Protein-cleaving catalyst selective for protein substrate

Article abstract of DOI:10.1021/ol0269300

(matrix presented) A protein-cleaving catalyst specific for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective for a protein substrate, a catalyst for myoglobin was designed by attaching Cu(II) or C

Full text of DOI:10.1021/ol0269300

ORGANIC
LETTERS
2002
Vol. 4, No. 23
4155-4158
Protein-Cleaving Catalyst Selective for
Protein Substrate
Joong Won Jeon,† Sang Jun Son,† Chang Eun Yoo,† In Seok Hong,‡
,†
Jung Bae Song,† and Junghun Suh*
School of Chemistry and Center for Molecular Catalysis, Seoul National UniVersity,
Seoul 151-747, Korea, and Artzyme Biotech Corporation, 403-1, Silim-2-Dong,
Seoul 151-012, Korea
jhsuh@snu.ac.kr
Received September 19, 2002
ABSTRACT
A protein-cleaving catalyst specific for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective
for a protein substrate, a catalyst for myoglobin was designed by attaching Cu(II) or Co(III) complex of cyclen to a binding site searched by
a combinatorial method using peptide nucleic acid monomers as building units.
In search of new drugs, efforts are made to design molecules
that specifically block the activity of disease-related proteins
such as enzymes, receptors, and ion channels.1-4 Even if a
drug molecule is bound to its target protein very strongly,
at least an equivalent amount of the drug is needed to
inactivate the protein. If a disease-related protein is deacti-
vated upon cleavage of its polypeptide backbone by a
synthetic catalyst, the catalyst can be used as a drug. Because
a catalytic amount of the drug can destroy the protein, the
dosage can be reduced substantially by using the catalyst.
Designing protein-cleaving catalysts specific for target
proteins, therefore, would become a powerful tool in drug
discovery.
functional groups and metal complexes have been exploited
as the catalytic groups in those artificial proteases. A protein-
cleaving catalyst with high substrate selectivity and catalytic
rate would be obtained by connecting a binding site that
recognizes the target protein to a catalytic group. On
complexation of the protein-cleaving catalyst to the target
protein, the effective molarity18 of the catalytic group toward
a peptide linkage of the target protein can increase to a
sufficiently high level to allow facile cleavage of the peptide
bond. To suppress cleavage of nontarget proteins, the
(6) Suh, J.; Oh, S. J. Org. Chem. 2000, 65, 7534.
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1999, 121, 8663.
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Soc. 2000, 122, 7742.
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(17) Suh, J. Synlett 2001, 9, 1343.
Several synthetic catalysts with proteolytic activity lacking
substrate selectivity have been reported.5-17 Both organic
† Seoul National University.
‡ Artzyme Biotech Corporation.
(1) Silverman, R. B. The Organic Chemistry of Drug Design and Drug
Action; Academic: San Diego, 1992; pp 52-97, 146-219.
(2) Ganellin, C. R., Roberts, S. M., Ed. Medicinal Chemistry, 2nd ed.;
Academic Press: London, 1993.
(3) Veerapandian, P., Ed. Structure-based drug design; Marcel Dekker:
New York, 1997.
(4) Leff, P.; Ed. Receptor-based drug design; Marcel Dekker: New York,
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10.1021/ol0269300 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/25/2002

catalytic center should have very low protein-cleaving
activity when unattached to the binding site. In the present
study directed toward the first protein-cleaving catalyst
selective for a target protein, we chose metal complexes of
cyclen (Cyc) as the catalytic center since a previous study11
indicated that the Cu(II) complex of Cyc has very low
proteolytic activity unless activated by additional catalytic
elements.
The PNA derivatives were synthesized by automated
synthetic procedures using an Expedite model 8909 Nucleic
Acid Synthesis System with the fmoc-derivatives of A*, T*,
G, C, and L-Lys as well as (boc)3CycAcOH. PNA monomers
G and C as well as L-lysine protected with the fmoc group
were purchased from commercial sources whereas A* (1)
and G* (2) protected with the fmoc group as well as
(boc)3CycAcOH (3) were synthesized according to the
synthetic pathways summarized in Scheme 1. In the con-
Scheme 1
As the binding site of the specific protein-cleaving catalyst,
small organic compounds such as inhibitors or antagonists
already reported to have high affinity toward the target
protein may be used. Alternatively, a new binding site may
be searched by the combinatorial approach. The catalyst
obtained by the latter method does not necessarily bind to
the active site of the protein.
In the present study, we constructed a combinatorial library
of Cyc derivatives containing analogues of peptide nucleic
acid (PNA)19,20 (Figure 1) to search the binding site of a
struction of the library, it was assumed that the carboxyl
groups of the fmoc derivatives of A*, T*, G, and C have
identical reactivity in coupling with the amino ends of PNA
oligomers attached to the polymer support. Purity of various
PNA derivatives was checked by MALDI-TOF MS analysis
by using a Voyager-DE STR biospectrometry workstation
model.
The library of CycAc(Q)nLysNH2 (total concentration: ca.
70 µM) was mixed with an aqueous solution of Cu(II)Cl2
(350 µM) to generate the library of Cu(II)CycAc(Q)nLysNH2
where Cu(II) is bound to the Cyc moiety. Cu(II) forms a
strong complex with Cyc (log Kf ) 16.8 at pH 7).23 With
the Cu(II)Cyc library containing 7- or 8-mer PNAs, no
evidence was obtained for cleavage of proteins (10 µM) such
as bovine serum albumin, γ-globulin, elongation factor P,
Figure 1. Structures of the combinatorial library of Cyc derivatives
containing PNA oligomers, ligand (4) of the catalyst selected from
the library, and an analogue (4a) of 4.
protein-cleaving catalyst. PNA was employed as the potential
binding site in view of protein recognition21 by derivatives
of purine. The library can be presented as CycAc(Q)nLysNH2
where Q is PNA monomer A*, G, T*, or C. PNA contains
nucleobases that can be used for base-pairing with nucleo-
bases of DNA. We used modified nucleobases A* and T*
instead of A and T. A* and T* recognize T and A,
respectively. A* and T*, however, do not recognize each
other.22 Base-pairing among PNA mixtures present in the
library, therefore, can be suppressed by using A* and T*.
4156
Org. Lett., Vol. 4, No. 23, 2002

gelatin A, gelatin B, and horse heart myoglobin (Mb) at 37
°C and pH 7 as checked by electrophoresis (SDS-PAGE).
Methods for kinetic measurement of protein cleavage by
SDS-PAGE are described elsewhere.5-7,11 The Cu(II)Cyc
library containing 9-mer PNAs clearly showed activity for
cleavage of Mb. Four groups of library with the known PNA
monomers positioned next to Cu(II)Cyc were subsequently
synthesized and tested for their activity and A* was identified
as the best monomer for that position. By repeating the search
for the rest of the nine positions occupied by PNA monomers,
4 (Figure 1) was chosen as the ligand of the best catalyst.
The binding site for Mb was, therefore, obtained by using
the pyrimidine and purine bases of 4.
to metal-abstracting materials in living body should be
substantially slower for the Co(III) complexes.
An example of degradation of Mb by Co(III)4 is illustrated
in Figure 2. Although the structure of 4 was obtained by
using the Cu(II) complex, detailed kinetic analysis was
performed with the Co(III) complex due to the higher
catalytic activity of the Co(III) complex. The dependence
of ko on Co (the initially added concentration of the catalyst)
measured at pH 7.5 is illustrated in Figure 3. Although the
The stock solution of Cu(II) complex of 4 was prepared
by adding an aqueous solution of CuCl2 to 4 (1.2 equiv) at
pH 6.0. The degradation of Mb by Cu(II)4 was followed by
electrophoresis (SDS-PAGE). An example of the plot of
[Mb] (total concentration of uncleaved Mb; estimated from
the electrophoretic bands of Mb) against time is illustrated
in Figure 2. The time-dependent decrease in [Mb] was fitted
Figure 3. Plot of ko against Co for cleavage of Mb ([Mb]o ) 4.7
µM) by Co(III)4 at pH 7.5 and 37 °C (0.05 M buffer; addition of
0.5 M NaCl did not affect the rate data appreciably). Straight lines
a (Co < So) and b (Co g So) stand for Vo/So (Vo: initial velocity)
and ko, resepctively, predicted by Michaelis-Menten scheme under
the condition of Co . Km.
plot of ln [Mb] against time was fitted to a straight line to
obtain ko, Michaelis-Menten scheme predicts that the kinetic
behavior does not conform to first-order kinetics when Co
is smaller than [Mb]o (the initially added concentration of
Mb) even when Co . Km. Moreover, rate data based on
electrophoretic measurement are not very accurate. These
may be related to the scattered data points of Figure 3 at Co
< [Mb]o.
Although the kinetic data are somewhat scattered, the two
straight lines drawn in Figure 3 intersect at Co ) [Mb]o. This
intersection agrees with strong binding of Mb to Co(III)4:
in terms of Michaelis-Menten parameters, Km , Co and,
thus, Km , 5 µM. Furthermore, ko measured with Co greater
than [Mb]o corresponds to kcat.
Figure 2. Decrease in [Mb] during incubation of Mb with Cu-
(II)4 (b; curve a, [Mb]o ) 7.9 µM, [Cu(II)4]o ) 2.0 µM) or Co-
(III)4 (O; curve b, [Mb]o ) 4.7 µM, [Co(III)4]o ) 0.47 µM) at pH
7.5 and 37 °C. The curves were obtained as indicated in the text:
ko ) 5.7 × 10-3 h-1 for curve a and 9.4 × 10-3 h-1 for curve b.
to pseudo-first-order kinetic equations to obtain pseudo-first-
order rate constant (ko).
The kcat values thus measured at various pHs are illustrated
in Figure 4. If ionization of Mb or 4 is disregarded, the pKa
values (5.50, 8.68) estimated from analysis of the bell-shaped
pH profile may be assigned to the ionization of water
To check whether other metal ions complexed to 4 cleave
Mb, metal ions such as Co(III), Fe(III), Hf(IV), Pt(IV),
Zr(IV), Pd(II), and Ce(IV) were added to 4 to generate the
respective complexes. Due to the kinetic inertness24 of Co(III)
complexes, direct insertion of Co(III) ion to the chelating
ligands is not easy. Instead, the Co(III) complex of 4 was
obtained by incorporating Co(II) ion to 4 and then oxidizing
the complexed Co(II) ion in methanol according to the
literature25 procedure: for Co(III)4, MS (MALDI-TOF) m/z
2908.44 (M + H)+ (C111H153N64O25S2Co calcd 2908.51).
Among the metal ions tested, Co(III) manifested the protein-
cleaving activity upon complexation to 4. It is noteworthy
that Co(III) complexes may be more suitable for medical
uses compared with Cu(II) complexes since metal transfer
(19) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem.,
Int. Ed. 1998, 37, 2796.
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S.; Norman, T. C.; Sarohia, R.; Leost, M.; Meijer, L.; Schultz, P. G. Chem.
Biol. 1999, 6, 361.
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1999, 96, 10804.
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Org. Lett., Vol. 4, No. 23, 2002
4157

different Co(III)4-Mb complexes or originate from an
identical one. More information on binding mode of the PNA
portion of the catalysts would be obtained by crystallographic
studies.
When other proteins such as albumin, γ-globulin, elonga-
tion factor P, gelatin A, and gelatin B were incubated with
Cu(II)4 or Co(III)4, protein cleavage was not observed. When
Mb was treated with CuCl2, Cu(II)Cyc, or Co(III)Cyc instead
of Cu(II)4 or Co(III)4, Mb was not degraded. An analogue
of Co(III)4 was prepared where the PNA residue next to the
CycAc unit is C instead of A* as indicated by 4a. No
catalytic activity was observed for Co(III)4a and Cu(II)4a
in the cleavage of Mb. The degree of intramolecular cleavage
of amide bonds of Cu(II)4 or Co(III)4 was not significant
when followed for several days by measuring the MALDI-
TOF mass spectrum and the catalytic activity, probably due
to the steric strain involved in the attack of the metal center
at an internal amide bond.
Figure 4. pH dependence of kcat for cleavage of Mb by Co(III)4
at 37 °C. The bell-shaped curve was obtained by analyzing the
data by treating the Co(III)4-Mb complex as a diprotonic acid
(pKa1 ) 5.50, pKa2 ) 8.68) and by assuming that the monoproto-
nated species is reactive.
Up to 2.5 or 6 molecules of Mb were cleaved by each
molecule of Cu(II)4 or Co(III)4, respectively, for the data
of Figure 2, indicating the catalytic nature of the action of
Cu(II)4 and Co(III)4. The number of Mb molecules cleaved
per catalyst molecule was small due to the small value of
kcat as a consequence of the low effective molarity of the
catalytic center in the Mb-catalyst complex. Catalytic
turnover by Co(III)4, despite the exchange-inertness of
Co(III) ion, indicates that the protein-cleavage products
dissociate from the Co(III) ion effectively. In this regard,
carboxylates bound to Co(III) complexes can be freed
hydrolytically through C-O bond cleavage.27
molecules coordinated to Co(III) ion of Co(III)4-Mb
complex. It is noteworthy that the catalyst is most active at
the physiological pH. The kcat measured at pH 7.5 and 37
°C corresponds to the half-life of 30 h. This may be
compared with the half-life26 of 200 y for spontaneous
hydrolysis of unactivated amides measured under identical
conditions. On optimization of the linker connecting Co(III)-
Cyc and the PNA moiety of 4, kcat would be raised further.
The MALDI-TOF mass spectrum (Figure 5) of a reaction
The rate for cleavage of Mb by Cu(II)4 or Co(III)4 was
unaffected by the removal of O2 from the reaction mixtures.
These results and previous observations8,13,15 for hydrolytic
cleavage of peptide bonds by Cu(II) complexes of tetraaza
ligands and Co(III) complexes suggest the hydrolytic nature
of cleavage of Mb by Cu(II)4 and Co(III)4.
The present study demonstrates that protein-cleaving
catalysts specific for target proteins can be designed by
choosing appropriate binding sites, linkers, and catalytic
units. The activity of a protein-cleaving catalyst would be
improved by the increase in its affinity to the target protein
and by proper orientation of the catalytic group in the
catalyst-protein complex. By following the general principles
discovered here, it would be possible to design protein-
cleaving catalysts specific for selected proteins. If the selected
targets are disease-related proteins, the protein-cleaving
catalysts can serve as catalytic drugs.
Figure 5. MALDI-TOF mass spectrum taken after incubation of
Mb (12 µM) with Co(III)4 (3.5 µM) at pH 6.0 and 37 °C for 85 h.
Mass values are 16953 (A), 9892 (B), 8909 (C), 16953/2 (D), 8045
(E), and 7074 (F); A and D are peaks for Mb (MW 16953), and
two pairs (B/F and C/E) are for proteins formed by cleavage of
Mb.
Acknowledgment. This work was supported by Artzyme
Biotech Corporation. J.W.J., S.J.S., and C.E.Y. were recipi-
ents of BK21 fellowships.
mixture obtained by incubation of Mb with Co(III)4 disclosed
that Mb was dissected into two pairs of proteins. Possible
sites of the protein cleavage by Co(III)4 are: Leu89-Ala90
(producing fragments with MW 7077 and 9894) and Leu72-
Gly73 (producing fragments with MW 8057 and 8914).
Attempts to identify the N-terminal sequences of the cleavage
products were unsuccessful since individual protein frag-
ments were not separated cleanly by electrophoresis. It is
not clear at present whether the two cleavage sites involve
Supporting Information Available: Experimental pro-
cedures for synthesis of 1-3. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL0269300
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