Convergent and sequential synthesis of dendritic, multivalent complexing agents

Article abstract of DOI:10.1055/s-2002-33116

Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized as dendritic ligands containing two, three, four, five or six terminal nitrilotriacetic acid (NTA) groups through convergent and sequential pathways. Tetrafluorophenyl esters were employed as activating reagents in the coupling step of assembling NTA groups to the cyclic polyamine head. A key step of the sequential synthesis is the use of the coupling reagent, 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) for the formation of amide bonds between the cyclic polyamine and pendant groups. Finally, two representative compounds were used to demonstrate the formation of stoichiometric protein assemblies.

Full text of DOI:10.1055/s-2002-33116

1398
PAPER
Convergent and Sequential Synthesis of Dendritic, Multivalent Complexing
Agents
Synthesis of
Den
u
,
Multiv
i
alent
C
h
omplexing A
u
gents a Liu, Robert H. E. Hudson, Nils O. Petersen*
Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7 Canada
Fax +1(19)6613022; E-mail: petersen@uwo.ca
Received 4 November 2001; revised 5 April 2002
cients of these molecules are size dependent.5 It is well
known that aggregation of membrane proteins plays a key
role in signal transduction,6 so it is of interest to prepare
membrane protein aggregates with defined sizes and
Abstract: Two series of macrocyclic polyamine derivatives with
various length of linkers were synthesized as dendritic ligands con-
taining two, three, four, five or six terminal nitrilotriacetic acid
(NTA) groups through convergent and sequential pathways. Tet-
rafluorophenyl esters were employed as activating reagents in the shapes. To generate aggregates of proteins, two series of
coupling step of assembling NTA groups to the cyclic polyamine
complexing agents containing multiple NTA functional
head. A key step of the sequential synthesis is the use of the cou-
groups were designed as shown in Figure 1. These com-
pling reagent, 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-
pounds have dendritic arms, which can chelate to histi-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) for the for-
dine-tagged proteins through nickel ions. Changing the
mation of amide bonds between the cyclic polyamine and pendant
size of the central ring will give a corresponding change
in the number of dendritic arms. Each dendritic chelating
arm can bind to a protein so that different sizes of protein
clusters can be created with the various reagents. It may
also be possible to control the tightness of the clusters
formed by varying the length of the linker chain. In this
paper, we report two synthetic strategies for making den-
groups. Finally, two representative compounds were used to dem-
onstrate the formation of stoichiometric protein assemblies.
Key words: complexing agents, polyamines, coupling, nitrilotri-
acetic acid
Immobilized metal affinity chromatography (IMAC) has
become a widely used technique in molecular biology and
protein engineering since it was first reported by Porath in
1975.1 In particular, it has been used for the isolation of
proteins, peptides, and nucleic acids. During IMAC, a
metal ion, such as Ni2+, Zn2+, or Cu2+, is complexed and
immobilized by a chelator, which is covalently linked to a
solid matrix. Our interest is based on the use of nitrilotri-
acetic acid (NTA) which has been employed as a chelator
to purify proteins since 1987.2 NTA chelates a Ni(II) ion
through four of its potential six coordination sites, the re-
maining sites bind two histidine residues forming an octa-
hedral complex. The formation of this type of complex
relies on incorporation of 2–6 histidines at one terminus of
a protein. The histidine-tagged protein is bound by the
metal immobilized on the NTA-derivatized stationary
phase, while other proteins are washed off the column.
The binding of Ni(II) by NTA is reversible,3 and the re-
versibility can be achieved by the use of ethylenediamine
tetraacetic acid (EDTA) which has a higher binding con-
stant for Ni(II) ions or imidazole which competes with the
histidine binding.
In the course of our study on biomembrane dynamics, we
reported a series of macrocyclic polyamine amphiphiles
both with and without fluorescent nitrobenzoxadiazole
(NBD) labels.4 These compounds have varied, but defined
sizes and shapes and have been useful for investigation of
diffusion in biological membranes. The diffusion coeffi-
Figure 1 Complexing agents containing multiple NTA functional
groups
Synthesis 2002, No. 10, Print: 30 07 2002.
Art Id.1437-210X,E;2002,0,10,1398,1406,ftx,en;M04401SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

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Synthesis of Dendritic, Multivalent Complexing Agents
1399
dritic ligands with a macrocyclic polyamine head group ride and hexacyclen trisulfate, respectively. To avoid the
and multiple nitrilotriacetic acid pendant groups. The use formation of byproducts with incomplete carboxymethyl-
of these ligands to assemble dimers, trimers, tetramers, ation during the preparation of 1a,b, we used lysine-
pentamers, or hexamers of histidine-tagged proteins will NTA2a,b (8), to couple with tetrafluorophenyl activated es-
be reported separately, but we illustrate the principle here ters of 4a,b as shown in Scheme 1. However, carboxyme-
with making dimers and trimers.
thylation of the polyamino acids (10a–e) as shown in the
sequential pathway (Scheme 2) yielded, under conditions
of controlled pH the fully substituted species, 2a–e, as the
major products. The synthesis of polyamides rather than
polyamines is chosen to control the shape of the central
ring. The rotation about the amide bond is restricted at
room temperature decreasing the conformational mobility
of the ring. This has been observed previously4 and will be
evident from the NMR data reported here as well. Also, a
polyamine ring may acts as a chelator as was demonstrat-
ed previously in the use as a radionuclide carrier attached
to monoclonal antibodies (mAbs) for cancer chemothera-
py.7 In the case of the macrocyclic system, introduction of
The macrocyclic polyamine derivatives with multiple
NTA functional groups shown in Figure 1 were synthe-
sized as outlined in Scheme 1 and Scheme 2. Their struc-
tures were confirmed by mass spectrometry (MS), NMR
spectroscopy and FTIR spectroscopy. The synthesis was
achieved by both a convergent and a sequential pathway
to provide versatility. All of the syntheses started from cy-
clic polyamines, some of which are not commercially
available as free bases. For example, 1,4,7,10,13-pen-
taazacyclopentadecane (3d) and 1,4,7,10,13-hexaazacy-
clooctadecane (3e) can be isolated as such easily from
1,4,7,10,13-pentaazacyclopentadecane pentahydrochlo-
Scheme 1 Convergent pathway to target molecules 1a,b
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C. Liu et al.
PAPER
Scheme 2 The pathway to target molecules 2a–e
TBTU: 2-(1H-benzotriazole-1,1,3,3,-tetramethyluronium tetrafluoroborate
Z-Glu-OBzl: L-glutamic acid N-(benzyloxycarbonyl)-1-benzyl ester
the side chain was achieved efficiently and in good yields The key step in Scheme 2 is the coupling of the polyamine
by direct amidation with succinic anhydride or L-glutamic with the commercially available derivative of glutamic
acid N-benzyloxycarbonyl-1-benzyl ester (Z-Glu-OBzl) acid, which was protected by a benzyloxycarbonyl (Z)
using coupling agents.
group at the N-terminus and benzyl at the C-terminus [L-
glutamic acid N-benzyloxycarbonyl-1-benzyl ester or N-
-Cbz-L-glutamic acid -benzyl ester]. The coupling
agent, 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluroni-
um tetrafluoroborate (TBTU), was first used in peptide
synthesis by Knorr in 1989,10 and was reported to be very
effective for couplings that involve a proline nitrogen,
which is similar to the secondary, cyclic polyamines used
in this study. The subsequent cleavage of protecting
groups (Z and benzyl) in compounds 9a–e by hydrogenol-
ysis (10 bar H2, room temperature, Pd/C) afforded
polyamino acids. The crude polyamino acids 10a–e were
obtained by filtration of the catalyst and evaporation of
the solvent. The pure compounds 10a–e were collected in
80–90% yields by recrystallization from ethanol. The last
step of the sequential pathway was the carboxymethyla-
tion of the polyamino acids 10a–e to provide dendritic
ligands 2a–e by a standard procedure in good yields.
Compounds 4a and 4b could be obtained quantitatively by
reacting 3a or 3b with succinic anhydride using the meth-
od of Kung and Izatt,8 and the products were easily puri-
fied (Scheme 1) in 45–65% yields. However, only two
polysuccinoyl amines were successfully obtained. This
route failed for the larger cyclic polyamines presumably
due to steric factors and/or decreased reactivity. The req-
uisite activated macrocyclic polysuccinoyl amine deriva-
tives 5a and 5b were prepared as the tetrafluorophenyl
(TFP) esters in 76–86% yields by the procedure described
by Wilbur et al.9 They are preferred in our work since they
are stable to hydrolysis and give good aminolysis yields
for products 1a,b while other activated esters, such as N-
hydroxysuccinicimide (NHS) esters, are more sensitive to
ambient moisture. The benzyloxycarbonyl protecting
group of compound 7 was removed by hydrogenolysis un-
der basic conditions. Purification of product 8 was
achieved by recrystallization. For the last step of the syn- The final compounds 1a,b and 2a–e are extremely polar
thesis of 1a and 1b, an acetonitrile–water mixture was and isolation by standard chromatographic techniques
used as a solvent to address the problem of the differential such as silica gel column chromatography was difficult.
solubility of TFP ester and lysine-NTA. Triethylamine Anion ion exchange chromatography was used for the pu-
was found to be the best base for this reaction, even rification of 2a,b and 1a,b. It was noted that as the num-
through it was difficult to remove afterwards.
ber of NTA groups increases, the solubility decreases. So
compounds 2c–e precipitate out from the mixture of etha-
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Synthesis of Dendritic, Multivalent Complexing Agents
1401
nol and water, and their purification can be performed by protein in the mixtures move more slowly and that the
simple filtration. The crude products 2c–e were further complex with 1b moves most slowly. The relative rate of
purified by recrystallization from methanol and collected migration is consitent with the majority of the protein
in 32–56% yields. Being concerned about the polarity of moving as monomers in lanes 1 and 4, as dimers in lane 5
the second set of dendritic compounds 2a–e, we initially and as trimers in lane 2. Lanes 3 and 6 confirm that forma-
tried another route to obtain 2e as shown in Scheme 3. We tion of the stoichiometric assemblies is mediated by the
aimed to make a representative methyl ester, i.e. 10e, fol- NTA-Ni-His complex since addition of excess EDTA
lowed by carboxymethylation with methyl bromoacetate causes the proteins to move as monomers. It should be
to give its complete methyl ester 11e. Subsequent hydrol- noted that the mixtures in lanes 3 and 6 were prepared by
ysis of all the methyl groups under basic conditions would adding EDTA to solutions after the formation of the pro-
give the desired compound 2e. However, it was difficult tein assemblies but prior to running the gels showing that
to limit the reaction to tertiary alkylation of the amine 11e
since the quaternary ammonium product formed easily in
organic solvents. In contrast, direct alkylation of the ami-
no acid was successfully carried out using bromoacetic
acid in aqueous solution under basic conditions (pH 11–
12) for a few days and followed by acidification with 6 M
hydrochloric acid to pH 1.
As a preliminary demonstration of the utility of these den-
dritic reagents for formation of stoichiometric protein as-
semblies, we used compounds 1a,b to aggregate His-
tagged thioredoxin in solution. Thioredoxin was chosen as
a model monomeric protein since it is water-soluble, heat-
stable, of low molecular weight and locally available (Dr.
Eric Ball, Department of Biochemistry, The University of
Western Ontario). Figure 2 shows the native polyacryla-
Figure 2 Native PAGE illustration of the aggregation of His-tagged
mide gel electrophersis analysis of solutions containing
mixtures of 1a and 1b with His-tagged thioredoxin (lanes
5 and 2, respectively) compared to a solution of protein
alone (lanes 1 and 4). It is evident that the majority of the
thioredoxin by compounds 1b (lane 2) and 1a (lane 5) and the subse-
quent dissociation by EDTA (lanes 3 and 6 respectively). Lanes 1 and
4 show monomeric protein in the absence of complexing agents. The
image of the original gel has been contrast enhanced for clarity.
Scheme 3
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1402
C. Liu et al.
PAPER
mass spectrometer. Reactions were monitored by TLC (precoated
plates 0.25 mm silica gel 60 F254) and visualized by UV (Z and ben-
zyl groups), ninhydrin (amino group), bromocresol blue (acids and
bases), or iodine vapour.
complex of E. coli His-tagged thioredoxin is observed
predominantly as a single oligomeric structure as long as
appropriate ratios of ligand:Ni2+:protein were used in so-
lutions. This is consistent with the expectation that the
dendritic ligands bind to the His-tagged protein with a
high affinity. Although the molecular weight (MW) of the
complexes were not determined directly, the gels provide
direct evidence that the size of the His-tagged thioredoxin
oligomers can be controlled in a reversible fashion by
changing the number of NTA groups on the complexing
reagents.
1,4-Bis(3 -carboxypropanoyl)diazacyclohexane (4a); Typical
Procedure
Piperazine (3a; 0.87 g, 10 mmol) was dissolved in benzene (10 mL)
and heated to 60–70 °C. Succinic anhydride (2.10 g, 20 mmol) in
benzene (20 mL) was added at this temperature and then the solu-
tion was refluxed for 48 h. After this period, the benzene was re-
moved under vacuum. The product 4a (1.85 g, 6.5 mmol) was
purified by crystallization from EtOH; yield: 65%; mp 156.6–158.5
°C.
In this work, only intermediates 4a,7, and 8 have previ-
ously been reported in the literature. We have detailed
IR (KBr): 3030 (OH stretch), 1720 (C=O stretch for carboxylic ac-
straightforward methods for the synthesis of two sets of id), 1600 (C=O stretch for tertiary amide), 1490 cm–1 (CH2 defor-
mation).
polyfunctional chelators 1a,b and 2a–e. We have tested
1H NMR (D2O):
= 2.56 (deformed t, J = 8 Hz, 4 H,
these compounds to show their ability to form stoichio-
metric complexes. These and other applications will be re-
ported separately. Access to appropriate ligands with
multiple binding features may be a useful step in many as-
pects of molecular biology or cell biology and protein en-
gineering. The formation constants for 1:1 trivalent metal
CH2CH2CO2H), 2.66 (deformed t, J = 8 Hz, 4 H, NCOCH2CH2),
3.62–3.48 (t, at r.t.; br s, at 55 °C, due to the existence of rotamers,
8 H, OCNCH2CH2NCO).
13C NMR (DMSO-d6): = 27.7, 29.3, 44.5, 52.4, 170.3, 174.3.
HRMS (FAB, oxalic acid/glycerol): m/z calcd for C12H18N2O6:
complexes of NTA (typically 108–1012) may be inade- 287.1243 [M + H]+; found: 287.1236 [M + H]+.
quate for in vivo applications, but the ability to form pro-
tein-NTA-metal-NTA-protein linkages may provide a
non-denaturing and readily reversible means for cross-
linking two proteins, such as antibodies and enzymes, for
1,4,7-Tris(3 -carboxypropanoyl)triazacyclononane (4b)
Compound 4b (0.21 g, 0.49 mmol) was prepared from 1,4,7-triaza-
cyclononane (3b; 0.14 g, 1.08 mmol) and succinic anhydride (0.33
g, 3.24 mmol) according to the procedure used for 4a in 45% yield
in vitro use. The bifunctional NTA ligands may also prove
useful for tethering ternary metal complexes to proteins,
as was done by Kline.11 The ability to introduce a protein-
reactive substituent into polyamino polycarboxylate che-
lator by the two pathways described here, should facilitate
the identification of additional areas where an interface
between coordination chemistry and protein biochemistry
may bear fruit.
as a white solid; mp 126–130 °C.
IR (KBr): 3010 (OH stretch), 1718 (C=O stretch for carboxylic ac-
id), 1602 (C=O stretch for tertiary amide), 1486 cm–1 (CH2 defor-
mation).
1H NMR (DMSO-d6):
= 2.46 (t, J = 6 Hz,
6
H,
OCCH2CH2CO2H), 2.58 (t, J = 8 Hz, 6 H, NOCCH2CH2), 3.68–
3.38 (2 br, 12 H, OCNCH2CH2NCO, conformational isomer).
13C NMR (DMSO-d6): = 27.6, 29.2, 40.4, 52.1, 170.2, 174.1.
HRMS (FAB, oxalic acid/glycerol): m/z calcd for C18H27N3O9:
430.1825 [M + H]+; found: 430.1848 [M + H]+.
Piperazine, 1,4,7-triazacyclononane, 1,4,7,10-tetraazacyclodode-
cane, hexacylen trisulfate, 2,3,5,6-tetrafluorophenol (TFP-OH), di-
cyclohexylcarbodiimide (DCC), bromoacetic acid, and succinic
anhydride were obtained from Aldrich Chemical Co. 1,4,7,10,13-
Pentaazacyclopentadecane pentahydrochloride was purchased from
Parish Chemical Co. TBTU and Z-Glu-OBzl were from Novabio-
chem. N( )-Z-L-Lysine was obtained from Fluka. Acrylamide,
N,N -methylene-bis(acrylamide), Coomassie brilliant blue R250,
tris(hydroxymethylaminomethane) hydrogen chloride (Tris-HCl)
and glycine were purchased from Life Technologies Inc. Ammoni-
um persulfate, N,N,N ,N -tetramethylenediamine (TEMED), bro-
mophenol blue, were bought from Sigma Chemical Co. CaCl2,
NaCl, sodium dodecyl sulfate (SDS), KCl and ethylenediamine tet-
raacetic acid (EDTA) were bought from BDH Chemicals. Bio-Rad
dye reagent and Mini-Protein II sets were obtained from Bio-Rad
laboratories. Native gel electrophoresis was run with a Bio-Rad
Model 200/2.0 power supply. All solvents are reagent grade and
were used without purification. Column chromatography was per-
formed with 230–400 mesh silica gel. Preparative TLC silica gel
1,4-Bis[3 -(2,3,5,6-tetrafluorophenoxycarbonyl)propanoyl]di-
azacyclohexane (5a); Typical Procedure
To a stirred solution of compound 4a (0.28 g, 0.98 mmol) in DMF
(10 mL) under argon was added 2,3,5,6-tetrafluorophenol (TFP-
OH) (0.41 g, 2.50 mmol) at 50 °C. A solution of dicyclohexylcar-
bodiimide (DCC, 0.45 g, 2.04 mmol) in DMF (10 mL) was added
dropwise at this temperature. The reaction mixture was stirred over-
night, and the solution was cooled to r.t. The white precipitate of di-
cyclohexylurea was filtered out, and the filtrate was evaporated
under vacuum. The residue of reaction was triturated with MeCN–
EtOAc (1:1, 15 mL). A white solid (0.49 g) was collected by vacu-
um filtration; yield: 86%; mp140–142 °C.
IR (KBr): 3422, 3086 (CH stretch for aromatic compound), 2970,
2930, (CH stretch), 1778 (C=O stretch for ester), 1696 (C=C
stretch), 1651 (C=O stretch for tertiary amide), 1485, 1451 (CH2 de-
formation), 1216 (C–F stretch), 851 cm–1 (CH out of plane deforma-
tion).
1H NMR (CDCl3): = 2.80 (t, J = 6 Hz, 4 H, NCOCH2CH2COO),
3.08 (t, J = 6 Hz, 4 H, NCOCH2CH2COO), 3.76–3.52 (2 m, J = 6
Hz, 8 H, CONCH2CH2NCO, conformational isomer), 6.98 (m,
J = 5 Hz, 2 H, 2 C6HF4).
F
254 plates were obtained from EM Separation Technology. Melting
points (uncorrected) were determined using a Thomas-Hoover
model capillary melting points apparatus. 1H and 13C NMR spectra
were recorded on a Varian Gemini-300 spectrometer operating at
300 MHz (1H) and 75 MHz (13C). IR spectra were recorded as KBr
pellets on a Perkin-Elmer System 2000 FTIR spectrometer. High
resolution mass spectra were obtained on a Finigan MAT 8320
13C NMR (CDCl3): = 26.4, 30.3, 41.1, 48.3, 128.5, 135.2, 172.0,
173.2.
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Synthesis of Dendritic, Multivalent Complexing Agents
1403
HRMS (EI, 70 V): m/z calcd for C24H18F8N2O6: 582.1037 [M]+;
13C NMR (D2O): = 26.3, 28.9, 32.6, 42.9, 59.1, 70.8, 174.7, 175.8.
found: 582.1040 [M]+.
1,4-Bis[5 -N,N-di(carboxymethyl)amino-5 -carboxypentylami-
no-3-oxopropanoyl]diazacyclohexane (1a); Typical Procedure
Lysine-NTA (8; 0.26 g, 0.99 mmol) was dissolved in H2O (4 mL).
To this solution was added Et3N (0.3 mL), followed by a solution of
5a (0.29 g, 0.49 mmol) in warm MeCN (5 mL). The reaction solu-
tion turned cloudy immediately and was stirred at r.t. for 12 h. The
reaction was monitored by TLC, the developed component was vi-
sualized by ninhydrin spray. When the reaction was complete, it
was quenched by the addition of aq 1 M HCl to pH 1. After extrac-
tion with CHCl3, the aqueous layer was evaporated to dryness under
vacuum and the resulting product redissolved in H2O. The resulting
residue was purified twice on by anion exchange column chroma-
tography and collected to give 1a (0.13 g) in 35% yield; mp 170 °C
(dec.).
1,4,7-Tris[3 -(2,3,5,6-tetrafluorophenoxycarbonyl)propanoyl]-
triazacyclononane (5b)
Compound 5b (0.15 g, 0.17 mmol) was obtained from 4b (0.11 g,
0.25 mmol), TFP-OH (0.15 g, 0.88 mmol) and DCC (0.18 g, 0.82
mmol) according to the procedure used for 5a as a white solid in
67% yield; mp 120–122 °C.
IR (KBr): 3412, 3085 (CH stretch for aromatic compound), 2950
(CH stretch), 1776 (C=O stretch for ester), 1686 (C=C stretch),
1650 (C=O stretch for tertiary amide), 1480, 1450 (CH2 deforma-
tion), 1216 (C–F stretch), 850 cm–1 (CH out of plane deformation).
1H NMR (CDCl3): = 2.82 (t, J = 8 Hz, 6 H, NOCCH2CH2COO),
3.07 (t, J = 8 Hz, 6 H, NOCCH2CH2COO), 3.72–3.53 (2 m, J = 7
Hz, 12 H, CONCH2CH2NCO, conformational isomer), 6.97 (m,
J = 5 Hz, 3 H, 3 C6HF4).
IR (KBr): 3330 (OH stretch), 2948, 2854 (CH stretch), 1730 (C=O
stretch for carboxylic acid), 1680 (C=O stretch for tertiary amide),
1436 (CH2 deformation), 1380 cm–1 (C–N stretch).
1H NMR (D2O): = 1.85–1.37 (br, 12 H, CH2CH2CH2CH2CH),
2.38 (t, J = 7 Hz, 4 H, CH2CH2CO), 2.58 (t, J = 7 Hz, 4 H,
NCOCH2CH2), 3.10 (t, J = 6 Hz, 4H, NHCH2CH2), 3.56–3.46 (two
b, 8 H, CH2CH2NCO), 3.89–3.80 (br s, 10 H, NCH2CO2H and
CH2CHCO2H).
13C NMR (CDCl3): = 27.3, 30.8, 41.5, 45.5, 128.0, 135.3, 172.3,
174.2.
HRMS (EI, 70 V): m/z calcd for C36H27F12N3O9: 873.1556 [M]+;
found: 873.1558 [M]+.
N-(5-Benzyloxycarbonylamino-1-carboxypentyl)iminodiacetic
Acid [N( )-Z-Lys-NTA, 7]
13C NMR (D2O): = 23.0, 26.7, 28.2, 30.8, 38.8, 46.8, 54.4, 63.9,
66.8, 168.1, 170.5, 171.6, 172.5.
Bromoacetic acid (4.17 g, 30 mmol) was dissolved in NaOH solu-
tion (15 mL, 2 M) and cooled to 0 °C. N( )-Benzyloxycarbonyl-L-
lysine (6, 4.20 g, 15 mmol) in aq 2 M NaOH solution (22.5 mL) was
added dropwise at 0 °C with stirring. After 2 h, the ice bath was re-
moved and the mixture was stirred overnight at r.t. The temperature
was then raised to 50 °C for 2 h and aq 1 M HCl solution (45 mL)
was added. After the mixture had been cooled, the crystals that
formed were filtered off, and redissolved again in aq 1 M NaOH so-
lution. The product precipitated out by the dropwise addition of aq
1 M HCl solution and was collected by filtration. Compound 7 (5.28
g) was obtained as a white solid in 89% yield; mp 172–174 °C.
HRMS (FAB, oxalic acid/glycerol): m/z calcd for C32H50N6O16:
775.3362 [M + H]+; found 775.3386 [M + H]+.
1,4,7-Tris[5 -N,N-di(carboxymethyl)amino-5 -carboxypentyl-
amino-3 -oxopropanoyl]triazacyclononane (1b)
Compound 1b was obtained from 5b (0.25 g, 0.28 mmol) and
lysine-NTA (8; 0.28 g, 1.17 mmol) according to the method given
for 1a as a white solid (0.1 g, 32%); mp 200 °C (dec.).
IR (KBr): 3331 (OH stretch), 2945, 2857 (CH stretch), 1732 (C=O
stretch for carboxylic acid), 1685 (C=O stretch for tertiary amide),
1434 (CH2 deformation), 1381 cm–1 (C–N stretch).
1H NMR (D2O): = 1.85–1.37 (br, 18 H, CH2CH2CH2CH2CH),
2.44 (t, J = 6 Hz, 6 H, CH2CH2CONH), 2.80 (t, J = 6 Hz, 6 H,
NCOCH2CH2), 3.18 (t, J = 6 Hz, 6 H, NHCH2CH2), 3.55–3.45 (br
s, 12 H, OCNCH2CH2NCO), 3.89 (br, 15 H, NCH2CO2H and
CH2CHCO2H).
IR (KBr): 3206 (OH stretch), 1720 (C=O stretch for carboxylic ac-
id), 1652 [C=O stretch for O(C=O)NH], 1640 (C=O stretch for ter-
tiary amide), 1594 (C=C stretch), 1464 (CH2 deformation), 1431,
1287 cm–1 (C–N stretch).
1H
NMR
(DMSO-d6):
= 1.76–1.36
(br,
6
H,
NHCH2CH2CH2CH2CH), 2.02 (q, J = 6 Hz, 2 H, NHCH2CH2), 3.55
(br s, 5 H, NCH2CO2H and NCHCOO), 4.98 (s, 2 H, C6H5CH2),
7.33 (br s, 5 H, C6H5).
13C NMR (DMSO-d6): = 23.3, 27.8, 28.6, 46.4, 54.6, 59.9, 64.2,
13C NMR (D2O): = 22.9, 23.0, 27.0, 28.2, 30.8, 38.1, 46.8, 54.5,
64.8, 66.9, 168.6, 170.0, 171.4, 172.3.
65.2, 117.7, 118.3, 128.5, 158.5, 173.2, 174.8.
HRMS (FAB, oxalic acid/glycerol): m/z calcd for C48H75N9O24:
1162.5003 [M + 1]+; found 1162.4982 [M + 1]+.
5-Aminopentylnitrilotriacetic Acid or N ,N -Bis[carboxymeth-
yl]-l-lysine (8)
1,4-Bis[4 -(N-benzyloxycarbonyl)amino-4 -benzyloxycarbon-
ylbutanoyl]diazacyclohexane (9a); Typical Procedure
Z-Glu-OBzL (2.5 g, 6.0 mmol) and TBTU (1.926 g, 6.0 mmol) were
dissolved in DMF (4 mL), followed by the addition of Et3N (0.83
mL, 6.0 mmol). To this solution, was added a warm solution of pip-
erazine (0.206 g, 2 mmol) in DMF (2 mL). After stirring overnight,
the reaction mixture was evaporated to dryness. The viscous residue
was dissolved in CHCl3 (80 mL), the CHCl3 solution was washed
twice with aq 5% NaHCO3, brine, and H2O, sequentially. The or-
ganic layer was dried (Na2SO4), and the solvent was removed by ro-
tary evaporation. The residue was subjected to silica gel column
chromatography, and the desired product was isolated by prepara-
tive TLC; the fourth band was collected (Rf 0.32, developed by a
5:2:5:1 mixture of EtOAc, CHCl3, hexane, and MeOH). The prod-
uct (1.36 g) was collected as a white solid; yield: 86%; mp 39–42
°C.
N( )-Z-Lys-NTA (7; 3.02 g, 7.50 mmol) was dissolved in aq 1 M
NaOH (50 mL). After the addition of a spatula tip of Pd/C (10%),
the mixture was hydrogenated at r.t. and normal pressure. The reac-
tion was complete after 6 h as monitored by TLC (MeCN–H2O, 4:1)
and visualized with UV, I2, and ninhydrin. The catalyst was filtered
off, and solvent was removed in vacuo. The resulting residue was
redissolved in EtOH (25 mL), the product crystalized at 0 °C within
2 d. The crystals (1.49 g) were collected and dried in vacuum; yield:
76%; mp 202–204 °C (dec.).
IR (KBr): 3016 (OH stretch), 1724 (C=O stretch for carboxylic ac-
id), 1710 (COO– antisym stretch), 1464 (CH2 deformation), 1381,
1287 cm–1 (C–N stretch).
1H NMR (D2O): = 1.91–1.52 (br, 6 H, NHCH2CH2CH2CH2CH),
2.88 (t, J = 7 Hz, 2 H, NHCH2CH2), 3.84–3.50 (br s, 5 H,
NCH2CO2H and NCHCOO).
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C. Liu et al.
PAPER
IR (KBr): 3300 (NH stretch), 3033 (CH stretch for aromatic com-
pound), 2947 (CH stretch for alkane), 1721 (C=O stretch for ester),
1636 (C=O stretch for tertiary amide), 1530 (C=C stretch), 1455
(CH2 deformation), 1215 (C–O–C stretch), 699 cm–1 (CH out of
plane deformation).
1H NMR (CDCl3): = 2.05 (br, 4 H, CH2CH2CH), 2.26 (t, J = 7 Hz,
4 H, COCH2CH2), 3.20 (br s, 4 H, OCNCH2CH2NCO, isomer), 3.48
(br s, 4 H, OCNCH2CH2NCO), 4.45 (t, J = 7 Hz, 2 H,
CH2CHCOO), 5.06 (s, 4 H, C6H5CH2OCO), 5.16 (q, J = 3 Hz, 4 H,
NCO2CH2C6H5), 5.70 (d, J = 8 Hz, 2 H, CHNHCO), 7.34–7.25 (br
s, 20 H, 4 C6H5).
6 M NaOH was added. The solution was extracted with chloroform
(4 40 mL) and dried (Na2SO4). Removal of the solvent gave 3d
(0.07 g) in 75% yield.
1,4,7,10,13-Pentakis[4 -(N-benzyloxycarbonyl)amino-4 -
benzyloxycarbonylbutanoyl]pentaazacyclopentadecane (9d)
Compound 9d (0.21 g, 0.11 mmol) was prepared as a white solid
from 3d (0.06 g, 0.28 mmol), Z-Glu-OBzl (0.77 g, 2.06 mmol) and
TBTU (0.66 g, 2.06 mmol) according to the procedure used for 9a
as a white solid in 38% yield; mp 56–58 °C.
IR (KBr): 3305 (NH stretch), 3030 (CH stretch for aromatic com-
pound), 2948 (CH stretch for alkane), 1719 (C=O stretch for ester),
1630 (C=O stretch for tertiary amide), 1528 (C=C stretch), 1456
(CH2 deformation), 1210 (C–O–C stretch), 697 cm-1 (CH out of
plane deformation).
1H NMR (CDCl3): = 2.05 (br, 10 H, CH2CH2CH), 2.45 (br, 10 H,
COCH2CH2), 3.55–3.23 (2 br s, 20 H, OCNCH2CH2NCO), 4.43 (br,
5 H, CH2CHCOO), 5.04 (s, 10 H, C6H5CH2COO), 5.15 (br, 10 H,
C6H5CH2O), 5.96 (d, J = 8 Hz, 5 H, CHNHCO), 7.35–7.25 (br s, 50
H, 10 C6H5).
13C NMR (CDCl3): = 27.6, 29.1, 41.4, 45.0, 53.7, 67.1, 128.2,
128.5, 128.7, 135.3, 136.3, 156.8, 170.4, 171.7.
HRMS (EI, 70 V): m/z calcd for C44H48N4O10: 792.3370 [M]+;
found: 792.3305 [M]+.
1,4,7-Tris[4 -N-benzyloxycarbonylamino-4 -benzyloxycarbon-
ylbutanoyl]triazacyclononane (9b)
Compound 9b (0.95 g, 0.52 mmol) was obtained as a white solid
from 1,4,7-triazacyclononane (3b; 0.10 g, 0.80 mmol), Z-Glu-OBzl
(1.35 g, 3.60 mmol) and TBTU (1.15 g, 3.60 mmol) according to the
procedure used for 9a in 65% yield; mp 40–43 °C.
13C NMR (CDCl3): = 27.0, 29.8, 38.7, 49.9, 53.5, 66.8, 67.3,
128.2, 128.9, 136.1, 157.5, 171.8, 172.0.
IR (KBr): 3305 (NH stretch), 3031 (CH stretch for aromatic com-
pound), 2948 (CH stretch for alkane), 1725 (C=O stretch for ester),
1634 (C=O stretch for tertiary amide), 1528 (C=C stretch), 1458
(CH2 deformation), 1215 (C–O–C stretch), 700 cm–1 (CH out of
plane deformation).
1H NMR (CDCl3): = 1.95 (br s, 6 H, COCH2CH2CH), 2.25 (br s,
6 H, COCH2CH2), 3.30 (br s, 9 H, OCNCH2CH2NCO, due to iso-
mer), 3.64 (br s, 3 H, OCNCH2CH2NCO), 4.38 (t, J = 8 Hz, 3 H,
CH2CHCOO), 5.05 (s, 6 H, C6H5CH2OCO), 5.15 (m, J = 2 Hz, 6 H,
C6H5CH2OCON), 6.01 (d, J = 8 Hz, 3 H, CHNHCO), 7.33–7.25 (br
s, 30 H, 6 C6H5).
HRMS (EI, 70 V): m/z calcd for C110H120N10O25: 1980.8246 [M]+;
found 1980.8265 [M]+.
1,4,7,10,13-Hexaazacycloocadecane or Hexacyclen (3e)
Hexacyclen trisulfate (0.46 g, 0.83 mmol) was dissolved in aq 6 M
NaOH (40 mL). The solution was extracted with CHCl3(4 40 mL)
and dried (Na2SO4). Removal of the solvent gave 3e (0.14 g) in 65%
yield.
1,4,7,10,13,16-Hexakis[4 -(N-benzyloxycarbonyl)amino-4 -ben-
zyloxycarbonylbutanoyl]hexaazacycloocadecane (9e)
Compound 9e (0.07 g, 0.03 mmol) was obtained as a white solid
from hexacyclen (3e) (0.024 g, 0.093 mmol), Z-Glu-OBzl (0.35 g,
0.93 mmol) and TBTU (0.30 g, 0.93 mmol) according to the proce-
dure used for 9a as a white solid in 33% yield; mp 56–58 °C.
13C NMR (CDCl3): = 26.5, 30.0, 46.0, 50.9, 54.0, 67.1, 67.3,
128.3, 128.6, 128.8, 136.3, 158.1, 172.2, 173.4.
HRMS (EI, 70 V): m/z calcd for C66H72N6O15: 1188.5056 [M]+;
found 1188.5067 [M]+.
IR (KBr): 3304 (NH stretch), 3030 (CH stretch for aromatic com-
pound), 2947 (CH stretch for alkane), 1720 (C=O stretch for ester),
1628 (C=O stretch for tertiary amide), 1521 (C=C stretch), 1452
(CH2 deformation), 1207 (C–O–C stretch), 699 cm–1 (CH out of
plane deformation).
1H NMR (CDCl3): = 2.05 (br, 12 H, CH2CH2CH), 2.45 (br, 12 H,
COCH2CH2), 3.54–3.27 (2 br s, 24 H, OCNCH2CH2NCO), 4.40 (br,
6 H, CH2CHCOO), 5.02 (s, 12 H, C6H5CH2COO), 5.12 (br, 12 H,
C6H5CH2OCON), 6.05 (br, 6 H, CHNHCO), 7.35–7.18 (br s, 60 H,
12 C6H5).
1,4,7,10-Tetrakis[4 -(N-benzyloxycarbonyl)amino-4 -benzyl-
oxycarbonylbutanoyl]tetraazacyclododecane (9c)
Compound 9c (0.17 g, 0.11 mmol) was obtained from cyclen or
1,4,7,10-tetraazacyclododecane (3c; 0.043 g, 0.24 mmol), Z-Glu-
OBzl (0.54 g, 1.44 mmol) and TBTU (0.46 g, 1.44 mmol) according
to the procedure used for 9a as a white solid in 44% yield; mp 60–
62 °C.
IR (KBr): 3310 (NH stretch), 3023 (CH stretch for aromatic com-
pound), 2946 (CH stretch for alkane), 1720 (C=O stretch for ester),
1638 (C=O stretch for tertiary amide), 1533 (C=C stretch), 1456
(CH2 deformation), 1205 (C–O–C stretch), 698 cm–1 (CH out of
plane deformation).
1H NMR (CDCl3): = 1.95 (br, 8 H, COCH2CH2CH), 2.50 (br, 8 H,
COCH2CH2), 3.71–3.13 (2 br s, 16 H, OCNCH2CH2NCO), 4.43 (br,
4 H, CH2CHCOO), 5.05 (s, 8 H, C6H5CH2OCO), 5.15 (m, J = 2 Hz,
8 H, C6H5CH2OCON), 6.04 (br s, 4 H, CHNHCO), 7.33–7.25 (br s,
40 H, 8 C6H5).
13C NMR (CDCl3): = 27.5, 29.0, 47.5, 53.9, 67.5, 67.7, 127.9,
128.8, 128.9, 136.9, 156.8, 172.5, 173.6.
HRMS (EI, 70 V): m/z calcd for C132H144N12O30: 2377.0111 [M]+;
found 2377.0178 [M]+.
1,4-Bis(4 -amino-4 -carboxybutanoyl)diazacyclohexane (10a);
Typical Procedure
Compound 9a (0.65 g, 0.82 mmol) was dissolved in MeOH (10
mL). A small amount of Pd/C (10%) was added to this solution. Af-
ter stirring the suspension for 2 min, H2 gas was introduced into the
flask at r.t. under normal pressure. The reaction progress was mon-
itored by TLC, visualized by illumination with UV light. The cata-
lyst was filtered off, washed with H2O, and the filtrate was dried in
vacuo. The resulting precipitate was redissolved in hot EtOH, and
allowed to crystallize at 0 °C. The crystals were filtered off and
dried in vacuo; yield: 90% (0.25 g); mp 168–170 °C.
13C NMR (CDCl3): = 26.9, 29.8, 38.7, 49.7, 53.5, 67.0, 67.4,
128.2, 128.9, 136.2, 156.5, 171.9, 172.5.
HRMS (EI, 70 V): m/z calcd for C88H96N8O20: 1584.6741 [M]+;
found 1584.6712 [M]+.
1,4,7,10,13-Pentaazacyclopentadecane (3d)
Pentaaza-15-crown-5 pentahydrochloride (0.17 g, 0.42 mmol) was
dissolved in aq 0.1 M HCl (1 mL), to which a large excess of aq
Synthesis 2002, No. 10, 1398–1406 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of Dendritic, Multivalent Complexing Agents
1405
IR (KBr): 3435 (NH and OH stretch), 2934 (CH stretch for alkane),
1716 (C=O stretch for carboxylic acid), 1630 (C=O stretch for ter-
1,4,7,10,13,16-Hexakis(4 -amino-4 -carboxybutanoyl)hexaaza-
cycloocadecane (10e)
Compound 10e (0.04 g, 0.04 mmol) was obtained from 9e (0.11 g,
0.05 mmol) according to the procedure used for 10a as a white solid
in 80% yield; mp 200 °C (dec.).
+
tiary amide), 1530 (NH3 deformation), 1443 (CH2 deformation),
1427 cm–1 (COO– sym stretch).
1H NMR (D2O): = 1.99 (q, J = 6 Hz, 4 H, CH2CH2CH), 2.48 (t,
J = 7 Hz, 4 H, COCH2CH2), 3.60–3.46 (2 br s, 8 H, CH2CH2NCO),
3.74 (t, J = 8 Hz, 2 H, CH2CHCOO).
IR (KBr): 3436 (NH and OH stretch), 2933 (CH stretch for alkane),
1717 (C=O stretch for carboxylic acid), 1635 (C=O stretch for ter-
+
tiary amide), 1447 (NH3 deformation), 1426 cm–1 (CH2 deforma-
13C NMR (D2O): = 25.2, 28.4, 41.7, 45.1, 173.0, 174.1.
tion).
HRMS (FAB, oxalic acid/glycerol): m/z calcd for C14H24N4O6:
1H NMR (D2O): = 2.02 (br, 12 H, CH2CH2CH), 2.64 (br, 12 H,
COCH2CH2), 3.56–3.46 (2 br, 24 H, OCNCH2CH2NCO), 3.78 (t,
J = 8 Hz, 6 H, CH2CHCOO).
345.1774 [M + H]+; found 345.1761 [M + H]+.
1,4,7-Tris(4 -amino-4 -carboxybutanoyl)triazacyclononane
(10b)
13C NMR (D2O): = 25.5, 28.5, 46.1, 51.0, 173.9, 174.7.
Compound 10b (0.11 g, 0.22 mmol) was obtained from 9b (0.30 g,
0.25 mmol) according to the procedure used for 10a as a white solid,
which was deliquescent, but could be recrystallized from hot EtOH;
yield: 86%; mp 210 °C (dec.).
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C42H72N12O18:
1033.5165 [M + H]+; found 1033.5158 [M + H]+.
1,4-Bis[4 -N,N-di(carboxymethyl)amino-4 -carboxylbutan-
oyl)]diazacyclohexane (2a)
IR (KBr): 3436 (NH and OH stretch), 2936 (CH stretch for alkane),
1723 (C=O stretch for carboxylic acid), 1638 (C=O stretch for ter-
A solution of bromoacetic acid (0.24 g, 1.72 mmol) in aq 1.5 M
NaOH (2 mL) was cooled to 0 °C and was added dropwise to a so-
lution of 10a (0.13 g, 0.38 mmol) in NaOH (2 mL, 2 M). The pH of
the resulting solution was adjusted to 12. Then the reaction mixture
was stirred at 45 °C for 7 d. The pH was maintained in the range of
11–12 by periodic addition of aq 2 M NaOH and the reaction
progress was monitored by TLC. After cooling to r.t., the mixture
was evaporated to dryness under vacuum to give a white solid. The
solid was redissolved in H2O, pH adjusted to10, and applied to a
Bio-Rad AG1-X4 anion-exchange column (formate ion). The col-
umn was eluted successively with H2O, aq 4 M formic acid and aq
5 M formic acid. The fraction containing the desired product deter-
mined by TLC and was applied to a second Bio-Rad AG1-X4 an-
ion-exchange column that was eluted with H2O, aq 5 M formic acid,
and aq 6 M formic acid. The desired product eluted slowly in the aq
5 M formic acid fractions and these were combined, evaporated to
dryness under vacuum, and redissolved in aq 4 M HCl. The result-
ing solution was again evaporated to dryness, and the residue was
repeatedly redissolved in H2O. The solution was dried to give a
white solid (0.12 g, 56%); mp 210 °C (dec.).
+
tiary amide), 1445 (NH3 deformation), 1429 cm–1 (COO– sym
stretch).
1H NMR (D2O): = 2.02 (br , 6 H, CH2CH2CH), 2.42 (br , 6 H,
COCH2CH2), 3.67–3.37 (2 br s, 12 H, OCNCH2CH2NCO), 3.69 (t,
J = 8 Hz, 3 H, CH2CHCOO).
13C NMR (D2O): = 25.7, 29.5, 44.1, 48.6, 50.5, 174.5, 175.3.
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C21H36N6O9:
517.2622 [M + H]+; found 517.2690 [M + H]+.
1,4,7,10-Tetrakis(4 -amino-4 -carboxybutanoyl)tetraazacyclo-
dodecane (10c)
Compound 10c (0.04 g, 0.06 mmol) was obtained from 9c (0.12 g,
0.07 mmol) according to the procedure used for 10a as a white solid
in 82% yield; mp 210 °C (dec.).
IR (KBr): 3432 (NH and OH stretch), 2933 (CH stretch for alkane),
1720 (C=O stretch for carboxylic acid), 1634 (C=O stretch for ter-
+
tiary amide), 1442 (NH3 deformation), 1430 cm–1 (COO– sym
stretch).
IR (KBr): 3436 (OH stretch), 1733 (C=O stretch for carboxylic ac-
id), 1631 (C=O stretch for tertiary amide), 1365 cm–1 (N–C stretch).
1H NMR (D2O): = 2.06 (br s, 4 H, CH2CH2CH), 2.54 (br s, 4 H,
NCOCH2CH2), 3.65–3.43 (2 br s, 8 H, OCNCH2CH2NCO), 3.89 (br
s, 10 H, NCH2CO2H and CH2CHCO2H).
1H NMR (D2O): = 2.05 (q, J = 6 Hz, 8 H, CH2CH2CH), 2.44 (t,
J = 7 Hz,
8 H, COCH2CH2), 3.63–3.43 (2 br, 16 H,
OCNCH2CH2NCO), 3.67 (t, J = 7 Hz, 4 H, CH2CHCOO).
13C NMR (D2O): = 25.6, 29.5, 45.7, 48.6, 51.4, 174.2, 175.0.
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C28H48N8O12:
13C NMR (D2O): = 22.5, 30.3, 40.6, 49.2, 54.1, 64.5, 170.1, 171.2,
175.3, 176.6.
689.3469 [M + H]+; found 689.3445 [M + H]+.
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C22H32N4O14:
1,4,7,10,13-Pentakis(4 -amino-4 -carboxybutanoyl)pentaazacy-
clopentadecane (10d)
577.1993 [M + H]+; found 577.1942 [M + H]+.
Compound 10d (0.10 g, 0.12 mmol) was obtained from 9d (0.30 g,
0.15 mmol) according to the procedure of 10a as white solid in 80%
yield; mp 200 °C (dec.).
1,4,7-Tris[4 -N,N-di(carboxymethyl)amino-4 -carboxybutan-
oyl]triazacyclononane (2b)
Compound 2b (0.017 g, 0.02 mmol) was obtained from 10b (0.02
g, 0.04 mmol) and bromoacetic acid (0.034 g, 0.25 mmol) according
to the procedure used for 2a as a white solid in 48% yield; mp 210
°C (dec.).
IR (KBr): 3430 (NH and OH stretch), 2931 (CH stretch for alkane),
1716 (C=O stretch for carboxylic acid), 1640 (C=O stretch for ter-
+
tiary amide), 1448 (NH3 deformation), 1430 cm–1 (COO– sym
stretch).
IR (KBr): 3435 (OH stretch), 1735 (C=O stretch for carboxylic ac-
id), 1630 (C=O stretch for tertiary amide), 1367 cm–1 (N–C stretch).
1H NMR (D2O): = 2.05 (br, 6 H, CH2CH2CH), 2.54 (br, 6 H,
NCOCH2CH2), 3.58–3.35 (2 br, 12 H, OCNCH2CH2NCO), 3.92–
3.74 (2 br, 15 H, NCH2CO2H and CH2CHCO2H).
1H NMR (D2O): = 2.01 (br, 10 H, CH2CH2CH), 2.42 (br , 10 H,
COCH2CH2), 3.63–3.43 (2 br, 20 H, OCNCH2CH2NCO), 3.67 (br,
5 H, CH2CHCOO).
13C NMR (D2O): = 25.2, 29.2, 45.6, 48.0, 50.9, 174.2, 175.1.
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C35H60N10O15:
13C NMR (D2O): = 24.5, 30.4, 48.7, 54.3, 66.6, 169.8, 175.0,
175.3.
861.4317 [M + H]+; found 861.4325 [M + H]+.
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C33H48N6O21:
865.2951 [M + H]+; found 865.2921 [M + H]+.
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1406
C. Liu et al.
PAPER
1,4,7,10-Tetrakis[4 -N,N-di(carboxymethyl)amino-4 -carboxy-
butanoyl]-tetraazacyclododecane (2c)
Compound 2c (0.04 g, 0.035 mmol) was obtained from 10c (0.056
g, 0.081 mmol) and bromoacetic acid (0.10 g, 0.73 mmol) according
to the procedure used for 2a as white solid in 43% yield; mp 220 °C
(dec.).
mophenol blue, 7 mL of H2O and 25 mL of glycerol). The protein
samples as described below were electrophoresized toward the an-
ode (+) for 30 minutes at 40 V, and followed by 210 mins at 70 V
until the dye front reached the bottom of the gel. The gel was then
placed in a staining solution containing 0.25% Coomassie Blue G-
250 (0.25 g Coomassie Blue G-250, 125 mL MeOH, 25 mL glacial
AcOH, and 100 mL H2O) overnight. Then the gel was destained in
a solution containing 10% MeOH and 10% AcOH for 4 h. The re-
sulting gel was dried and scanned.
IR (KBr): 3430 (OH stretch), 1723 (C=O stretch for carboxylic ac-
id), 1634 (C=O stretch for tertiary amide), 1387 cm–1 (N–C stretch).
1H NMR (D2O): = 2.01 (br, 8 H, CH2CH2CH), 2.54 (br, 8 H,
NCOCH2CH2), 3.85–3.45 (2 br, 36 H, NCH2CO2H, CH2CHCO2H
and OCNCH2CH2NCO).
13C NMR (D2O): = 24.7, 29.6, 47.4, 55.0, 66.8, 169.3, 171.0,
174.3, 175.4.
The model protein was loaded to lane 1 and lane 4. Lanes 2 and 5
contain the complex formed by a combination of ligand 1a and 1b,
NiCl2 and protein with molar ratio of 1:2.5:2.5 and 1:3.5:3.5, re-
spectively. Lanes 3 and 6 represent the complex (lanes 2 and 5) be-
ing dissociated by the addition of EDTA with molar ratios of 1/4
(Dimeric complex/EDTA) or 1/8 (trimeric complex/EDTA). The
same amount of protein (15 L of a 0.06 mg/mL solution) was load-
ed in each lane. The gel was formed at 17% polyacrylamide.
HRMS (FAB, oxalic acid): m/z calcd for C44H64N8O28: 1153.3908
[M + H]+; found 1153.3938 [M + H]+.
1,4,7,10,13-Pentakis[4 -N,N-di(carboxymethyl)amino-4 -car-
boxybutanoyl]pentaazacyclopentadecane (2d)
Compound 2d (0.044 g, 0.03 mmol) was obtained from 10d (0.07
g, 0.08 mmol) and bromoacetic acid (0.12 g, 0.88 mmol) according
to the procedure used for 2a as a white solid in 38% yield; mp 220
°C (dec.).
Acknowledgements
This work was supported by an operating grant from the National
Sciences and Engineering Research Council (NSERC), Canada
(NOP). Mr. D. Hairsine worked hard to find the optimal conditions
for mass spectrometry of these compounds and we appreciate his
patience. Dr. Eric Ball of the Department of Biochemistry, The Uni-
versity of Western Ontario, London ON generously provided sam-
ples of His-tagged thioredoxin.
IR (KBr): 3433 (OH stretch), 1730 (C=O stretch for carboxylic ac-
id), 1637 (C=O stretch for tertiary amide), 1364 cm–1 (N–C stretch).
1H NMR (D2O): = 2.01 (br, 10 H, CH2CH2CH), 2.70 (br, 10 H,
NCOCH2CH2), 3.86–3.45 (2 br, 45 H, NCH2CO2H, CH2CHCO2H
and OCNCH2CH2NCO).
13C NMR (D2O): = 26.5, 30.4, 48.6, 54.7, 64.8, 170.0, 174.8,
175.2.
References
(1) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature
(London) 1975, 258, 598.
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C55H80N10O35:
1441.4865 [M + H]+; found 1441.4856 [M + H]+.
(2) (a) Hochuli, E.; Doböli, H.; Schaher, A. J. Chrom. 1987,
411, 177. (b) Hochuli, E. Gen. Eng. 1990, 12, 87.
(c) Crowe, J.; Doböli, H.; Gentz, R.; Hochuli, E.; Stüber, D.;
Henco, K. Methods Mol. Biol. 1994, 31, 371.
(3) Schmitt, L.; Dietrich, C.; Tampe, R. J. Am. Chem. Soc. 1994,
116, 8485.
(4) Paprica, P. A.; Petersen, N. O. Langmuir 1994, 10, 4630.
(5) Liu, C.; Paprica, A.; Petersen, N. O. Biophys. J. 1997, 73,
258.
1,4,7,10,13,16-Hexakis[4 -N,N-di(carboxymethyl)amino-4 -car-
boxybutanoyl]hexaazacyclooctadecane (2e)
Compound 2e (0.13 g, 0.007 mmol) was obtained from 10e (0.023
g, 0.022 mmol) and bromoacetic acid (0.04 g, 0.30 mmol) according
to the procedure used for 2a as a white solid in 33% yield; mp 230
°C (dec.).
IR (KBr): 3430 (OH stretch), 1729 (C=O stretch for carboxylic ac-
id), 1680 (C=O stretch for tertiary amide), 1360 cm–1 (N–C stretch).
1H NMR (D2O): = 2.01 (br, 12 H, CH2CH2CH), 2.72 (br, 12 H,
NCOCH2CH2), 3.86–3.46 (2 br, 54 H, NCH2CO2H, CH2CHCO2H
and OCNCH2CH2NCO).
13C NMR (D2O): = 27.5, 31.4, 48.7, 54.2, 57.4, 65.8, 67.8, 169.6,
171.0, 174.8, 175.2.
(6) Moore, M. S.; Mahaffey, D. T.; Brodsky, F. M.; Anderson,
R. G. W. Science 1987, 236, 558.
(7) (a) Moreau, P.; Tinkl, M.; Tsukazaki, M.; Bury, P. S.;
Griffen, E. J.; Snieckus, V.; Maharajh, R. B.; Kwok, C. S.;
Somayaji, V. V.; Peng, Z.; Sykes, T. R.; Noujaim, A. A.
Synthesis 1997, 1010. (b) Kruper, W. J. Jr.; Pollock, D. K.;
Fordyce, W. A.; Fazio, M. J.; Inbasekaran, M. N.; Muthyala,
R. US Patent 5489425, 1996; Chem. Abstr. 1996, 124,
330781.
(8) (a) Kung, V. T.; Redemann, C. T. Biochim. Biophys. Acta
1986, 862, 435. (b) Izatt, M.; Christensen, J. J. J.
Heterocycl. Chem. 1977, 14, 85.
HRMS (FAB, glycerol/oxalic acid): m/z calcd for C66H96N12O42:
1729.5823 [M + H]+; found 1729.5801 [M + H]+.
Native Polyacrylamide Gel Electrophoresis (PAGE)
A solution of His-tagged thioredoxin obtained from Dr. E. Ball was
further dialysed against 20 mM HEPES buffer (20 mM Tris-HCl,
100 mM NaCL, and 2 mM CaCl2), pH 8.0 using a Spectra/por1 wet
tubing MWCO 1000. The resulting solution was concentrated with
lyophilizer (ETS System, Inc.), and the size and purity was deter-
mined by SDS-PAGE. The protein concentration was determined
from the absorbance at 595 nm using the Bradford method.12 The
conditions for native PAGE were adapted from Weber and Os-
born.13 The apparatus routinely used generates an 8.5 cm
(height) 7.5 cm separating gel, 1 mm thickness. The electrode
buffer contained 25 mM Tris base, 0.19 M glycine, and 20 mM KCl,
pH 8.0. Samples for native PAGE were prepared by mixing one part
of the protein sample with five parts of native PAGE sample buffer
(15.5 mL of 1 M Tris-HCl pH 8.0, 2.5 mL of a 1% solution of bro-
(9) (a) Wilbur, D. S.; Hamlin, D. K.; Pathare, P. M.;
Weerawarna, S. A. Bioconjugate Chem. 1997, 8, 572.
(b) Wibur, D. S.; Pathare, P. M.; Hamlin, D. K.;
Weerawarna, S. A. Bioconjugate Chem. 1997, 8, 819.
(c) Pathare, P. M.; Wilbur, D. S.; Hamlin, D. K.; Heusser, S.;
Quadros, E.; McLoughlin, P.; Morgan, A. C. Bioconjugate
Chem. 1997, 8, 161.
(10) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927.
(11) Kline, S. J.; Betebenner, D. A.; Johnson, D. K. Bioconjugate
Chem. 1991, 2, 26.
(12) Bradford, M. M. Anal. Biochem. 1976, 72, 248.
(13) Weber, K.; Osborn, M. J. Biol. Chem. 1969, 244, 4406.
Synthesis 2002, No. 10, 1398–1406 ISSN 0039-7881 © Thieme Stuttgart · New York