Amino acid and peptide bioconjugates of copper(II) and zinc(II) complexes with a modified N,N-bis(2-picolyl)amine ligand

Article abstract of DOI:10.1021/ic048343b

Four chelating nitrogen ligands 2-5 derived from N,N-bis(2-picolyl)amine (bpa, 1) were synthesized, namely, (PyCH₂)₂N-CH ₂-p-C₆H₄-CO₂R (R = Me, 2, and R = H, 3) and (PyCH₂)₂N-(CH₂)_n-CO ₂H (n = 2, 4, and n = 5, 5). Amino acid conjugates 6 and 7 were formed by condensation of 3 with H-Phe-OMe and H-βAla-OMe, respectively. Cu(II) and Zn(II) complexes of 1-7 were prepared and fully characterized. The X-ray structures of 1_zn, 2_Zn, 4_cu, and 7 _Cu were determined. The Zn complexes 1_Zn and 2 _Zn as well as 7_Cu show a distorted trigonal bipyramidal coordination environment in the solid state. An octahedral complex is observed for 4_cu which forms chains along the crystallographic b axis by intermolecular coordination of the carboxylic acid to the metal ion of a neighboring complex. Ligand 3 was used to prepare the peptide bioconjugate 8 (3-Ahx-Pro-Lys-Lys-Lys-Arg-Lys-Phe-NH₂) with a nuclear localization signal (nls) heptapeptide by solid phase synthesis. Cu(II) and Zn(II) complexes of 8 were synthesized in situ and studied by FAB-MS, ESI-MS, UV/vis, and EPR (for 8_Cu), and FAB-MS, ESI-MS, and NMR (for 8_zn). All spectroscopic results clearly support metal coordination to the bpa ligand in the bioconjugates 8_M, even in the presence of other potential ligands from amino acid side chains of the peptide. We suggest metal-peptide conjugates like 8_M as artificial metallochaperones because they have the potential to deliver metal ions to specific compartments in the cell as determined by the peptide moieties.

Full text of DOI:10.1021/ic048343b

Inorg. Chem. 2005, 44, 5405−5415
Amino Acid and Peptide Bioconjugates of Copper(II) and Zinc(II)
Complexes with a Modified N,N-Bis(2-picolyl)amine Ligand
Srec´ko I. Kirin,† Pierre Dubon,† Thomas Weyhermuller,‡ Eckhard Bill,‡ and Nils Metzler-Nolte*,†
1 1
Institute of Pharmacy and Molecular Biotechnology, UniVersity of Heidelberg,
Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany, and Max Planck Institute for
Bioinorganic Chemistry, Stiftstrasse 34-36, D-45470 Mu¨lheim an der Ruhr, Germany
Received November 23, 2004
Four chelating nitrogen ligands 2
(PyCH2)2N-CH2-p-C6H4-CO2R (R
Amino acid conjugates 6 and 7 were formed by condensation of 3 with H-Phe-OMe and H-
Cu(II) and Zn(II) complexes of 1 7 were prepared and fully characterized. The X-ray structures of 1Zn, 2Zn, 4Cu
−
)
5 derived from N,N-bis(2-picolyl)amine (bpa, 1) were synthesized, namely,
Me, 2, and R H, 3) and (PyCH2)2N-(CH2)n-CO2H (n 2, 4, and n 5, 5).
Ala-OMe, respectively.
)
)
â
)
−
,
and 7Cu were determined. The Zn complexes 1Zn and 2Zn as well as 7Cu show a distorted trigonal bipyramidal
coordination environment in the solid state. An octahedral complex is observed for 4Cu which forms chains along
the crystallographic b axis by intermolecular coordination of the carboxylic acid to the metal ion of a neighboring
complex. Ligand 3 was used to prepare the peptide bioconjugate 8 (3-Ahx-Pro-Lys-Lys-Lys-Arg-Lys-Phe-NH2) with
a nuclear localization signal (nls) heptapeptide by solid phase synthesis. Cu(II) and Zn(II) complexes of 8 were
synthesized in situ and studied by FAB-MS, ESI-MS, UV/vis, and EPR (for 8Cu), and FAB-MS, ESI-MS, and NMR
(for 8Zn). All spectroscopic results clearly support metal coordination to the bpa ligand in the bioconjugates 8M,
even in the presence of other potential ligands from amino acid side chains of the peptide. We suggest metal
−
peptide conjugates like 8M as artificial metallochaperones because they have the potential to deliver metal ions to
specific compartments in the cell as determined by the peptide moieties.
Introduction
X-ray single crystal structures and a NMR solution structure
of a Cu chaperone was first reported by Rosenzweig,
O’Halloran, and Wu¨thrich.6-8 Evidently, control of cellular
uptake and metal ion localization is a challenge for medicinal
inorganic chemistry and inorganic biochemistry. For such
systems, we propose to use bioconjugates of metal-chelating
ligands linked to physiologically active peptides.9,10 This
concept has been successfully applied to radioimaging with
99mTc complexes, including the organometallic Tc(CO)3
core.11,12 It has not been, however, widely applied to
nonradioactive metals.
The cellular concentration and localization of metal ions
is an important parameter for the correct function of a cell.
In analogy to genome and proteosome, the term “metallo-
some” has been coined.1 Many metal ions are therefore under
strict control inside cells. For example, the concentration of
free Ca2+ inside the cytoplasm of eukaryote cells is only
10-8 M.1 On the other hand, it was shown that there is not
one single free copper ion in certain cells.2 Any Cu2+ ion is
sequestered by so-called Cu chaperones, proteins which also
serve to deliver the metal to specific Cu enzymes.3-5 The
* Author to whom correspondence should be addressed. E-mail:
nils.metzler-nolte@urz.uni-heidelberg.de. Fax: (international) +49-6221/
54-6441.
(4) Rosenzweig, A. C. Acc. Chem. Res. 2001, 34, 119-128.
(5) Rosenzweig, A. C. Chem. Biol. 2002, 9, 673-677.
(6) Lamb, A. L.; Wernimont, A. K.; Pufahl, R. A.; O’Halloran, T. V.;
Rosenzweig, A. C. Nat. Struct. Biol. 1999, 6, 724-729.
(7) Rosenzweig, A. C.; Huffman, D. L.; Hou, M. Y.; Wernimont, A. K.;
Pufahl, R. A.; O’Halloran, T. V. Structure 1999, 7, 605-617.
(8) Wimmer, R.; Herrmann, T.; Solioz, M.; Wu¨thrich, K. J. Biol. Chem.
1999, 274, 22597-22603.
† University of Heidelberg.
‡ Max Planck Institute for Bioinorganic Chemistry.
(1) Frausto da Silva, J. J. R.; Williams, R. J. P. The biological chemistry
of the elements: The inorganic chemistry of life; Oxford University
Press: Oxford, 2001.
(2) Changela, A.; Chen, K.; Xue, Y.; Holschen, J.; Outten, C. E.;
O’Halloran, T. V.; Mondrago´n, A. Science 2003, 3001, 1383-1387.
(3) Rosenzweig, A. C.; O’Halloran, T. V. Curr. Opin. Chem. Biol. 2000,
4, 140-147.
(9) Liu, S.; Edwards, D. S.; Barrett, J. A. Bioconjugate Chem. 1997, 8,
621-636.
(10) Boschi, A.; Uccell, L.; Bolzati, C.; Marastoni, M.; Tomatis, R.; Spisani,
S.; Traniello, S.; Piffanelli, A. Nucl. Med. Biol. 2000, 27, 791-795.
10.1021/ic048343b CCC: $30.25
Published on Web 06/22/2005
© 2005 American Chemical Society
Inorganic Chemistry, Vol. 44, No. 15, 2005 5405

Kirin et al.
Scheme 1. Synthesis of Aromatic Side Chain Bpa Ligands 2 and 3
and Their Amino Acid Conjugates 6 and 7
Our group has recently published conjugates of the neuro-
peptide enkephalin with Mo complexes.13 In this work, a
Mo(histidinato)(allyl)(CO)2 derivative was used as well as
Mo(CO)3 complexes of the bis(picolyl)amine (bpa) ligand.
The bpa ligand, in particular, is a versatile chelating ligand.
Metal(bpa) complexes of many transition metals were
reported.14,15 Most importantly, Cu(bpa) complexes show
good activity in the hydrolysis of phosphate esters and DNA
plasmids.16-21 Very recently, two groups reported the use
of bpa conjugates with carbohydrates22,23 and peptides24,25
for radioimaging with 99mTc.
In this paper, we present the synthesis and characterization
of metal(bpa) complexes linked to amino acids and a cellular
localization signal peptide, namely, a nuclear localization
signal (nls).26,27 The nls peptide used in this work is a
heptapeptide with primary sequence H-Pro-Lys-Lys-Lys-
Arg-Lys-Phe-OH (H-PKKKRKF-OH). It is a derivative of
the original SV 40 viral nls sequence,28,29 in which the
C-terminal valine has been exchanged for phenylalanine to
permit easier detection of the conjugate in HPLC. The nls
peptide serves as a tag to proteins, indicating their destination
in the nucleus of cells.30 The bpa-nls conjugate is prepared
by solid phase peptide synthesis (SPPS). Metal complexation
is carried out in solution with bpa-nls. We present two metal
complexes, Cu(II) and Zn(II), which allow for complemen-
tary methods of detection, i.e., EPR and UV/vis spectroscopy
Scheme 2. Synthesis of Aliphatic Side Chain Bpa Ligands 4 and 5
(11) Bullok, K. E.; Dyszlewski, M.; Prior, J. L.; Pica, C. M.; Sharma, V.;
Piwnica-Worms, D. Bioconjugate Chem. 2002, 13, 1226-1237.
(12) Langer, M.; La Bella, R.; Garcia-Garayoa, E.; Beck-Sickinger, A. G.
Bioconjugate Chem. 2001, 12, 1028-1034.
for the Cu(II) complexes and NMR spectroscopy for the
Zn(II) species.
(13) van Staveren, D. R.; Metzler-Nolte, N. J. Chem. Soc., Chem. Commun.
2002, 1406-1407.
Results
(14) Glerup, J.; Goodson, P. A.; Hodgson, D. J.; Michelsen, K.; Nielsen,
K. M.; Weihe, H. Inorg. Chem. 1992, 31, 4611-4616.
(15) He, Z.; Craig, D. C.; Colbran, S. B. J. Chem. Soc., Dalton Trans.
2002, 4224-4235.
(16) Kobayashi, T.; Okuno, T.; Suzuki, T.; Kunita, M.; Ohba, S.; Nishida,
Y. Polyhedron 1998, 17, 1553-1559.
(17) Humphreys, K. J.; Karlin, K. D.; Rokita, S. E. J. Am. Chem. Soc.
2001, 123, 5588-5589.
(18) Komiyama, M.; Kina, S.; Matsumura, K.; Sumaoka, J.; Tobey, S.;
Lynch, V. M.; Anslyn, E. J. Am. Chem. Soc. 2002, 124, 13731-13736.
(19) Zhu, L.; dos Santos, O.; Koo, C. W.; Rybstein, M.; Pape, L.; Canary,
J. W. Inorg. Chem. 2003, 42, 7912-7920.
(20) Gunnlaugsson, T.; Nieuwenhuyzen, M.; Nolan, C. Polyhedron 2003,
22, 3231-3242.
(21) Kirin, S. I.; Happel, C. M.; Hrubanova, S.; Weyhermu¨ller, T.; Klein,
C.; Metzler-Nolte, N. J. Chem. Soc., Dalton Trans. 2004, 1201-1207.
(22) Storr, T.; Fisher, C. L.; Mikata, Y.; Yano, S.; Adam, M. J.; Orvig, C.
J. Chem. Soc., Dalton Trans. 2005, 654-655.
(23) Storr, T.; Sugai, Y.; Barta, C. A.; Mikata, Y.; Adam, M. J.; Yano, S.;
Orvig, C. Inorg. Chem. 2005, 44, 2698-2705.
(24) Stephenson, K. A.; Zubieta, J.; Ray Banerjee, S.; Levadala, M. K.;
Taggart, L.; Ryan, L.; McFarlane, N.; Boreham, D. R.; Maresca, K.
P.; Babich, J. W.; Valliant, J. F. Bioconjugate Chem. 2004, 15, 128-
136.
(25) Stephenson, K. A.; Banerjee, S. R.; Besanger, T.; Sogbein, O. O.;
Levadala, M. K.; McFarlane, N.; Lemon, J. A.; Boreham, D. R.;
Maresca, K. P.; Brennan, J. D.; Babich, J. W.; Zubieta, J.; Valliant, J.
F. J. Am. Chem. Soc. 2004, 126, 8598-8599.
(26) Go¨rlich, D.; Mattaj, I. W. Science 1996, 271, 1513-1518.
(27) Nakielny, S.; Dreyfuss, G. Cell 1999, 99, 677-690.
(28) Feldherr, C. M.; Lanford, R. E.; Akin, D. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 11002-11005.
Ligand Synthesis. Starting from commercially available
bpa 1 and methyl 4-(bromomethyl)benzoate, aromatic side
chain ligands 2 and 3 were prepared (Scheme 1).31 The
aliphatic side chain derivatives 4 and 5 were synthesized by
reaction of 2-picolyl chloride hydrochloride with â-alanine
(âAla) or ω-aminohexanoic acid (Ahx), respectively (Scheme
2). As a first step in the synthesis of oligomers, the coupling
reaction of acid 3 with amino acid esters was explored. To
this end, a solution of 3 in acetonitrile was reacted with
H-Phe-OMe or H-âAla-OMe after activation by TBTU.
Products 6 or 7 were isolated and purified by colum
chromatography.
Solid Phase Synthesis. The nls-bpa bioconjugate 8 was
prepared by Fmoc solid phase synthesis using ligand 3. A
Rink amide resin with an acid labile linker and acid labile
side chain protecting groups for amino acids Lys (Boc) and
Arg (Pbf) were used (Scheme 3). The peptide synthesis cycle
was composed of Fmoc deprotection by piperidine and
TBTU coupling. Final cleavage from the resin and side chain
deprotection was performed with TFA:TIS:H2O (95:2.5:2.5).
Synthesis of Metal Complexes. Metal complexes were
prepared by mixing alcoholic solutions (MeOH or EtOH)
(29) Conti, E.; Uy, M.; Leighton, L.; Blobel, G.; Kuriyan, J. Cell 1998,
94, 193-204.
(30) Noor, F.; Wu¨stholz, A.; Kinscherf, R.; Metzler-Nolte, N. Angew.
Chem., Int. Ed. 2005, 44, 2429-2432.
(31) van Staveren, D. R.; Bothe, E.; Weyhermuller, T.; Metzler-Nolte, N.
Eur. J. Inorg. Chem. 2002, 1518-1529.
5406 Inorganic Chemistry, Vol. 44, No. 15, 2005

Bioconjugates of Cu(II) and Zn(II) Complexes
Scheme 3. Solid Phase Synthesis of Bioconjugate 8
Scheme 4. Synthesis of Metal Bpa Complexes 1M-8M, M ) Cu(II)
or Zn(II)
Figure 1. ORTEP plot of 1Zn (50% probability). Most hydrogen atoms
have been omitted for clarity.
of ligands 1-7 and metal nitrates, final concentrations being
in the mM range (Scheme 4). If no precipitation occurred,
the product was isolated by diffusion of ether into the reaction
mixture. The bioconjugate metal complexes 8M were pre-
pared in situ by mixing equimolar amounts of 8 and Cu(II)
or Zn(II) nitrate, with final concentration 10 mM in water
(Scheme 4). In the complexes, ligands 1, 2, and 6-8 are
neutral with two nitrate ions bound to the metal center, while
acids 3-5 are monoanions and contain only one nitrate
counterion to form neutral complexes with the divalent metal
cations.
Crystal Structures. Single crystals which were suitable
for an X-ray analysis could be obtained for four compounds
in this study. Diffusion of ether in the methanol solution of
the complex was successful for 1Zn, 2Zn, and 7Cu. Compound
4Cu was crystallized by slow evaporation of an ethanol
solution. Single crystals of the complexes were mounted
directly from the reaction mixture at room temperature.
ORTEP diagrams of these complexes are shown in Figures
1-4. Selected bond lengths and angles are collected in Tables
1 and 2.
Figure 2. ORTEP plot of 2Zn (50% probability). Hydrogen atoms have
been omitted for clarity.
conditions. The reaction of 3 with H-Phe-OMe and H-âAla-
OMe gives high yields of conjugates 6 and 7 (Scheme 1).31
On the other hand, we were not able to isolate coupling
products of 4 or 5 with H-Phe-OMe and TBTU/DIPEA. This
Discussion
Reactions of ligands 3-5 with amino acid esters in
solution were carried out using standard peptide coupling
Inorganic Chemistry, Vol. 44, No. 15, 2005 5407

Kirin et al.
Table 2. Selected Bond Lengths (Å) and Angles (deg) in 4Cu
M(1)-N(1)
M(1)-N(8)
M(1)-N(15)
1.990(2)
2.040(2)
1.993(2)
M(1)-O(19)
M(1)-O(20A)
2.3358(14)
1.9525(14)
N(1)-M(1)-N(8)
82.85(7) N(8)-M(1)-O(19)
91.63(6)
176.14(6)
91.50(6)
85.44(5)
N(1)-M(1)-N(15) 164.93(7) N(8)-M(1)-O(20A)
N(1)-M(1)-O(19)
N(8)-M(1)-N(15)
94.38(6) N(15)-M(1)-O(19)
83.13(7) O(19)-M(1)-O(20A)
synthesis protocol.32 However, Kawai and co-workers re-
ported the synthesis of bpa-CH(CH3)-CO-Gly-OEt, but
without yield or any experimental details.33 In a very recent
paper, Orvig et al. described the coupling of bpa-CH2-CO2H
with glucosamine using DCC/HOSu and dimethylaminopy-
ridine at 0 °C.22
Our results show that aliphatic side chain ligands 4 and 5
are not suitable for use in solid phase synthesis, since the
coupling efficiency is not sufficient. Therefore, we prepared
the bioconjugate 8 using ligand 3.31 In order to increase the
flexibility between the nls peptide and the rather rigid benzyl
group, ω-aminohexanoic acid was introduced. It should be
noted that the crude product obtained by this procedure does
not require further purification.
Figure 3. ORTEP plot of 7Cu (50% probability). Most hydrogen atoms
have been omitted for clarity.
Metal complexes 1M-7M were prepared from the corre-
sponding ligands and M(NO3)2, with M ) Cu(II) and Zn-
(II). All complexes were comprehensively characterized
spectroscopically. FAB-MS and ESI-MS of both Cu and Zn
complexes reveal signals of mononitrate [LMNO3]+ and
[LM]+, after the loss of one or both nitrate anions. An
absorption maximum at about 650 nm and axial EPR spectra
with g| ) 2.251, g ) 2.068, and magnetic hyperfine
interaction at g| of A| ) 189 × 10-4 cm-1 are characteristic
for the blue paramagnetic Cu compounds. The diamagnetic
Zn complexes can be characterized by NMR spectroscopy
(see below).
The crystal structures in this paper can be divided into
two groups: dinitrates without coordination of the side chain,
such as 1Zn, 2Zn, and 7Cu, and the mononitrate 4Cu with side
chain coordination. Mononuclear discrete molecules are
present in the amine 1Zn and the esters 2Zn and 7Cu. The bpa
acts as meridional tridentate ligand, and both nitrate coun-
terions are coordinated to the metal, resulting in a distorted
trigonal bipyramid. It is interesting to note that the metal
coordination sphere remains the same, regardless of the
substituent on the aliphatic nitrogen: H in 1Zn, p-benzylic
acid ester in 2Zn, and the p-CH2C6H4CO-âAla-OMe conju-
gate in 7Cu. It can be expected that a comparable metal
coordination is present in the metal nls bioconjugate 8M. The
structure of complex 1Cu has been reported, showing similar
features.34
Figure 4. ORTEP plot of 4Cu (50% probability). Most hydrogen atoms
have been omitted for clarity.
Table 1. Selected Bond Lengths (Å) and Angles (deg) in 1Zn, 2Zn, and
7Cu
1Zn
2Zn
7Cu
M(1)-N(1)
M(1)-N(8)
M(1)-N(15)
M(1)-O(41)
M(1)-O(51)
2.097(2)
2.144(3)
2.087(2)
2.085(2)
2.068(2)
2.0591(9)
2.3069(9)
2.0508(9)
2.0971(8)
2.1083(8)
1.977(4)
2.080(4)
1.969(4)
2.210(4)
2.082(3)
N(1)-M(1)-N(8)
N(1)-M(1)-N(15)
N(8)-M(1)-N(15)
N(1)-M(1)-O(41)
N(8)-M(1)-O(41)
N(15)-M(1)-O(41)
N(1)-M(1)-O(51)
N(8)-M(1)-O(51)
N(15)-M(1)-O(51)
O(41)-M(1)-O(51)
78.37(10)
156.70(9)
78.37(10)
96.01(8)
146.97(11)
99.82(9)
77.40(3)
154.51(3)
77.27(3)
96.83(4)
139.92(3)
100.66(4)
101.25(4)
140.89(3)
100.15(3)
79.18(3)
82.31(16)
165.11(17)
82.81(16)
94.99(15)
145.77(15)
97.08(15)
97.46(16)
138.10(15)
93.86(15)
76.13(14)
In 4Cu the ligand is pentadentate in that three bpa nitrogen
atoms and two carboxylic oxygen atoms coordinate to
96.70(8)
(32) Niklas, N.; Walter, O.; Alsfasser, R. Eur. J. Inorg. Chem. 2000, 1723-
1731.
127.59(11)
101.54(9)
85.24(8)
(33) Kawai, M.; Yamamura, H.; Izuhara, N.; Kawaguchi, T.; Tanaka, R.;
Akasaka, R.; Takahashi, Y.-i.; Yamada, K.; Araki, S. In 17th American
Peptide Symposium Peptides: The WaVe of the Future; Lebl, M.,
Houghten, R. A., Eds.; American Peptide Society: San Diego, 2001;
pp 516-517.
(34) Palaniandavar, M.; Mahadevan, S.; Ko¨ckerling, M.; Henkel, G. J.
Chem. Soc., Dalton Trans. 2000, 1151-1154.
finding correlates to the work of Alsfasser et al., who isolated
only a very low yield of bpa-CH2-CO-Phe-OMe using
conditions that are hardly applicable to a solid phase
5408 Inorganic Chemistry, Vol. 44, No. 15, 2005

Bioconjugates of Cu(II) and Zn(II) Complexes
Figure 5. UV/vis spectra of Cu(NO3)2 (dotted line) and 8Cu (solid line)
in water.
copper. As in all other structures in this paper, the bpa ligand
binds to the metal in a meridional manner. One of the
carboxy oxygen atoms coordinates intramolecularly to form
a six membered chelate ring, and the other one forms an
intermolecular bond to the neighboring metal ion in the
lattice, thus producing chains along the crystallographic b
axis. One more metal coordination site in 4Cu is occupied
by a water molecule, resulting in an octahedral coordination
polyhedron with a Jahn-Teller distortion, as expected for
d9-Cu(II) ions. The nitrate counterion is not bound to the
metal, and an ethanol solvent molecule is present in the
Figure 6. 1H NMR (DMSO-d6) of 8 (top) and 8Zn (bottom).
The proton NMR spectra of the in situ prepared zinc
bioconjugate complex 8Zn also supports metal bpa binding.
A comparison of the proton spectra of 8 and 8Zn is shown
in Figure 6. In the spectrum of the free ligand 8, two sharp
singlets can be assigned to the four HPy-R and the two H1
protons, respectively. Coordination of the metal ion to the
tertiary amine nitrogen in 8Zn causes an upfield shift of the
H1 singlet by about 0.3 ppm. In addition, the symmetry of
the HPy-R methylene groups is lost and they become two
separate doublets at 4.21 ppm (HPy-R1) and 3.73 ppm
(HPy-R2), with a characteristic large geminal coupling (J )
17 Hz). A similar observation is made for all other zinc
complexes in this paper and has also been reported in the
literature.21,32
35
crystal. In recent literature, the triflate salt of 4Cu and the
perchlorate salt of 4Zn36 were described and form comparable
zigzag polymer cations linked via the coordinating side chain.
In addition, X-ray data of a similar complex, namely, a
copper(II) bromide derivative of bpa-(CH2)3-CO2H, were
published.37 In this case, intramolecular coordination of the
metal to a carboxylic oxygen atom would cause an unfavor-
able seven-membered ring. Therefore, both carboxy oxygen
atoms coordinate to the neighboring molecule.
Metal binding to the bpa ligand in the bioconjugates 8
could also be investigated by EPR spectroscopy in the case
of the paramagnetic d9-Cu(II) system (Figure 7). The X-band
EPR spectrum of 8Cu in water (ca. 50 µM, Figure 7a) shows
a well-resolved axial Cu(II) signal with large separation of
If 1 equiv of Cu(NO3)2 is added to an aqueous solution of
bioconjugate 8, the formation of the complex 8Cu is im-
mediately apparent by the deep blue color of the solution.
This color is due to a blue shift of the Cu d-d transition
g
and g| and a resolved hyperfine splitting at g|. This
spectrum is distinctly different from that of aquated Cu(II)
ions, which shows a much wider g splitting. By analogy,
we expected the spectrum of 1Cu to be very similar to that
of 8Cu. However, the EPR spectrum of 1Cu in H2O at the
same concentration shows much broader resonances and the
g values cannot be easily extracted (Figure 7b). We reasoned
that the signal broadening might be due to intermolecular
metal ion spin-spin interactions. Therefore, the solution was
“magnetically diluted” by addition of diamagnetic 1Zn (ca.
2.5 mM, Figure 7c). Indeed, the resulting spectrum is now
almost identical to the EPR spectrum of 8Cu. Clearly,
compound 8Cu is sufficiently bulky to prevent intermolecular
Cu-Cu spin-spin interactions in the same way that an
excess of 1Zn works on 1Cu. The almost exact similarity of
the spectrum of dilute 1Cu with that of 8Cu is a strong
indication that the Cu ion is indeed coordinated to the bpa
ligand in 8Cu.
from λmax ) 808 nm for the hydrated Cu2+ ion to λmax
)
651 nm in the Cu(bpa) complex (Figure 5). In addition, the
color intensifies upon complexation as seen by a 7-fold
increase of ꢀ. This low-energy region of the UV/vis spectrum
is identical for 8Cu and 3Cu, strongly suggesting an identical
Cu coordination into the bpa ligand pocket and ruling out a
coordination to Lys or Arg side chains of the nls peptide.
MS spectra of 8Cu and 8Zn show the characteristic fragments
after the loss of one nitrate [8‚MNO3]z+ or both nitrates [8‚
M]z+, as known for the simple metal complexes 1M-7M
discussed above.
(35) So, K. W.; Yang, C.-T.; Vittal, J. J.; Ranford, J. D. Inorg. Chim. Acta
2003, 349, 135-141.
(36) Long, L.-S.; Zheng, L.-S.; Chen, X.-M.; Ng, S. W. Main Group Met.
Chem. 2001, 24, 465-466.
(37) Stearns, D. M.; Hoffman, L. R.; Armstrong, W. H. Acta Crystallogr.
C 1992, C48, 253-256.
Inorganic Chemistry, Vol. 44, No. 15, 2005 5409

Kirin et al.
chains by intermolecular coordination of the carboxylic acid
to the metal ion of a neighboring complex. Ligand 3 was
used to prepare the bpa-nls bioconjugate 8 with a heptapep-
tide by solid phase synthesis. Cu(II) and Zn(II) complexes
of 8 were synthesized in situ and studied by FAB-MS, ESI-
MS, and UV/vis (for 8Cu), EPR (for 8Cu), and NMR (for
8Zn). The results indicate similar structural features of the
isolated well-defined simple metal bpa complexes 1M-7M
and the in situ prepared metal bioconjugate complex 8M.
This work demonstrates the use of simple chelating
nitrogen heterocycles as ligands for late first-row metal ions
in peptide bioconjugates. The conjugates are readily prepared
by solid phase peptide synthesis (SPPS) techniques. Metal
ion coordination is achieved in solution. The conjugates show
characteristic spectroscopic features that match exactly those
of smaller bioconjugates with simple amino acids. We
suggest metal-peptide conjugates like 8M as artificial
metallochaperones because they have the potential to deliver
metal ions to specific compartments in the cell as determined
by the peptide moieties. Investigations along those lines are
in progress in our laboratory.
Experimental Section
General Remarks. Reactions were carried out in ordinary
glassware and chemicals were used without further purification
except where indicated. Mass spectra were measured on a Mat 8200
instrument (EI, HR-EI, and FAB), on a Joel JMS-700 (HR-FAB),
or on a Finnigan TSQ 700 (ESI-MS). Only characteristic fragments
with possible composition are given in brackets. For fragments
containing metals only the isotopomer with highest intensity was
described. Infrared spectra were recorded on a Brucker Equinox55
FT-IR spectrometer between NaCl windows, or as KBr disks.
Wavenumbers, ν, are given in cm-1. UV/vis spectra were measured
on a Varian CARY 100 instrument in 1 cm quartz Suprasil cells
thermostated at 20 °C. Absorption maxima, λmax, and molar
absorption coeficients, ꢀmax, are given in nm and M-1 cm-1
,
respectively. NMR spectra were determined on a Brucker AM 360
spectrometer, 1H at 360.14 MHz and 13C at 90.56 MHz. Chemical
shifts, δ/ppm, indicate a downfield shift from tetramethylsilane,
TMS, the internal standard. Coupling constants, J, are given in Hz.
X-band CW EPR spectra were recorded on a Bruker ELEXSYS
E500 spectrometer equipped with a helium flow cryostat (Oxford
Instruments ESR 910). The spectra were simulated with a program
Figure 7. X-band EPR of 8Cu (a), and 1Cu (b), and 1Cu “magnetically
diluted” with 1Zn (c) recorded from frozen solutions at 32 K. Experimental
conditions: microwave frequency 9.6449 GHZ (a), (9.6450 GHz (b, c);
power 5 mW, modulation 1 mT/100 kHz. Spectra a and c could be fitted
with g| ) 2.2507 and g ) 2.0667, 2.0687 and magnetic hyperfine coupling
constant A| ) 189 × 10-4 cm-1 (a), and g| ) 2.2502 and g ) 2.0579,
2.0906 and magnetic hyperfine coupling constant A| ) 189 × 10-4 cm-1
(c) (simulations not shown).
1
developed in-house for powder samples with S ) /2. Hyperfine
3
interaction with I ) /2 was taken into account in second-order
perturbation treatment for 63Cu only (100%). HPLC was performed
using a Varian Pro Star PDA detector (model 330), a Varian solvent
delivery system (model 210), and an analytical C-18 Microsorb
column (4.6 mm × 250 mm, 60 Å/8 µm). Water (A) and acetonitrile
(B) were used as solvents, both containing 0.1% TFA. The flow
rate was 1 mL min-1, and peaks were detected at 254 and 220 nm.
The ascii-data processing and figure drawing for UV/vis, NMR,
and EPR spectra, as well as HPLC chromatograms, was done with
ORIGIN software.
Ligands. 2, Bpa-p-CH2C6H4CO2Me. Ligand 1 (0.90 mL, 5.02
mmol, Aldrich) and methyl p-(bromomethyl)benzoic acid (1.15 g,
5.02 mmol) were dissolved in THF (35 mL). DIPEA (0.83 mL,
4.99 mmol) was added and the mixture refluxed for 2 h. After
cooling to room temperature the insoluble precipitate was separated
by filtration. The filtrate was evaporated in Vacuo, and the residue
was dissolved in ether, filtered again, and evaporated. The crude
Conclusion
A series of bpa ligands 1-7 and their Cu(II) and Zn(II)
complexes were synthesized and extensively characterized,
including X-ray structures of 1Zn, 2Zn, 4Cu, and 7Cu. Metal
coordination is immediately evident from characteristic
1
changes in the H NMR spectra of the Zn complexes. Cu
coordination, on the other hand, can be followed by changes
in the UV/vis and EPR spectra. Distinct structural differences
were observed for the ester derivatives compared to com-
pounds with a free carboxylic acid side chain. Both structur-
ally characterized Zn complexes and 7Cu show a distorted
trigonal bipyramidal coordination environment in the solid
state. An octahedral complex is observed for 4Cu which forms
5410 Inorganic Chemistry, Vol. 44, No. 15, 2005

Bioconjugates of Cu(II) and Zn(II) Complexes
Figure 8. Structure of metal bioconjugates 8M, M ) Cu(II) or Zn(II).
product was purified by column chromatography on silica (50 g,
L ) 2.5 cm, ethyl acetate:methanol ) 9:1), Rf(2) ) 0.19. Yield:
1.40 g (4.02 mmol, 80%) of colorless oil. Mr (C21H21N3O2) )
347.42, Mexact ) 347.1633. HRMS (EI, 70 eV): 347.1633. MS (EI,
271 [1%, M+], 198 [1%, bpa+], 135 [17%, PyCH2N(CH2)2+], 119
[12%, PyCH2NHCH2+], 107 [11%, PyCH2NH+], 93 [100%,
PyCH2+], 78 [7%, Py+]. 1H NMR (DMSO-d6, 360 MHz): δ 8.46-
8.43 (m, 2H, HPy-6), 7.73 (dt, 2H, HPy-4, J ) 7.6 and 1.8), 7.53 (d,
2H, HPy-3, J ) 8.0), 7.23-7.18 (m, 2H, HPy-5), 3.69 (s, 4H, HPy-R),
2.64 (t, 2H, H2, J ) 7.8), 2.15 (m, 2H, H1, J ) 7.8). 13C NMR
(DMSO-d6, 90 MHz): δ 175.1 (CdO), 159.8 (CPy-2), 148.6 (CPy-6),
136.4 (CPy-4), 122.5 (CPy-3), 121.9 (CPy-5), 59.7 (CPy-R), 51.4 (C1),
30.7 (C2). IR (NaCl): ν 3380, 3083, 3011, 2954, 2816, 2346, 1577,
1459, 1410, 1149, 856, 764, 598.
70 eV): m/z 347 [4%, M+], 316 [M+ - OMe], 255 [100%, M+
-
PyCH2], 198 [23%, bpa+], 149 [18%, BzlCOOMe+], 121 [11%,
PyCH2NHCH2+], 107 [1%, PyCH2NH+], 93 [84%, PyCH2+], 78
[6%, Py+]. 1H NMR (DMSO-d6, 300 MHz): δ 8.48-8.46 (m, 2H,
HPy-6), 7.90 (d, 2H, HBzl-3, J ) 7.7), 7.77 (dt, 2H, HPy-4, J ) 7.2
and 1.2), 7.57-7.54 (m, 4H, HPy-3 and HBzl-2), 7.28-7.23 (m, 2H,
H
Py-5) 3.82 (s, 3H, HOMe), 3.70 (2s, 6H, HPy-R and H1). 13C NMR
5, Bpa-(CH2)5CO2H. Ligand 5 was prepared using the method
described for ligand 4 with 656 mg (4.00 mmol) of 2-picolyl
chloride hydrochloride and 256 mg (1.95 mmol) of ω-aminohex-
anoic acid. After evaporation of 2-propanol, the crude product was
purified by column chromatography on silica (50 g, L ) 3 cm,
dichloromethane:methanol ) 9:1), Rf(5) ) 0.23. Yield: 320 mg
(1.02 mmol, 51%) of a pale yellow oil. Mr (C18H25N3O2) ) 313.42,
Mexact ) 313.1790. HRMS (EI, 70 eV): 313.1790. MS (EI, 70
(DMSO-d6, 90 MHz): δ 166.3 (CdO), 158.9 (CPy-2), 149.0 (CPy-6),
144.9 (CBzl-1), 136.9 (CPy-4), 129.3 (CBzl-3), 129.0 (CBzl-2), 128.5
(CBzl-4), 122.8 (CPy-3), 122.4 (CPy-5), 59.3 (CPy-R), 57.3 (C1), 52.2
(COMe). IR (NaCl): ν 3420, 2950, 2822, 1721, 1611, 1589, 1670,
1474, 1434, 1280, 1111, 1047, 1020, 977, 764.
3, Bpa-p-CH2C6H4CO2H. Ester 2 (695 mg, 2.00 mmol) was
dissolved in methanol and a solution of NaOH (5 mL, 2 M) in
water was added. After 2 h of stirring at room temperature the
solution was neutralized with HCl (10 mL, 1 M) and the solvent
evaporated in Vacuo (at 50 °C). To the residue was added
dichloromethane (30 mL), and the insoluble precipitate and the
filtrate were dried over Na2SO4. After filtration and evaporation
the yellow oily residue was dissolved in acetonitrile (15 mL). After
crystallization overnight at 4 °C, acid 3 was isolated by filtration.
Yield: 370 mg (1.11 mmol, 71%) of an off white solid. Mr
(C20H19N3O2) ) 333.39, Mexact ) 333.1479. HRMS (EI, 70 eV):
333.1477. MS (EI, 70 eV): m/z 333 [3%, M+], 241 [100%, M -
PyCH2+], 198 [21%, bpa+], 135 [17%, PyCH2N(CH2)2+], 119 [5%,
PyCH2NHCH2+], 107 [14%, PyCH2NH+], 93 [87%, PyCH2+], 78
[7%, Py+]. 1H NMR (DMSO-d6, 360 MHz): δ 8.52-8.49 (m, 2H,
HPy-6), 7.93 (d, 2H, HBzl-3, J ) 8.3), 7.80 (dt, 2H, HPy-4, J ) 7.6
and 1.8), 7.60-7.54 (m, 4H, HPy-3, HBzl-2), 7.29-7.25 (m, 2H,
eV): m/z 313 [3%, M+], 235 [2%, M+ - Py], 221 [100%, M+
-
PyCH2], 198 [2%, bpa+], 121 [3%, PyCH2NHCH2+], 107 [4%,
PyCH2NH+], 93 [45%, PyCH2+], 78 [3%, Py+]. 1H NMR (DMSO-
d6, 360 MHz): δ 8.47-8.44 (m, 2H, HPy-6), 7.75 (dt, 2H, HPy-4
,
J ) 7.6 and 1.8), 7.50 (d, 2H, HPy-3, J ) 7.9), 7.25-7.19 (m, 2H,
HPy-5), 3.70 (s, 4H, HPy-R), 2.41 (t, 2H, H1, J ) 7.2), 2.13 (t, 2H,
H5, J ) 7.2), 1.49-1.34 (m, 4H, H4 and H2), 1.26-1.14 (m, 2H,
H3). 13C NMR (DMSO-d6, 90 MHz): δ 174.4 (Cpy-6), 159.5
(Cpy-2), 148.7 (CPy-6), 136.4 (CPy-4), 122.5 (CPy-5), 122.0 (CPy-3),
59.8 (CPy-R), 53.4 (C1), 33.6 (C5), 26.2, 26.2, 24.3 (C2, C3, and
C4). IR (NaCl): ν 3420, 2936, 2860, 1716, 1592, 1475, 1435, 1365,
1236, 1124, 765.
6, Bpa-p-CH2C6H4CO-Phe-OMe. Acid 3 (333 mg, 1.00 mmol)
was suspended in acetonitrile (10 mL), and phenyl-
alanine methyl ester hydrochloride (216 mg, 1.00 mmol), TBTU
(323 mg, 1.00 mmol), and DIPEA (1.20 mL, 7.20 mmol) were
added. The yellow reaction mixture was stirred for 1 h at room
temperature, and thereafter the solvent was evaporated in Vacuo.
To the residue was added dichloromethane (75 mL), and the mixture
was washed with saturated NaHCO3 solution (75 mL) and water
(2 × 75 mL). After drying of the organic phase over Na2SO4 and
filtration, the solvent was removed in Vacuo and the crude product
purified by colum chromatography on silica (35 g, L ) 2.5 cm,
ethyl acetate:acetonitrile ) 9:1), Rf(6) ) 0.12. Yield: 400 mg (80.9
mmol, 80.9%) of colorless oil. Mr (C30H30N4O3) ) 494.60, Mexact
) 494.2317. HRMS (EI, 70 eV): 494.2317. MS (EI, 70 eV): m/z
494 [6%, M+], 463 [M+ - OMe], 402 [100%, M+ - Bzl], 198
[31%, bpa+], 134 [5%, PyCH2N(CH2)2+], 121 [1%, PyCH2-
NHCH2+], 107 [1%, PyCH2NH+], 93 [64%, PyCH2+], 78 [2%,
Py+]. 1H NMR (DMSO-d6, 360 MHz): δ 8.82 (d, 1H, Hamide, J )
7.7), 8.50-8.47 (m, 2H, HPy-6), 7.87-7.69 (m, 4H, HBzl-3 and
H
Py-5), 3.74 (s, 4H, HPy-R), 3.72 (s, 2H, H1). 13C NMR (DMSO-
d6, 90 MHz): δ 167.2 (CdO), 158.9 (CPy-2), 148.8 (CPy-6), 144.1
(CBzl-1), 136.6 (CPy-4), 129.6 (CBzl-3), 129.3 (CBzl-2), 128.6 (CBzl-4),
122.6 (CPy-3), 122.2 (CPy-5), 59.1 (CPy-R), 57.1 (C1). IR (KBr): ν
3429, 2822, 1701, 1599, 1436, 1310, 1278, 1013, 764.
4, Bpa-(CH2)2CO2H. 2-Picolyl chloride hydrochloride (1.97
g, 12.0 mmol) and â-alanine (526 mg, 5.91 mmol) were dissolved
in water (15 mL) and stirred at room temperature for 5 days. The
pH of the solution was maintained between 8 and 10 by addition
of 5 M NaOH. The dark red solution was washed with chlorofom
(3 × 30 mL). The pH of the water phase was adjusted to 3 with 1
M HCl, and the solution was again washed with chlorofom (3 ×
30 mL). After neutralization with 2 M NaOH, the aqueous phase
was evaporated to dryness (at 40 °C). 2-Propanol (30 mL) was
added to the residue and the insoluble NaCl separated by filtration.
The 2-propanol was evaporated and the crude product recrystallized
from 2-propanol (5 mL). Yield: 740 mg (2.73 mmol, 46%) of a
brownish solid. Mr (C15H17N3O2) ) 271.31. MS (EI, 70 eV): m/z
HPy-4), 7.57 (d, J ) 7.6, 2H, HPy-3), 7.49 (d, J ) 7.9, 2H, HBzl-2),
7.31-7.22 (m, 7H, HPhe-o, HPhe-m, HPhe-p and HPy-5), 4.68-4.60
Inorganic Chemistry, Vol. 44, No. 15, 2005 5411

Kirin et al.
(m, 1H, HPhe-R), 3.70 (s, 4H, HPy-R), 3.67 (s, 2H, H1), 3.62 (s, 3H,
OMe), 3.19-3.03 (m, 2H, HPhe-â). 13C NMR (DMSO-d6, 90
MHz): δ ) 172.2 (CdOPhe), 166.4 (CdOBzl), 158.9 (CPy-2), 148.9
(CPy-6), 142.6 (CBzl-1), 137.7 (CPhe-i), 136.7 (CPy-4), 132.5 (CBzl-4
129.1 (CBzl-2), 128.4 (CBzl-3), 128.3 (CPhe-o), 127.4 (CPhe-m), 126.5
(CPhe-p), 122.6 (CPy-3), 122.2 (CPy-5), 59.1 (CPy-R), 57.1 (C1), 54.3
(CPhe-R), 52.0 (COMe), 36.2 (CPhe-â). IR (NaCl): ν 3311, 2951, 2824,
1743, 1652, 1590, 1538, 1498, 1434, 1217, 1149, 995, 849, 765,
736.
(dt, 2H, Hpy-4, J ) 7.7 and 1.5), 7.52-7.48 (m, 4H, HPy-3 and
H
HPy-5), 5.16 (br s, 1H, NH), 4.45 (dd, 2H, HPy-Ra, J ) 16.8 and
6.8), 3.95-3.89 (m, 2H, HPy-Rb). 13C NMR (DMSO-d6, 90 MHz):
δ 155.4 (CPy-2), 146.9 (CPy-6), 139.9 (CPy-4), 124.2 (CPy-3), 123.7
(CPy-5), 52.2 (CPy-R). IR (KBr): ν 3436, 3266, 3070, 2916, 1608,
1574, 1443, 1384, 1321, 1289, 1025, 774. UV (H2O): λmax (ꢀ) 260
nm (7770).
)
2Cu, [(Bpa-p-CH2C6H4CO2Me)Cu(NO3)2]. Ligand 2 (86.8 mg,
0.25 mmol), Cu(NO3)2‚3H2O (60.4 mg, 0.25 mmol), and methanol
(2 × 10 mL) were used, method B. After 2 days, product 2Cu was
collected by filtration. Yield: 73.0 mg (0.14 mmol, 55%) of blue
crystals. Mr (C21H21N5O8Cu) ) 534.99. MS (ESI): m/z 472 [M -
NO3]+, 455 [M - Py]+, 441 [M - PyCH2]+, 427 [M - PyCH2 -
Me]+, 410 [M - 2 NO3]+, 346 [M - Cu(NO3)2]+, 317 [M - 2
NO3 - PyCH2]+, 260 [Cu-bpa]+. MS (FAB, glycerol): m/z 472
[M - NO3]+, 410 [M - 2 NO3]+, 346 [M - Cu(NO3)2]+, 317 [M
7, Bpa-p-CH2C6H4CO-âAla-OMe. Ligand 7 was prepared
using the method described for ligand 6 with acid 3 (1.00 g, 3.00
mmol), â-alanine methyl ester hydrochloride (419 mg, 3.00 mmol),
TBTU (963 mg, 3.00 mmol), and DIPEA (3.85 mL, 22.5 mmol)
in acetonitrile (30 mL). After extraction the crude product was
purified by column chromatography on silica (ethyl acetate:
acetonitrile ) 1:1). Yield: 791 mg (1.89 mmol, 63%) as a colorless
oil. Mr (C24H26N4O3) ) 418.50, Mexact ) 418.2005. HRMS (EI, 70
eV): 418.2005. MS (EI, 70 eV): m/z 418 [6%, M+], 387 [11%,
M+ - OCH3], 326 [100%, M+ - PyCH2], 198 [24%, bpa+], 119
[4%, PyCH2NHCH2+], 107 [1%, PyCH2NH+], 93 [49%, PyCH2+],
- 2NO3 - PyCH2]+, 260 [Cu-bpa]+. HRMS (FAB, glycerol,
63
PEG300): m/z exp 472.0819 and calcd 472.0808 for [C21H21N4O5
-
Cu]+, exptl 410.0915 and calcd 410.0930 for [C21H21N3O263Cu]+.
IR (KBr): ν 3421, 3033, 2947, 1720, 1611, 1420, 1281, 1108, 767.
UV (H2O): λmax (ꢀ) 652 (110), 240 nm (12270).
1
78 [2%, Py+]. H NMR (DMSO-d6, 360 MHz): δ 8.53 (t, 1H,
Hamide, J ) 5.5) 8.50-8.48 (m, 2H, HPy-6), 7.81-7.75 (m, 4H, HPy-4
2Zn, [(Bpa-p-CH2C6H4CO2Me)Zn(NO3)2]. Ligand 2 (86.8 mg,
and HBzl-3), 7.57 (d, 2H, HPy-3, J ) 7.7), 7.49 (m, 2H, HBzl-2, J )
8.1), 7.27-7.21 (m, 2H, HPy-5), 3.70 (s, 4H, HPy-R), 3.67 (s, 2H,
H1), 3.59 (s, 3H, HOMe), 3.51-3.44 (m, 2H, HâAla-1), 2.58 (t, 2H,
HâAla-2, J ) 7.0). 13C NMR (DMSO-d6, 90 MHz): δ 171.8 (CdO
0.25 mmol), Zn(NO3)2‚6H2O (74.4 mg, 0.25 mmol), and methanol
(2 × 10 mL) were used, method B. After 3 days, product 2Zn was
collected by filtration. Yield: 92.0 mg (0.17 mmol, 69%) of white
crystals, X-ray quality. Mr (C21H21N5O8Zn) ) 536.81. MS (ESI):
m/z 473 [M - NO3]+, 456 [M - Py]+, 442 [M - PyCH2]+, 428
[M - PyCH2 - Me]+, 410 [M - 2 NO3]+, 348 [M - Zn(NO3)2]+.
MS (FAB, glycerol): m/z 473 [M - NO3]+, 410 [M - 2 NO3]+,
348 [M - Zn(NO3)2]+, 319 [M - 2 NO3 - PyCH2]+, 262 [Zn-
bpa]+. HRMS (FAB, glycerol, PEG300): m/z exptl 473.0855 and
calcd 473.0803 for [C21H21N4O564Zn]+, exptl 410.0836 and calcd
410.0847 for [C21H20N3O264Zn]+. 1H NMR (DMSO-d6, 300
â
Ala), 166.2 (CdOBzl), 158.9 (CPy-2), 148.9 (CPy-6), 142.2 (CBzl-1),
136.6 (CPy-4), 133.2 (CBzl-3), 128.4 (CBzl-2), 127.2 (CBzl-4), 122.6
(CPy-3), 122.2 (CPy-5), 59.1 (CPy-R), 57.1 (C1), 51.4 (COMe), 35.5
(CâAla-2), 33.6 (CâAla-1). IR (KBr): ν 3316, 2950, 2824, 1738, 1652,
1544, 1435, 1307, 1256, 1150, 995, 852.
Metal Complexes 1M-7M. General Procedure. The corre-
sponding ligand was dissolved in the indicated solvent in a beaker.
The metal salt was dissolved in the indicated solvent in another
beaker. Both beakers were shortly heated to boiling. The ligand
solution was added to the metal salt solution, and the reaction
mixture was filtered hot in a vial (which was silanized by shaking
for 5 min with Me3Si-SiMe3 in order to avoid sticking of the
crystals on the walls of the vial) and left to cool to room temperature
during 1 h. If precipitation occurred, the vial was closed and left at
room temperature for the period indicated (method A). If no
precipitation occurred, the vial was placed in a container with ether
(10 mL), closed, and left at room temperature for the period
indicated (method B). After filtration, the product was dried in air.
MHz): δ 8.66-8.64 (m, 2H, HPy-6), 8.13-8.03 (m, 4H, HBzl-3
,
H
H
Py-4), 7.66-7.56 (m, 6H, HPy-5, HPy-3, and HBzl-2), 4.26 (d, 2H,
Py-Ra, J ) 16.0), 3.90 (s, 3H, HOMe), 3.84 (s, 2H, H1), 3.70 (d,
2H, HPy-Rb, J ) 16.0). 13C NMR (DMSO-d6, 90 MHz): δ 166.0
(CdO), 154.1 (CPy-2), 147.9 (CPy-6), 140.8 (CPy-4), 137.2 (CBzl-1),
132.0 (CBzl-3), 129.7 (CBzl-4), 129.3 (CBzl-2), 124.9 (CPy-3), 124.8
(CPy-5), 56.3 (C1), 55.4 (CPy-R), 52.3 (COMe). IR (KBr): ν 3422,
3035, 2939, 1721, 1609, 1465, 1310, 1281, 1193, 1027, 766. UV
(H2O): λmax (ꢀ) 261 (11530), 238 (6650).
3Cu, [(Bpa-p-CH2C6H4CO2)CuNO3]. Ligand 3 (83.4 mg, 0.25
mmol), Cu(NO3)2‚3H2O (60.4 mg, 0.25 mmol), and methanol (2
× 10 mL) were used, method A. After standing overnight, product
3Cu was collected by filtration. Yield: 89 mg (0.17 mmol, 68%)
of blue crystals. Mr (C20H18N4O5Cu) ) 457.93. MS (ESI): m/z 396
[M - NO3]+. MS (FAB, glycerol): m/z 458 [M]+, 396 [M -
NO3]+, 352 [M - NO3 - CO2]+, 303 [M - NO3 - PyCH2]+ 260
[Cu-bpa]+. HRMS (FAB, glycerol, PEG300): m/z exptl 458.0585
and calcd 458.0651 for [C20H19N4O563Cu]+, exptl 396.0780 and
calcd 396.0773 for [C20H19N3O263Cu]+. IR (KBr): ν 3536, 3031,
2939, 1700, 1611, 1481, 1430, 1384, 1289, 1254, 1018, 787. UV
(H2O): λmax (ꢀ) 652 (110), 240 nm (12270).
1Cu, [(Bpa)Cu(NO3)2]. Ligand 1 (90.0 µL, 0.50 mmol, Aldrich),
Cu(NO3)2‚3H2O (121 mg, 0.50 mmol), and methanol (2 × 20 mL)
were used, method A. After standing overnight, product 1Cu was
collected by filtration. Yield: 154 mg (0.40 mmol, 80%) of blue
crystals. Mr (C12H13N5O6Cu) ) 386.82. MS (ESI): m/z 324 [M -
NO3]+, 261 [M - 2 NO3]+. MS (FAB, glycerol): m/z 324 [M -
NO3]+, 262 [Cu-bpa]+, 198 [bpa]+. HRMS (FAB, glycerol,
PEG300): m/z exptl 324.0289 and calcd 324.0284 for [C12H13N4O363
-
Cu]+, exptl 262.0387 and calcd 262.0405 for [C12H13N363Cu]+. IR
(KBr): ν 3435, 3221, 3080, 2931, 1612, 1384, 1298, 1099, 1035,
782. UV (H2O): λmax (ꢀ) 650 nm (100), 252 (11100).
3Zn, [(Bpa-p-CH2C6H4CO2)ZnNO3]. Ligand 3 (83.4 mg, 0.25
mmol), Zn(NO3)2‚6H2O (74.4 mg, 0.25 mmol), and methanol (2
× 10 mL) were used, method A. After standing overnight, product
3Zn was collected by filtration. Yield: 68.0 mg (0.13 mmol, 52%)
of white powder. Mr (C20H19N4O5Zn) ) 460.78. MS (ESI): m/z
398 [M - 2 NO3]+. MS (FAB, glycerol): m/z 459 [M]+, 396 [M
- NO3]+, 334 [M - ZnNO3]+. HRMS (FAB, glycerol): m/z exptl
1Zn, [(Bpa)Zn(NO3)2]. Ligand 1 (90.0 µL, 0.50 mmol, Aldrich),
Zn(NO3)2‚6H2O (148.7 mg, 0.50 mmol), and methanol (2 × 30
mL) were used, method B. After 2 days, product 1Zn was collected
by filtration. Yield: 106 mg (0.27 mmol, 55%) of white crystals,
X-ray quality. Mr (C12H13N5O6Zn) ) 388.64. MS (ESI): m/z 325
[M - NO3]+, 262 [M - 2 NO3]+, 198 [bpa]+. MS (FAB,
glycerol): m/z 325 [M - NO3]+, 262 [M - 2 NO3]+, 198 [bpa]+.
1H NMR (DMSO-d6, 300 MHz): δ 8.56-8.54 (m, 2H, Hpy-6), 7.98
459.0676 and calcd 459.0647 for [C20H19N4O564Zn]+. H NMR
1
(DMSO-d6, 360 MHz): δ 8.69-8.67 (m, 2H, HPy-6), 8.14-8.03
5412 Inorganic Chemistry, Vol. 44, No. 15, 2005

Bioconjugates of Cu(II) and Zn(II) Complexes
(m, 4H, HBzl-3 and HPy-4), 7.67-7.63 (m, 4H, HBzl-2, HPy-3), 7.54-
7.52 (m, 2H, HPy-5), 4.26 (m, 2H, HPy-Ra, J ) 16.1), 3.83 (bs, 2H,
H1), 3.73 (m, 2H, HPy-Rb, J ) 16.1), 13C NMR (DMSO-d6, 90
MHz): δ 154.2 (CPy-2), 148.0 (CPy-6), 140.8 (CPy-4), 136.7 (CBzl-1),
131.9 (CBzl-3), 131.0 (CBzl-4), 129.5 (CBzl-2), 124.9 (CPy-3), 124.8
(CPy-5), 56.0 (C1), 55.4 (CPy-R). IR (KBr): ν 3554, 3029, 2941,
1703, 1609, 1471, 1293, 1027, 787. UV (H2O): λmax (ꢀ) 261 (8840).
4Cu, [(Bpa-(CH2)2CO2)Cu(H2O)]NO3‚EtOH. Ligand 4 (67.8
mg, 0.25 mmol), Cu(NO3)2‚3H2O (60.4 mg, 0.25 mmol), and
ethanol (2 × 12.5 mL) were used. After slow evaporation of the
solvent for 3 days, product 4Cu was collected by filtration. Yield:
70 mg (0.15 mmol, 61%) of blue crystals, X-ray quality. Mr
(C17H24N4O7Cu) ) 459.95. MS (ESI): m/z 397 [M]+, 334 [M -
NO3]+, 289 [M - NO3 - CO2]+. MS (FAB, glycerol): m/z 396
[M]+, 334 [M - NO3]+, 260 [Cu-bpa]+. IR (KBr): ν 3429, 1610,
1559, 1384, 1355, 1306, 1052, 769. UV (H2O): λmax (ꢀ) 650 (100),
252 (9590).
ethanol (2 × 5 mL) were used, method A. After 4 days, product
6Cu was collected by filtration. Yield: 23 mg (34 µmol, 34%) of
blue powder. Mr (C30H30N6O9Cu) ) 682.17. MS (ESI): 619 [M -
NO3]+, 602 [M - Py]+, 588 [M - NO3 - OMe]+, 557 [M - 2
NO3]+, 496 [M - Cu(NO3)2]+. MS (FAB, glycerol): m/z 619 [M
- NO3]+, 557 [M - 2 NO3]+, 497 [M - Cu(NO3)2]+, 351 [Cu-
bpaBzl]+, 260 [Cu-bpa]+. HRMS (FAB, NBA, PEG600): m/z
exptl 619.1451 and calcd 619.1492 for [C30H30N5O663Cu]+, exptl
557.1575 and calcd 557.1614 for [C30H30N4O363Cu]+. IR (KBr):
ν 3423, 3032, 2953, 1744, 1663, 1611, 1447, 1424, 1291, 1030,
768, 703. UV (H2O): λmax (ꢀ) 651 (130), 242 (19970).
6
Zn, [(Bpa-pCH2C6H4CO-Phe-OMe)Zn(NO3)2]. Ligand 6
(49.5 mg, 0.10 mmol), Zn(NO3)2‚6H2O (29.8 mg, 0.10 mmol), and
ethanol (2 × 5 mL) were used, method A. After standing overnight,
product 6Zn was collected by filtration. Yield: 30 mg (44 µmol,
44%) of white crystals. Mr (C30H30N6O9Zn) ) 683.99. MS (ESI):
620 [M - NO3]+, 603 [M - Py]+, 589 [M - PyCH2]+, 575 [M -
PyCH2 - Me]+, 495 [M - Zn(NO3)2]+. MS (FAB, glycerol): m/z
682 [M]+, 620 [M - NO3]+, 559 [M - 2 NO3]+, 262 [Zn-bpa]+.
HRMS (FAB, NBA, PEG600): m/z exptl 620.1464 and calcd
620.1488 for [C30H30N5O664Zn]+. 1H NMR (DMSO-d6, 360
MHz): δ 8.95 (d, 1H, Hamide, J ) 8.0), 8.65-8.63 (m, 2H, HPy-6),
8.11-8.05 (m, 2H, HPy-4), 7.91 (d, 2H, HBzl-3, J ) 8.0), 7.64-
7.58 (m, 4H, HPy-3, HPy-5), 7.49 (d, 2H, HBzl-2, J ) 8.4), 7.33-
7.18 (m, 5H, HPhe-o, HPhe-m, and HPhe-p), 4.75-4.68 (m, 1H,
4Zn, [(Bpa-(CH2)2CO2)Zn(NO3)]. Ligand 4 (67.8 mg, 0.25
mmol), Zn(NO3)2‚6H2O (74.4 mg, 0.25 mmol), and ethanol (2 ×
12.5 mL) were used, method B. After standing overnight, product
4Zn was collected by filtration. Yield: 49.0 mg (0.11 mmol, 43%)
of white powder. Mr (C15H17N4O5Zn) ) 396.70. MS (ESI): m/z
398 [M]+, 334 [M - NO3]+. MS (FAB, glycerol): m/z 397 [M]+,
334 [M - NO3]+, 262 [Zn-bpa]+. 1H NMR (DMSO-d6, 360
MHz): δ 8.56-8.54 (m, 2H, HPy-6), 8.12 (dt, 2H, HPy-4, J ) 7.7
and 1.7), 7.64 (d, 2H, HPy-3, J ) 7.7), 7.58-7.54 (m, 2H, HPy-5),
4.35 (d, 2H, HPy-Ra, J ) 16.4), 4.14-4.06 (m, 2H, HPy-Rb, J )
16.4), 3.47-3.38 (m, 2H, H1, J ) 5.7), 2.27 (t, 1H, H2, J ) 5.7).
13C NMR (DMSO-d6, 90 MHz): δ 174.8 (CdO), 155.4 (CPy-2),
147.8 (CPy-6), 140.8 (CPy-4), 124.6 (CPy-3), 124.1 (CPy-5), 58.4
(CPy-R), 52.9 (C1), 33.2 (C2). IR (KBr): ν 3435, 2923, 1609, 1576,
1540, 1384, 1363, 1026, 761. UV (H2O): λmax (ꢀ) 261 (7970).
5Cu, [(Bpa-(CH2)5CO2)CuNO3]. Ligand 5 (78.4 mg, 0.25
mmol), Cu(NO3)2‚3H2O (60.4 mg, 0.25 mmol), and ethanol (2 ×
10 mL) were used, method B. After 5 days, product 5Cu was
collected by filtration. Yield: 75 mg (0.17 mmol, 69%) of blue
crystals. Mr (C18H22N4O5Cu) ) 437.94. MS (ESI): m/z 375 [M -
NO3]+. MS (FAB, glycerol): m/z 438 [M]+, 376 [M - NO3]+,
330 [M - NO3 - CO2]+, 283 [M - NO3 - PyCH2]+, 260 [Cu-
bpa]+, 239 [CuPyCH2N(CH2)5]+. HRMS (FAB, glycerol,
H
Phe-1), 4.25 (dd, 2H, HPy-Ra, J ) 16.1 and 6.0), 3.81 (s, 2H, H1),
3.71-3.67 (m, 2H, HPy-Rb), 3.66 (s, 3H, HOMe), 3.23-3.09 (m, 2H,
Phe-2). 13C NMR (DMSO-d6, 90 MHz): δ 171.7 (CdOPhe), 165.7
(CdOBzl), 154.0 (CPy-2), 147.7 (CPy-6), 140.6 (CPy-4), 137.7
(CPhe-i), 134.8 (CBzl-1), 134.2 (CBzl-4), 131.5 (CBzl-2), 131.4 (CBzl-3
H
)
129.3 (CPhe-o), 127.4 (CPhe-m), 127.2 (CPhe-p), 124.7 (CPy-3), 124.6
(CPy-5), 56.3 (C1), 55.5 (CPy-R), 55.4 (CPhe-R), 51.3 (COMe), 35.5
(CPhe-â). IR (KBr): ν 3445, 3031, 2943, 1747, 1674, 1609, 1460,
1384, 1295, 1026, 766. UV (H2O): λmax (ꢀ) 250 (15720).
7Cu, [(Bpa-pCH2C6H4CO-âAla-OMe)Cu(NO3)2]. Ligand 7
(41.9 mg, 0.1 mmol), Cu(NO3)2‚3H2O (41.9 mg, 0.1 mmol), and
methanol (2 × 5 mL) were used, method A. After standing
overnight, product 7Cu was collected by filtration. Yield: 24 mg
(40 µmol, 40%) of blue crystals, X-ray quality. Mr (C24H26N6O9-
Cu) ) 606.07. MS (ESI): m/z 543 [M - NO3]+, 481 [M - 2
NO3]+, 353 [Cu(PyCH2)2Bzl]+. MS (FAB, glycerol): m/z 606 [M]+,
543 [M - NO3]+, 481 [M - 2 NO3]+, 421 [M - Cu(NO3)2]+, 388
[M - 2 NO3 - PyCH2]+, 351 [Cu-bpaBzl]+, 260 [Cu-bpa]+.
HRMS (FAB, NBA, PEG600): m/z exptl 543.1194 and calcd
543.1179 for [C24H26N5O663Cu]+, exptl 481.1321 and calcd 481.1301
[C24H26N4O363Cu]+. IR (KBr): ν 3380, 3033, 2953, 1735, 1646,
1611, 1540, 1503, 1440, 1384, 1298, 1082, 1032, 770. UV (H2O):
λmax (ꢀ) 653 (130), 240 (19650).
PEG300): m/z exptl 376.1028 and calcd 376.1086 for [C18H23N3O263
-
Cu]+. IR (KBr): ν 3435, 2931, 1628, 1611, 1420, 1384, 1288, 1033,
769. UV (H2O): λmax (ꢀ) 649 (110), 254 (10900).
5Zn, [(Bpa-(CH5)5CO2)ZnNO3]. Ligand 5 (78.4 mg, 0.25
mmol), Zn(NO3)2‚6H2O (74.4 mg, 0.25 mmol), and methanol (2
× 10 mL) were used, method B. After 3 days, product 5Zn was
collected by filtration. Yield: 42.7 mg (97 µmol, 39%) of white
crystals, X-ray quality. Mr (C18H22N4O5Zn) ) 439.78. MS (ESI):
m/z 376 [M - NO3]+, 283 [M - NO3 - PyCH2]+, 239 [ZnPyCH2N-
(CH2)5]+. MS (FAB, glycerol): m/z 376 [M - NO3]+, 332 [M -
NO3 - CO2]+. HRMS (FAB, glycerol, PEG300): m/z exptl
376.0984 and calcd 376.1003 for [C18H22N3O264Zn]+, exptl 332.1105
and calcd 332.1105 for [C17H22N364Zn]+. 1H NMR (DMSO-d6, 360
MHz): δ 8.63-8.61 (m, 2H, HPy-6), 8.08 (dt, 2H, HPy-4, J ) 7.7
7
Zn, [(Bpa-pCH2C6H4CO-âAla-OMe)Zn(NO3)2]. Ligand 7
(104.6 mg, 0.25 mmol), Zn(NO3)2‚6H2O (74.4 mg, 0.25 mmol),
and ethanol (2 × 12.5 mL) were used, method B. After 5 days,
product 7Zn was collected by filtration. Yield: 23 mg (38 µmol,
15%) of white crystals. Mr (C24H26N6O9Zn) ) 607.89. MS (ESI):
m/z 419 [M - Zn(NO3)2]+. MS (FAB, glycerol): m/z 544 [M -
NO3]+, 528 [M - Py]+, 483 [M - 2 NO3]+, 419 [M - Zn(NO3)2]+,
262 [Zn-bpa]+. HRMS (FAB, NBA, PEG600): m/z exptl 544.1151
and 1.7), 7.63-7.58 (m, 4H, HPy-3 and HPy-5), 4.26 (d, 2H, HPy-Ra
,
J ) 16.2), 3.96 (d, 2H, HPy-Rb, J ) 16.2), 2.65-2.60 (m, 2H, H1),
2.18 (t, 2H, H5, J ) 7.4), 1.59-1.45 (m, 4H, H4 and H2), 1.21-
1.13 (m, 2H, H3). 13C NMR (DMSO-d6, 90 MHz): δ 174.8 (Cd
O), 154.9 (Cpy-2), 147.6 (CPy-6), 130.6 (CPy-4), 124.6 (CPy-3), 124.3
(CPy-5), 56.7 (CPy-R), 55.0 (C1), 33.5 (C5), 26.3, 24.2, 22.4 (C2, C3
and C4,). IR (KBr): ν 3421, 3070, 2949, 1708, 1608, 1468, 1295,
1226, 1027, 767.
and calcd 544.1175 for [C24H26N5O664Zn]+. H NMR (DMSO-d6,
1
360 MHz): δ 8.69-8.63 (m, 3H, HPy6 and Hamide), 8.11-8.06 (m,
2H, HPy-4), 7.94 (d, 2H, HBzl-3, J ) 7.7), 7.65-7.60 (m, 4H, HPy-5
and HPy-3), 7.49 (d, 2H, HBzl-2, J ) 8.6), 4.25 (m, 2H, HPy-Ra, J
) 16.0), 3.81 (s, 2H, H1), 3.68 (m, 2H, HPy-Rb, J ) 16.0), 3.61 (s,
3H, HOMe), 3.55-3.50 (m, 2H, HAla-1), 2.61 (t, 2H, CH2, HAla-2, J
) 7.0). 13C NMR (DMSO-d6, 90 MHz): δ 171.8 (CdOAla), 165.8
6
Cu, [(Bpa-pCH2C6H4CO-Phe-OMe)Cu(NO3)2]. Ligand 6
(49.5 mg, 0.10 mmol), Cu(NO3)2‚3H2O (24.2 mg, 0.10 mmol), and
Inorganic Chemistry, Vol. 44, No. 15, 2005 5413

Kirin et al.
Table 3. Crystallographic Data for 1Zn, 2Zn, 4Cu, and 7Cu
1Zn
2Zn
4Cu
7Cu
empirical formula
formula weight
T [K]
C12H13N5O6Zn
388.64
100(2)
C21H21N5O8Zn
536.80
100(2)
C17H24CuN4O7
459.94
100(2)
C24H28CuN6O10
624.06
100(2)
λ [Å]
0.71073
0.71073
0.71073
0.71073
crystal size [mm]
crystal system
space group
a [Å]
0.05 × 0.04 × 0.01
monoclinic
P21/n (No. 14)
7.5977(6)
12.9881(8)
14.9203(10)
90
100.48(1)
90
1447.77(17)
4
0.24 × 0.14 × 0.14
0.35 × 0.06 × 0.04
monoclinic
C2/c (No. 15)
26.547(2)
9.1410(6)
21.0208(14)
90
127.87(1)
90
4026.8(5)
8
0.09 × 0.08 × 0.02
monoclinic
P21/n (No. 14)
8.1755(6)
37.912(3)
8.8352
90
105.46(1)
90
2639.4(3)
4
1.570
0.897
2.15 e θ e 23.50
22878
triclinic
P1h (No. 2)
8.4052(3)
8.4879(3)
17.0262(6)
100.09(1)
99.23(1)
108.20(1)
1105.62(7)
2
1.612
1.171
3.22 e θ e 31.46
31389
b [Å]
c [Å]
R [deg]
â [deg]
γ [deg]
V [Å3]
Z
F
calcd [g cm-3
]
]
1.783
1.740
3.14 e θ e 30.00
34873
1.517
1.132
4.40 e θ e 33.07
23181
µ
MoKR [mm-1
θ range [deg]
data collected
independent data
4213 [R(int) ) 0.0768]
4213/0/217
1.068
0.0483
0.0953
7316 [R(int) ) 0.0264]
7316/0/317
1.055
0.0244
0.0633
7586 [R(int) ) 0.0668]
7553/4/268
1.020
0.0498
0.1108
3823 [R(int) ) 0.0891]
3823/0/380
1.134
0.0655
0.1293
data/restraints/parameters
GOF on F2 a
final R1 (obsd data)b
final wR2 (all data)c
largest diff peak/hole [e Å-3
]
0.791 / -0.516
0.481 / -0.323
0.715 / -0.649
0.737 / -0.539
2
2
2
a GOF ) [∑[w(Fo - Fc )2]/(n - p)]1/2 where n ) no. of reflections and p ) no. of refined parameters. b R1 ) ∑||Fo| - |Fc||/∑|Fo|. c wR2 ) [∑[w(Fo
- Fc )2]/∑[w(Fo )2]1/2, where w ) 1/σ2(Fo ) + (aP)2 + bP, P ) (Fo + 2Fc )/3.
2
2
2
2
2
(CdOBzl), 156.1 (CPy-2), 147.7 (CPy-6), 140.6 (CPy-4), 134.9
(CBzl-1), 134.3 (CBzl-4), 131.6 (CBzl-2), 127.3 (CBzl-3), 124.8 (CPy-3),
124.7 (CPy-5), 56.4 (C1), 55.5 (CPy-R), 51.4 (COMe), 35.5 (CAla-2),
33.6 (CAla-1). IR (KBr): ν 3435, 2937, 1735, 1653, 1609, 1473,
1292, 1025, 768. UV (H2O): λmax (ꢀ) 261 (10620).
mg, 9.93 mmol) was added a solution of Cu(NO3)2‚3H2O in water
(136.4 µL, 72.8 mM, prepared by dissolving 177.3 mg in 10 mL
of water), and the volume was brought to 1 mL with water. Mr
(C70H107N21O15Cu) ) 1546.28. MS (ESI): m/z 742.1 [M + 2 H -
NO3]2+; 710.7 [M + 2 H - 2 NO3]2+, 495.2 [M + 3 H - NO3]3+
MS (FAB, glycerol): m/z 1422 [M + H - 2 NO3]+.
.
Bioconjugate 8 and Bioconjugate Metal Complexes 8M. 8,
Bpa-p-CH2C6H4CO-Ahx-PKKKRKF-NH2. The synthesis was
performed manually in a syringe equipped with a porous filter (5
mL, MultiSynTech) using Fmoc protected Rink amide resin (159
mg, 0.1 mmol, loading 0.63 mmol/g, Novabiochem), Fmoc
protected amino acid monomers (Novabiochem), bpa ligand 3, and
amine free DMF (Roth) as solvent. Side chain protecting groups
were Boc(Lys) and Pbf(Arg). Synthetic cycle: (A) Fmoc-depro-
tection using two times about 3 mL of a 20% piperidine solution
in DMF, first 2 min and then 10 min, without washing in between;
(B) DMF wash 5× with about 3 mL; (C) coupling using 2.2 mL
of coupling cocktail during 20 min (see below); and (D) DMF wash
5× with about 3 mL. The coupling mixture contained the
corresponding monomer (protected amino acid or bpa ligand 3, 0.5
mmol, 5-fold excess), TBTU (157.3 mg, 0.49 mmol, 4.9-fold
excess), and HOBt (75.0 mg, 0.49 mmol, 4.9-fold excess) dissolved
in DMF (2 mL); then DIPEA (0.2 mL, 1.2 mmol, 11.7-fold excess,
activation period 1 min) was added. Workup: After the resin was
dried (1 h, 10 mbar), final deprotection and cleavage from the resin
was performed by a TFA mixture (TFA:H2O:TIS ) 95:2.5:2.5, 3
mL, 3 h). The suspension was filtered, and the resin washed with
TFA (2 × 1.5 mL). The combined TFA solutions were poured into
cold ether (10 mL, -30 °C), and the suspension was centifuged
(5000 rpm, 10 min). After the supernatant was decanted, the crude
product was washed with cold ether (2 × 10 mL), dissolved in
water, filtered, and lyophilized, yielding a white solid. Analytical
HPLC (5% B f 40% B in 30 min): 19.6 min, purity 97.2% (254
nm). Mr (C70H107N19O9) ) 1358.72, Mexact ) 1357.8499. MS
(ESI): m/z 680.2 [M + 2 H]2+, 454.0 [M + 3 H]3+, 340.8 [M +
8
Zn, [(Bpa-p-CH2C6H4CO-Ahx-PKKKRKF-NH2)Zn(NO3)2].
This complex was prepared in situ: To a solution of bioconjugate
8 (32 mg, 23.5 µmol) in DMSO-d6 (450 µL) in an NMR tube was
added a solution of Zn(NO3)2‚6H2O in DMSO-d6 (34.3 µL, 686
mM, prepared by dissolving 48.8 mg in 200 µL of DMSO-d6). Mr
(C70H107N21O15Zn) ) 1545.75. MS (ESI): m/z 711.1 [M + 2 H -
2 NO3]2+, 495.5 [M + 3 H - NO3]3+. MS (FAB, glycerol): m/z
1423 [M + H - 2 NO3]+.
X-ray Crystallographic Data Collection and Refinement of
the Structures. Colorless single crystals of 1Zn and 2Zn and blue
crystals of 4Cu and 7Cu were coated with perfluoropolyether and
mounted in the nitrogen cold stream of a Nonius Kappa-CCD
diffractometer equipped with a Mo-target rotating-anode X-ray
source and a graphite monochromator (Mo KR, λ ) 0.71073 Å).
Final cell constants were obtained from least-squares fits of all
integrated reflections of each data set. Crystal faces of 2Zn, 4Cu
,
and 7Cu were determined, and the corresponding intensity data were
corrected for absorption using the Gaussian-type routine embedded
in XPREP.38 The data set of 1Zn was not corrected for absorption.
The Siemens ShelXTL38 software package was used for solution
and artwork of the structure, and ShelXL9739 was used for the
refinement. The structures were readily solved by direct and
Patterson methods and subsequent difference Fourier techniques.
All non-hydrogen atoms were refined anisotropically, except
disordered parts in 1Zn and 4Cu. Hydrogen atoms were placed at
calculated positions and refined as riding atoms with isotropic
displacement parameters.
4 H]4+
Cu, [(Bpa-p-CH2C6H4CO-Ahx-PKKKRKF-NH2)Cu(NO3)2].
.
8
(38) ShelXTL; Siemens Analytical X-ray Instruments, Inc.: 1994.
This complex was prepared in situ: To the bioconjugate 8 (13.5
(39) Sheldrick, G. M. ShelXL97; University of Go¨ttingen: Go¨ttingen, 1997.
5414 Inorganic Chemistry, Vol. 44, No. 15, 2005

Bioconjugates of Cu(II) and Zn(II) Complexes
The bpa ligand in 1Zn was found to be disordered by inversion
of the ligating nitrogen atom N(8) and its attached C atoms C(7)
and C(9). A split atom model with occupation factors of 0.68 and
0.32 was used to account for the disorder. An isotropic displacement
parameter was refined for N(8) since the split atom was not
positively defined when it was refined anisotropically.
An ethanol solvate molecule in 4Cu was found to be disordered
on two positions. The corresponding atoms were isotropically
refined and the C-C and C-O distances were restrained to be equal
within certain errors using the SADI instruction of ShelXL97.
Crystallographic data of the compounds are listed in Table 3.
carbonyl. HOBt: 1-hydroxybenzotriazole. HOSu: N-hy-
droxysuccinimide. Nls: nuclear localization signal. Pbf:
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.
TBTU: O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
tetrafluoroborate. TIS: triisopropylsilane. TFA: trifluoro-
acetic acid.
Acknowledgment. Financial support from the Volks-
wagen Foundation and the Fonds der Chemischen Industrie
is gratefully acknowledged. The authors are grateful to H.
Rudy (MS), A. Seith (MS), U. Hertle (NMR), and H. Schucht
(X-ray) for technical assistance.
CCDC reference numbers are 249568-249571 (1Zn, 2Zn, 4Cu
Cu).
,
7
Supporting Information Available: Spectroscopic character-
ization of the bioconjugate 8. X-ray crystallographic file in CIF
format for the structures of 1Zn, 2Zn, 4Cu, and 7Cu. This material is
available free of charge via the Internet at http://pubs.acs.org.
Abbreviations. Aaa: any amino acid. Ahx: ω-aminohexa-
noic acid. Bpa: N,N-bis(2-picolyl)amine. Boc: tert-butoxy-
carbonyl. DCC: N,N′-dicyclohexylcarbodiimide. DIPEA:
N,N-diisopropylethylamine. Fmoc: 9-fluorenylmethoxy-
IC048343B
Inorganic Chemistry, Vol. 44, No. 15, 2005 5415