Oxidation of l -serine and l -threonine by bis(hydrogen periodato)argentate(III) complex anion: A mechanistic study

JOURNAL OF

Inorganic Biochemistry Journal of Inorganic Biochemistry 101 (2007) 165–172 www.elsevier.com/locate/jinorgbio

Oxidation of L-serine and L-threonine by bis(hydrogen periodato)argentate(III) complex anion: A mechanistic study Hongmei Shi *, Shigang Shen *, Hanwen Sun, Zhanfeng Liu, Liqing Li College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, PR China Received 17 April 2006; accepted 6 September 2006 Available online 19 September 2006

Abstract Oxidation of L-serine and L-threonine by a silver(III) complex anion, [Ag(HIO6)2]5, has been studied in aqueous alkaline medium. The oxidation products of the amino acids have been identified as ammonia, glyoxylic acid and aldehyde (formaldehyde for serine and acetaldehyde for threonine). Kinetics of the oxidation reactions has been followed by the conventional spectrophotometry in the temperature range of 20.0–35.0 C and the reactions display an overall second-order behavior: first-order with respect to both Ag(III) and the amino acids. Analysis of influences of [OH] and [periodate] on the second-order rate constants k 0 reveals an empirical rate 3 expression: k 0 ¼ ðk a þ k b ½OH ÞK 1 =ð½H2 IO3 6 e þ K 1 Þ, where ½H2 IO6 e is equilibrium concentration of periodate, and where 1 1 2 1 ka = 6.1 ± 0.5 M s , kb = 264 ± 6 M s , and K1 = (6.5 ± 1.3) · 104 M for serine and ka = 12.6 ± 1.7 M1 s1, kb = (5.5 ± 0.2) · 102 M2 s1, and K1 = (6.2 ± 1.5) · 104 M for threonine at 25.0 C and ionic strength of 0.30 M. Activation parameters associated with ka and kb have also been derived. A reaction mechanism is proposed to involve two pre-equilibria, leading to formation of an Ag(III)-periodato-amino acid ternary complex. The ternary complex undergoes a two-electron transfer from the coordinated amino acid to the metal center via two parallel pathways: one pathway is spontaneous and the other is assisted by a hydroxide ion. Potential applications of the Ag(III) complex as a reagent for modifications of peptides and proteins are implicated.  2006 Elsevier Inc. All rights reserved. Keywords: L-serine; L-threonine; Ag(III); Oxidation; Kinetics and mechanism

1. Introduction L-serine and L-threonine are non-charged hydrophilic amino acids; the hydrophilic property confers that they are often found on the surfaces of proteins and are often the residues of epitopes for molecular recognition. In biochemistry, metal ion-promoted photooxidative cleavages of some enzymes at some specific serine sites have been reported [1–3]. In bioconjugate chemistry, oxidation of Lserine and/or L-threonine residues at certain positions in proteins and peptides by IO 4 to create an aldehyde group is one of the approaches to modify the proteins and pep-

*

Corresponding authors. Fax: +86 312 5079525. E-mail addresses: [email protected] (H. Shi), shensg@ mail.hbu.edu.cn (S. Shen). 0162-0134/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.09.008

tides [4–9]. Subsequently, the created aldehyde group is used to link other functional molecules. This modification approach appears to be successful in acidic and neutral media [6–9], but cares must be taken to the reaction conditions such as reaction time and temperature. For instance, a long reaction time could lead to the oxidations of most tyrosine and methionine residues in the proteins [10]. It is perhaps not surprising that oxidations of L-serine and L-threonine by various oxidants have been investigated extensively in view of the important biological consequences of the amino acids. Some of the oxidants are metal ions/complexes [11–16] and others are non-metallic agents [17–22]. Although these oxidation reactions display diverse reaction mechanisms, concurrent deamination and decarboxylation to the two amino acids are virtually found in almost all the cases [11–22].

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We have undertaken a careful study of oxidations of L-serine and L-threonine by a silver(III) complex, [Ag(HIO6)2]5, in aqueous alkaline medium. [Ag(HIO6)2]5 is a moderately stable complex and has a square-planner geometry around the metal center [23–26]. The aims of the present study are (a) to identify the oxidation products; (b) to establish a rate law through kinetic measurements and data analysis; (c) to propose a reasonable and convincing reaction mechanism; and (d) to implicate potential applications of the Ag(III) complex in bioconjugate chemistry.

this work. Kinetic traces were followed at 362 nm by the same spectrophotometer mentioned above. Pseudo firstorder reaction conditions were fulfilled by making [amino  acid] P 10[Ag(III)], ½IO 4 tot P 10½AgðIIIÞ, where ½IO4 tot denotes the total concentration of periodate added externally (vide infra). Indeed, under those conditions, kinetic traces could be well described by a single exponential equation, confirming that the reaction is first-order with respect to Ag(III). The observed pseudo first-order rate constants, kobsd, were computed from the kinetic traces, and are reported as average values of three repeated runs. Standard deviations associated with kobsd are usually within 5%.

2. Experimental section 2.3. Analysis of oxidation products 2.1. Chemicals and solutions L-Serine

and L-threonine were obtained from Sigma–Aldrich. AgNO3, KIO4, K2S2O8, KOH, KNO3, formaldehyde, acetaldehyde, glyoxylic acid, 2,4-dinitrophenylhydrazine (DNPH), acrylonitrile and Nessler’s reagent were obtained either from Beijing Chemical Reagent Company (Beijing) or from Tianjin Chemical Reagent Company (Tianjin, China). All the above reagents were of either analytical grade or reagent grade and used as received without further purification. All solutions were prepared with doubly distilled water. An equivalent amount of KOH was used to convert the protonated amine group of serine/threonine to the free amine group in preparation of amino acid stock solutions. Bis(hydrogen periodato)argentate(III) complex anion, [Ag(HIO6)2]5, was synthesized according to the procedure described previously [23]. Electronic spectra of aqueous solution of the Ag(III) complex gave rise to two absorption bands centering at 362 nm and 253 nm, which are in excellent agreement with those reported earlier [24]. Stock solutions of the Ag(III) complex, prepared from the solid state compound obtained [23], was used freshly and daily; their concentrations were determined spectrophotometrically at 362 nm by use of the molar absorptivity of e = 1.26 · 104 M1 cm1 [24]. 2.2. Spectral and kinetic measurements UV–visible spectra were recorded on a TU-1901 spectrophotometer (Beijing, China) and quartz cells with a 1.00 cm optical pathlength were used. The spectrophotometer was equipped with a cell compartment which was thermostated by circulation of water from a thermostat (BG-chiller E10, Beijing Biotech Inc., Beijing). Temperature of solutions in cells can be controlled to ±0.2 C when cells are put in the compartment. Two reaction solutions, one containing known concentrations of Ag(III), KIO4, KOH, and KNO3 and the other containing desired concentrations of serine/threonine and KNO3, were thermostated for at least 20 min before mixing each other. The function of KNO3 was to adjust the ionic strength (l) in the reaction solutions and l = 0.30 M was kept for all the kinetic measurements in

A 100 mL solution containing 0.1 mM Ag(III) and 0.06 M KOH was mixed with another 100 mL solution containing 1 mM L-serine (or L-threonine) and 0.06 M KOH. After disappearance of yellow color from the initial Ag(III), an excess of KI and Na2SO3 was added to the reaction mixture. Adjust pH of the above solution to 5–6 with 1 M HCl; under these conditions, periodate was rapidly reduced to iodate with formation of I2 [5], which was quickly reduced to iodide by sulfite. After aging the solution for a while, precipitates, presumably AgI and AgCl, appeared. The precipitates were filtered off and the filtrate was collected. Take 10 mL the above filtrate and then mixed with several drops of Nessler’s reagent; a yellow color was developed in minutes, indicating that ammonium ion was involved in the filtrate. This observation suggests that a deamination reaction has occurred in the oxidation process. In another test, 50 mL saturated DNPH solution in 2 M HCl was added to 100 mL of the above filtrate; yellow precipitates appeared after aging the mixture for several hours in a refrigerator. Thus, the oxidation reaction produces aldehyde(s) and/or ketone(s), as indicated by the DNPH test. The above Nessler’s test and DNPH test were also carried out against a mixture solution as blank runs; this mixture solution was prepared by the same procedure used to prepare the above filtrate except that 0.2 mM periodate solution was used instead of 0.1 mM Ag(III). Both Nessler’s and DNPH blank tests showed negative results. Since oxidation of serine/threonine by the Ag(III) complex could lead to different aldehede(s)/ketone(s) depending on the mode of interactions, further analysis of the oxidation products by HPLC was carried out. An HPLC system (Shimadzu LC-6 A, Japan) equipped with a UV detector (set at 220 nm), and a C18 column of 0.46 cm · 25 cm (Shim-Pack CLC-ODS, 5 lm in particle size, Shimadzu, Japan) were used. The mobile phase consisted of 20% methanol/80% H2O and the flow rate was set at 1.0 mL/ min. For the filtrate obtained from the Ag(III)-serine reaction, two peaks appeared on the chromatogram at retention times of 2.9 min and 11.7 min, which were identified as formaldehyde and glyoxylic acid, respectively, by comparing the retention times with those obtained from the

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authentic samples. For the filtrate obtained from the Ag(III)-threonine reaction, there were also two peaks shown on the chromatogram with retention times of 6.9 min and 11.7 min. The two peaks were similarly identified as acetaldehyde and glyoxylic acid, respectively. 2.4. Free radical trapping experiment Under the reaction conditions used for kinetic measurements, a 100 mL solution of serine/threonine contained 8% acrylonitrile was mixed with a 100 mL Ag(III) solution in a 3-neck flask; both solutions were flushed for 30 min with nitrogen gas before mixing. By stirring the reaction mixture for 3 h under the protection of nitrogen gas, no precipitates of polyacrylonitrile could be noticed. This observation implies that involvement of free radicals in the reaction course is not likely. 3. Results and discussion 3.1. Oxidation products Oxidation of L-serine and L-threonine by the Ag(III) complex in alkaline medium gives rise to the products described by reactions (1a) and (1b), respectively. Moreover, AgðIIIÞ þ H2 NCHðCH2 OHÞCOO ! AgðIÞ þ NH3 þ CH2 O þ OHCCOO

ð1aÞ

AgðIIIÞ þ H2 NCHðCHðOHÞMeÞCOO ! AgðIÞ þ NH3 þ MeCHO þ OHCCOO

ð1bÞ

a common character in these oxidation reactions is that Ag(III) breaks down the Ca–Cb bond of the amino acids, and accompanied by a deamination reaction. This character is in contrast to the periodate oxidation reactions in which simultaneous deamination and decarboxylation have been observed [19]. 3.2. Second-order kinetics Observed pseudo first-order rate constants, kobsd, as functions of [amino acid], [OH], ½IO 4 tot , and temperature, have been collected and are summarized in Supporting tables (Tables S1–S6) in supplementary material. Plots of kobsd versus [amino acid] are linear and passing through the origin, as shown in Fig. 1, indicating that the redox reactions are also first-order with respect to serine/ threonine. Thus, an overall second-order rate law, described by Eq. (2), is established here, where k 0 represents observed second-order d½AgðIIIÞ=dt ¼ k obsd ½AgðIIIÞ ¼ k 0 ½amino acid½AgðIIIÞ rate constants and k 0 = kobsd/[amino acid].

ð2Þ

Fig. 1. Pseudo first-order rate constants, kobsd, as a function of [amino acid]. Reaction conditions for the top panel: [Ag(III)] = 0.03 mM,  ½IO 4 tot ¼ 1:00 mM, [OH ] = 0.060 M, and l = 0.30 M. Reaction conditions for the bottom panel: [Ag(III)] = 0.03 mM, ½IO 4 tot ¼ 1:00 mM, [OH] = 0.040 M, and l = 0.30 M.

Kinetics and mechanism for oxidation of L-serine and by periodate in acidic media (pH 2.10–5.0) have been reported [19], and the oxidation reactions also follow an overall second-order rate law, first-order in both periodate and the amino acids. The second-order rate constants, derived at 10.0 C, are estimated to be much smaller than those we obtained for the Ag(III) oxidations in the present work. Since variation of the concentration of periodate, the coordinated ligand of the silver(III) complex, has a significant influence on the reaction rates for the Ag(III) reactions (vide infra), the interaction between periodate and the amino acids should be checked in the alkaline medium we used in the present work. Reactions between periodate (1–2 mM) and L-serine/L-threonine (10–20 mM) at [OH] = 0.020 M and 25.0 C were followed spectrophotometrically at 310 nm, the wavelength used for kinetic measurements in acidic media by Pascual and Herraez [19]. Following the reactions for about 4 h, no significant absorbance changes were observed, indicating that the reactions L-threonine

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between periodate and the amino acids were either extremely slow or did not proceed at all in the alkaline medium. 3.3. Protolytic equilibria Under the reaction conditions used in the present work, i.e., [OH]  [amino acid], ½OH   ½IO 4 tot and 0.020 M < [OH] < 0.15 M, we ensure that [OH] remains constant during the reaction course. Protolysis constants of amino acids at 25.0 C and l = 0.1 M have been reported to be: pKa (–COOH) = 2.13 and pK a ð–NHþ 3 Þ ¼ 9:06 for Lserine, and pKa (–COOH) = 2.21 and pK a ð–NHþ 3 Þ ¼ 8:97 for L-threonine [27]. Moreover, in the ionic strength region 0.1 M 6 l 6 1.0 M, these protolysis constants remain essentially the same [27]. It can be calculated from those protolysis data that more than 99.9% of amino acids are existing in the form of H2NCH(CH(OH)R)COO (R = H, Me) in the alkaline medium we used and the contribution of the zwitterionic form to the total concentration of serine/threonine is very trifling and negligible. Several protolytic equilibria have been described for aqueous periodate chemistry [28,29]. The following three equilibria, together with equilibrium constants, were reported at 25.0 C and l = 0.50 M by Aveston when potassium periodate was dissolved in aqueous alkaline medium [29].  4 2IOH 4 þ 2OH  H2 I2 O10

IO 4



þ OH þ

H2 O  H3 IO2 6

 3 IO 4 þ 2OH  H2 IO6

log b1 ¼ 15:05

ð3Þ

log b2 ¼ 6:21

ð4Þ

log b3 ¼ 8:67

ð5Þ

Based on the above equilibrium constants, calculations reveal that under our experimental conditions H2 IO3 6 is the predominant species whereas the dimeric form H2 I2 O4 10 makes a very minor contribution to the total periodate speciation and is negligible. Thus, the total concentration of  periodate, ½IO 4 tot , can be expressed by: ½IO4 tot ¼  2 3 ½IO4 e þ ½H3 IO6 e þ ½H2 IO6 e , where subscript e denotes the equilibrium concentrations. In turn, Eq. (6) can be de rived to express ½H2 IO3 6 e as a fraction of ½IO4 tot from equilibria (3) and (4).   ½H2 IO3 6 e ¼ fð½OH Þ½IO4 tot

ð6aÞ

where

Fig. 2. Second-order rate constants k 0 (=kobsd/[amino acid]) as a function of [OH] at four temperatures. Reaction conditions: [Ag(III)] = 0.03 mM, ½IO 4 tot ¼ 1:00 mM, and l = 0.30 M.

½IO 4 tot at variable temperatures are displayed in Fig. 3. This kind of reaction trend, observed repeatedly in the oxidation of different substrates by [Ag(HIO6)2]5 [30–32], has been interpreted in terms of a pre-equilibrium in which [Ag(HIO6)2]5 equilibrates with a mono-periodate coordinated silver(III) complex. In a previous study of oxidation of triethanolamine by Ag(III) [31], reaction (7) was K1



 2



 2

f ð½OH Þ ¼ b3 ½OH  =ð1 þ b2 ½OH  þ b3 ½OH  Þ

ð6bÞ

3.4. Influence of [OH] and ½IO 4 tot on the reaction rates  0 At constant ½IO 4 tot , plots of k versus [OH ] are linear at different temperatures (shown in Fig. 2), and moreover, well-defined intercepts can be obtained from the linear plots. These characteristics are indicative that the oxidation reactions proceed via two reaction routes: one route is independent of OH and the other is facilitated by OH. At constant [OH], oxidation rates decrease with 0 increasing ½IO 4 tot ; the rate constants k as a function of

½AgðHIO6 Þ2 5 þ 2H2 O  ½AgðHIO6 ÞðOHÞðH2 OÞ2 þ H2 IO3 6 ð7Þ

suggested to be the pre-equilibrium (structure of [Ag(HIO6)2]5 [25] and the proposed structure of [Ag(HIO6)(OH)(H2O)]2 [31] are shown in Scheme 1 below). Moreover, it was assumed that [Ag(HIO6)(OH)(H2O)]2 was the reactive species whereas [Ag(HIO6)2]5 was virtually non-reactive. Provided that this assumption is also valid in the present work, we have found that correlations between k 0 and ½IO 4 tot at different temperatures can be simulated by an expression k 0 ¼ CK 1 =ð½H2 IO3 6 e þ K 1 Þ, where K1 is defined by reaction

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169

these curve-fittings, it is assumed that the fraction f([OH]) defined by Eq. (6b) remains unchanged in the temperature range used in the present work. Values of K1 at variable temperatures derived from the curve-fittings are listed in Table 1. After obtaining K1 values, rearrangement of Eq. (8b) renders:  k 0 ff ð½OH Þ½IO 4 tot þ K 1 g=K 1 ¼ k a þ k b ½OH 

ð9Þ

Fig. 4 shows the linear dependence of k 0 ff ð½OH Þ½IO 4 tot þ K 1 g=K 1 on [OH] according to Eq. (9); values of ka and kb have been calculated from the intercepts and slopes, respectively, by least-squares routine and are summarized in Table 1. Subsequently, activation parameters associated with ka and kb have been calculated from the Eyring’s plots shown in Fig. 5, and values of DH6¼ and DS6¼ are listed in Table 1 as well. 3.5. Mechanistic interpretations

Fig. 3. Second-order rate constants k 0 as a function of ½IO 4 tot at temperatures of 20.0 C (a), 25.0 C (b), 30.0 C (c) and 35.0 C (d). Reaction conditions: [Ag(III)] = 0.03 mM, [OH] = 0.060 M, and l = 0.30 M for the L-serine reaction; [Ag(III)] = 0.03 mM, [OH] = 0.040 M, and l = 0.30 M for the L-threonine reaction. The solid curves represent the best fits of Eq. (8b) to the experimental data by use of a nonlinear least-squares method.

It is clear from the data in Table 1 that the K1 values at variable temperatures are actually the same within the standard deviations. Moreover, the K1 values derived from the L-serine reaction are well agreed by those calculated from the L-threonine reaction. The K1 values are also in excellent agreement with those reported previously (K1 = 6.15 · 104 M at 25.0 C and 6.18 · 104 M at 30.0 C and l = 0.30 M) in the oxidation of triethanolamine by Ag(III) [31]. The consistency of the K1 values obtained from different reaction systems supports the pre-equilibrium, i.e., reaction (7) proposed earlier [31]. Based on the empirical rate expression, a straightforward reaction mechanism is put forward which involves reaction (7), and two ratedetermining reactions (10) and (11), where R = H for L-serine and R = Me for L-threonine. ½AgðHIO6 ÞðOHÞðH2 OÞ

2 ki

þ H2 NCHðCHðOHÞRÞCOO ! Products ½AgðHIO6 ÞðOHÞðH2 OÞ

ð10Þ

2

þ H2 NCHðCHðOHÞRÞCOO þ OH k ii

! Products



(7) and C is a constant. By combining the effects of [OH ] and ½IO 4 tot on the reaction rates together, Eq. (8) is deduced as an empirical rate expression, where ka and kb are rate parameters and will be discussed below. k 0 ¼ ðk a þ k b ½OH ÞK 1 =ð½H2 IO3 6 e þ K 1 Þ

ð8aÞ

Eq. (12) is the rate law derived from the reaction mechanism in terms of the total concentration of Ag(III). Eq. (13) is equivalent to the empirical rate expression/Eq. (8a) if we assume ki = ka and kii = kb.

ð8bÞ

d½AgðIIIÞ ðk i þ k ii ½OH ÞK 1 ½amino acid½AgðIIIÞ ¼ dt ½H2 IO3 6 e þ K 1

or k 0 ¼ ðk a þ k b ½OH ÞK 1 =ff ð½OH Þ½IO 4 tot þ K 1 g

ð11Þ

Eq. (8b) was utilized to fit the k 0  ½IO 4 tot dependence data by non-linear least-squares method with K1 and the term, ka + kb[OH], as two adjustable parameters. The resultant fittings are good and displayed also in Fig. 3; in

ð12Þ or k 0 ¼ ðk i þ k ii ½OH ÞK 1 =ð½H2 IO3 6 e þ K 1 Þ

ð13Þ

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O

O I O

O

O

O I

O

O

O

OH

3-

OH H2N-CH-COO-

OH2

K2

+

Ag

+ H2 IO63-

O 2-

OH O

OH2 Ag

I

O

O

O

O

K1

+ 2H2 O

I O

2-

OH

O

O Ag

O

5-

OH

OH

OH

O

O I

HO-CHR

O

O

NH2

CH-COO

+ 2H2 O

Ag O

O

CHR

O 3-

OH O

O I

NH2

CH-COO

k1

Ag

O

O

O

+

Ag(I) + RCHO + H2N-CH-COO-

CHR

O 3-

OH O

O I

NH2

CH-COO

Ag

O

O

O

+ OH-

k2

Ag(I) + NH3 + RCHO + -OOC-CHO

CHR

O

fast

+

H2N-CH-COO- + OH-

NH3 + -OOC-CHO

(R = H for L-serine; R = Me for L-threonine) Scheme 1. Proposed reaction mechanism.

Table 1 Rate constants, equilibrium constants and activation parameters for oxidation of L-serine and L-theronine by Ag(III) at l = 0.30 M Amino acid

t (C)

104K1 (M)

L-serine

20.0 25.0 30.0 35.0

7.1 ± 1.0 6.5 ± 1.3 6.3 ± 1.0 6.6 ± 0.4

1 DH 6¼ a ¼ 29:2  0:8 kJ mol

L-threonine

1 DH 6¼ a ¼ 39  5 kJ mol

1 DS 6¼ mol1 a ¼ 132  5 J K

20.0 25.0 30.0 35.0 1 DS 6¼ mol1 a ¼ 63  15 J K

ka (M1 s1) 4.8 ± 0.3 6.1 ± 0.5 7.4 ± 0.9 9.1 ± 0.8

1 DH 6¼ b ¼ 32  3 kJ mol

5.3 ± 0.8 6.2 ± 1.5 5.8 ± 0.7 4.8 ± 0.6

Although this reaction mechanism can explain the experimental rate law, it is disfavored by two concerns. The first concern comes from the third-order reaction route described by reaction (11). This reaction route requires a concurrent collision by the three reacting species described in the reaction. This kind of collision is, however, rarely happened in reality, making this reaction route unlikely. The second concern stems from considerations of the oxidation products. In fact, the two reaction routes described by reactions (10) and (11) are required to result

194 ± 4 264 ± 6 331 ± 10 386 ± 10

1 DS 6¼ mol1 b ¼ 92  11 J K

9.6 ± 0.4 12.6 ± 1.7 16.7 ± 1.0 25.4 ± 3.1

1 DH 6¼ b ¼ 45  4 kJ mol

kb (M2 s1)

(3.7 ± 0.2) · 102 (5.5 ± 0.2) · 102 (6.4 ± 0.2) · 102 (8.8 ± 0.5) · 102

1 DS 6¼ mol1 b ¼ 71  14 J K

in a deamination action; this requirement needs that [Ag(HIO6)(OH)(H2O)]2 directly attacks amine groups of the amino acids. This mode of deamination will lead probably to formation of ketones at the a-carbons of the amino acids rather than to the breaking down of Ca–Cb bond as we found. Alternatively, a more reasonable reaction mechanism is proposed and illustrated in Scheme 1 in which equilibrium (7) is incorporated. In this mechanism, another pre-equilibrium in which [Ag(HIO6)(OH)(H2O)]2 reversibly forms

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Fig. 5. Eyring’s plots for the rate parameters, ka and kb, as defined by Eq. (8).

 Fig. 4. Plots of k 0 ff ð½OH Þ½IO 4 tot þ K 1 g=K 1 versus [OH ] at four temperatures. Reaction conditions are the same as those described in Fig. 2.



d½AgðIIIÞ ðk 1 þ k 2 ½OH ÞK 1 K 2 ½amino acid½AgðIIIÞ ¼ dt ½H2 IO3 6 e þ K 1 ð15Þ

k 0 ¼ ðk 1 K 2 þ k 2 K 2 ½OH ÞK 1 =ð½H2 IO3 6 e þ K 1 Þ chelate ternary complexes with the amino acids is suggested. The ternary complexes undergo an inner-sphere two-electron transfer from the coordinated amino acids to the metal center through two parallel pathways; one pathway (described by k1) is spontaneous and the other (described by k2) is assisted by a hydroxide ion. This onestep two-electron transfer mode is substantiated by our free radical trapping experiment. According to Scheme 1, a rate law in terms of the total concentration of Ag(III) can be deduced as Eq. (14). 

d½AgðIIIÞ ðk 1 þ k 2 ½OH ÞK 1 K 2 ½amino acid½AgðIIIÞ ¼ dt ½H2 IO3 6 e þ K 1 þ K 1 K 2 ½amino acid ð14Þ

Eq. (14) can be simplified to Eq. (15) if K1K2[amino acid]  K1, i.e., K2[amino acid]  1 (cf. discussions below); the second-order rate constants k 0 are expressed consequently by Eq. (16).

ð16Þ

If we assume that k1K2 = ka and k2K2 = kb, Eq. (16) will be identical to the empirical rate expression/Eq. (8a). Apparently, the two concerns raised earlier are no longer problematic any more for the mechanism described in Scheme 1. In fact, this reaction mechanism is very similar to that proposed earlier for Ag(III)-triethanolamine reaction [31]. However, the kinetic data for the triethanolamine reaction strictly fit Eq. (14) when [amino acid] in the equation is replaced by [triethanolamine]; in other words, there was kinetic evidence supporting formation of the ternary chelate complex and the equilibrium constant K2 was thus derived from the kinetic data (K2 = 537 M1 at 25.0 C and l = 0.30 M). In the present reaction systems, K2 values are probably much smaller, validating the assumption of K2[amino acid]  1. Since k1K2 = ka and k2K2 = kb, we know that 6¼ 6¼ 6¼ 6¼ 0 0 DH 6¼ a ¼ DH 1 þ DH 2 and DS a ¼ DS 1 þ DS 2 ; and DH b ¼ 6¼ 6¼ 6¼ 0 0 0 DH 2 þ DH 2 and DS b ¼ DS 2 þ DS 2 , where DH 2 and DS 02

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are standard reaction enthalpy and entropy, respectively, associated with pre-equilibrium constant K2. Moreover, DH 02 and DS 02 are probably having negative values as expected from the reaction nature. This can explain why 6¼ our measured values of DH 6¼ a and DH b are relative small ¼ 6 whereas DS 6¼ a and DS b have relatively large negative values. 3.6. Concluding remarks Undoubtedly, the proposed reaction mechanism, illustrated in Scheme 1, can explain convincingly all the experimental observations. Moreover, oxidation reactions of L-serine and L-threonine by the Ag(III) complex have displayed two attractive features. These include (1) the reactions take place in alkaline medium and (2) the Ag(III) complex selectively breaks down the Ca–Cb bonds, leading to formation of two types of aldehydes. These features implicate that the Ag(III) complex might be used as a reagent for modifications of peptides and proteins in alkaline medium, complementing to the current methodologies [6–9]. Studies are in progress toward this direction. Acknowledgement Financial support of this work in part by a grant from the Natural Science Foundation of Hebei Province (B2006000962) is gratefully acknowledged. Appendix A. Supplementary material Supporting tables S1–S6 summarize the pseudo firstorder rate constants measured under various reaction conditions. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jinorgbio.2006.09.008. References [1] C.R. Cremo, J.C. Grammer, R.G. Yount, J. Biol. Chem. 264 (1989) 6608–6611.

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