An unprecedented trans-oriented product from the cleavage of a dipeptide.

Article abstract of DOI:10.1039/b212587k

An unusual trans cleavage reaction was observed when trans-[Co(3,2,3-tet)Cl2]Cl (3,2,3-tet = N,N'-bis(3-aminopropyl)ethylenediamine), was allowed to react with beta-alanyl-L-histidine (a bioactive dipeptide) in an aqueous medium at pH approximately 7.5 and 45 degrees C for 6 h.

Full text of DOI:10.1039/b212587k

An unprecedented trans-oriented product from the cleavage of a
dipeptide
Manas K. Saha and Ivan Bernal*
Depatment of Chemistry, University of Houston, Houston, Texas 77204-5641, USA. E-mail: ibernal@uh.edu
Received (in Purdue, IN, USA) 18th December 2002, Accepted 8th January 2003
First published as an Advance Article on the web 5th February 2003
An unusual trans cleavage reaction was observed when
trans-[Co(3,2,3-tet)Cl2]Cl {3,2,3-tet = N,NA-bis(3-amino-
propyl)ethylenediamine}, was allowed to react with b-
nine)2]+, which illustrates the exclusive formation of 1 in this
reaction.
In a control experiment an aqueous solution of b-alanyl- -
L
alanyl-L-histidine (a bioactive dipeptide) in an aqueous
histidine was kept at similar conditions (at pH ~ 7.5 and 45 °C
for 6 h) without any trans-[Co(3,2,3-tet)Cl2]Cl. No cleavage
products were formed as evidenced by 1H NMR spectroscopy,
TLC work and electron spray mass spectroscopy. To check the
possibility of a peptide bond cleavage, under the influence of
silica gel, used for column chromatography, we further ran the
medium at pH ~ 7.5 and 45 °C for 6 h.
Metal complexes that can cleave peptide bonds under mild
condition are of potential interest due to their usefulness for
structure–activity studies of proteins, investigation of protein
structural domains and in converting large proteins into smaller
fragments that are more amenable for sequencing.1,2 Moreover,
such reagents are also useful for the chemical modification or
heavy atom labeling of proteins.2,3 The cleavage reaction either
follows an oxidative or a hydrolytic pathway and the latter is
preferred because the oxidative process involves the high risk of
damaging the protein chain. Cobalt(III) complexes are known to
induce the hydrolysis of peptides and model studies were
carried out to investigate the mechanism of the peptide cleavage
reaction by using small peptide molecules.4 Those studies
involve cis-Co(III) complexes at a slightly basic pH which
proceed by a cis-coordination to the peptide molecule and,
subsequently, the peptide bond undergoes cleavage by the
attack of the hydroxide anions.5 The unsuccessful attempt to
cleave the peptide bond by the use of trans-Co(III) complexes
are reported in literature.6 In this communication, we report a
peptide bond cleavage reaction which is promoted by an
1
solution through the column and eluted it with water. The H
NMR shows the identical spectrum as that known for b-alanyl-
L
-histidine.10 The intact b-alanyl-
-histidine was further
L
checked by electron spray mass spectroscopy which shows a
single peak at [M + 1] = 227.2.
It is noteworthy, that the hydrolytic cleavage reaction occurs
irrespective of a 1+1 or a 1+2 molar ratio between trans-
[Co(3,2,3-tet)Cl2]Cl and b-alanyl- -histidine. In the latter case,
L
a higher yield of the crystalline material [Co(3,2,3-tet)(H-b-
alanine)2](ClO4)3 is obtained.
The X-ray analysis11 of the perchlorate salt of 1 shows the
trans-coordination of the (bis-b-alanine) residues to the cobalt
center through oxygen atoms of the carboxylate groups, while
the protonated amine groups remain dangling. The basal plane
is constructed by the four nitrogen atoms of the 3,2,3-tet ligand.
An ORTEP12 diagram of 1 is presented in Fig. 1. The Co–N
bond distances span the range of 1.952–1.992 Å. One of the Co–
O bonds (1.952 Å) is longer than the other (1.909 Å). The bond
angles and the torsion angle are in normal ranges.
unusual trans-Co(III) complex (trans-[Co(3,2,3-tet)Cl2]Cl
{3,2,3-tet = N,NA-bis(3-aminopropyl)ethylenediamine} (see
ref. 7 for structure) and follows a different reaction pathway.†
To the best of our knowledge, this is the first trans-oriented
product obtained from the cleavage of peptide bonds. These
results are important for a better understanding of the cleavage
reaction mechanism(s) and should provide useful ideas for
designing new active metal complexes for sequential and
specific cleavage of protein matrices.
b-Alanyl- -histidine possesses multiple coordinating centers,
L
and the type of complex formation depends on the metal ion,
ligand+metal ratios and pH of the solution.13 Dihalo complexes
of Co(III), are known to be highly labile and react with water to
form aquo-hydroxo complexes, specially if the solutions are
neutral or slightly basic.4–6 The crystal structure 1 indicates the
substitution of OH2/H2O by the carbonyl oxygen of the peptide
linkage leading to the cleaved product (Scheme 1). The
coordination of the carbonyl oxygen to the metal centre
promotes attack by the hydroxide ion to the carbonyl carbon
atom and, thus, causes the cleavage of the peptide bond.
b-Alanyl- -histidine (carnosine, found in relatively high
L
concentrations in several body tissues—most notably in skeletal
muscle, heart muscle, and brain, acts as an antioxidant and
promotes wound healing)8 was allowed to react with a half
molar amount of trans-[Co(3,2,3-tet)Cl2]Cl at pH ~ 7.5 and 45
°C for 6 h. The reaction mixture was spotted on a silica gel plate
and eluted with CH3OH–H2O (1+1) mixture; the cobalt
containing species remained on the starting line but the
histidine fragment moved in the normal manner, which has been
compared with the movement of known -histidine solutions.
L-
L
The trans oriented cleavage product [Co(3,2,3-tet)(H-b-ala-
nine)2]3+ 1 was isolated as the perchlorate salt from the solution.
The -histidine residue was collected by column chromato-
L
graphic separation techniques and was charaterised by 1H
NMR9 and electron spray mass spectroscopy [M + 1 = 156.1].
The reaction yield (checked by 1H NMR) of the cleavage
product based on the isolated -histidine is 75%; however, we
L
could isolate only 30% of the [Co(3,2,3-tet)(H-b-alanine)2]-
(ClO4)3 in crystalline form due to its high solubility. Continued
concentration of the mother liquor results to a highly viscous
material. This was redissolved in water and subjected to
electron spray mass spectroscopy. The spectrum shows only
one signal at 409 that corresponds to [Co(3,2,3-tet)(b-ala-
Fig. 1 An ORTEP view of 1.
612
CHEM. COMMUN., 2003, 612–613
This journal is © The Royal Society of Chemistry 2003

complex, then run through silica column, shows a signal at [M + 1] = 227.2,
corresponding to uncleaved b-alanyl-L
-histidine. The 1H NMR spectra is
the same as that reported in the literature.10
1 (a) P. B. Dervan, Nature, 1992, 359, 87; (b) A. H. Krotz, L. Y. Kuo, T.
P. Shields and J. K. Barton, J. Am. Chem. Soc. 1993,, 115, 3877; (c) C.
C. Cheng, S. E. Rokita and C. J. Burrows, Angew. Chem., Int. Ed. Eng.,
1993, 32, 277; (d) N. Gupta, N. Grover, G. A. Neyhart, P. Singh and H.
H. Thorp, Inorg. Chem., 1993, 32, 310; (e) D. P. Mack and P. B. Dervan,
Biochemistry, 1992, 31, 9399.
2 (a) A. Schepartz and B. Cuenoud, J. Am. Chem. Soc., 1990, 112, 3247;
(b) D. Hoyer, H. Cho and P. G. Schultz, J. Am. Chem. Soc., 1990, 112,
3249; (c) T. M. Rana and C. F. Meares, J. Am. Chem. Soc., 1990, 112,
2457.
3 (a) N. Ettner and W. Hillen, J. Am. Chem. Soc., 1993, 115, 2546; (b) N.
Ettner, J. W. Metzger, T. Lederer, J. D. Hulmes, C. Kisker, W. Hinrichs,
G. Ellestad and W. Hillen, Biochemistry, 1995, 34, 22; (c) S. A. Strobel,
L. A. Doucette-Stamm, L. Riba, D. E. Housman and P. B. Dervan,
Science, 1991, 254, 1639; (d) D. Rehder, Angew. Chem., Int. Ed. Engl.,
1991, 30, 148; (e) C. V. Kumar and A. Buranaprapuk, Angew. Chem.,
Int. Ed. Engl., 1997, 36, 2085.
4 (a) D. A. Buckingham, J. P. Collman, D. A. R. Happer and L. G.
Marzilli, J. Am. Chem. Soc., 1967, 89, 1082; (b) D. A. Buckingham and
J. P. Collman, Inorg. Chem., 1967, 6, 1803; (c) E. Kimura, S. Young and
J. P. Collman, Inorg. Chem., 1970, 9, 1183.
5 D. A. Buckingham and Charles R. Clark, in Metal ions in Biological
Systems, ed. A. Sigel and H. Sigel, Basel, New York, 1976, vol. 38, pp.
43–98.
6 C. J. Boreham, D. A. Buckingham and F. R. Keene, Inorg. Chem., 1979,
18, 28.
Scheme 1 Reaction pathway for formation of [(3,2,3-tet)Co(H-b-ala-
nine)2]3+
.
7 (a) I. Bernal, J. Cetrullo, J. Cai and S. S. Massoud, Struct. Chem., 1995,
6, 99 and references therein (b) N. C. Payne, Inorg. Chem., 1973, 12,
1151.
8 (a) L. Bonfanti, P. Peretto, S. De Marchis and A. Fasolo, Prog.
Neurobiol., 1999, 59, 333; (b) G. I. Klebanov, Y. O. Teselkin and I. V.
Babenkova, Membr. Cell Biol., 1998, 12, 89; (c) A. R. Hipkiss, Int. J.
Biochem. Cell Biol., 1998, 30, 863; (d) P. R. Roberts, K. W. Black, J. T.
Satamauro and G. P. Zaloga, Nutrition, 1998, 14, 266.
9 (a) C. Tessier, F. D. Rochon and A. L. Beauchamp, Inorg. Chem., 2002,
41, 6527; (b) R. B. Martin and R. Mathur, J. Am. Chem. Soc., 1965, 87,
1065; (c) R. E. Wasylishen and G. Tomlinson, Biochem. J., 1975, 147,
605.
10 J. O. Friedrich and R. E. Wasylishen, Can. J. Chem., 1986, 64, 2132.
11 Crystal data: C14H36N6O16Cl3Co, Mw = 709.8, orthorhombic, space
group Pbn21, a = 11.696(3), b = 14.531(3), c = 16.561(4), V =
2815.03 Å3, Z = 4, Dc = 1.67 g cm23, m = 0.975 mm21, Mo-Ka ,
F(000) = 1471.7, q range of data collection 2.1–26.0, final R = 0.034,
wR2 = 0.086, GOOF = 1.015 for 352 parameters and 3327 unique
reflections, [R(int) = 0.039] of which 2148 are observed, positive and
negative peaks in DF map 0.340 and 20.464 e Å23. the data set was
collected on a Enraf Nonius CAD4 system equipped with Mo-Ka
radiation (l = 0.71073 Å) at room temperature. The structure was
solved by Patterson and Fourier analyses and refined by full-matrix least
squares based on F2 using SHELX 97.16 All the hydrogen atoms were
placed in their geometrically ideal positions with isotropic temperature
factors 1.2 times those of the attached non-hydrogen atoms. All
calculations were performed using the WinGX system (ver 1.64).17
CCDC 187487. See http://www.rsc.org/suppdata/cc/b2/b202587k/ for
crystallographic data in CIF or other electronic format.
Coordination through other sites of the dipeptide requires a cis
coordination to the metal ion through one of peptide linkage
atoms in order to form a chelate ring, a process that will induce
polarity to the peptide bond. However, a cis attack to the metal
centre is restricted by the rigidity of the 3,2,3-tet ligand. The
above results demonstrate that a trans-oriented cleavage
reaction can take place when a polydentate ligand to the metal
complex shows strong rigidity towards the trans-orientation.
The cis orientation and the chelate formation are not an essential
condition to polarize the peptide linkage. Further investigations
are in progress to provide more information on the trans-
oriented cleavage reaction. The fact that the 3,2,3-tet ligand can
be forced to the rearrange to a cis-b- product by a strong enough
cis-binding ligand has been observed in this group.14
Notes and references
† Experimental section: for column chromatography, the silica used came
from the Davison Chemical Division of Grace Chemical with its pH in water
at ca. 5.5. [(3,2,3-tet)CoCl2]Cl was prepared by the known literature
method.15 0.17 g (0.05 mol) of [(3,2,3-tet)CoCl2]Cl was dissolved in 100
cm3 water. The pH of the solution was adjusted to 7.5 by adding an aqueous
solution of LiOH. A 10 cm3 aqueous solution of 0.26 g (0.1 mol) of b-
alanyl-L-histidine was added slowly and the reaction mixture was set to 45
°C, with continuous stirring, for 4 h. The mixture was allowed to cool and
the solution was added to a silica gel chromatographic column and eluted by
water. The histidine fragment was eluted out first, followed by the single
dark red band, which was collected. The solution was reduced to 25 cm3. 3
cm3 of 10% H(ClO4) was added to it and after seven days crystals were
obtained. 1H NMR spectra were recorded at 300 MHz on a GE QE-300
spectrometer in deuterated water. The solvent signal (d 4.8 ppm) was used
as internal standard. Electron spray mass spectra in aqueous solution were
recorded with an LCQ DECA XP mass spectrometer. The cleavage product,
12 M. N. Burnett and C. K. Johnson, ORTEP-III Oak Ridge Thermal
Ellipsoid Plot Program for Crystal Structure Illustrations: Oak Ridge
National Laboratory, ORNL 6895, 1996.
13 E. J. Baran, Biochemistry (Moscow), 2000, 65, 789.
14 I. Bernal, J. Cetrullo and J. Cai, Transtion Met. Chem., 1994, 19, 221.
15 I. Bernal, F. Somoza, Y. Chen and S. S. Massoud, J. Coord. Chem.,
1997, 41, 233.
16 G. M. Sheldrick, SHELXS 97, Programs for Crystal Structure Analysis
(Release 97.2), University of Gottingen (Germany) 1997.
17 L. J. Farrugia, WinGX- A Windows Program for Crystal Structure
Analysis, University of Glasgow, Glasgow, 1998.
L
-histidine, shows the same peak positions for 1H NMR spectra as recorded
in the literature9 and shows a single molecular ion peak at [M + 1] = 156.1.
The solution obtained by mixing b-alanyl- -histidine in absence of metal
L
CHEM. COMMUN., 2003, 612–613
613