into ligands. Considering only seven
monoisotopic rare earth elements, 19 different mass
tags are produceable with mass differences from 2 Da
for 139La/141Pr to 86 Da for 89Y/175Lu. Thereby more
than two samples can be investigated in parallel, or am-
biguous analytical results can be verified in an inde-
pendent run.
N-substituted tetraazacyclododecanes are generally
synthesized starting with diethylenetriamine (1) and
diethanolamine as common educts via bimolecular
cyclization reactions using toluenesulfonyl protecting
groups, with subsequent deprotection. To improve our
recently published procedure [21], we used a modified
way (Scheme 1). The first step, the selective tosyla-
tion of the two primary amino groups of 1 is possi-
ble at −45 ◦C in dichloromethane. Thus, the protection
and deprotection of the terminal amino groups with
phthalic anhydride can be avoided and the yield of 1,7-
bis(p-toluenesulfonyl)diethylenetriamine (2) [22] is
increased. The tri-tosylated by-product, 1,4,7-tris(p-
toluenesulfonyl)diethylenetriamine (7) [23], can easily
be separated by filtration and used for further prepa-
rations. Alkylation of 2 with benzyl bromide and an
excess of K2CO3 results in the formation of 4-benzyl-
1,7-bis(p-toluenesulfonyl)diethylenetriamine (3) [21],
which crystallizes after a few weeks as colorless
crystals. Cyclization with 1,5-bis(methylsulfonyloxy)-
3-aza-3-(p-toluenesulfonylamido)pentane (4) [24] ac-
cording to [21] and elimination of the protecting
groups by sodium amalgam yields the monosubstituted
cyclen 6 [19] in 80 % yield.
For about 15 years, several research groups have
been engaged in the synthesis of mono-functionalized
DOTA derivatives [15 – 18]. Meanwhile, N- and
C-functionalized DOTA derivatives are commercially
available, but still very expensive. To make these
important compounds more readily available, we
describe in this paper two suitable, cost-efficient syn-
thetic routes to 1-benzyl-1,4,7,10-tetraazacyclododec-
ane (6) [19], 1,4,7,10-tetraazacyclododecane-1-acetic
acid ethyl ester (11) [20] and 1,4,7,10-tetraazacyclo-
dodecane-1-acetic acid-4,7,10-tris-(acetic acid tert-
butyl ester) (tris-tBu-DOTA) (15) [17], the start-
ing materials for the synthesis of N-functionalized
DOTA ligands, as well as the synthesis of 1-
Route II for the synthesis of 6 starts with the
{2-[4-(maleimido-N-propylacetamidobutyl)amino]-2- complete tosylation of 1 at 0 ◦C yielding 7 [23]
oxoethyl}-1,4,7,10-tetraazacyclododecane-4,7,10-tri-
as a white powder which forms colorless single
acetic acid (17) and its Y, Ho, Tm, and Lu com- crystals from acetone suitable for X-ray analysis
plexes. (Scheme 2). Cyclization of 7 with the bis-methylsulf-
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S. W. Kohl et al. · Important Starting Materials for Metal-coded DOTA-Based Affinity Tags
399
Scheme 2. Route II for the
synthesis of 1-benzyl-1,4,7,10-
tetraazacyclododecane (6) and
1,4,7,10-tetraazacyclododecane-
1-acetic acid ethyl ester (11).
onyloxy compound 4 yields 1,4,7,10-tetrakis(p-tolu-
enesulfonyl)-1,4,7,10-tetraazacyclododecane (8) [25],
which is converted into 1,4,7,10-tetraazacyclododec-
ane (cyclen) (9) by heating in concentrated H2SO4 for
three days [26]. Reaction with Mo(CO)6 following the
procedure described by Patinec et al. [27, 28] resulted
in η3-1,4,7,10-tetraazacyclododecanemolybdenumtri-
carbonyl (10) [26], which was alkylated with benz-
ylbromide and bromoacetic acid ethyl ester in DMF
followed by decoordination from the Mo(CO)3 frag-
ment by HCl yielding 6 and 1,4,7,10-tetraazacyclodo-
decane-1-acetic acid ethyl ester (11) [20], respectively.
Molecular structure of 4-benzyl-1,7-bis(p-toluenesulf-
onyl)diethylenetriamine (3) and 1,4,7-tris(p-toluene-
sulfonyl)diethylenetriamine (7)
Fig. 1. ORTEP [29] presentation of the molecular structure
of 3 (displacement ellipsoids at the 30 % probability
level); all hydrogen atoms except H(1) and H(2) have
˚
been omitted for clarity; selected bond lengths (A) and
angles (deg): N(1)–C(1) 1.476(3), N(1)–C(8) 1.460(3),
N(1)–C(10) 1.476(3), N(2)–C(9) 1.472(3), N(2)–S(1)
1.613(2), N(3)–C(11) 1.458(4), N(3)–S(2) 1.608(3),
N(2)–O(1) 3.165(3), N(3)–O(3) 3.090(3); C(9)–N(2)–S(1)
The structure of monoclinic crystals of the dito-
sylated benzylated triamine 3 (Fig. 1) shows non-
˚
exceptional averaged bond lengths C–N (1.47 A) and
˚
C–C (1.50 A) along the chain of the triamine. Sim- 118.11(17), C(11)–N(3)–S(2) 120.4(2).
˚
˚
ilar distances C–N (1.46 A) and C–C (1.52 A) were
found in the structure of the monoclinic crystals of the
tritosylated diethylenetriamine 7 (Fig. 2). The mean
N–S distances in the structures of 3 and 7 are 1.61 and
the distances between the nitrogene atoms N(2) and
N(3) and the oxygen atom O(1) of the adjacent
˚
molecule are 3.17 and 3.09 A, respectively. The
˚
two molecules are linked via intermolecular hydro-
gen bonds. The bonding of two nitrogen atoms to only
one oxygen atom is the reason for the close proximity
1.62 A, respectively. The conformation of the dimethy-
lene units in the amine chain is N(1)–C(8)–C(9)–
N(2) 66◦ ((+)-synclinal) and N(1)–C(10)–C(11)–N(3)
−58◦ ((–)-synclinal) for compound 3 and N(2)–C(3)–
C(4)–N(3) 179◦ (antiperiplanar) and N(1)–C(1)–
C(2)–N(2) 69◦ ((+)-synclinal) for compound 7. For 3
˚
(3 – 4 A) of the two tosyl groups in this molecule, com-
pared with 7, where the terminal tosyl-groups are lo-
˚
cated far away from each other (9 – 10 A). Intermolec-
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S. W. Kohl et al. · Important Starting Materials for Metal-coded DOTA-Based Affinity Tags
Fig. 2. ORTEP [29] presentation of the molecular structure
of 7 (30 % probability ellipsoids); all hydrogen atoms
except H(1) and H(2) have been omitted for clarity; selected
˚
bond lengths (A): N(1)–C(1) 1.462(5), N(1)–S(1) 1.620(3),
N(2)–C(2) 1.473(4), N(2)–C(3) 1.467(4), N(2)–S(2)
1.473(4), N(3)–C(4) 1.526(5), N(3)–S(3) 1.610(3),
N(1)–O(2) 3.008(4), N(3)–O(3) 2.923(4).
ular hydrogen bonds located in 7 between N(1) and
˚
˚
O(2) (3.01 A) and between N(3) and O(6) (2.92 A) lead
to a network in the crystal.
Synthesis of 1-{2-[4-(maleimido-N-propylacetamido-
butyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclodo-
decane-4,7,10-triacetic acid (17) and its Y, Ho, Tm,
and Lu complexes
Compounds 6 and 11 are the key compounds
for the synthesis of 1,4,7,10-tetraazacyclododec-
ane-4,7,10-tris(acetic acid tert-butyl ester)-1-acetic
acid (tris-tBu-DOTA) (15) [17], which in turn is
the starting material for the synthesis of N-func-
tionalized DOTA ligands, which are commercially
available, but very expensive. Following published
routes [16, 30, 31], 15 is prepared either starting from 6
by alkylation of the unprotected amine functions with
BrCH2COOtBu to yield 1,4,7,10-tetraazacyclododec-
ane-1-benzyl-4,7,10-tris(acetic acid tert-butyl ester)
(12), followed by removal of the protecting benz-
yl group with H2/Pd/C to produce 1,4,7,10-tetraaza-
cyclododecane-4,7,10-tris(acetic acid tert-butyl ester)
(13) [32] and finally by incorporation of an acetate
group or from 11 in two steps via 1,4,7,10-tetra-
azacyclododecane-1-acetic acid ethyl ester-4,7,10-tris
(acetic acid tert-butyl ester) (14) (Scheme 3).
Scheme 3. Synthesis of tris-tBu-DOTA (15).
deprotected and cleaved from the resin by trifluo-
roacetic acid (TFA), water and triisopropylsilane (TPS)
yielding 16 in 84 % yield as a white solid (Scheme 4).
Compound 16 reacts with β-maleimidopropionic
acid N-hydroxysuccinimid ester in the presence
of triethylamine in DMF yielding 1-{2-[4-(male-
imido-N-propylacetamidobutyl)amino]-2-oxoethyl}-
1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid
(17) in 73 % yield as a white solid. Its reaction with
lanthanide trichlorides in water at pH 7.0 results in the
formation of lanthanide(III) complexes. The yttrium,
The triply protected DOTA-derivative 15 reacts holmium, thulium, and lutetium complexes 18, 19,
with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetrameth- 20, and 21 have been characterized by MALDI-TOF
yluronium hexafluorophosphate (HATU), Hu¨nig’s mass spectrometry. Fig. 3 shows the MALDI-TOF
base (DIEA) and 1,4-diaminobutane trityl resin in mass spectrum of 17 chelating Y3+, Ho3+, Tm3+ and
DMF with formation of the resin-fixed tris-tert-butyl Lu3+ ions. The spectrum demonstrates the utility of
ester of 2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymeth- rare earths embedded in peptide specific labels as
yl)-1-cyclododecyl)-acetyl-diaminobutane, which is internal standards for quantitative proteomics based
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401
% intensity
100
Tm(III)
792.1676
6.4E+4
Y(III)
Ho(III)
788.1628
80
712.1332
Lu(III)
60
40
20
0
798.1712
mass (m/z)
691.0
717.2
744.4
770.6
797.2
Fig. 3. MALDI-TOF Mass spectrum of the potential thiol-
specific MECAT ligand 17 chelating Y, Ho, Tm, and Lu ions.
Further investigations concerning structural analysis of
the lanthanide complexes and their application are in
progress.
Experimental Section
Unless noted otherwise, all reactions were carried out
at r. t. in dried solvents under dry dinitrogen, using stan-
dard Schlenk techniques. Chemicals were purchased from
Aldrich, Acros, Chempur, and Macrocyclics and used with-
out further purification. p-CH3C6H4SO2N(CH2CH2OSO2
1
CH3)2 (4) was prepared according to the literature [24]. H
and 13C NMR spectra were recorded with Bruker ARX 200
and Bruker AV 400 spectrometers. Chemical shifts δ were
references to TMS or 3-(trimethylsilyl)-propionic acid-D4
sodium salt (TSP) for measurements in D2O. Signs of cou-
pling constants were not determined. The MALDI-TOF
spectra were recorded with a MALDI-TOF/TOF 4700 Pro-
teomics Analyzer (Applied Biosystems, Framingham, MA,
USA). Elemental analyses were carried out using a Thermo
Finnigan, Flash EA, 1112 Series analyzer.
Scheme 4. Synthesis of 1-{2-[4-(maleimido-N-propylacet-
amidobutyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclodode-
decane-4,7,10-triacetic acid (17) and the lanthanide com-
plexes 18 – 21.
HN(CH2CH2NHSO2C6H4CH3-p)2 (2). Diethylenetri-
amine (10.0 g, 0.097 mol) and triethylamine (19.2 g,
on mass spectrometry. Here, mass tags with differ-
0.190 mol) were dissolved in CH2Cl2 (300 mL) and
ences from 4 Da for 165Ho/169Tm-17 to 86 Da for cooled to −48 C. To this solution p-toluenesulfonylchlor-
◦
89Y/175Lu-17 are shown as an example for the variety
of different combinations. The use of rare earth ele-
ide (36.2 g, 0.190 mol) in CH2Cl2 (100 mL) was added over
a period of 4 h. The temperature did not exceed −45 ◦C. Af-
ter that, the mixture was stirred for 4 h at r. t., and washed
ments in addition has the advantage that quantitation
three times with water. The organic layer was dried with
can be accomplished by means of ICP MS with very
Na2SO4 and the solvents evaporated to give a colorless oil,
high efficiency and sensitivity. Furthermore, the mass
which was further dried in vacuum. The residue was crys-
differences between the heavy rare earth-containing
tallized from CH2Cl2/CH3OH (1 : 3). Beside the favored di-
tags is useful for the peptide and protein identification
tosylated oily product (2), the crystalline tri-tosylated prod-
in complex mixtures [33].
uct 7 is formed (m. p. 59 – 61 ◦C). Yield: 30.0 g (75 %) for 2
The lutetium complex 21 was also characterized by
elemental analysis and 1H and 13C NMR spectroscopy.
These spectra as well as those of 16 and 17 are compli-
cated and very hard to assign because of internal hy-
drogen bonds which cause very broad signals for the
and 6.0 g (10 %) for 7. – 1H NMR (25 ◦C, 200 MHz, CDCl3):
δ = 2.35 (s, 6 H, Ts-CH3), 2.51 (m, 4 H, Ts-NHCH2CH2N),
2.86 (m, 4 H, Ts-NHCH2CH2N), 4.56 (br, 3 H, NH), 7.20
(m, 4 H, SO2CCHCH), 7.66 (m, 4 H, SO2CCHCH). –
13C NMR (25 ◦C, 100.64 MHz, CDCl3): δ = 21.0 (Ts-
macrocyclic CH2 protons at low pH values [20, 34]. CH3), 41.9 (NHCH2CH2NH-Ts), 47.3 (NHCH2CH2NH-
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S. W. Kohl et al. · Important Starting Materials for Metal-coded DOTA-Based Affinity Tags
Ts), 126.6 (2 × SO2CCHCH), 129.3 (2 × SO2CCHCH), less mixture changed to white, and mercury precipitated
136.3 (2 × SO2CCH), 142.9 (2 × SO2CCHCHCCH3). – which was separated. The solvent was removed on a ro-
C18H25N3O4S2(411.53): calcd. C 52.53, H 6.12, N 10.21, tary evaporator and the grey residue was dissolved in CHCl3
S 15.58; found C 52.45, H 5.99, N 10.12, S 15.55.
(80 mL) and washed three times with water (55 mL). The
organic phases were combined and dried with Na2SO4.
The solvent was removed and the crude product was
dried under vacuum. Recrystallization from CH2Cl2/CH3OH
(10 : 1) yielded 6 as a bright yellow solid. M. p. 83 –
85 ◦C. Yield: 1.6 g (80 %). Route II: 5.1 g (14 mmol)
of 10 and 6.8 g (49 mmol) of K2CO3 were suspended in
DMF (150 mL) and stirred for 30 min at 75 ◦C. After-
wards C6H5CH2Br (1.7 mL, 14 mmol) was added drop-
wise and the mixture was refluxed for 2 h precipitating
a white solid. After cooling to r. t., filtering and evaporat-
ing the solvent, the yellow residue was treated with HCl
(35 mL, 10 %) and stirred at r. t. on air for further 16 h.
After raising the pH to 8 a brown solid was formed and
removed by centrifugation. The resulting clear blue solu-
tion was extracted with CHCl3 (4 × 35 mL). The com-
bined organic layers were dried with Na2SO4. A yellow
solid was obtained after evaporation of all volatiles and
drying under vacuum. Yield: 2.49 g (68 %). – 1H NMR
(25 ◦C, 400 MHz, CDCl3): δ = 2.44 – 2.77 (m, 16 H,
macrocyclic CH2), 3.52 (s, 2 H, PhCH2), 7.12 – 7.25 (m,
C6H5CH2N(CH2CH2NHSO2C6H4CH3-p)2 (3). The to-
sylated trisamine 2 (5.8 g, 14 mmol) was dissolved in CH3
CN (200 mL) and 5.5 g (39 mmol) of dried K2CO3 were
added. The mixture was heated to 80 ◦C and after rapid
dropwise addition of C6H5CH2Br (1.7 g, 14 mmol) re-
fluxed for 24 h. The resulting precipitate was filtered and
washed 3 times with CH2Cl2 (30 mL). The organic lay-
ers were washed with water (4 × 40 mL) and dried with
Na2SO4. The solvent was removed by rotary evaporation to
leave 3 as a pale yellow oil, which crystallizes after sev-
eral ◦days. M. p. 52 ◦C. Yield: 6.9 g (98 %). – 1H NMR
(25 C, 400 MHz, CDCl3): δ = 2.30 (s, 6 H, CH3), 2.35 –
2.40 (m, 4 H, NCH2CH2NH), 2.85 (br, 4 H, NCH2CH2NH),
3.50 (s, 2 H, benzyl-CH2), 5.64 (br; 2 H, NH), 7.13 – 7.26
(m, 5 H, benzyl-H), 7.27 – 7.29 (m, 4 H, SO2CCHCH),
7.63 – 7.75 (m, 4 H, SO2CCHCH). – 13C NMR (25 ◦C,
100.64 MHz, CDCl3): δ = 21.3 (Ts-CH3), 44.9 (NH-
CH2CH2NH-Ts), 51.3 (NHCH2CH2NH-Ts), 59.3 (PhCH2-),
126.8 (2 × SO2CCHCH), 127.35 (CH2CCHCHCH), 128.34
(CH2CCHCH), 129.92 (CH2CCHCH), 130.1 (2 × SO2C-
CHCH), 136.1 (2 × SO2CCH), 136.31 (CH2CCH), 142.7
(2 × SO2CCHCHC). – C25H31N3O4S2 (501.66): calcd.
C 59.86, H 6.23, N 8.38, S 12.78; found C 59.55, H 6.13,
N 8.20, S 12.71.
◦
5 H, benzyl-H). – 13C-NMR (25 C, 100.64 MHz, CDCl3):
δ = 44.54 (benzyl-N(CH2)2NCH2CH2), 45.92 (benzyl-
N(CH2)2NCH2), 46.70 (benzyl-NCH2CH2), 50.90 (benz-
yl-NCH2), 58.91 (PhCH2-), 126.82 (CCHCHCH), 128.06
(CCHCHCH), 129.06 (CCHCHCH), 138.78 (CCHCHCH). –
C15H26N4 (262.40): calcd. C 68.66, H 9.99, N 21.35; found
C 68.28, H 9.80, N 21.28.
C6H5CH2N[CH2CH2N(SO2C6H4CH3-p)]3CH2CH2 (5)
was prepared according to [21] from 10.7 g◦(21 mmol) of 3
and 8.9 g (21 mmol) of◦4. M. p. 161 – 164 C. Yield: 9.9 g
1
(65 %). – H NMR (25 C, 400 MHz, CDCl3): δ = 2.39 (s,
p-CH3C6H4SO2N(CH2CH2NHSO2C6H4CH3-p)2
(7).
6 H, Ts-CH3), 2.44 (s, 3 H, Ts-CH3), 2.73 (dd, 4 H, benz-
yl-NCH2), 3.08 (dd, 4 H, benzyl-NCH2CH2), 3.34 (dd, 4 H,
benzyl-NCH2CH2NCH2), 3.46 (dd, 4 H, benzyl-NCH2CH2-
NCH2CH2), 3.61 (s, 2 H, PhCH2-), 7.14 – 7.20 (m, 5 H, Ph),
7.26 (m, 4 H, SO2CCHCH), 7.33 ( m, 2 H, SO2CCHCH),
7.57 (m, 4 H, SO2CCHCH), 7.72 (m, 2 H, SO2CCHCH).
2.0 g (19 mmol) of 1 and NEt3 (7.9 mL, 57 mmol) were
dissolv◦ed in CH2Cl2 (100 mL) and the mixture cooled
to −2 C. A solution of p-toluenesulfonylchloride (10.9 g,
57 mmol) in CH2Cl2 (50 mL) was added dropwise keep-
ing the temperature at 0 ◦C. The mixture was stirred at
this temperature for 24 h and then washed with water
(3 × 55 mL). The organic layer was dried with Na2SO4.
Evaporation and drying under vacuum resulted in 7 as
a white solid. M. p. 59 – 61 ◦C. Yield: 10.2 g (95 %). –
1H NMR (25 ◦C, 200 MHz, [D6]DMSO): δ = 2.37 (s,
9 H, Ts-CH3), 2.81 – 3.02 (m, 8 H, NHCH2CH2N), 5.37
(br, 2 H, NH), 7.33 – 7.40 (m, 6 H, SO2CCHCH), 7.54
(d, 3J = 8.2 Hz, 2 H, SO2CCH), 7.66 (m, 6 H, (d, 3J =
8.2 Hz, 4 H, NHSO2CCH). – 13C NMR (25 ◦C, 50.32 MHz,
[D6]DMSO): δ = 20.97 (Ts-CH3), 41.59 (2× NHCH2),
48.40 (2× NHCH2CH2), 126.54 (2× NHSO2CCHCH),
126.83 (NSO2CCHCH), 129.68 (2× NHSO2CCHCH),
–
13C NMR (25 ◦C, 100.64 MHz, CDCl3): δ = 21.48
(Ts-CH3), 21.53 (2 × Ts-CH3), 48.62 (benzyl-NCH2CH2),
50.93 (benzyl-NCH2CH2NCH2CH2), 51.68 (benzyl-NCH2-
CH2NCH2), 55.12 (benzyl-NCH2CH2), 59.52 (PhCH2-),
127.42 (2 × SO2CCHCH), 127.57 (CH2CCHCHCH),
128.25 (CH2CCHCH), 129.70 (2 × SO2CCHCH), 129.92
(CH2CCHCH), 134.68 (2 × SO2CCH), 135.62 (SO2CCH),
136.31 (CH2CCH), 143.45 (SO2CCHCHC), 143.58 (2 ×
SO2CCHCHC). – C36H44N4O6S3 (724.95): calcd. C 59.65,
H 6.12, N 7.73, S 13.27; found C 59.08, H 6.14, N 7.94,
S 13.22.
C6H5CH2N(CH2CH2NH)3CH2CH2 (6). Route I: 5.8 g 129.88 (NSO2CCHCH), 135.31 (NSO2CCH), 137.36 (2×
(8.0 mmol) of 5, 6.8 g (48 mmol) of anhydrous Na2HPO4, NHSO2CCH), 142.76 (2× NHSO2CCHCHCCH3), 143.46
and sodium amalgam (2 %, 9.5 g, 48 mmol) were stirred (NSO2CCHCHCCH3). – C25H31N3O6S3 (565.73): calcd.
in CH3CN (250 mL) at 80 ◦C for one day. The colour- C 53.08, H 5.52, N 7.43, S 17.00; found C 53.15, H 5.45,
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S. W. Kohl et al. · Important Starting Materials for Metal-coded DOTA-Based Affinity Tags
403
N 17.29, S 17.29.
89 – 91 ◦C. – 1H NMR (25 ◦C, 400 MHz, CDCl3): δ =
3
1.20 (t, J = 8.9 Hz, 3 H, CH2CH3), 2.48 – 2.84 (m, 16 H,
(p-CH C H SO NCH CH ) (8). Cs2CO3 (50.6 g, 0.150
3
6
4
2
2
2 4
macrocyclic CH2), 3.30 (s, 2 H, NCH2CO), 4.10 (q, 3J =
mol) was suspended in a solution of 7 (29.3 g, 0.052 mol)
in CH3CN (300 mL) and heated to 80 ◦C. Afterwards
21.5 g (0.052 mol) of 4, dissolved in CH3CN (250 mL),
were added over a period of 1 h and the mixture stirred
at this temperature for 2 d. After cooling to r. t. 500 mL
of water were added in order to separate the partially pre-
cipitated product from the excess carbonate by filtration.
The remaining solution was extracted with CH2Cl2 (5 ×
80 mL), the organic layers were combined, the solvent was
reduced in volume to about one third and CH3OH (100 mL)
was added. After storing the solution for 3 d at 5 ◦C◦, 8
was obtained as a white solid. Decomposition > 260 C.
Yield: 27.9 g (68 %). – 1H NMR (25 ◦C, 200 MHz, CDCl3):
δ = 2.45 (s, 12 H, Ts-CH3), 3.43 (s, 16 H, macrocyclic CH2),
7.28 – 7.35 (m, 8 H, SO2CCHCH), 7.61 – 7.71 (m, 8 H,
SO2CCHCH). – 13C NMR (25 ◦C, 50.32 MHz, CDCl3): δ =
21.38 (Ts-CH3), 44.41 (CH2), 126.43 (SO2CCHCH), 129.68
(SO2CCHCH), 135.41 (SO2CCH), 142.75 (SO2CCHCHC).
– C36H44N4O8S4 (789.01): calcd. C 54.80, H 5.62, N 7.10,
S 16.25; found C 54.45, H 5.44, N 7.29, S 16.00.
◦
8.9 Hz, 2 H, CH2CH3). – 13C NMR (25 C, 100.64 MHz,
CDCl3): δ = 13.95 (CH2CH3), 51.05 (NCH2CO), 46.30
(ester-N(CH2)2NCH2CH2), 47.03 (ester-N(CH2)2NCH2),
50.34 (ester-NCH2CH2), 55.70 (ester-NCH2), 60.65
(CH2CH3), 172.34 (NCH2CO). – C12H26N4O2 (258,36):
calcd. C 55.79, H 10.14, N 21.69; found C 55.95, H 10.30,
N 21.80.
C H CH NCH CH [N(CH COOtBu)CH CH ] (12).
6
5
2
2
2
2
2
2 3
1.1 g (4 mmol) of 6 and 1.7 g (12 mmol) of dried K2CO3
were suspended in DMF (180 mL) and heated to 80 ◦C
for 30 min. Afterwards BrCH2COOtBu (2.34 mL, 12 mmol)
was added dropwise and the mixture refluxed for 20 h, pre-
cipitating KBr as a white solid. The solvent was evaporated,
the residue dissolved in CH2Cl2 (50 mL) and filtered. The
colorless solution was then washed with water (3 × 45 mL)
and the organic layer was dried with Na2SO4. A yellow solid
was obtained after evaporation of the volatiles and drying in
vacuum. Yield: (1.81 g, 75 %). M. p. 95 – 98 ◦C. – 1H NMR
(25 ◦C, 200 MHz, CDCl3): δ = 1.39 (s, 18 H, tBu), 1.43 (s,
(HNCH CH ) (9). 65.3 g (0.083 mol) of 8 was stirred 9 H, tBu), 2.57 – 2.81 (m, 16 H, macrocyclic CH2), 3.17 (s,
2
2 4
with 100 mL of concentrated sulphuric acid for 3 d at 130 ◦C. 4 H, CH2COt Bu), 3.29 (s, 2 H, CH2COt2Bu), 3.49 (s, 2 H,
The initially colorless solution changed to brown after a benzyl-CH2),27.20 – 7.25 (m, 5 H, benzyl-H). – 13C NMR
◦
few h and a black precipitate occurred. The mixture was (25 C, 50.32 MHz, CDCl3): δ = 28.72 (C(CH3)3), 52.41
◦
cooled to 0 C, diluted with 150 mL of water and then the (benzyl-N(CH2)2NCH2CH2), 52.60 (benzyl-NCH2CH2),
pH was adjusted to > 13 by addition of solid KOH (130 g, 52.72 (benzyl-NCH2CH2), 56.68 (NCH2CO), 60.11 (NCH2-
2.32 mol). The filtered precipitate was washed with EtOH benzyl), 81.75 (C(CH3)3), 127.32 (benzyl-Cpara), 128.51
(2 × 90 mL) and the aqueous and the organic phases were (benzyl-Cmeta), 128.97 (benzyl-Cortho), 136.01 (benzyl-
t
combined and evaporated. The brownish residue was dis- Cquart), 169.68 (NCH2CO2 Bu). – C33H56N4O6 (604.82):
solved in 80 mL of 0.1 M HCl and washed with CH2Cl2 (4 × calcd. C 65.53, H 9.33, N 9.26; found C 65.83, H 9.51,
30 mL). The pH of the aqueous phase was adjusted again to N 9.40.
HNCH CH [N(CH COOtBu)CH CH ] (13). Hydro-
> 13 and the solution extracted with CHCl3 (4 ×30 mL). Af-
ter combining and drying of the organic phases with K2CO3
the solvent was removed and the white solid of 9 was dried in
a vacuum. M. p. 113 – 114 ◦C. Yield: 8.6 g (60 %). – 1H NMR
2
2
2
2
2 3
gen gas was bubbled through a suspension of 12 (1.2 g,
2.0 mmol) and the catalyst Pd/C (10 % Pd, 200 mg) in a mix-
ture of CH3OH and THF (1 : 1, 300 mL) at r. t. over night. Af-
ter removing the catalyst by filtration over celite, the solvent
was evaporated and the brownish residue dried in vacuum.
Crystallization from a mixture of acetone/diisopropylether
(2 : 1) y◦ielded a light yellow solid (0.41 g, 40 %). M. p.
47 – 50 C. – IR (KBr): ν = 1733 (s, C=O, ester), 1158 (s,
C–O, ester), 1059 (m, C–N) cm−1. – 1H NMR (400 MHz,
CDCl3, r. t.): δ = 1.39 (s, 27 H, tBu), 2.52 (m, 4 H,
NHCH2CH2), 2.68 (s, 8 H, NRCH2CH2NR), 2.77 (m, 4 H,
◦
(25 C, 400 MHz, CDCl3): δ = 2.17 (s, 4 H, NH), 2.68 (s,
16 H, CH2). – 13C NMR (25 ◦C, 100.64 MHz, CDCl3): δ =
46.12 (CH2). – C8H20N4 (127.27): calcd. C 55.78, H 11.70,
N 32.52; found C 55.13, H 11.99, N 32.50.
(CO) Mo(HNCH CH ) (10). 2.7. g (16 mmol) of 9 and
3
2
2 4
4.6 g (16 mmol) of Mo(CO)6 were suspended in n-dibut-
◦
ylether (80 mL) and heated to 140 C for 2 h. The yellow
precipitate was filtered off and washed with diethyl ether
(3 ×15 mL) to yield 5.2 g (92 %) of 10. – C11H20N4O3Mo
(352.24): calcd. C 37.51, H 5.72, N 15.91; found C 36.93,
H 5.83, N 15.61.
t
1
NHCH2), 3.26 (s, 6 H, CH2CO2 Bu). – 13C{ H} NMR
(100.64 MHz, CDCl3, r. t.): δ = 28.24 (C(CH3)3), 47.56
(NHCH2CH2), 50.70 (NH(CH2)2NRCH2), 52.16 (NHCH2),
C H OC(O)CH N(CH CH NH) CH CH (11). 11 was 52.24 (NH(CH2)2NRCH2CH2), 52.80 (NH(CH2)2N-
2
5
2
2
2
3
2
2
prepared in analogy to the synthesis of 6 following route II R(CH2)2NCH2CO), 57.23 (s, NH(CH2)2NCH2CO), 81.78
using 1.1 g (3.0 mmol) of 10, 1.4 g (49 mmol) of K2CO3, (C(CH3)3), 171.14 (NCH2CO). – MS (EI, 70 eV): m/z
t
0.33 mL (3.0 mmol) of BrCH2COOEt, and 80 mL of DMF. (%) = 514 (24.10) [M]+, 413 (100) [M – CO2 – Bu]+. –
Yield: 0.40 g (55 %) of 11 as a light yellow solid. M. p. C26H50N4O6 (514.70): calcd. C 60.67, H 9.79, N 10.89;
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404
S. W. Kohl et al. · Important Starting Materials for Metal-coded DOTA-Based Affinity Tags
found C 60.38, H 9.62, N 10.39.
was agitated at r. t. overnight and the solvent removed in
EtOC(O)CH NCH CH [N(CH COOtBu)CH CH ]
vacuum. Afterwards the cleavage from the resin was carried
out with 50 mL of TFA, 5 % water and 1 % tri-iso-prop-
ylsilane for 2 h. The mixture was filtered and the filtrate
was evaporated in vacuum. The residue was washed with
ether yielding 16 (935 mg, 84 %). Further purification was
achieved by preparative HPLC (Agilent-Prep-C18 column;
solvent A: 0.1 % TFA in water; solvent B: 10 % of aq.
0.1 % TFA, 90 % aq. CH3CN). Removal of the mobile phase
gave the product as a lyophilized solid. M. p. 168 – 170 ◦C. –
1H NMR (400 MHz, D2O): δ = 1.40 (m, 2 H, NH2CH2CH2),
1.48 (m, 2 H, NHCH2CH2), 2.89 (t, 2 H, NH2CH2), 3.08 (m,
2 H, CONHCH2), 2.70 – 3.50 (broad, 16 H, NCH2CH2N),
2
2
2
2
2
2 3
(14) was prepared as described for 12 from 11 (0.25 g,
1.0 mmol), K2CO3 (0.41 g, 3.0 mmol), BrCH2COOtBu
(0.44 mL, 3.0 mmol), and 25 mL of DMF as a white
solid. Yield: 0.44 g (74 %). Decomposition > 140 ◦C. –
1H NMR (200 MHz, CDCl3, r. t.): δ = 1.21 (t, 3J = 8.85 Hz,
t
t
3 H, OCH2CH3), 1.39 (s, 18 H, Bu), 1.41 (s, 9 H, Bu),
2.50 – 2.86 (m, 16 H, macrocyclic CH2), 3.20 – 3.31 (m,
6 H, CH2CO2 Bu), 3.34 (s, 2 H, NCH2CO2Et), 4.10 (q, 3J =
t
8.85 Hz, 2 H, OCH2CH3). – 13C{ H} NMR (50.32 MHz,
1
CDCl3, r. t.): δ = 14.03 (OCH2CH3), 27.72 (C(CH3)3), 51.59
(NCH2CO), 55.91 (EtO2CCH2N(CH2)2NCH2CH2), 56.25
(EtO2CCH2N(CH2)2NCH2), 56.41 (EtO2CCH2NCH2CH2),
56.78 (EtO2CCH2NCH2), 60.85 (OCH2CH3), 81.75
1
3.60 – 4.20 (broad, 8 H, NHCH2CO). – 13C{ H} NMR
(100.64 MHz, D2O):
δ = 24.2 (NHCH2CH2), 25.4
t
(NH2CH2CH2), 38.6 (CONHCH2), 39.1 (NH2CH2), 47.0 –
53.5 (broad, NCH2CH2N), 53.5 – 57.0 (broad, NHCH2CO),
174.0 – 175.0 (broad, CO) – MALDI-TOF MS: m/z = 457
[M+H]+. – C20H38N6O7 (474.56): calcd. C 50.62, H 8.07,
N 17.71; found C 50.53, H 8.01, N 17.81.
(C(CH3)3), 171.98 (NCH2CO2 Bu), 172.64 (NCH2CO2Et).
– C30H56N4O8 (600.80): calcd. C 59.98, H 9.39, N 9.33;
found C 59.83, H 9.31, N 9.20.
HOC(O)CH NCH CH [N(CH COOtBu)CH CH ]
2 3
2
2
2
2
2
(15). a) 15 was prepared as described above for 12 from 13
(0.1 g, 0.2 mmol), K2CO3 (28 mg, 0.2 mmol), BrCH2COOH
C H O N(CH ) C(O)NH(CH ) NHC(O)CH NCH -
4
2
2
2 2
2 4
2
2
◦
CH [N(CH COOH)CH CH ] (17). To a solution of 16
(0.014 mL, 0.2 mmol) and 10 mL of DMF at 70 C (2 ◦h),
2
2
2
2 3
(500 mg, 1.05 mmol) in 25 mL of DMF, 0.75 mL of NEt3
and a solution of 560 mg (2.1 mmol) of β-maleimidoprop-
ionic acid N-hydroxysuccinimide ester in 10 mL of DMF
were added. The mixture was allowed to stand for 4 h at r. t.
with occasional stirring. The precipitate was filtered and the
filtrate was evaporated to dryness. Impurities were removed
by washing with CHCl3 and CH3OH. Yield: 480 mg (73 %)
of 17. Further purification was achieved by preparative
HPLC (Agilent-Prep-C18 column; solvent A: 0.1 % TFA
in water; solvent B: 10 % of aq. 0.1 % TFA, 90 % aq.
CH3CN). M. p. 183 – 185 ◦C. – 1H NMR (400 MHz,
D2O): δ = 1.42 (m, 2 H, NHCH2CH2CH22), 1.46 (m,
2 H, NHCH2CH2CH2), 2.38 (t, 2 H, NCH2CH2CO),
3.05 (t, 2 H, CONHCH2), 3.10 (m, 2 H, CONHCH2),
3.12 – 3.54 (broad, 16 H, NCH2CH2N), 3.69 (t, 2 H,
NCH2CH2CO), 3.64 – 4.23 (broad, 8 H, NHCH2CO), 6.88
as a white solid. Yield: 74 mg (65 %). M. p. 127 – 130 C.
b) 14 (0.3 g, 0.5 mmol) was suspended in aqueous KOH
solution (1 M, 5 mL) and stirred for one day at 30 C. The
◦
mixture was brought to dryness, the residue suspended
in C2H5OH (10 mL) and then filtered. This process was
repeated five times. The combined organic layers were
dried with Na2SO4. A white microcrystalline solid was
obtained after evaporation of the solvents and drying in
vacuum. Yield: 0.17 g (59 %). M. p. 129 – 131 ◦C. – IR
(KBr): ν = 1738 (s, C=O, ester), 1644 (s, C=O, acid),
1161 (s, C–O, ester), 1120 (m, C–N) cm−1. – 1H NMR
(400 MHz, CDCl3, r. t.): δ = 1.42 (s, 27 H, tBu), 2.76 (s, 8 H,
acid-N(CH2)2NCH2CH2), 3.04 (m, 4 H, acid-NCH2CH2),
3.29 (s, 4 H, acid-N(CH2)2NCH2CO), 3.37 (s, 2 H, acid-
N{(CH2)2N}2CH2CO), 3.60 (m, 4 H, acid-NCH2), 3.69 (s,
1
2 H, CH2COOH). – 13C{ H} NMR (100.64 MHz, CDCl3,
1
(s, 2 H, CH=CH). – 13C{ H} NMR (100.64 MHz, D2O):
r. t.): δ = 28.13 (C(CH3)3), 48.47 (acid-NCH2CH2), 50.28
(acid-N(CH2)2NCH2), 53.51 (acid-NCH2CH2NCH2CH2),
55.75 (NCH2COOH), 56.07 (acid-N{(CH2)2N}2CH2CO),
56.75 (acid-N(CH2)2NCH2CO), 81.80 (C(CH3)3), 166.95
(COOH), 169.93 (acid-N(CH2)2NCH2CO), 170.69 (acid-
N{(CH2)2N}2CH2CO). – MS (EI, 70 eV): m/z (%) = 572
δ = 26.4 (NHCH2CH2CH2), 27.1 (NHCH2CH2CH2), 34.0
(NCH2CH2CO), 37.9 (NCH2CH2CO), 39.6 (CH2NHCO),
40.2 (CONHCH2), 47.0 – 54.0 (broad, NCH2CH2N), 54.0 –
57.0 (broad, NHCH2CO), 135.6 (CH=CH), 167.0 – 174.0
(broad, CO) – MALDI-TOF MS: m/z = 626 [M+H]+. –
C27H43N7O10 (625.68): calcd. C 51.83, H 6.93, N 15.67;
found C 51.23, H 7.12, N 15.71.
(3.10) [M]+, 471 (100) [M – CO2 – Bu]+. – C28H52N4O8
t
(572.74): calcd. C 58.72, H 9.15, N 9.78; found C 58.61,
H 9.07, N 9.51.
LnC H O N(CH ) C(O)NH(CH ) NHC(O)CH NCH -
4
2
2
2 2
2 4
2
2
H N(CH ) NHC(O)CH NCH CH [N(CH COOH)CH - CH [N(CH COO)CH CH ] (18) – (21). Using a 0.1
2 3
M
2
2 4
2
2
2
2
2
2
2
2
2 3
CH ] (16). To a solution of 15 (1.34 g, 2.35 mmol) in DMF Na2CO3/HCl solution (pH = 7.5 buffer), 0.15 mmol
(40 mL), 0.983 g (2.585 mmol) of HATU and 0.5 mL of of YCl3, HoCl3, TmCl3 or LuCl3 were dissolved and
Hu¨nig’s base were added. The mixture was stirred for 5 min combined with 60 mg (0.096 mmol) of 17. The pH was
and added to 5 g of 1,4-diaminobutane trityl resin (loading adjusted to 7.0 and the samples were kept at r. t. over
0.47 mmol/g, 2.35 mmol) in DMF. The reaction mixture night. Analytical HPLC was carried out to verify the
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405
Table 1. Parameters of the sin-
gle crystals, data collection and
structure refinement of 3 and 7.
Compound
3
7
Empirical formula
Formula weight [g mol−1
Crystal size [mm3]
Crystal system
Space group
C25H31N3O4S2
501.65
C25H31N3O6S3
565.71
]
0.75 ×0.52 ×0.35
monoclinic
P21/c
0.52 ×0.24 ×0.15
monoclinic
P21/n
Z
a [A]
b [A]
c [A]
4
4
˚
˚
˚
11.9855(4)
10.9267(4)
20.0058(7)
97.530(1)
2597.40(16)
1.283
0.240
0.9207 / 0.8404
1064
5.1910 (1)
27.4576(5)
18.9445(4)
93.168(1)
2696.08(9)
1.394
0.320
0.8880 / 0.4807
1192
β [deg]
3
˚
V [A ]
Dcalcd [g cm−3
]
Absorption coefficient [mm−1
Min./max. transmission
F(000) [e]
]
2θ Range for data collection [deg]
1.71 ≤ θ ≤ 26.00
−14 ≤ h ≤ 12
−13 ≤ k ≤ 13
−22 ≤ l ≤ 24
17564
5095 (Rint = 0.0773)
5095 / 0 / 317
0.988
R1 = 0.0516
wR2 = 0.1180
R1 = 0.0971
wR2 = 0.1394
0.234 / −0.382
1.48 ≤ θ ≤ 27.50
−6 ≤ h ≤ 6
−30 ≤ k ≤ 35
−24 ≤ l ≤ 21
20467
6177 (Rint = 0.0859)
6177 / 1 / 345
1.081
R1 = 0.0798
wR2 = 0.1410
R1 = 0.1404
wR2 = 0.1625
0.334 / −0.338
Data set
Reflections, collected
Reflections, unique
Data / restraints / parameter
Goodness-of-Fit (F2)
Final R indices (I ≥ 2σ(I))
R indices (all data)
−3
˚
Largest diff. peak and hole [e A
]
coordination to the Ln(III) ions. The complexes were (1× CO, CO), 181.2 (3× CO, CO). – C27H40N7O10Lu
purified on an Agilent-Prep-C18 column (solvent A: (797.65): calcd. C 40.66, H 5.05, N 12.29; found C 40.39,
0.1 % TFA in water; solvent B: 10 % of 0.1 % TFA, 90 % aq. H 5.12, N 12.12.
CH3CN). Yield ∼ 30 mg. – MALDI-TOF MS: m/z = 712
(C27H40N7O10Y, [M+H]+); m/z = 788 (C27H40N7O10Ho,
[M+H]+); m/z = 792 (C27H40N7O10Tm, [M+H]+); m/z =
798 (C27H40N7O10Lu, [M+H]+). The analytical HPLC
method for the metal-DOTA-conjugates used an Agilent
1100 HPLC system and was performed on a Zorbax
300SB-C18 4.6 × 150 mm column (Agilent) with a flow
rate of 1 mL/min and a linear gradient of 100 % solution A
to 60 % solution B in 30 min (A: 0.1 % TFA in water;
solvent B: 0.1 % TFA, 90 % aq. CH3CN) with spectropho-
tometric monitoring at λ = 220 nm. The retention time
was the same (9.05 min) for◦ all complexes. 18, 19, 20
Crystal structure determination
Crystals suitable for X-ray diffraction were obtained
by crystallization of 3 from the pure oil and of 7 from
acetone. The data were collected on a Siemens SMART
CCD diffractometer (graphite monochromated MoKα ra-
˚
diation, λ = 0.71070 A) by use of ω scans at 293 K
(3) and 173 K (7). The structures were solved by Direct
Methods using SHELXS-97 [35] and refined on F2 us-
ing all reflections with SHELXL-97 [36]. All non-hydrogen
atoms were refined anisotropically and the carbon-bound hy-
drogen atoms were placed in calculated positions and as-
1
and 21: decomposition > 220 C. 21: H NMR (400 MHz,
signed to an isotropic displacement parameter of Uiso
=
D2O): δ = 1.40 (m, 2 H, NHCH2CH2CH2), 1.45 (m, 2 H,
NHCH2CH2CH2), 2.23 – 2.82 (broad, 12 H, NCH2CH2N;
2 H, NCH2CH2CO), 3.09 (t, 2 H, CONHCH2), 3.12 (m,
2 H, CONHCH2), 3.12 – 3.73 (broad, 8 H, NHCH2CO;
broad 4 H, NCH2CH2N), 3.75 (broad, 2 H, NCH2CH2CO),
6.74 (s, 2 H, CH=CH). – 13C NMR (100.64 MHz, D2O):
δ = 26.6 (NHCH2CH2CH2), 26.7 (NHCH2CH2CH2), 34.3
(NCH2CH2CO), 38.2 (NCH2CH2CO), 39.8 (CH2NHCO),
40.0 (CONHCH2), 54.3 – 56.8 (8× CH2, NCH2CH2N), 63.4
(1× CH2, NHCH2CO), 65.7 (3× CH2, NHCH2CO), 134.8
(CH=CH), 171.4 (NCH2CH2CO), 174.2 (=CHCO), 180.9
2
˚
0.08 A . Hydrogen atoms bonded to nitrogen were found.
SADABS [37] was used to perform area-detector scaling
and absorption corrections. Important parameters of the sin-
gle crystals, data collection and the refinement of the struc-
ture are listed in Table 1. Further crystallographic data were
deposited as supplementary publication no. CCDC 608991
(7) und CCDC 608992 (3) and can be obtained free of
charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data request/cif.
Acknowledgements
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406
S. W. Kohl et al. · Important Starting Materials for Metal-coded DOTA-Based Affinity Tags
This work was supported by the Deutsche Forschungsge- Dr. Peter Henklein, Universita¨tsklinikum Charite´, Humboldt-
meinschaft (SPP “Lanthanoidspezifische Funktionalita¨ten in Universita¨t zu Berlin, for the purification of compounds
Moleku¨l und Material”) and the Fonds der Chemischen In- 16 – 21.
dustrie. We thank Barbara Brecht-Jachan, Prisca Kunert and
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