Document [original]

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 M¨uggeb, Katharina Janekc, and Hardy Weißhoffb

aInstitut f¨ur Chemie, Technische Universit¨at Berlin, Straße des 17. Juni 135, D-10623 Berlin,

Germany

bInstitut f¨ur Chemie, Humboldt-Universit¨at zu Berlin, Brook-Taylor-Straße 2, D-12489 Berlin,

Germany

cUniversit¨atsklinikum Charit´e, Humboldt-Universit¨at zu Berlin, Monbijoustraße 2, D-10098 Berlin,

Germany

Reprint requests to Prof. Dr. H. Schumann. Fax +49 30 31422168.

E-mail: [email protected]u-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 1Hand13C 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

been intensively studied because of their numerous

applications, which often require selective functional-

ization [1, 2]. Metal ion-conjugated peptides with 1,4,

7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

(DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid

(NOTA) or 1,4,8,11-tetraazacyclotetradecane-1,4,8,

11-tetraacetic acid (TETA) ligands are ideal agents

for a spectrum of applications in biomedicine,

as therapeutic radiopharmaceuticals, luminescent

probes for biochemical analysis, or MRI contrast

agents [3 – 6]. Recently a new class of DOTA con-

jugates was introduced, the so-called element- or

metal-coded affinity tags (MECAT) [7, 8]. These

reagents can be used in quantitative proteomics, as

an additional or alternative method to established

2D-GE and recently developed methods employing

isotope-coded affinity tags (ICAT) and isobaric

tags for relative and absolute quantitation (ITRAQ)

[9 – 13].

Metal-coded affinity tags are reagents composed

of a chelating ligand, a monoisotopic metal ion, pre-

dominantly rare earth cations, and a reactive group

with specificity towards thiol or amino groups. The

affinity can be achieved for example by an incorpo-

rated group like biotin [11] or by interaction with

antibodies [4, 14]. The principle of MECATs is de-

rived from ICAT, but instead of the stable isotope la-

beling of proteins or peptides a metal ion labeling

is applied. The protein mixture of two or more sets

of cell states is independently labeled with MECAT

reagents containing different metal ions; the sam-

ples are combined, and then conventionally cleaved.

The MECAT labeled peptides are isolated by affin-

ity chromatography and analyzed by LC-ESI-MS/MS.

Peptide sequence information is obtained by tandem

mass spectrometry and computer searches of pro-

tein data banks. Quantitation of proteins in two cell

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398 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

the identical peptide peak pair from the samples de-

fined by the mass difference of the complex ions

chosen.

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.

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-

{2-[4-(maleimido-N-propylacetamidobutyl)amino]-2-

oxoethyl}-1,4,7,10-tetraazacyclododecane-4,7,10-tri-

acetic acid (17) and its Y, Ho, Tm, and Lu com-

plexes.

Results and Discussion

Synthesis of 1-benzyl-1,4,7,10-tetraazacyclododecane

(6) and 1,4,7,10-tetraazacyclododecane-1-acetic acid

ethyl ester (11)

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 1is 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 2with benzyl bromide and an

excess of K2CO3results 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.

Route II for the synthesis of 6starts with the

complete tosylation of 1at 0 ◦C yielding 7[23]

as a white powder which forms colorless single

crystals from acetone suitable for X-ray analysis

(Scheme 2). Cyclization of 7with 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 4yields 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 H2SO4for

three days [26]. Reaction with Mo(CO)6following the

procedure described by Patinec et al. [27, 28] resulted

3-1,4,7,10-tetraazacyclododecane molybdenumtri-

carbonyl (10) [26], which was alkylated with benz-

ylbromide and bromoacetic acid ethyl ester in DMF

followed by decoordination from the Mo(CO)3frag-

ment by HCl yielding 6and 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)

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-

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 3and 7are 1.61 and

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 3and N(2)–C(3)–

C(4)–N(3) 179◦(antiperiplanar) and N(1)–C(1)–

C(2)–N(2) 69◦((+)-synclinal) for compound 7.For3

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)

118.11(17), C(11)–N(3)–S(2) 120.4(2).

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

(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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400 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 7between 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 6and 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).

The triply protected DOTA-derivative 15 reacts

with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetrameth-

yluronium hexafluorophosphate (HATU), H¨unig’s

base (DIEA) and 1,4-diaminobutane trityl resin in

DMF with formation of the resin-fixed tris-tert-butyl

ester of 2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymeth-

yl)-1-cyclododecyl)-acetyl-diaminobutane, which is

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

-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,

holmium, thulium, and lutetium complexes 18,19,

20,and21 have been characterized by MALDI-TOF

mass spectrometry. Fig. 3 shows the MALDI-TOF

mass spectrum of 17 chelating Y3+,Ho

3+,Tm

3+and

Lu3+ions. The spectrum demonstrates the utility of

rare earths embedded in peptide specific labels as

internal standards for quantitative proteomics based

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S. W. Kohl et al. ·Important Starting Materials for Metal-coded DOTA-Based Affinity Tags 401

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.

on mass spectrometry. Here, mass tags with differ-

ences from 4 Da for 165Ho/169Tm-17 to 86 Da for

89Y/175Lu-17 are shown as an example for the variety

of different combinations. The use of rare earth ele-

ments in addition has the advantage that quantitation

can be accomplished by means of ICP MS with very

high efficiency and sensitivity. Furthermore, the mass

differences between the heavy rare earth-containing

tags is useful for the peptide and protein identification

in complex mixtures [33].

The lutetium complex 21 was also characterized by

elemental analysis and 1Hand13C 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

macrocyclic CH2protons at low pH values [20,34].

100 792.1676

798.1712

788.1628

mass (m/z)

intensity

Tm(III)

Y(III) Ho(III)

Lu(III)

691.0 717.2 744.4 770.6 797.2

712.1332

6.4E+4

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

CH3)2(4) was prepared according to the literature [24]. 1H

and 13C NMR spectra were recorded with Bruker ARX 200

and Bruker AV 400 spectrometers. Chemical shifts

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.

HN(CH2CH2NHSO2C6H4CH3-p)2(2). Diethylenetri-

amine (10.0 g, 0.097 mol) and triethylamine (19.2 g,

0.190 mol) were dissolved in CH2Cl2(300 mL) and

cooled to −48 ◦C. To this solution p-toluenesulfonylchlor-

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

three times with water. The organic layer was dried with

Na2SO4and the solvents evaporated to give a colorless oil,

which was further dried in vacuum. The residue was crys-

tallized from CH2Cl2/CH3OH (1 : 3). Beside the favored di-

tosylated oily product (2), the crystalline tri-tosylated prod-

uct 7is formed (m. p. 59 – 61 ◦C). Yield: 30.0 g (75 %) for 2

and6.0g(10%)for7.–1HNMR(25◦C, 200 MHz, CDCl3):

=2.35(s,6H,Ts-CH

3), 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). –

13CNMR(25◦C, 100.64 MHz, CDCl3):

= 21.0 (Ts-

CH3), 41.9 (NHCH2CH2NH-Ts), 47.3 (NHCH2CH2NH-

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402 S. W. Kohl et al. ·Important Starting Materials for Metal-coded DOTA-Based Affinity Tags

Ts), 126.6 (2 ×SO2CCHCH), 129.3 (2 ×SO2CCHCH),

136.3 (2 ×SO2CCH), 142.9 (2 ×SO2CCHCHCCH3). –

C18H25N3O4S2(411.53): calcd. C 52.53, H 6.12, N 10.21,

S 15.58; found C 52.45, H 5.99, N 10.12, S 15.55.

C6H5CH2N(CH2CH2NHSO2C6H4CH3-p)2(3).Theto-

sylated trisamine 2(5.8 g, 14 mmol) was dissolved in CH3

CN (200 mL) and 5.5 g (39 mmol) of dried K2CO3were

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 3as a pale yellow oil, which crystallizes after sev-

eral days. M. p. 52 ◦C. Yield: 6.9 g (98 %). – 1HNMR

(25 ◦C, 400 MHz, CDCl3):

=2.30(s,6H,CH

3), 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,4H,SO

2CCHCH). – 13CNMR(25◦C,

100.64 MHz, CDCl3):

= 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.

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

(65 %). – 1HNMR(25◦C, 400 MHz, CDCl3):

=2.39(s,

6H,Ts-CH

3), 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).

–13CNMR(25◦C, 100.64 MHz, CDCl3):

= 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

(8.0 mmol) of 5, 6.8 g (48 mmol) of anhydrous Na2HPO4,

and sodium amalgam (2 %, 9.5 g, 48 mmol) were stirred

in CH3CN (250 mL) at 80 ◦C for one day. The colour-

less mixture changed to white, and mercury precipitated

which was separated. The solvent was removed on a ro-

tary evaporator and the grey residue was dissolved in CHCl3

(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 6as 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 K2CO3were 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 %). – 1HNMR

(25 ◦C, 400 MHz, CDCl3):

= 2.44 – 2.77 (m, 16 H,

macrocyclic CH2), 3.52 (s, 2 H, PhCH2), 7.12 – 7.25 (m,

5 H, benzyl-H). – 13C-NMR (25 ◦C, 100.64 MHz, CDCl3):

= 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.

p-CH3C6H4SO2N(CH2CH2NHSO2C6H4CH3-p)2(7).

2.0 g (19 mmol) of 1and NEt3(7.9 mL, 57 mmol) were

dissolved 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 7as

a white solid. M. p. 59 – 61 ◦C. Yield: 10.2 g (95 %). –

1HNMR(25◦C, 200 MHz, [D6]DMSO):

=2.37(s,

9H,Ts-CH

3), 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=

8.2Hz,4H,NHSO

2CCH). – 13CNMR(25◦C, 50.32 MHz,

[D6]DMSO):

= 20.97 (Ts-CH3), 41.59 (2×NHCH2),

48.40 (2×NHCH2CH2), 126.54 (2×NHSO2CCHCH),

126.83 (NSO2CCHCH), 129.68 (2×NHSO2CCHCH),

129.88 (NSO2CCHCH), 135.31 (NSO2CCH), 137.36 (2×

NHSO2CCH), 142.76 (2×NHSO2CCHCHCCH3), 143.46

(NSO2CCHCHCCH3). – C25H31N3O6S3(565.73): calcd.

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.

(p-CH3C6H4SO2NCH2CH2)4(8).Cs

2CO3(50.6 g, 0.150

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 %). – 1HNMR(25◦C, 200 MHz, CDCl3):

=2.45(s,12H,Ts-CH

3), 3.43 (s, 16 H, macrocyclic CH2),

7.28 – 7.35 (m, 8 H, SO2CCHCH), 7.61 – 7.71 (m, 8 H,

SO2CCHCH). – 13CNMR(25◦C, 50.32 MHz, CDCl3):

21.38 (Ts-CH3), 44.41 (CH2), 126.43 (SO2CCHCH), 129.68

(SO2CCHCH), 135.41 (SO2CCH), 142.75 (SO2CCHCHC).

–C

36H44N4O8S4(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.

(HNCH2CH2)4(9). 65.3 g (0.083 mol) of 8was stirred

with 100 mL of concentrated sulphuric acid for 3 d at 130 ◦C.

The initially colorless solution changed to brown after a

few h and a black precipitate occurred. The mixture was

cooled to 0 ◦C, diluted with 150 mL of water and then the

pH was adjusted to >13 by addition of solid KOH (130 g,

2.32 mol). The filtered precipitate was washed with EtOH

(2 ×90 mL) and the aqueous and the organic phases were

combined and evaporated. The brownish residue was dis-

solved in 80 mL of 0.1 MHCl and washed with CH2Cl2(4 ×

30 mL). The pH of the aqueous phase was adjusted again to

>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 9was dried in

a vacuum. M. p. 113 – 114 ◦C. Yield: 8.6 g (60 %). – 1HNMR

(25 ◦C, 400 MHz, CDCl3):

= 2.17 (s, 4 H, NH), 2.68 (s,

16 H, CH2). – 13CNMR(25◦C, 100.64 MHz, CDCl3):

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)3Mo(HNCH2CH2)4(10). 2.7. g (16 mmol) of 9and

4.6 g (16 mmol) of Mo(CO)6were 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.–C

11H20N4O3Mo

(352.24): calcd. C 37.51, H 5.72, N 15.91; found C 36.93,

H 5.83, N 15.61.

C2H5OC(O)CH2N(CH2CH2NH)3CH2CH2(11).11 was

prepared in analogy to the synthesis of 6following route II

using1.1g(3.0mmol)of10, 1.4 g (49 mmol) of K2CO3,

0.33 mL (3.0 mmol) of BrCH2COOEt,and80mLofDMF.

Yield: 0.40 g (55 %) of 11 as a light yellow solid. M. p.

89–91 ◦C. – 1HNMR(25◦C, 400 MHz, CDCl3):

1.20 (t, 3J= 8.9 Hz, 3 H, CH2CH3), 2.48 – 2.84 (m, 16 H,

macrocyclic CH2), 3.30 (s, 2 H, NCH2CO), 4.10 (q, 3J=

8.9Hz,2H,CH2CH3). – 13CNMR(25◦C, 100.64 MHz,

CDCl3):

= 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.

C6H5CH2NCH2CH2[N(CH2COOtBu)CH2CH2]3(12).

1.1 g (4 mmol) of 6and 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. – 1HNMR

(25 ◦C, 200 MHz, CDCl3):

=1.39(s,18H,tBu), 1.43 (s,

9H,tBu), 2.57 – 2.81 (m, 16 H, macrocyclic CH2), 3.17 (s,

4H,CH2COt

2Bu), 3.29 (s, 2 H, CH2COt

2Bu), 3.49 (s, 2 H,

benzyl-CH2),7.20–7.25(m,5H,benzyl-H). – 13CNMR

(25 ◦C, 50.32 MHz, CDCl3):

= 28.72 (C(CH3)3), 52.41

(benzyl-N(CH2)2NCH2CH2), 52.60 (benzyl-NCH2CH2),

52.72 (benzyl-NCH2CH2), 56.68 (NCH2CO), 60.11 (NCH2-

benzyl), 81.75 (C(CH3)3), 127.32 (benzyl-Cpara), 128.51

(benzyl-Cmeta), 128.97 (benzyl-Cortho), 136.01 (benzyl-

Cquart ), 169.68 (NCH2CO2tBu). – C33H56N4O6(604.82):

calcd. C 65.53, H 9.33, N 9.26; found C 65.83, H 9.51,

N 9.40.

HNCH2CH2[N(CH2COOtBu)CH2CH2]3(13). Hydro-

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) yielded a light yellow solid (0.41 g, 40 %). M. p.

47–50 ◦C. – IR (KBr):

= 1733 (s, C=O, ester), 1158 (s,

C–O, ester), 1059 (m, C–N) cm−1.–1H NMR (400 MHz,

CDCl3,r.t.):

=1.39(s,27H,tBu), 2.52 (m, 4 H,

NHCH2CH2), 2.68 (s, 8 H, NRCH2CH2NR), 2.77 (m, 4 H,

NHCH2), 3.26 (s, 6 H, CH2CO2tBu). – 13C{1H}NMR

(100.64 MHz, CDCl3,r.t.):

= 28.24 (C(CH3)3), 47.56

(NHCH2CH2), 50.70 (NH(CH2)2NRCH2), 52.16 (NHCH2),

52.24 (NH(CH2)2NRCH2CH2), 52.80 (NH(CH2)2N-

R(CH2)2NCH2CO), 57.23 (s, NH(CH2)2NCH2CO), 81.78

(C(CH3)3), 171.14 (NCH2CO). – MS (EI, 70 eV): m/z

(%) = 514 (24.10) [M]+, 413 (100) [M – CO2–tBu]+.–

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.

EtOC(O)CH2NCH2CH2[N(CH2COOtBu)CH2CH2]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.):

=1.21(t,3J=8.85Hz,

3H,OCH

2CH3), 1.39 (s, 18 H, tBu), 1.41 (s, 9 H, tBu),

2.50 – 2.86 (m, 16 H, macrocyclic CH2), 3.20 – 3.31 (m,

6H,CH2CO2tBu), 3.34 (s, 2 H, NCH2CO2Et), 4.10 (q, 3J=

8.85 Hz, 2 H, OCH2CH3). – 13C{1H}NMR (50.32 MHz,

CDCl3,r.t.):

= 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

(C(CH3)3), 171.98 (NCH2CO2tBu), 172.64 (NCH2CO2Et).

–C

30H56N4O8(600.80): calcd. C 59.98, H 9.39, N 9.33;

found C 59.83, H 9.31, N 9.20.

HOC(O)CH2NCH2CH2[N(CH2COOtBu)CH2CH2]3

(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

(0.014 mL, 0.2 mmol) and 10 mL of DMF at 70 ◦C(2h),

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):

= 1738 (s, C=O, ester), 1644 (s, C=O, acid),

1161 (s, C–O, ester), 1120 (m, C–N) cm−1.–1HNMR

(400 MHz, CDCl3,r.t.):

=1.42(s,27H,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,

2H,CH2COOH). – 13C{1H}NMR (100.64 MHz, CDCl3,

r. t.):

= 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

(3.10) [M]+, 471 (100) [M – CO2–tBu]+.–C

28H52N4O8

(572.74): calcd. C 58.72, H 9.15, N 9.78; found C 58.61,

H 9.07, N 9.51.

H2N(CH2)4NHC(O)CH2NCH2CH2[N(CH2COOH)CH2-

CH2]3(16). To a solution of 15 (1.34 g, 2.35 mmol) in DMF

(40 mL), 0.983 g (2.585 mmol) of HATU and 0.5 mL of

H¨unig’s base were added. The mixture was stirred for 5 min

and added to 5 g of 1,4-diaminobutane trityl resin (loading

0.47 mmol/g, 2.35 mmol) in DMF. The reaction mixture

was agitated at r. t. overnight and the solvent removed in

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):

=1.40(m,2H,NH

2CH2CH2),

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),

3.60 – 4.20 (broad, 8 H, NHCH2CO). – 13C{1H}NMR

(100.64 MHz, D2O):

= 24.2 (NHCH2CH2), 25.4

(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]+.–C

20H38N6O7(474.56): calcd. C 50.62, H 8.07,

N 17.71; found C 50.53, H 8.01, N 17.81.

C4H2O2N(CH2)2C(O)NH(CH2)4NHC(O)CH2NCH2-

CH2[N(CH2COOH)CH2CH2]3(17). To a solution of 16

(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

-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 CHCl3and 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):

=1.42(m,2H,NHCH

2CH2CH22), 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,2H,

NCH2CH2CO), 3.64 – 4.23 (broad, 8 H, NHCH2CO), 6.88

(s, 2 H, CH=CH). – 13C{1H}NMR (100.64 MHz, D2O):

= 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.

LnC4H2O2N(CH2)2C(O)NH(CH2)4NHC(O)CH2NCH2-

CH2[N(CH2COO)CH2CH2]3(18)–(21).Usinga0.1M

Na2CO3/HCl solution (pH = 7.5 buffer), 0.15 mmol

of YCl3,HoCl

3,TmCl

3or LuCl3were dissolved and

combined with 60 mg (0.096 mmol) of 17. The pH was

adjusted to 7.0 and the samples were kept at r. t. over

night. Analytical HPLC was carried out to verify the

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S. W. Kohl et al. ·Important Starting Materials for Metal-coded DOTA-Based Affinity Tags 405

Compound 37

Empirical formula C25H31N3O4S2C25H31N3O6S3

Formula weight [g mol−1] 501.65 565.71

Crystal size [mm3]0.75 ×0.52 ×0.35 0.52 ×0.24 ×0.15

Crystal system monoclinic monoclinic

Space group P21/cP21/n

Z44

a[˚

A] 11.9855(4) 5.1910 (1)

b[˚

A] 10.9267(4) 27.4576(5)

c[˚

A] 20.0058(7) 18.9445(4)

[deg] 97.530(1) 93.168(1)

V[˚

A3] 2597.40(16) 2696.08(9)

Dcalcd [g cm−3] 1.283 1.394

Absorption coefficient [mm−1] 0.240 0.320

Min./max. transmission 0.9207 / 0.8404 0.8880 / 0.4807

F(000) [e] 1064 1192

Range for data collection [deg] 1.71 ≤

≤26.00 1.48 ≤

≤27.50

Data set −14 ≤h≤12 −6≤h≤6

−13 ≤k≤13 −30 ≤k≤35

−22 ≤l≤24 −24 ≤l≤21

Reflections, collected 17564 20467

Reflections, unique 5095 (Rint = 0.0773) 6177 (Rint = 0.0859)

Data / restraints / parameter 5095 / 0 / 317 6177 / 1 / 345

Goodness-of-Fit (F2) 0.988 1.081

Final Rindices (I≥2

(I))R1 = 0.0516 R1 = 0.0798

wR2 = 0.1180 wR2 = 0.1410

Rindices (all data) R1 = 0.0971 R1 = 0.1404

wR2 = 0.1394 wR2 = 0.1625

Largest diff. peak and hole [e ˚

A−3] 0.234 / −0.382 0.334 / −0.338

Table 1. Parameters of the sin-

gle crystals, data collection and

structure refinement of 3and 7.

coordination to the Ln(III) ions. The complexes were

purified on an Agilent-Prep-C18 column (solvent A:

0.1 % TFA in water; solvent B: 10 % of 0.1 % TFA, 90 % aq.

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

= 220 nm. The retention time

was the same (9.05 min) for all complexes. 18,19,20

and 21: decomposition >220 ◦C. 21:1H NMR (400 MHz,

D2O):

= 1.40 (m, 2 H, NHCH2CH2CH2), 1.45 (m, 2 H,

NHCH2CH2CH2), 2.23 – 2.82 (broad, 12 H, NCH2CH2N;

2H,NCH

2CH2CO), 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):

= 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

(1×CO, CO), 181.2 (3×CO, CO). – C27H40N7O10Lu

(797.65): calcd. C 40.66, H 5.05, N 12.29; found C 40.39,

H 5.12, N 12.12.

Crystal structure determination

Crystals suitable for X-ray diffraction were obtained

by crystallization of 3from the pure oil and of 7from

acetone. The data were collected on a Siemens SMART

CCD diffractometer (graphite monochromated MoK

ra-

diation,

= 0.71070 ˚

A) by use of

scans at 293 K

(3) and 173 K (7). The structures were solved by Direct

Methods using SHELXS-97 [35] and refined on F2us-

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-

signed to an isotropic displacement parameter of Uiso =

0.08 ˚

A2. 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-

meinschaft (SPP “Lanthanoidspezifische Funktionalit¨aten in

Molek¨ul und Material”) and the Fonds der Chemischen In-

dustrie. We thank Barbara Brecht-Jachan, Prisca Kunert and

Dr. Peter Henklein, Universit¨atsklinikum Charit´e, Humboldt-

Universit¨at zu Berlin, for the purification of compounds

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