A substrate-specific α-hydoxylation of dipeptides mediated upon a Co(III)-terpyridine complex: A functional model for peptidylglycine α-hydroxylating monooxygenase

Article abstract of DOI:10.1246/cl.2001.30

A substrate-specific α-hydroxylation of dipeptides has been found out as a functional model for peptidylglycine α-hydroxylating monooxygenase (PHM), in the reaction of the Co(III) ternary complexes containing terpyridine and dipeptide ligands under aerobic and slightly alkaline conditions.

Full text of DOI:10.1246/cl.2001.30

30
Chemistry Letters 2001
A Substrate-Specific α-Hydoxylation of Dipeptides Mediated upon a Co(III)-terpyridine
Complex: A Functional Model for Peptidylglycine α-Hydroxylating Monooxygenase
Koichiro Jitsukawa,* Tsuyoshi Irisa, Hisahiko Einaga, and Hideki Masuda*
Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555
(Received October 19, 2000; CL-000952)
A substrate-specific α-hydroxylation of dipeptides has
been found out as a functional model for peptidylglycine α-
hydroxylating monooxygenase (PHM), in the reaction of the
Co(III) ternary complexes containing terpyridine and dipeptide
ligands under aerobic and slightly alkaline conditions.
The biologically active peptides, such as hormones, neuro-
transmitters and growth factors, carry an amide group at their
carbonyl termini, of which nitrogen atom is derived from the
terminal glycine residue of the precursor.1–3 Peptidylglycine α-
amidating monooxygenase (PAM) catalyzes oxidative removal
of the two carbon atoms from a glycine-extended precursor
through a two-step process.4 At the first step, peptidylglycine
α -hydroxylating monooxygenase (PHM) catalyzes an α -
hydroxylation of C-terminal glycine and the second enzyme,
peptidylamido-glycolate lyase (PAL), promotes decomposition
of the hydroxylated amimo acid residue to a peptide amide.
Recently, an X-ray analysis of the active site of PHM with a
substrate revealed an intriguing picture of its catalytic machin-
ery.5 There have been several reports on the catalytic activities
of PAM in the transformation of the biological compounds.6–8
However, the PHM model reaction for oxidative hydroxylation
of C-terminal peptide by a metal complex has scarcely been
reported on account of difficulty of control of molecular oxy-
gen.9 In the course of study on the transformation of amino
acid, we previously discovered some interesting hydroxylation
mediated on the Co(III) complex under aerobic condition.10–12
Moreover, the novel type transamination from salicylidene–ala-
nine to α-hydroxy salicylidene-o-hydroxybenzylamine was pro-
moted by the ternary complexes with 2,2':6',2"-terpyridine
(terpy) ligand.13 On the basis of these findings, we have carried
out an α-hydroxylation of dipeptide (dp), which is expected to
be accelerated by the π-orbital character of terpy on the ternary
cobalt complex.14 Here, we describe the substrate-specific α-
hydoxylation of dipeptide mediated upon the Co(III)-terpy
complex as a functional model for PHM.
1
lows: In the H NMR spectra, a quartet peak of alanine α-pro-
ton for 1a observed at 4.94 ppm disappeared completely in 2a
and a doublet peak of the alanine methyl protons for 1a at 1.95
ppm was replaced to a singlet one at 2.24 ppm, although the
other proton peaks did not show any significant difference
between them. Clearly, the α-position of C-terminal amino
acid residue was substituted by the other functional group after
the reaction. In order to elucidate its functional group, FAB-
mass spectrum of 2a was measured and compared with that of
1a. Although a parent peak of complex 1a was observed at m/z
= 436, that in 2a was detected at 452. The increase of 16 mass
numbers suggests that an oxygen atom was incorporated in the
product complex. Interestingly, this hydroxylation has exhibit-
ed a substrate-specificity for dipeptides employed, as shown in
Figure 1. The reactions of 1a, 1d, 1e, and 1f gave the Co(III)
complexes containing glycyl-α-hydroxy amino acid (2a, 2d, 2e,
and 2f), while those of 1b and 1c with a bulky aliphatic side
chain scarcely afforded such a hydroxylated complex. Also, the
complexes with aromatic side chain (1d and 1e) were quickly
oxidized, and the formation rate of 2f was the fastest of all.
Notably, this hydroxylation proceeds neither under argon
atmosphere in slightly alkaline media nor under an aerobic con-
dition in slightly acidic or neutral media. Under higher pressure
of oxygen, the yield of the hydroxylated complexes increased;
those of 2a after 12 h under oxygen pressures of 1 and 2 atms at
pH 10.5 were 53 and 73%, respectively, although it was only 20
% under atmospheric condition. The reaction was also depend-
ent on the concentration of hydroxide ion; the yield of 2a under
atmospheric condition was 8% at pH 10.0, whereas those were
raised up to 20 and 29 % at pH 10.5 and 11.0, respectively. At
pH 9.5, no oxidized product was obtained, whereas at higher
pH under aerobic condition, the proportion of [Co(terpy)2]3+
complex in the products increased. It is sure that this reaction
requires both molecular oxygen and hydroxide ion, although the
detailed mechanism is not clear yet. In addition, the character-
istic CD spectrum (∆ε = –0.9 at 19kcm–1, ∆ε = –0.3 at 27kcm–1,
and ∆ε = +1.7 at 33kcm–1) observed in 1a disappeared in the
course of hydroxylation to 2a, indicating that the C-terminal α-
position of 1a, an asymmetric center, was attacked by some
species during the reaction. Such a reaction did not proceed in
the Co(III) ternary complexes with N-methyldipicolylamine
The starting Co(III) ternary complexes, [Co(dp)(terpy)]+
(1), were prepared by the reaction of [Co(CO3)(OH)(terpy)]15
(2 mmol) with an equimolar amount of dp (dp = gly–ala (a),
gly–val (b), gly–leu (c), gly–phe (d), gly–trp (e), and gly–phg
(f)) at pH 8.5 and 40°C for 6 h in an aqueous solution (50 mL).
After treating with SP Sephadex C-25 column (H+ form, 3.6
cmφ × 28 cm) with 0.1 M NaCl solution and desalting with
methanol, red colored complexes, [Co(dp)(terpy)]+ (1a–1f),
1
were obtained.16 Their structures were confirmed by the H
NMR and electronic absorption spectra.
The treatment of 1a at pH 10.5 and 40 °C under aerobic
condition for 12 h gave a ternary Co(III) complex (2a) contain-
ing glycyl-α-hydroxy-alanine (gly-Oala) ligand, whose struc-
ture was determined by 1H NMR and FAB-mass spectra as fol-
Copyright © 2001 The Chemical Society of Japan

Chemistry Letters 2001
31
We thank Chemical Material Centre, the Institute for
Molecular Science, for measurement of mass spectra. This
research was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and
Culture.
References and Notes
1
2
3
4
5
6
7
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8
9
instead of terpy, which may be attributable to the terpy ligand
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The most interesting finding in this reaction is a substrate-
specific α-hydroxylation of dipeptides as mentioned above.
We can consider that the interligand interaction between the C-
terminal amino acid sidechain and terpy ring is one of the possi-
ble control factors. Because the solvent effect of the NMR
chemical shift has been observed for the starting complexes of
1b and 1c. That is, the α-H peak of leucine residue of 1c
observed at 4.88 ppm in D2O exhibited a higher-field shift to
4.74 ppm in less polar solvent (D2O:dioxane-d8 = 1:3) although
α-H at 2.19 pm and δ-Hs at 1.08 and 1.16 ppm shifted to lower-
field region (2.29 ppm and 1.08, 1.19 ppm), respectively. This
finding indicates that the α- and β-Hs in 1c are located at the
just side of terpy ring and the γ- and δ-Hs of leucine residue are
situated over the ring. Such a characteristic interaction was not
detected in the complexes 1a, 1d, 1e, and 1f. The hydrophobic
interaction in an aqueous solution will be much stronger and the
shielding effect will be enhanced.21 This substrate-specific
hydroxylation will be explainable that an intramolecular inter-
action between the alkyl sidechain of C-terminal amino acid
and terpy ligand prevents the approach of nucleophilic reagent
such as OH– that can abstract the α-H of dp.
In conclusion, the PHM model reaction, the oxidative trans-
formation of C-terminal amino acid residue, has been modeled on
the ternary Co(III) complexes with terpy and dipeptide under the
presence of molecular oxygen in slightly alkali media, which fur-
thermore demonstrates the substrate specificity.
21 M. Mizutani, S. Tomosue, H. Kinoshita, K. Jitsukawa, H.
Masuda, and H. Einaga, Bull. Chem. Soc. Jpn., 72, 981
(1999).