The crystal structures of glycylglycine and glycine complexes of cis,cis-1,3,5-triaminocyclohexane-copper(II) as reaction intermediates of metal-promoted peptide hydrolysis

Article abstract of DOI:10.1039/a901569h

The glycylglycine and glycine complexes of cis,cis-1,3,5-triaminocyclohexane-copper(II), model reaction intermediates of peptide hydrolysis by Cu(II)-triamine complexes, have been synthesized and characterized by X-ray crystallography.

Full text of DOI:10.1039/a901569h

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The crystal structures of glycylglycine and glycine complexes of
cis,cis-1,3,5-triaminocyclohexane–copper(ii) as reaction intermediates of
metal-promoted peptide hydrolysis
Xiang Shi Tan,a Yuki Fujii,*a Tsuyoshi Sato,a Yoshiharu Nakanoa and Morio Yashirob
a Department of Chemistry, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito 310-8512, Japan.
E-mail: yuki@mito.ipc.ibaraki.ac.jp
b Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo,
Bunkyo-ku, Tokyo 113-8656, Japan
Received (in Cambridge, UK) 26th February 1999, Accepted 6th April 1999
The glycylglycine and glycine complexes of cis,cis-1,3,5-tri-
aminocyclohexane–copper(ii), model reaction intermediates
of peptide hydrolysis by CuII–triamine complexes, have been
synthesized and characterized by X-ray crystallography.
prefered to the peptide nitrogen N(5) as a metal binding site at
pHs where the amide proton is not dissociated, as in the
tripeptide complexes of Cu(gly-gly-gly)+,9 Cu(gly-leu-tyr)+,10
and Zn(gly-gly-gly)+,11 or the CoIII–tetraamine complex of the
glycylglycine O-ethyl ester.12 This mode of co-ordination
accounts for the ease of hydrolysis of the dipeptide. The
positively charged copper(ii) ion renders the carbonyl carbon
atom C(8) more susceptible to nucleophilic reagents (OH2) and
the peptide chelate ring is not required to break for hydrolysis to
occur, so the NH2-terminal glycine remains attached to the
Cu(tach) residue to form [glycinato–copper(ii)–tach] complex
There has been great interest in designing artificial metal-
lopeptidases that hydrolyze unactivated amides under mild
conditions.1,2 Up to now a variety of metal complexes of CuII,1,3
ZnII,3c,d NiII,3c,d PdII,4 CoIII2,5 and CeIV6 have been used to
study the hydrolysis of amides. Recently Burstyn and co-
worker1 reported the discovery that a macrocyclic copper(ii)
complex, Cu[9]aneN3, can hydrolyze both the unactivated
dipeptide glycylglycine and proteins at near physiological pH.
Although no reaction mechanism has been shown for Cu[9]-
aneN3, in such a reaction, the activation of the carbonyl group
of the amide by the metal centre seems to play an important role,
and it is necessary to get information about the reaction
intermediate to advance the hydrolysis reaction. We have been
studying some copper(ii)–triamine complexes which effectively
promote the hydrolytic cleavage of phage DNA,7 as well as
peptide bonds. To understand the mechanism of the hydrolytic
reaction of peptides by CuII–triamine complexes, we present
here the crystal structures of glycylglycine and glycine
complexes of cis,cis-1,3,5-triaminocyclohexane–copper(ii) as
model reaction intermediates.
We have investigated the hydrolysis of peptide with cis,cis-
1,3,5-triaminocyclohexane–copper(ii) complex7 1 by HPLC
and found that glycylglycine is hydrolyzed to glycine when
incubated with 1.† The hydrolysis of glycylglycine (Gly-Gly) (2
mM) to glycine (Gly) with 1 (2 mM) at 70 °C and pH 8.1 ± 0.1
(50 mM HEPES) is ca. 18% for 24 h, which is similar to that of
Cu[9]aneN3, and the conversion is dependent on metal complex
concentration, reaction time and solution pH.
Fig. 1 ORTEP plot with 30% probability thermal ellipsoids showing the
structure of the cation of 2. Selected bond distances (Å) and angles (°): Cu–
N(1) 1.990(3), Cu–N(2) 2.015(2), Cu–N(3) 2.232(3), Cu–N(4) 2.017(3),
Cu–O(1) 2.039(2), O(1)–Cu–N(1) 89.38(9), O(1)–Cu–N(2) 162.32(10),
O(1)–Cu–N(3) 104.52(9), O(1)–Cu–N(4) 80.32(9), N(1)–Cu–N(2)
92.61(9), N(1)–Cu–N(3) 91.5(1), N(1)–Cu–N(4) 167.49, N(2)–Cu–N(3)
93.00(9), N(2)–Cu–N(4) 95.09(9), N(3)–Cu–N(4) 97.9(1).
The tetraphenylborate salts of [Cu(tach)(gly-gly)]+ 2 and
[Cu(tach)(gly)]+ 3 (tach = cis,cis-1,3,5-triaminocyclohexane,
gly = glycine anion, gly-gly = glycylglycine anion) were
synthesized by stirring a mixture of CuSO4.5H2O (1 mmol),
tach (1 mmol) and Gly-Gly (1 mmol) for 2 or Gly (1 mmol) for
3 in MeOH–water at pH 8 (in the presence of NaBPh4) in 40%
yield for 2 and 60% yield for 3, respectively. Blue crystals of the
two complexes were grown by slow evaporation of a MeOH–
water solution and the structures were characterized by X-ray
crystallography.‡
The crystal structures (Figs. 1 and 2) of complexes 2 and 3
both show a five co-ordinate copper centre ligated to the three
face capping nitrogens of the tach ligand. In each case the
copper co-ordination geometry is square pyramidal distorted
along the apical Cu(1)–N(3) bond with a distance of 2.232(3) Å
in 2 and 2.152(6) Å in 3. The other Cu–N distances, Cu(1)–N(1)
and Cu(1)–N(2), are 1.990(3) and 2.015(2) Å in 2 and 2.019(6)
and 2.042(6) Å in 3, respectively, as in other copper-tach
complexes.8 The peptide (gly-gly) is coordinated through the
terminal NH2 group and the carbonyl oxygen atom from the
same glycine residue. The carbonyl oxygen atom O(1) is here
Fig. 2 ORTEP plot with 30% probability thermal ellipsoids showing the
structure of the cation of 3. Selected bond distances (Å) and angles (°): Cu–
O(1) 1.974(4), Cu–N(1) 2.019(6), Cu–N(2) 2.042(6), Cu–N(3) 2.152(6),
Cu–N(4) 2.007(6), O(1)–Cu–N(1) 173.6(2), O(1)–Cu–N(2) 87.5(2), O(1)–
Cu–N(3) 97.0(2), O(1)–Cu–N(4) 82.6(2), N(1)–Cu–N(2), 92.8(3), N(1)–
Cu–N(3) 89.4(2), N(1)–Cu–N(4) 93.2(2), N(2)–Cu–N(3) 92.7(2), N(2)–
Cu–N(4) 141.4(2), N(3)–Cu–N(4) 125.5(2).
Chem. Commun., 1999, 881–882
881

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1.355 g cm23, m = 0.707 mm21, 7778 reflections measured, 5756 observed
[I > 3.00 s(I)]. Solution by direct methods with SIR92, expanded using
Fourier Techniques with DIRDIF94. Full-matrix least-square refinement on
F with all non-hydrogen atoms anistropic and hydrogens included but not
refined. Final R and Rw values on observed data were 0.050 and 0.051.
For 3: C32H39N4O2BCu, M
= 586.04, orthorhombic, space group
P212121, a = 9.879(2), b = 30.374(9), c = 9.769(2) Å, U = 2931(1) Å3,
l = 0.71069 Å, Z = 4, Dc = 1.328 g cm23, m = 0.781 mm21. 3808
reflections measured, 2857 observed [I > 3.00 s(I)]. Solution methods and
refinements as for 2. Final R and Rw values on observed data were 0.052 and
0.051. CCDC 182/1212. See http://www.rsc.org/suppdata/cc/1999/881/ for
crystallographic files in .cif format.
§
The pH dependency is independent on the pKa curve of [Cu-
(tach)(H2O)2]2+ [ref. 7(b)], indicating that the attack of internal coordinated
OH2 can be neglected.
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Scheme 1
ion, as shown in Fig. 2. Thus the complex cation of 2,
[Cu(tach)(gly-gly)]+, is an intermediate of the hydrolysis of the
N-terminal peptide bond promoted by CuII–triamine complex,
and is clear evidence for the activation of the CNO group by the
metal centre.
On the basis of the intermediate structure a reasonable
mechanism may be postulated for the peptide hydrolysis at near
physiological pH, as shown in Scheme 1. The initial step
involves the rapid replacement of the water molecule by the N-
terminal amino group of the peptide, which becomes chelated to
the copper(ii) ion. The carbonyl group is then activated to attack
by external OH2. The mechanism is supported by the fact that
the rate of hydrolysis increases with increasing hydroxide
concentration.§
Experiments to prove this mechanism via solution chemistry
studies and kinetic measurements of the hydrolysis of peptides
by CuII–triamine complexes are presently being conducted.
The support of this work by a JSPS postdoctoral fellowship to
X. S. T. from the Japan Society for the Promotion of Science
(No. P97370) and by a Grant-in-Aid for Science Research (No.
09440224) from the Ministry of Education, Science, Sports and
Culture of Japan is gratefully acknowledged.
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Notes and references
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† Glycylglycine (2 mM) was dissolved in water and incubated with 1 (2
mM) at pH 8.1 ± 0.1 (50 mM HEPES) and 70 °C for 24 h. Samples were
analyzed in a JASCO Model FP-920 Intelligent Fluorescence Detector.
‡ Crystal data for 2: C35H46N5O4BCu, M = 675.14, triclinic, space group
¯
P1, a = 10.238(4), b = 18.026(7), c = 10.065(3) Å, a = 92.29(3), b =
113.71(3), g = 101.13(3)°, U = 1654(1) Å3, l = 0.71069 Å, Z = 2, Dc
=
Communication 9/01569H
882
Chem. Commun., 1999, 881–882