Zinc(II) complexes of tripodal peptides mimicking the zinc(II)-coordination structure of carbonic anhydrase

Article abstract of DOI:10.1016/S0968-0896(98)00180-1

Two new tripodal peptide ligands with histidine side chains have been synthesized and were shown to form stable zinc(II) complexes. Their NMR and mass spectra indicate a structure that is analogous to the active center of carbonic anhydrase. Both the liga

Full text of DOI:10.1016/S0968-0896(98)00180-1

Bioorganic & Medicinal Chemistry 7 (1999) 699±707
Zinc(II) Complexes of Tripodal Peptides Mimicking the
Zinc(II)-Coordination Structure of Carbonic Anhydrase
Ulrike Herr,a Werner Spahl,a Gunter Trojandt,a Wolfgang Steglich,a,*
Florian Thalerb and Rudi van Eldikb
aInstitut fur Organische Chemie, Universitat Munchen, Karlstraûe 23, D-80333 Munchen, Germany
È
È
È
È
bInstitut fur Anorganische Chemie, Universitat Erlangen-Nurnberg, Egerlandstraûe 1, D-91058 Erlangen, Germany
È
È
È
Received 14 April 1998; accepted 13 July 1998
AbstractÐTwo new tripodal peptide ligands with histidine side chains have been synthesized and were shown to form stable zinc(II)
complexes. Their NMR and mass spectra indicate a structure that is analogous to the active center of carbonic anhydrase. Both the
ligands and the zinc complexes were titrated potentiometrically in order to obtain the pKa values for the coordinated water of the zinc
complexes; due to the low solubility of the complexes only estimates could be obtained. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
To mimic the zinc(II)-coordination structure or the
function of the zinc(II) ion at the active site, several
mononuclear model zinc(II) complexes have been
designed and investigated (Fig. 2).19±37
The enzyme carbonic anhydrase (CAII) catalyzes the
reversible hydration of carbon dioxide with an impress-
ively high turnover number (106 s 1, eq (1)).1,2
Up to now, the best functional models of CAII are the
zinc(II) complexes of 1,5,9-triazacyclododecane ([12]aneN3)
(1)24±27 and 1,4,7,10-tetraazacyclododecane([12]aneN4)
‡
CO2 ‡ H2O „ HCO3 ‡ H
ꢀ1†
This crucial and rather simple reaction has fascinated
numerous research groups. Extensive studies have been
carried out to elucidate the chemical and structural
aspects of its mechanism.2±8 The results of these inves-
tigations help to shed light upon the evolution of CAII
into biochemically important noncatalytical functions,
such as signal transduction.9±13
(2)28±30 in the forms ZnL(H2O)2+ and ZnL(OH )2+
.
Woolley's enzyme model 331 was also successful in
reproducing catalysis of carbon dioxide hydrolysis. The
pKa values of complexes 1a±3a were reported to
approximate that of CAII very closely. The Zn(II)±OH
complex 432±34 was the ®rst structural model of the
active site of CAII to be isolated. Recently, Parkin et
al.37 synthesized and characterized the mononuclear
zinc(II)-complex 5, in which the zinc(II) ion is coordi-
nated by three imidazole moieties.
The structures of CAII in the zinc±water, zinc±hydroxide,
and zinc±bicarbonate forms that are involved in the
reaction, are well characterized.2,5,14,15 The active site of
CAII consists of a zinc(II) ion pseudotetrahedrally
coordinated by three histidine imidazoles (His-96, -94,
and -119) and either a water molecule or an OH ion in
the fourth position as depicted in Figure 1.14
However, there have been relatively few attempts to
design and prepare zinc(II)-binding peptides.38 Here, we
report the synthesis of two peptide ligands with three
histidine side chains featuring an environment analo-
gous to the active center of CAII.
The zinc(II)-bound water molecule is deprotonated to
form a zinc(II)-bound hydroxide species, which is
widely accepted to be the catalytically active com-
plex.13,16,17 Thus, the ability of CAII to hydrate CO2 is
characterized by a pKa value (ꢀ7) of this deprotonation.18
Results and Discussion
Synthesis of the tripodal histidine ligands
C3-Symmetrical molecules are of particular interest as
model compounds for natural ionophores and as
ligands for metal complexation.39,40 The optically pure
C3-symmetrical triacid (SSS)-N(BzGly*ValOH)3 (6)
Key words: Peptides; mimetics; histidine ligand; carbonic anhydrase;
metal complex.
*Corresponding author. Tel.: +49-89-5902-528; fax: +49-89-5902-604;
e-mail: wos@org.chemie.uni-muenchen.de
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00180-1

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U. Herr et al. / Bioorg. Med. Chem. 7 (1999) 699±707
Synthesis of the zinc(II) complexes
The reaction of the histidine ligands 8 and 11 with
.
equimolar amounts of Zn(ClO4)2 6H2O in ethanol a€orded
the zinc(II) complexes 12 and 13, respectively, as white
precipitates that could be separated by centrifugation.
Structural assignment for the zinc(II) complexes was
achieved by FAB/ESI HRMS and NMR spectra.
Figure 1. Active center of carbonic anhydrase.
The FAB mass spectrum of the zinc(II) complex of
N(BzGly*ValHisOMe)3 (12) exhibited peaks at m/z
1400 [M H+K]+, 1362 [M H]+ and 1299 [L]+. The
peak at m/z 1362 has been con®rmed by FAB HRMS.
In the ESI mass spectrum peaks at m/z 1400
[M H+K]+, 1299 [L]+ and 650 (100) [L]2+ were
proved to be an ideally preorganized template for build-
ing up longer tripodal peptides.41,42 We therefore chose
6 as starting material for the synthesis of zinc(II)-binding
pseudo-peptides. To introduce the histidine moiety we
used a p-Bom protected histidine ester since its deriva-
tives have been successfully applied in suppressing race-
mization during coupling of histidine.43 The side chain
protected histidine ester 7 was obtained by coupling the
1
found. The H NMR spectra of the tripodal histidine
ligand 8 and its resulting zinc(II) complex 12 (Fig. 3)
show close similarities except for the imidazole 2-H and
4-H protons. For the peptide ligand 8, a broad signal at
d=6.90 for the His 4-H protons and a sharp singlet at
d=7.53 for the His 2-H protons were obtained. These
.
template (SSS)-6 with H-His(p-Bom)OMe 2HCl, which
could be easily obtained by deprotection of commer-
.
cially available Boc-His(p-Bom)OMe HCl with HCl in
1
ethyl acetate.44 Subsequent cleavage of the Bom groups
by hydrogenolysis in 80% acetic acid45,46 yielded the
chiral tripodal histidine ligand 8 (Scheme 1).
data are comparable with H NMR data for histidine
derivatives reported in the literature.48,49 In the 1H
NMR spectrum of the zinc(II) complex 12, the reso-
nances of these protons are signi®cantly shifted down-
®eld (d=7.0 and d=8.08, respectively) and broadened.
Thus, these data support coordination of the zinc(II)
ion by the three histidine ligands and a close structural
analogy to the active center of carbonic anhydrase.
In order to increase the water solubility of the histidine
ligand, we elongated the three peptide chains by a serine
residue (Scheme 2). Saponi®cation47 of the pseudo-
nonapeptide 7 with lithium hydroxide yielded the pseudo-
nonapeptide 9. The synthesis of the p-Bom protected
ligand 10 could be achieved by coupling of 9 with l-
serine methyl ester. In contrast to the synthesis of 7, the
reaction had to be performed in DMF. Furthermore,
the workup of 10 was only possible by using chloroform
for extraction, which indicates a higher water solubility
of the serine derivative. In order to cleave the Bom
groups by hydrogenolysis to generate the more polar
histidine ligand 11, pressure (4 bar) had to be applied.
The ESI mass spectrum of the zinc(II) complex of
N(BzGly*ValHisSerOMe)3 (13) showed peaks at m/z
1625 [M+H]+, 1623 [M H]+, 1583 [L+Na]+, 1561
[L+H]+, and 812 [M]2+. The base peak at m/z 812
strongly supports the complex formation. The molecular
composition C72H93N19O21Zn at m/z 1625 has been
1
con®rmed by ESI HRMS. In the H NMR spectrum of
the zinc(II) complex 13, we also observed a remarkable
Figure 2. Mononuclear model complexes for carbonic anhydrase.

U. Herr et al. / Bioorg. Med. Chem. 7 (1999) 699±707
701
Scheme 1. Synthesis of the tripodal histidine ligand 8.
Scheme 2. Preparation of the tripodal histidine ligand 11.
Scheme 3. Preparation of the zinc(II) complexes 12 and 13.

702
U. Herr et al. / Bioorg. Med. Chem. 7 (1999) 699±707
down®eld shift of the imidazole 2-H and 4-H protons
(Fig. 4). For the peptide ligand 11, the resonances of the
His 4-H and the His 2-H protons were found at d=6.88
and 7.55, respectively. The corresponding signals of the
zinc(II) complex 13 were signi®cantly broadened and
appeared at d=6.94 and d=8.13, respectively. Interest-
ingly, the resonance of the NH-imidazole protons of 13
was shifted signi®cantly to lower ®eld (Ád=0.83).
to the insolubility of the ligand in pure water. The
titration curve of 8 is depicted in Figure 5(a).
The titration curve shows an in¯ection at 3 equiv of
base. Titration data were analyzed for equilibria 1±3
(eqs (2±4)). The calculated protonation constants (log
Kn) are summarized in Table 1. The protonation con-
stants are de®ned as follows:
‡
‡
‡
‡
H ‡ L „ HL K1 ˆ ‰HL Š=‰H Š‰LŠ
ꢀ2†
Protonation and complex-formation constants
H ‡ HL „ H2L2‡K2 ˆ ‰H2L2‡Š=‰H Š‰HL Š ꢀ3†
‡
‡
‡
‡
The protonation constants (Kn) of the ligand
N(BzGly*ValHisOMe)3 (8) were determined by potentio-
H ‡ H2L2‡ „ H3L3‡K3 ˆ ‰H3L3‡Š=‰H Š‰H2L2‡Š ꢀ4†
For determination of the complex stability constant and
‡
‡
metric pH-titration of 8 3H+ using 0.10 M NaOH at an
.
ionic strength of I=0.10 (NaClO4) at 25.0 ꢁC. The
titrations were performed in 33% aqueous ethanol due
+
.
the pKa of the coordinated water molecule, 8 3H was
Figure 3. Aromatic region of the 300 MHz 1H NMR spectra (DMSO-d6) of histidine ligand 8 and its zinc(II) complex 12.
Figure 4. Aromatic region of the 600 MHz 1H NMR spectra (DMSO-d6) of histidine ligand 11 and its zinc(II) complex 13.

U. Herr et al. / Bioorg. Med. Chem. 7 (1999) 699±707
703
Figure 6. pH-Titration curꢁves for N(BzGly*ValHisSerOMe)3 (11) at
(b) solution a+1.00 mM Zn2+SO4. eq(OH) is the moles of base
(0.10 M NaOH) per mole of ligand.
Figure 5. pH-Titration curves for N(BzGly*ValHisOMe)3 (8) at
ꢁ
(b) solution a+1.00 mM Zn2+SO4. eq(OH ) is the moles of base
(0.10 M NaOH) per mole of ligand.
+ 3+
.
I=0.10 (NaClO4) and 25.0 C in 52% EtOH: (a) 1.00 mM [L 3H
]
;
+ 3+
.
I=0.10 (NaClO4) and 25.0 C in 33% EtOH: (a) 1.00 mM [L 3H
]
;
Table 1. Comparison of the protonation constants of histidine ligands
8 (33% EtOH) and 11 (52% EtOH) and zinc(II) complexation
constants of 11 (25.0 ꢁC, I=0.10 M, NaClO4)
bation in the pKa of amino acids can also be found in
other enzyme systems.53
Titration data were analyzed for equilibria 1±4 (eqs (2)±
(4), and (7)). The calculated protonation constants (log
K1 3) and the deprotonation constant log KLÃ are sum-
marized in Table 1. The mixed protonation constants
K1 are de®ned in the eqs (2)±(4). In addition, the
3
deprotonation of the serine hydroxy group has to be
considered (eq (7)).
Ligand 8
Ligand 11
log K1
log K2
log K3
log KLÃ
log KZnL
log Ka
7.32‹0.03
6.78‹0.03
5.04‹0.02
6.73‹0.01
6.13‹0.01
5.80‹0.01
7.58‹0.01
5.59‹0.05
6.19‹0.05
‡
‡
L „ Là ‡ H KL ˆ ‰LʉH Š=‰LŠ
ꢀ7†
Ã
when L*=deprotonated ligand.
titrated in the presence of an equimolar amount of zinc(II)
using 0.10 N NaOH. The equilibria are de®ned as fol-
lows (eqs (5) and (6)):
For determination of the complex-formation constant
+
.
and the pKa of the coordinated water molecule, 11 3H
was titrated in the presence of an equimolar amount of
zinc(II) using 0.10 N NaOH. Even though the serine
groups have increased the polarity of 11 compared to
the histidine ligand 8 the complex started to precipitate
at pH 6, (i.e., after 4 equiv of base had been consumed).
Three equivalents of protons are released from the his-
tidine units when the complex is formed and the fourth
equivalent of base is neutralized either by the coordi-
nated water of the zinc complex or is due to the OH
group of serine. In the latter case this would mean that
the pKa value of 7.58‹0.01 for the free ligand
decreases towards a pKa value of 6.19‹0.05 for the
complex. In contrast to 8, the complex was dissolved
again at a pH of about 11.5. The equilibria are de®ned
as follows (eqs (8) and (9)):
2‡
2‡
2‡
ZnꢀH2O† ‡ L „ ZnLꢀH2O†
ꢀ5†
ꢀ6†
2‡
KZnL ˆ ‰ZnLꢀH2O† Š=‰ZnꢀH2O† Š‰LŠ
2‡
‡
‡
ZnLꢀH2O† „ ZnLꢀOH† ‡ H
‡
2‡
‡
Ka ˆ ‰ZnLꢀOH† Š‰H Š=‰ZnLꢀH2O†
Š
Since during the titration the complex precipitated at a
pH of about 5, the constants could not be calculated.
The titration curve is shown in Figure 5(b).
The titration curves of 11 are depicted in Figure 6.
The protonation constants (Kn) of the histidine ligand
11 were determined by potentiometric pH-titration of
11 3H using 0.10 M NaOH at an ionic strength of
2‡
2‡
2‡
ZnꢀH2O† ‡ L „ ZnLꢀH2O†
+
ꢀ8†
ꢀ9†
.
2‡
KZnL ˆ ‰ZnLꢀH2O† Š=‰ZnꢀH2O† Š‰LŠ
I=0.10 (NaClO4) at 25.0 ꢁC (Fig. 6(a)). In contrast to
the titration curve of 8, an in¯ection occurred at 4 equiv
of base. Consequently, an additional consumption of 1
equiv of base is taking place. We assume therefore that
the fourth proton could possibly be released from one
of the serine hydroxy groups. The pKa value of the
hydroxy group in serine is about 12±13, but in the case
of the protease chymotrypsin a signi®cant perturbation
of the pKa value of serine 195 can be observed. The pKa
value is shifted dramatically to a value of about 7 due to
the coordination to histidine 57.50±52 The large pertur-
2‡
‡
‡
ZnLꢀH2O† „ ZnLꢀOH† ‡ H
‡
2‡
‡
Ka ˆ ‰ZnLꢀOH† Š‰H Š=‰ZnLꢀH2O†
Š
or
2‡
‡
ZnLꢀH2O† „ ZnLÃꢀH2O† ‡ H
‡
‡
2‡
Ka ˆ ‰ZnLÃꢀH2O† Š‰H Š=‰ZnLꢀH2O†
Š
‡
when L*=deprotonated ligand

704
U. Herr et al. / Bioorg. Med. Chem. 7 (1999) 699±707
The calculated complex formation constant (log
KꢂZnL†) and the deprotonation constant (log Ka) are
summarized in Table 1.
3.54 mmol) and EDCI (566 mg, 2.95 mmol) were added.
Stirring was continued for 1 h at 0 ꢁC, then the reaction
mixture was allowed to warm to room temperature
(23 h). Ethyl acetate (75 mL) was added, and the organic
layer was washed with saturated NaHCO3 (2Â70 mL)
and 10% citric acid (5Â70 mL). The combined citric
acid extracts were neutralized with satd NaHCO3 and
extracted with ethyl acetate (5Â70 mL). The ethyl acet-
ate extracts were combined and washed with brine.
Drying (MgSO4) and evaporation gave a colorless solid,
which was recrystallized from ethyl acetate/petroleum
ether to a€ord 694±823 mg 7 (71±84%); mp 104±106 ꢁC;
Rf 0.70 (H2O/methanol/CHCl3, 2/20/80); [a]2d5 +10.5
(c 1.00, MeOH); 1H NMR (300 MHz, CDCl3): d 0.89 (d,
J=7 Hz, 9H, CH(CH3)2), 0.93 (d, J=7 Hz, 9H,
CH(CH3)2), 2.12 (m, 3H, CH(CH3)2), 3.24 (m, 6H,
CH2-His), 3.69 (s, 9H, OCH3), 4.26 (dd, J=7 Hz, 3H,
CH-Val), 4.41 (d, J=12 Hz, 3H, OCH2), 4.48 (d,
J=12 Hz, 3H, OCH2), 4.98 (m, 3H, CH-His), 5.34 (d,
J=11 Hz, 3H, NCH2), 5.49 (d, J=11 Hz, 3 H, NCH2),
5.82 (d, J=9 Hz, 3H, NHCHN), 6.81 (s, 3 H, His4H),
7.11 (dd, J=8 Hz, 6H, mPh), 7.28±7.35, 7.40 (m, 18H,
d, J=7 Hz, 6H, Ph), 7.49 (s, 3H, His2H), 8.08 (d,
J=9 Hz, 3H, NH-Val), 8.33 (d, J=7 Hz, 3H, NH-His),
8.62 (d, J=9 Hz, 3H, NHCHN); 13C NMR (75.44 MHz,
CDCl3): d 18.59, 19.31, 26.36, 30.73, 51.62, 52.44, 60.30,
63.49, 70.12, 73.49, 126.65, 127.42, 128.13, 128.27,
128.67, 129.94, 131.72, 132.89, 136.20, 138.69, 167.81,
Conclusion
The structural features of the zinc(II) complexes 12 and
1
13 that can be concluded from the H NMR and mass
spectra indicate that both peptide ligands 8 and 11 fea-
ture an environment comparable to that of the active
center of CAII. The potentiometric titrations of the
ligands 8 and 11 could be carried out only in a water±
ethanol mixture since both ligands have a low solubility
in pure water. An unexpected observation was made in
the case of the pseudo-peptide 11, where there is possi-
ble evidence for the perturbation of the pKa of the
hydroxy group of one serine. While the zinc(II) complex
of 8 has a low solubility at the given experimental con-
ditions and no complex stability constant could be
obtained, the zinc(II) complex has a higher solubility
and a ®rst deprotonation constant of the complex could
be obtained. The pKa value of 6.19‹0.05 is somewhat
lower than for CAII (pKa of about 7), but it is not
completely certain that the proton is released from the
coordinated water on the zinc or from the ligand itself
(i.e., the hydroxy groups of serine).
~
169.00, 171.30, 171.36; IR (KBr): n 3300, 3020, 2960,
2860, 1740, 1660, 1600, 1580, 1510, 1480, 1340, 1280,
1210, 1090, 1070, 930, 750, 700, 660; FAB MS (NBA);
m/z (%): 1600 (96) [M]+; C87H102N16O18 (1659.87),
calcd C 62.95, H 6.19, N 13.50; found, C 62.81, H 6.08,
N 13.58.
Experimental
Instrumental analyses
THF was distilled from potassium under an atmosphere
of dry argon. All common reagents and solvents were
purchased from commercial suppliers and used without
further puri®cation unless otherwise stated. TLC was
performed on aluminum-backed plates coated with
silica gel 60 with F254 indicator. Optical rotations were
determined with a Perkin±Elmer 241 polarimeter using a
Na lamp; data are reported as follows: [a]2d5 (con-
centration in g per 100 mL, solvent). Melting points
were determined with a Reichert thermovar apparatus
and a Buchi 350 and are uncorrected. IR spectra were
recorded on a Perkin±Elmer 1420 spectrometer and a
N,N0,N00 -[(SSS)-Nitrilotris[2-benzoylamino)-1-oxo-2,1-
ethandiyl]]tris-[L-valyl-L-histidine] trimethyl ester (8).
The protected histidine ligand 7 (500 mg, 0.30 mmol)
was dissolved in 80% aqueous acetic acid (40 mL) and
treated with palladium on carbon (10%, 500 mg). The
suspension was hydrogenated for 24 h (TLC control).
The palladium on carbon was removed by ®ltration and
the solvent was evaporated. The residue was neutralized
with saturated NaHCO3 and extracted with ethyl acet-
ate (5Â70 mL). The combined ethyl acetate extracts
were washed with brine, dried (MgSO4) and evaporated
to yield 8 as a colorless solid (365 mg, 93 %); mp 145±
Bruker IFS 45 FT-IR spectrometer. H and 13C NMR
1
spectra were measured on a Bruker ARX-300 or a Bru-
ker ARM-600 instrument. All chemical shifts were
recorded in ppm down®eld from TMS on the d scale.
Mass spectra were recorded on a Finnigan MAT90 and
a Finnigan MAT95Q (high resolution mass spectra).
Elemental analyses were performed by the University of
Munich Microanalytical Laboratory.
147 ꢁC; [a]2d5
(300 MHz, DMSO-d6):
3.2 (c 1.00, methanol); 1H NMR
0.92 (d, J=6 Hz, 18H,
d
CH(CH3)2), 2.11±2.18 (m, 3H, CH(CH3)2), 2.94 (s, br,
6H, CH2-His), 3.52 (s, 9H, OCH3), 4.24 (dd, J=6 Hz,
3H, CH-Val), 4.51 (m, 3H, CH-His), 5.65 (d, J=9 Hz,
3H, NHCHN), 6.90 (s, br, 3H, His4H), 7.22 (dd,
J=8 Hz, 6H, mPh), 7.37±7.44 (m, 9H, Ph), 7.54 (s, br,
His2H), 8.50 (m, br, 9H, NHCHN, NH-His, NH-Val),
11.81 (s, br, NH-Im); 13C NMR (75.44 MHz, DMSO-
d6): d 17.97, 19.06, 28.78, 29.98, 51.53, 52.62, 58.83,
59.65, 63.23, 126.81, 128.05, 131.75, 132.57, 166.41,
Synthesis of the histidine peptide ligands
N,N0,N00 -[(SSS)-Nitrilotris[2-benzoylamino)-1-oxo-2,1-
ethandiyl]]tris-[L-valyl-N(ꢀ)-benzyloxymethyl-L-histidine]
trimethylester (7). Compound (SSS)-6 (499 mg, 0.59 mmol)
and N(p)-benzyloxymethyl-l-histidine methyl ester
dihydrochloride (851 mg, 2.35 mmol)42 were suspended
in THF (70 mL) and treated with NEM (0.74 mL,
5.90 mmol). After cooling to 0 ꢁC, HOBt (478 mg,
~
167.75, 170.76, 171.44; IR (KBr): n 3260, 3060, 2950,
1740, 1660, 1600, 1580, 1520, 1490, 1440, 1340, 1320,
1210, 1170, 1080, 980, 930, 800, 710, 690, 620; FAB MS
(NBA); m/z (%): 1322 (9) [M+Na]+, 1300 (53)
[M+H]+, 1299 (71) [M]+, 105 (100) [Ph-CꢃO]+; FAB

U. Herr et al. / Bioorg. Med. Chem. 7 (1999) 699±707
705
HRMS (NBA) calcd for C63H79N16O15 [M+H]+:
1299.5911, found (m/z) 1299.5987.
(m, 3H, CH2OH), 5.43 (s, 6H, N-CH2), 5.70 (d,
J=9 Hz, 3H, NHCHN), 6.83 (s, 3H, His4H), 7.21 (t,
J=8 Hz, 6H, mPh), 7.25±7.33 (m, 18H, Ph), 7.40±7.43
(m, 6H, Ph), 7.71 (s, 3H, His2H), 8.38 (d, J=7 Hz, 3H,
NH), 8.41 (d, J=7 Hz, 3H, NH), 8.45 (d, J=8 Hz, 3H,
NH), 8.65 (d, J=9 Hz, 3H, NH); 13C NMR:
(150.91 MHz, DMSO-d6): d 18.18, 19.21, 25.95, 30.40,
51.78, 51.86, 54.65, 58.90, 61.12, 63.81, 69.20, 73.40,
127.00, 127.30, 127.62, 127.86, 128.07, 128.25, 131.75,
132.80, 137.20, 138.18, 166.73, 167.91, 170.49, 170.58,
N,N0,N00 -[(SSS)-Nitrilotris[2-benzoylamino)-1-oxo-2,1-
ethandiyl-]]tris-[L-valyl-N(ꢀ)-benzyloxymethyl-L-histidine]
trimethyl ester (9). To a cooled solution of the triester 7
(400 mg, 0.241 mmol) in THF (9 mL) and water (3 mL),
lithium hydroxide (24 mg, 0.963 mmol, 4 equiv) was
added. After 2.5 h at 0 ꢁC the solution was adjusted to
pH 4.5 by addition of 1 N HCl. The volatile components
(THF) were evaporated and the water removed by lyo-
philization. The resulting yellow solid was puri®ed on a
column of Sephadex LH 20, swollen and eluted with
methanol to a€ord a colorless solid (361 mg, 93%); mp
168±170 ꢁC.; [a]2d5+ 29.92ꢁ (c 1.00, methanol); 1H
NMR (300 MHz, DMSO-d6): d 0.87 (d, J=7 Hz, 18H,
CH(CH3)2), 2.14 (m, 3H, CH(CH3)2), 2.94±3.19 (m, 6H,
CH2-His), 4.26 (dd, J=7 Hz, 3H, CH-Val), 4.45 (s, 6H,
OCH2), 5.44 (dd, J=17, 11 Hz, 9H, CH-His, N-CH2),
5.69 (d, J=9 Hz, 3H, NHCHN), 6.75 (s, 3H, His4H),
7.20 (t, J=8 Hz, 6H, mPh), 7.24±7.43 (m, 24H, Ph), 7.70
(s, 3H, His2H), 8.50 (m, br, 9H, NHCHN, NH-His,
NH-Val); 13C NMR (75.44 MHz, DMSO-d6): d 17.95,
19.22, 25.83, 28.08, 29.99, 53.06, 59.33, 63.62, 69.15,
73.36, 126.90, 127.24, 127.48, 128.12, 128.51, 131.73,
132.54, 132.66, 137.25, 137.79, 166.57, 166.19, 170.34,
~
170.73; IR (KBr) n 3412, 3062, 2963, 1744, 1659, 1578,
1523, 1488, 1438, 1372, 1293, 1222, 1158, 1076, 1029,
931, 748, 698, 660, 607; ESI MS; m/z (%): 1943 (5)
[M+Na]+, 1921 (8) [M+H]+, 961 (100) [M+2H]2+
;
ESI HRMS: calcd for C96H118N19O24 [M+H]+
1920.8597, found (m/z) 1920.8623.
N,N0,N00 -[(SSS)-Nitrilotris[2-benzoylamino)-1-oxo-2,1-
ethandiyl-]]tris-[L-valyl-L-histidyl-L-serine] trimethyl ester
(11). The protected histidine ligand 10 (372 mg,
0.194 mmol) was dissolved in 80% aqueous acetic acid
(50 mL) and treated with palladium on carbon (10%,
750 mg). The suspension was hydrogenated under pres-
sure (4 bar) for 24 h (TLC control). The palladium on
carbon was removed by ®ltration and the solvent was
evaporated. The crude product was dissolved in water
(5 mL) and methanol (5 mL), and the pH of the solution
(initial pH 5) was adjusted to pH 9 by addition of aqu-
eous ammonia. The volatile components (THF) were
evaporated. Water and the resulting ammonium acetate
were removed by lyophilization. The yellowish residue
was puri®ed on a column of Sephadex LH 20, swollen
and eluted with acetone/methanol (4/1) to a€ord a col-
orless solid (245 mg, 81%); mp 161±163 ꢁC; [a]d25 5.9ꢁ (c
~
172.77; IR (KBr): n 3429, 3063, 2965, 2932, 1969, 1654,
1579, 1522, 1488, 1392, 1221, 1100, 1028, 930, 747, 698,
628; FAB MS (NBA); m/z (%): 1641 (0.81) [M+Na]+;
ESI MS; m/z (%): 1618 (5.12) [M+H]+, 810 (100)
[M+2 H]2+; ESI HRMS: calcd for C84H97N16O18
[M+H]+ 1617.7167, found (m/z) 1617.7138.
N,N0,N00 -[(SSS)-Nitrilotris[2-benzoylamino)-1-oxo-2,1-
ethandiyl-]]tris-[L-valyl-N(ꢀ)-benzyloxymethyl-L-histidyl-
L-serine] trimethyl ester (10). To a stirred solution of 9
(700 mg, 0.44 mmol) in DMF (40 mL), H-l-Ser-
1
1.00, MeOH); H NMR (600 MHz, DMSO-d6): d 0.89
(m, 18 H, CH(CH3)2), 2.13 (m, 3 H, CH(CH3)2), 2.88
(m, 3H, CH2-His), 2.97 (m, 3H, CH2-His), 3.59 (s, 9H,
OCH3), 3.57±3.60 (m, 3H, CH2OH), 3.69±3.71 (m, 3H,
CH2OH), 4.22 (m, 3 H, CH-Val), 4.28 (m, 3H, CH-Ser),
4.57 (m, 3H, CH-His), 5.31 (s, br, 3 H, CH2OH), 5.69
(d, J=9 Hz, 3H, NHCHN), 6.88 (s, 3H, His4H), 7.23 (t,
J=8 Hz, 6H, mPh), 7.37±7.44 (m, 9H, o,p-Ph), 7.55 (s,
3H, His2H), 8.19 (s, br, 3H, NH), 8.34 (s, br, 3H, NH),
8.49 (s, br, 3H, NH), 8.58 (s, br, 3H, NH), 11.82 (s, br,
3H, NH-Im); 13C NMR: (150.91 MHz, DMSO-66): d
18.10, 19.10, 30.05, 51.62, 52.62, 54.60, 58.99, 60.85,
63.58, 126.87, 128.04, 131.74, 132.65, 134.58, 166.66,
.
OMe HCl (275 mg, 1.77 mmol) and NEM (0.28 mL,
2.21 mmol) were added consecutively. After cooling to
0 ꢁC, HOBt (358 mg, 2.65 mmol) and EDCI (423 mg
2.21 mmol) were added. Stirring was continued for 1h at
0 ꢁC, and the reaction mixture was then allowed to
warm to room temperature (23 h). The solvent was
evaporated and the resulting yellow oil dissolved in
CHCl3 (40 mL). The solution was washed with
saturated NaHCO3 (2Â40 mL) and 10% citric acid
(6Â40 mL). The combined citric acid extracts were
neutralized with saturated NaHCO3 and extracted
with CHCl3 (6Â40 mL). The CHCl3 extracts were
combined and washed with brine. Drying (MgSO4)
and evaporation gave a colorless solid which was
puri®ed on a column of Sephadex LH 20, swollen
and eluted with acetone/methanol (4/1) to a€ord a
colorless solid (547 mg, 65%); mp 118±120 ꢁC; Rf
~
167.86, 170.22, 170.74; IR (KBr) n 3420, 2964, 1741,
1657, 1578, 1522, 1487, 1438, 1226, 1182, 1143, 1081,
935, 714, 694, 619; ESI MS; m/z (%):1583 (47)
[M+Na]+, 1561 (100) [M+H]+, 781 (22) [M+2H]2+
;
ESI HRMS: calcd for C72H94N19O21[M+H]+ 1560.6871,
found (m/z) 1560.6827.
0.70 (H2O/methanol/CHCl3, 2/20/80); [a]d25
2.94ꢁ
Synthesis of the zinc(II)-complexes
(c 1.00, MeOH); 1H NMR (600 MHz, DMSO-d6): d
0.89 (d, J=6 Hz, 9H, CH(CH3)2), 0.90 (d, J=6 Hz,
9H, CH(CH3)2), 2.11 (m, 3H, CH(CH3)2), 2.96 (dd,
J=15 Hz, 8 Hz, 3H, CH2-His), 3.11 (dd, J=15 Hz,
5 Hz, 3H, CH2-His), 3.58 (s, 9H, OCH3), 3.58±3.61
(m, 3H, CH2OH), 3.68±3.71 (m, 3H, CH2OH), 4.26
(dd, J=6 Hz, 3H, CH-Val), 4.34 (m, 3H, CH-Ser),
4.42 (s, 6H, OCH2), 4.72 (m, 3H, CH-His), 5.11
.
General procedure. A solution of Zn(ClO4)2 6H2O
(29 mg, 0.077 mmol) in ethanol (2 mL) was added care-
fully to a stirred solution of the histidine ligand
(0.077 mmol) in ethanol (5 mL). Upon the addition of
.
one drop of the solution of Zn(ClO4)2 6H2O in ethanol,
a colorless precipitate was formed that was separated by
centrifugation (3000 U/min, 10 min). The solution was

706
U. Herr et al. / Bioorg. Med. Chem. 7 (1999) 699±707
removed, the precipitate washed with ethanol (5 mL)
and centrifugated (3000 U/min, 10 min, 3Â). The pro-
duct was dried by lyophilization.
the ligands and its zinc complexes were performed as
follows: a 33% EtOH aqueous solution (50 mL) of the
ligand 8 (1.00 mM) with 3 equiv of HClO4 (3.00 mM) in
the absence or presence of equivalent amounts of
ZnSO4 (1.00 mM) was titrated with carbonate-free
0.100 M NaOH aqueous solutions. Before the titration,
the ionic strength of the solution was adjusted to
I=0.10 (NaClO4). Titration data for ligand 11 were
obtained by the same procedure but in contrast to 8, a
52% EtOH aqueous solution (50 mL) of the ligand 11
(1.00 mM) was chosen. At least three independent titra-
tions were performed for the determination of the con-
stants. The ligand protonation constants (K1 3), the
deprotonation constant of the ligand 11 (KL*), the
zinc(II) complexation constant (KZnL) and the deproto-
nation constant of the zinc(II) complex (Ka) were cal-
culated with the PSEQUAD program.54
Zinc(II) complex of 8 (12)
The title compound was obtained according to the gen-
eral procedure starting from 8 (100 mg, 0.077 mmol) and
.
Zn(ClO4)2 6H2O (29 mg, 0.077 mmol). Colorless solid
ꢁ
1
(85 mg, 70%); mp decp > 220 C; H NMR (300 MHz,
DMSO-d6): d 0.92 (s, br, 18 H, CH(CH3)2), 2.14 (s, br,
3H, CH(CH3)2), 2.90 (s, br, 6 H, CH2-His), 3.57 (s, 9 H,
OCH3), 4.26 (s, br, 3 H, CH-Val), 4.50 (s, br, 3H, CH-
His), 5.71 (s, br, 3H, NHCHN), 7.00 (s, br, 3H, His4H),
7.24 (dd, J=8 Hz, 6H, mPh), 7.43 (m, 9H, Ph), 8.08 (s,
br, His2H), 8.50 (m, br, 9H, NHCHN, NH-His, NH-
Val); 13C NMR (75.44 MHz, DMSO-d6): d 17.56, 19.27,
30.53, 52.01, 63.82, 127.01, 128.18, 131.99, 132.57,
~
137.05, 167.04, 168.37, 170.83, 171.18; IR (KBr) n 3431,
2964, 1741, 1658, 1578, 1520, 1487, 1440, 1219, 1121,
1108, 693, 635; FAB MS (NBA); m/z (%): 1400 (0.15)
[M H+K]+, 1362 (0.54) [M H]+, 1299 (3.31) [L]+;
ESI MS; m/z (%): 1400 (9) [M H+K]+, 1299 (31)
[L]+, 650 (100) [L]2+; FAB HRMS: calcd for C63H77
N16O15Zn [M H]+ 1361.5046, found (m/z) 1361.5126.
Acknowledgements
Financial support for this work was provided by the
Deutsche Forschungsgemeinschaft. Florian Thaler
acknowledges the Fonds der Chemischen Industrie for
providing a research scholarship leading to a PhD
degree. The authors would like to thank Professor
Colin D. Hubbard for helpful discussions and for help-
ing to prepare the manuscript.
Zinc(II) complex of 11 (13)
According to the general procedure starting from 11
.
(121 mg, 0.077 mmol) and Zn(ClO4)2 6H2O (29 mg,
0.077 mmol). Colorless solid (89 mg, 58%); mp 218±
References
220 ꢁC; H NMR (600 MHz, DMSO-d6): d 0.88 (s, br,
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