Recognition of flexible peptides in water by transition metal complexes.

Article abstract of DOI:10.1021/ol005555d

This paper describes the design, synthesis, and evaluation of transition metal complexes capable of recognizing flexible histidine-containing peptides in aqueous medium (25 mM HEPES buffer, pH = 7.0, 25 degrees C). When the pattern of metal ions on a comp

Full text of DOI:10.1021/ol005555d

ORGANIC
LETTERS
2000
Vol. 2, No. 7
911-914
Recognition of Flexible Peptides in
Water by Transition Metal Complexes
Shuguang Sun, Md. Abul Fazal, Bidhan C. Roy, and Sanku Mallik*
Department of Chemistry, North Dakota State UniVersity, Fargo, North Dakota 58105
sanku_mallik@ndsu.nodak.edu
Received January 14, 2000
ABSTRACT
This paper describes the design, synthesis, and evaluation of transition metal complexes capable of recognizing flexible histidine-containing
peptides in aqueous medium (25 mM HEPES buffer, pH ) 7.0, 25 °C). When the pattern of metal ions on a complex matches with the pattern
of histidine moieties on the peptide, strong interaction (K ) 1.2 × 106 M-1) can be achieved. The complex was highly selective (>200:1) in
discriminating similar flexible peptides differing only by one glycine unit.
Recognition of peptides is very important for hormone action,
immune response, and other biochemical events.1 Peptide
therapeutics rely on strong binding of the peptide drugs to
their biological targets. Detection of disease marker peptides
has the potential of early diagnosis of the disease. Thus,
recognition of peptides has attracted considerable attention
in recent years. Several groups have reported strong and
selective binding to peptides2 and proteins3 by designed
artificial receptors in aqueous media. Combinatorial ap-
proaches to peptide recognition have been demonstrated.4
Synthetic receptors are reported to recognize and bind to
specific sites of proteins and disturb biologically important
protein-protein interactions.5 To our knowledge, these
artificial receptors selectively bind to proteins or peptide
molecules through hydrogen bonding, ion-pair, or hydro-
phobic interactions and often the binding constants are low
(<50 000).
We are interested in the construction of metal complexes
capable of binding strongly to histidine patterns of flexible
peptides in water. Herein, we report that if the distribution
of the metal ions on a complex is complementary to the
histidine pattern of a peptide, a strong and selective binding
can be achieved. Recognition based on metal-ligand interac-
tions has several advantages compared to the approaches
reported in the literature for peptide recognition. Hydrogen
bonding and ion-pair interactions are particularly weak in a
competitive solvent, e.g, water.6 On the other hand, the
strength and kinetics of metal-ligand interactions in water
can be tuned by the choice of metal ions, the ligands
positioning the metal ions, and the pH of the medium.7 The
(1) Hecht, S. M., Ed. Bioorganic Chemistry: Peptides and Proteins;
Oxford University Press: New York, 1998.
(2) (a) Ngola, S. M.; Kearney, P. C.; Mecozzi, S.; Russell, K.; Dougherty,
D. A. J. Am. Chem. Soc. 1999, 121, 1192-1201. (b) Hossain, A. Md.;
Schneider, H.-J. J. Am. Chem. Soc. 1998, 120, 11208-11209. (c) Breslo,
R.; Yang, Z.; Ching, R.; Trojandt, G.; Obodel, F. J. Am. Chem. Soc. 1998,
120, 3536-3537. (d) Hioki, H.; Still, W. C. J. Org. Chem. 1998, 63, 904-
905. (e) Peczuh, M. W.; Hamilton, A. D.; Sanohez-Quesada, J.; Mendoza,
J.; Haack, T.; Giralt, E. J. Am. Chem. Soc. 1997, 119, 9327-9328.
(3) (a) Souers, A. J.; Virgill, A. A.; Rosenquist, A.; Fenuik, W.; Ellman,
J. A. J. Am Chem. Soc. 1999, 121, 1817-1825. (b) Bernardi, A.; Cocchia,
A.; Brocca, P.; Sonnino, S.; Zuccotto, F. J. Am. Chem. Soc. 1999, 121,
2032-2036. (c) Hamuro, Y.; Calama, M. C.; Park, H. S.; Hamilton, A. D.;
Angew. Chem., Int. Ed. Engl. 1997, 36, 2680-2683.
(4) (a) Hioki, H.; Clark Still, W. J. Org. Chem. 1998, 63, 904-905. (b)
Breslow, R.; Yang, Z.; Ching, R.; Trojandt, G.; Odobel, F. J. Am. Chem.
Soc. 1998, 120, 3536-3537. (c) Torneiro, M.; Clark Still, W. Tetrahedron
1997, 53, 8739-8750. (d) Sasaki, S.; Takagi, M.; Tanaka, Y.; Maeda, M.
Tetrahedron Lett. 1996, 37, 85-88.
(5) (a) Bishop, A.; Kung, C.; Shah, K.; Witucki, L.; Shokat, K. M.; Liu,
Y. J. Am. Chem. Soc. 1999, 121, 627-631. (b) Park, H. S.; Liu, Q.;
Hamilton, A. D. J. Am. Chem. Soc. 1999, 121, 8-13. (c) Hirota, S.; Endo,
M.; Hayamizu, K.; Tsukazaki, T.; Takabe, T.; Kohzuma, T.; Yamaguchi,
O. J. Am. Chem. Soc. 1999, 121, 849-855. (d) Zutshi, R.; Brickner, M.;
Chmielewski, J. Curr. Opin. Chem. Biol. 1998, 2, 62-66.
(6) Frey, P. A.; Clelard, W. W. Bioorg. Chem. 1998, 26, 175-192.
10.1021/ol005555d CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/09/2000

metal ions, depending on the oxidation state, will have
defined spectroscopic properties. These properties can be
used to obtain structural information about the recognition
process.8
To demonstrate the pattern-matching concept for peptide
recognition, we have designed and synthesized water-soluble
peptides with three histidine moieties at particular distances
apart (12-18 Å, t_H, t_GH, t_AH, t_QH, and t_GGH,
Figure 1). This corresponds to the inter-histidine distances
deprotection of the benzyl group (H2, Pd black), the resultant
free acid was combined with 1,3,5-tris(aminomethyl)ben-
zene.11 Finally the Boc groups of histidines are cleaved by
trifluoroacetic acid followed by ion exchange (Dowex
macroporous resin, MeOH-H2O as the eluant) to yield the
free peptides.
Scheme 1. Synthesis of the Peptides
Figure 1. Structures of the histidine-containing peptides used for
recognition in water (pH ) 7.0).
(His 3, His 10, and His 15) on the surface of the protein
carbonic anhydrase (bovine erythrocyte). The peptides were
designed using the Spartan software (Version 5.0.3, Wave-
function Inc., Irvine, CA). The molecules were first energy
minimized employing the Merck molecular mechanics force
field. Then conformational searches (systematic) were per-
formed to identify the lowest energy conformation(s) of the
molecules. The mono histidine peptide m_H (Figure 1) was
used as the control for the recognition studies.
The transition metal complexes 1 and 2 (structures and
synthesis are shown in Scheme 2) were designed and
synthesized for selective and strong binding to these peptides.
The distances among the three Cu2+ ions were estimated by
molecular modeling (using the software Spartan, energy
minimization employing the Merck molecular mechanics
force field followed by systematic conformational search)
and are also indicated in Scheme 2. Complex 1 can position
three metal ions ∼12 Å apart. Complex 2 is flexible, and
From modeling, the distance among the imidazole groups
of the histidines was found to increase by ∼4 Å when the
peptide length increased by a glycine unit. The inter-histidine
distances changed from 12 Å for t_H to 16-18 Å for t_GH.
Because of flexibility, the estimated distance for t_GGH was
measured to vary from 18 to 23 Å. The peptide t_GGH was
designed to probe the role of flexibility and length in the
recognition process. Insertion of another glycine unit
(t_GGGH) decreased the water solubility, rendering it
unsuitable for our studies. The peptide t_AH has the
hydrophobic methyl group; t_QH has the hydrophilic amide
moiety in the side chain. Replacing the alanine of t_AH with
a phenylalanine (t_FH) made the peptide insoluble in water
(pH ) 7.0).
Scheme 2. Structures of the Transition Metal Complexes and
the Synthesis of Complex 2
The syntheses of ligands9 are depicted in Scheme 1. Benzyl
ester derivatives of the appropriate amino acid were coupled
with N,N-di-Boc histidine using the BOP reagent.10 After
(7) Lippard, S. J.; Berg, J. M. Principles of Bioinorganic Chemistry;
University Science Books: Mill Valley, CA, 1994.
(8) Kendrick, M. J.; May, M. T.; Plishka, M. J.; Robinson, K. D. Metal
in Biological Systems; Ellis Horwood: Chichester, England, 1992; pp 17-
48.
912
Org. Lett., Vol. 2, No. 7, 2000

the distances among the cupric ions were estimated to be
14-18 Å. Complex C with one cupric ion was used as the
control in these studies. The imminodiacetate-Cu2+ moiety
was selected for these recognition studies in aqueous medium
because of its wide use in affinity chromatography purifica-
tion of proteins.12 Elemental analyses indicated that C
complexed one cupric ion while 1 and 2 incorporated three
cupric ions. Determination of Cu2+ by UV/vis spectrometry
(employing EDTA) for these complexes also indicated the
proper number of cupric ions (Supporting Information).
Interactions of the peptides and the metal complexes were
followed by isothermal titration microcalorimetry (ITC).13
ITC is a rapid method of determining the binding constant
(K), enthalpy (∆H), and stoichiometry (n) of the interactions
simultaneously. It has been used to study small-molecule
interactions (including metal-ligand interactions),14 protein-
ligand interactions,15 and protein-protein and other interac-
tions.16
ITC experiments (Model ITC-4200 from Calorimetry
Sciences, Provo, UT) were conducted in aqueous solutions
(25 mM HEPES buffer, pH ) 7.0, [peptide] ) 0.80-5.0
mM, [Cu2+ complex] ) 0.1-0.5 mM, 25.0 °C). The raw
data were corrected for the heats of dilution of the appropriate
peptides and processed using the software provided by the
manufacturer (Bindworks 3.0). Typical raw and processed
data are shown in Figure 2 (titration of t_GH with complex
2).
The peptides have two potential sites for metal coordina-
tion: the R-amino and the imidazole moieties of the histidine
residues. At pH ) 7.0, the R-amino groups remain mostly
protonated (pKa ) 9.2) while the imidazole nitrogen atoms
are unprotonated (pKa ) 6.8). Experimental conditions were
optimized with variation of pH, concentration of the buffer,
and the identity of the buffer. It was found that the affinity
was highest at pH ) 7.0 (compared to pH ) 6.0 and 8.0).
Buffer identity (HEPES, MES, MOPS) and concentration
(25 mM, 50 mM, 100 mM) did not affect the binding process.
Stability constants and the thermodynamic parameters of
the interactions are shown in Table 1. The mono-histidine
peptide m_H was also found to have low affinity for all the
metal complexes (C, 1, and 2). Strong and selective binding
Figure 2. Raw and processed data for the titration of t_GH with
complex 2 ([2] ) 50 µM; [t_GH] ) 600 µM; 25 mM HEPES
buffer, pH ) 7.0, 25.0 °C; 40 × 5 µL injections).
was achieved by employing the flexible metal complex 2.
With the distance-matched peptide (t_GH), a strong affinity
(1.2 × 106 M-1) was obtained. This appears to be due to a
relatively low loss of entropy upon binding. A shorter peptide
(t_H) or a longer peptide (t_GGH) was found to decrease
Table 1. Binding Constants, Enthalpy, Entropy, and the
Stoichiometry of Binding for Complexes C, 1, and 2 with the
Histidine Peptides (25 mM HEPES buffer, pH ) 7.0, 25.0 °C;
40 × 5 µL Injections)
-∆H,
kcal
-∆G, -T∆S,
kcal kcal
mol-1 mol-1
(9) Characterization data for the peptides and the metal complexes are
available as Supporting Information.
(10) Castro, B.; Dormoy, J.-R.; Dourtoglou, B.; Evin, G.; Selve, C.;
Zieglar, J.-C. Synthesis 1976, 751-757.
(11) Michcell, M. S.; Waller, D.-L.; Whelan, J.; Bosnich, B. Inorg. Chem.
1987, 26, 396-400.
system
n
10-3K, M-1
mol-1
C-m _H
C-t_H
C-t_GH
1-m _H
1-t_H
1-t_GH
1-t_GGH 1.25 ( 0.03
1.12 ( 0.1
(4.5 ( 0.8) 11.6 ( 0.8 5.0
6.6
15.7
17.8
1.5
0.31 ( 0.01
0.44 ( 0.06
3.89 ( 0.06
0.82 ( 0.01
0.92 ( 0.01
(56 ( 12)
(23.5 ( 2)
(9.5 ( 1)
22.1 ( 1.5 6.5
23.8 ( 0.5 6.0
6.9 ( 0.2 5.4
25.8 ( 0.9 6.8
(12) (a) Vunnum, S.; Natarajan, V.; Cramer, S. J. Chromatogr. A 1998,
818, 31-41. (b) Muller, K. M.; Arndt, K. M.; Bauer, K.; Pluckthun, A.
Anal. Biochem. 1998, 259, 54-61. (c) Mahiou, J.; Abastado, J. P.; Cabanie,
L.; Godeau, F. Biochem. J. 1998, 330, 1051-1058.
(104.6 ( 32)
18.9
24.1
15.4
23.8
26.1
14.0
23.2
12.6
20.7
46.3
64.7
(32.6 ( 3.7) 30.2 ( 0.7 6.1
(50.3 ( 7.6) 21.9 ( 0.9 6.4
(15.4 ( 1.3) 29.6 ( 0.8 5.7
1-t_AH
1-t_QH
2-m _H
2-t_H
1.06 ( 0.01
0.61 ( 0.02
2.63 ( 0.21
(13) (a) Doyle, M. L. Curr. Opin. Biotech. 1997, 8. 31-35. (b) Wadso,
I. Chem Soc. ReV. 1997, 79-86.
(77 ( 15)
32.7 ( 2.1 6.7
(1.7 ( 0.2) 18.4 ( 2.5 4.4
(14) (a) Berger, M.; Schmidtcher, F. P. Angew. Chem., Int. Ed. 1998,
37, 2694-2696. (b) Wu, C.; Chen, W.-Y.; Lee, J.-F. J. Colloid Interface
Sci. 1996, 183, 236-242.
0.80 ( 0.02a (104.4 ( 22.8) 30.0 ( 1.5 6.8
2-t_GH
1.02 ( 0.006
(1190 ( 130) 20.8 ( 0.2 8.3
(15) (a) Taquet, A.; Labarbe, R.; Houssier, C. Biochemistry 1998, 37,
9119-9126. (b) Hileman, R. E.; Jennings, R. N.; Linhardt, R. J. Biochem-
istry 1998, 37, 15231-15237.
2-t_GGH 0.68 ( 0.003a (243.6 ( 24.5) 28.0 ( 0.4 7.3
2-t_AH
2-t_QH
0.61 ( 0.04a
0.47 ( 0.03a
(5.1 ( 1)
51.5 ( 6.8 5.0
(41.1 ( 7.5) 71.0 ( 6.7 6.3
(16) (a) Ladbury, J. E.; Chowdhury, B. Z. Biocalorimetry: Applications
of Calorimetry in the Biological Sciences; John-Wiley & Sons: New York,
1998. (b) Jelesarov, I.; Bosshard, H. R. J. Mol. Recogn. 1999, 12, 3-18.
(c) Haq, I.; Trent, J. O.; Chowdhury, B. Z.; Jenkins, T. C. J. Am. Chem.
Soc. 1999, 121, 1768-1779.
a Possibly reflects the formation of oligomeric species as the distances
are not matched. No isobestic points were observed for these titrations using
UV spectrometry.
Org. Lett., Vol. 2, No. 7, 2000
913

the binding constant. Higher loss of entropy offsets the
enthalpy gain for these interactions.
A hydrophobic side chain on the peptide (t_AH) had a
devastating effect on the affinity for the metal complexes
(K ∼15 000 for 1; 5000 for 2), again due to unfavorable
entropy changes. Introduction of a hydrophilic side chain
on the peptide (t_QH) also led to large entropy losses and
consequently decreased the affinity for the metal complexes.
With the flexible metal complex 2, 1:1 binding was observed
only when the distances were matched (t_GH). The stoi-
chiometry was found to be less than 1 when the distances
were not matched. This possibly indicates the formation of
oligomeric species among the peptide and the metal complex
2 (high enthalpy but high entropy loss). On the other hand,
the more rigid metal complex 1 was prone to forming 1:1
complexes with the peptides.
The binding constants are graphically depicted in Figure
3. The binding parameters are given in Table 1. A proposed
Figure 4. Proposed structure of 2.t_GH.
to imidazole moieties of organic molecules.17 In addition,
ITC titrations between the peptides and ligands without Cu2+
ions failed to detect any binding.
The interactions between the metal complexes and the
histidine-containing peptides were also followed using UV-
vis spectrometry (25 mM HEPES buffer, 25 °C). The
absorption maxima for the metal complexes (0.5 mM) shifted
from 720 to 665 nm upon sequential addition of the peptides
(2 mM, 10 × 100 µL additions). Analysis of the resultant
titration curves18 corroborated the stoichiometry and the
binding constants obtained by ITC. The position of absorp-
tion maxima for the resultant complexes (665 nm) indicated
that each cupric ion was coordinating to one histidine unit.17
In conclusion, metal complex 2 binds to its matched
trihistidine (t_GH) strongly and selectively. Complex 2
favors t_GH compared to other peptides differing by only
one amino acid unit (selectivity 230:1 with t_AH, 30:1 with
t_QH), a shorter peptide (selectivity 10:1 with t_GH), or a
longer peptide (selectivity 5:1 for t_GGH).
Figure 3. Binding constants for complexes C, 1, and 2 with the
histidine peptides (25 mM HEPES buffer, pH ) 7.0, 25.0 °C).
structure of the complex 2.t_GH is shown in Figure 4. The
structure was generated by docking the individual structures
(2 and t_GH) using the MM3 force field of the modeling
software CAChe (Oxford Molecular Inc.)
To demonstrate that the cupric ions are involved in
binding, the systems were studied by EPR spectroscopy
(9.445 GHz, field 2000-4000 G, 25 °C) in the solution phase
(25 mM HEPES buffer, pH ) 7.0). As an illustrative
example, the value of g| was found to be 2.31 for complex
2 (0.8 mM solution). After addition of the peptide t_GH
(1.6 mM), g| decreased to 2.25. These values matched well
with the reported values for iminodiacetate-Cu2+ binding
Acknowledgment. This work was supported by an NSF-
CAREER award (CHE-9896083) and an NIGMS-AREA
grant (1R15 59594-01, NIH). The microcalorimeter was
purchased through an NSF-EPSCoR award to North Dakota.
Supporting Information Available: Characterization
data for the peptides (1H and 13C spectra) and metal
complexes (elemental analysis) and ITC titration data (raw
and processed) for the results reported in Table 1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Dhal, P. K.; Arnold, F. H. Macromolecules 1992, 25, 7051-7059.
(18) Connors, K. A. Binding Constants; Wiley: New York, 1987; pp
141-215.
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