Formation of Schiff Bases of O-Phosphorylethanolamine and O

Article pubs.acs.org/JPCA

Formation of Schiff Bases of O‑Phosphorylethanolamine and O‑Phospho‑D,L‑serine with Pyridoxal 5′‑Phosphate. Experimental and Theoretical Studies Bartolomé Vilanova,* Jessica M. Gallardo, Catalina Caldés, Miquel Adrover, Joaquín Ortega-Castro, Francisco Muñoz, and Josefa Donoso Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Departament de Química, Universitat de les Illes Balears, Ctra Valldemosa km 7.5, E-07122 Palma de Mallorca, Spain S Supporting Information *

ABSTRACT: Pyridoxal 5′-phosphate (PLP) is a B6 vitamer acting as an enzyme cofactor in various reactions of aminoacid metabolism and inhibiting glycation of biomolecules. Nonenzymatic glycation of aminophospholipids alters the stability of lipid bilayers and cell function as a result. Similarly to protein glycation, aminophospholipid glycation initially involves the formation of a Schiff base. In this work, we studied the formation of Schiff bases between PLP and two compounds mimicking the polar head of natural aminophospholipids, namely: O-phosphorylethanolamine and O-phospho-D,L-serine. Based on the results, the pH-dependence of the microscopic constants of the two PLP−aminophosphate systems studied is identical with that for PLP−aminoacid systems. However, the rate and equilibrium formation constants for the Schiff bases of the aminophosphates are low relative to those for the aminoacids. A theoretical study by density functional theory of the formation mechanism for the Schiff bases of PLP with the two aminophospholipid analogues confirmed that the activation energy of formation of the Schiff bases is greater with aminophosphates; on the other hand, that of hydrolysis is essentially similar with aminoacids and aminophosphates.

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Ravandi et al. 6,10 and Pamplona et al. 11 provided experimental evidence of aminophospholipid glycation in vivo. Subsequently, Fountain et al.12 quantified glucose-linked PE and PS in samples from diabetic patients. In 2001, BreitlingUtzmann et al.13 identified and quantified Schiff−PE and Amadori−PE adducts in erythrocytes from healthy and diabetic individuals by liquid chromatography−electrospray mass spectrometry. In recent years, Miyazawa and co-workers8,14,15 have improved the analysis of Amadori-glycated phosphatidylethanolamine in human erythrocytes and blood plasma by mass spectrometry. Glycation of lipid membranes can inactivate membranebound enzymes, induce their cross-linking and peroxidation, and eventually lead to cell death.16 The high biological significance of lipid glycation to various diseases has raised growing interest in finding effective inhibitors for the process.17−19 Pyridoxal 5′-phosphate is a B6 vitamer functioning as an enzyme cofactor in various reactions of aminoacid metabolism;20,21 also, it can inhibit biomolecular glycation.22 Taguchi et al.23 found the joint administration of PLP and aminoguanidine (an inhibitor of protein glycation) to

INTRODUCTION Nonenzymatic glycation of biomolecules by reducing sugars and other carbonyl compounds causes irreversible changes in their properties. Glycation occurs naturally in the human body during aging and is the main culprit of some hyperglycemiarelated diseases.1,2 Whereas the nonenzymatic glycation of proteins has been the subject of much study,1,3,4 that of aminophospholipids has received considerably less attention. In 1993, Bucala et al.5 showed the aminophospholipid phosphatidylethanolamine (PE), which is present in mammal cell membranes, to react with glucose and initiate its glycation. Subsequent in vitro studies showed the reactions of PE and phosphatidylserine (PS) with glucose to develop via the general glycation mechanism previously proposed for proteins (see Scheme 1).6,7 The first step in the process is the reversible formation of a Schiff base that subsequently rearranges to form more stable compound: a ketoamine known as an “Amadori compound”. Amadori compounds can give various reactions leading to the formation of a heterogeneous body of compounds known as “advanced glycation products” (AGEs). Oxidation of Schiff bases and/or Amadori compounds produce free radicals capable of peroxidizing lipids and giving advanced lipoxidation end-products (ALEs).5,6,8,9 © 2012 American Chemical Society

Received: December 2, 2011 Revised: January 26, 2012 Published: January 26, 2012 1897

dx.doi.org/10.1021/jp2116033 | J. Phys. Chem. A 2012, 116, 1897−1905

The Journal of Physical Chemistry A

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Scheme 1

substantially reduce AGE formation in vitro. Also, Miyazawa and co-workers14,24 found PLP to inhibit lipid glycation in vivo via the formation of protective Schiff bases. They detected PE− PLP adducts in red cells and showed the dietary administration of PLP to diabetic rats to reduce their levels of Amadori−PE adducts. Few studies have examined the mechanisms behind lipid glycation or its inhibition despite their significance. In recent work, we modeled the lipid monolayer in theoretical terms.25 To this end, we examined the formation mechanism of the Schiff

base between an acetaldehyde molecule and the amino group in phosphatidylethanolamine, and found the phosphate anion and amino group present in aminophospholipids to facilitate the formation of Schiff bases on the surface of the lipid membrane via a neighboring catalytic effect. A previous study on the formation of Schiff bases between compounds mimicking the polar head of biological aminophospholipids and various carbonyl compounds with glycating properties26 showed aminophosphates to react more efficiently with PLP than with glycating carbonyl compounds. 1898



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units. pH measurements were made by using a Crison pHmeter equipped with a glass electrode at 25 °C. Determination of Schiff Base Formation Rate Constants. The overall reaction between an aldehyde and an amine can be depicted as follows:

Accordingly, rather than evolving to Amadori compounds, aminophosphates tend to form stable Schiff bases with PLP and hence to prevent glycation. These results confirm the inhibitory role of PLP in lipid glycation. The high biological significance of B6 vitamers in inhibiting biomolecular glycation27,28 led us to examine the reactivity of aminophospholipids with PLP by using two structurally simplified derivatives, namely: O-phosphorylethanolamine and O-phospho-D,L-serine. We calculated the kinetic formation and hydrolysis constants for the Schiff bases of the two model compounds with PLP. The fact that the formation of a Schiff base substantially alters the UV−vis absorption spectrum for PLP29−32 facilitated the kinetic study of the process. Experimental tests were performed over a wide pH range to expose the reactivity of the different ionic forms of the reactants. The calculated constants were compared with reported values for PLP and primary amines or aminoacids. A theoretical study based on density functional theory (DFT) of the formation of Schiff bases between PLP and the model aminophospholipids allowed the results to be interpreted and the significance of intramolecular hydrogen bonds in some of the compounds involved in the process to be assessed.

k1

R1CHO + NH2R2 ↽ ⎯⎯⎯⇀ ⎯⎯⎯ R1CHNR2 + H2O k−1

(1)

where k1 and k−1 are the rate constants of formation and hydrolysis, respectively, of the Schiff base, which were calculated as described elsewhere.34−36 The equilibrium constant was calculated as their ratio: KpH = k1/k−1. The overall formation and hydrolysis rate constants for Schiff bases can be expressed in terms of the elemental constants for each chemical species present in the medium at a given pH. Scheme 2 shows the different chemical species for the Scheme 2

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EXPERIMENT AND METHODS Reagents. Pyridoxal 5′-phosphate hydrate (PLP), O-phosphorylethanolamine (PEA) and O-phospho-D,L-serine (PSer) were purchased from Sigma−Aldrich; potassium dihydrogen phosphate, potassium hydrogen carbonate, potassium chloride, sodium acetate and sodium hydroxide solutions were obtained from Across Organics. All reagents were used as received. Acetate, phosphate and carbonate buffers were used over appropriate pH ranges. The buffer concentration used was typically 0.04 M and the ionic strength kept at 0.4 M by adding appropriate amounts of KCl to the medium. PLP solutions were prepared in appropriate buffers and stored in the dark. Their exact concentrations, which ranged from 1 × 10−4 to 2 × 10−4 M, were determined by dilution in 0.1 M NaOH and subsequent measurement of the absorbance at 388 nm (ε = 6600 M−1 cm−1). Aminophosphate solutions spanning the concentration range 5 × 10−4−2 × 10−2 M were prepared on a daily basis by diluting the required amounts of previously made stock solutions in an appropriate buffer. Reaction Mixtures for the Kinetic Study of Schiff Base Formation between Aminophosphates and PLP. Kinetic measurements were made at a variable pH on a Shimadzu UV-2401 PC double-beam spectrophotometer. The background spectrum for the buffer solution was used as spectral reference. Quartz cells of 1 cm path length were used to obtain electronic spectra. The cell temperature was kept at 25 ± 0.1 °C by using a Shimadzu TCC-240A thermostat. The reaction was started by adding a known volume of PLP buffered solution to prethermostated aminophosphate solutions at the desired pH. Pseudo first-order kinetic curves were obtained by monitoring the absorbance at 420 nm during the formation of a Schiff base of PLP with a 10-fold concentration of the corresponding aminophosphate. The observed rate constants, kobs, were determined by using the software Sigma Plot33 for first-order reactions. The difference between the initial and final pH in the reaction cell never exceeded 0.03

aminophosphates, PLP and the corresponding Schiff bases over the pH range 5.0−10.0. PLP and SB in the Scheme denote pyridoxal 5′-phosphate and Schiff base, respectively. No hydrated forms of PLP were considered as they are only significant below pH 5.37 1899



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using 10 mL of a 0.01 M aqueous solution of each compound adjusted to a total ionic strength of 0.1 M at 25 °C. The titrant was a standardized 0.1 M NaOH solution. Titrations were performed in duplicate and equilibrium ionization constants calculated by using Sigma Plot v. 10.0.33 The resulting values for PEA were pK1 = 5.7 ± 0.1 (phosphate group) and pK2 = 10.4 ± 0.1 (amino group), and those for PSer pK0 = 2.25 ± 0.08 (carboxyl group), pK1 = 6.0 ± 0.1 (phosphate group), and pK2 = 10.30 ± 0.05 (amino group). Computational Details. DFT calculations were done with the Gaussian 09 software package.38 All structures were fully optimized at the M06-2X level,39,40 using the 6-31+G(d,p) basis set in combination with the solvation model density (SMD)41 to mimic the water solvent effect. The M06-2X functional is a hybrid meta-exchange correlation functional recently proposed by Zhao and Truhlar and recommended for applications involving kinetic and noncovalent interactions.39,40,42 SMD is a universal solvation model based on the polarized continuous quantum mechanical charge density of the solute that provides accurate estimates of solvation free energies. The structures obtained were subjected to vibrational analysis calculations toward their characterization as local minima (all positive force constants) or transition states (one imaginary force constant only). Intrinsic Reaction Coordinate (IRC) calculations were carried out at the SMD/M06-2X/631+G(d,p) level of theory and we confirm that the transition states structures connect to reactants and products (IRC curves are available in the Supporting Information). Besides, we found that the free energy of reactants and products obtained previously is lower than that obtained in the IRC calculations, because of the mobility of the system and in particular to the mobility of the water molecule within the system in all the mechanisms proposed in this study.

Scheme 2 was used to derive the following equations relating the overall formation or hydrolysis rate constants for the Schiff bases to the microscopic constants for each ionic species ⎧ ⎪ ⎪ [H O+] [H O+]2 [H3O+]3 ⎫ ⎬ + k12 3 + k11 k1 = ⎨k14 + k13 3 ⎪ K2P K2PK1P K2PK1PK 0P ⎪ ⎭ ⎩ ⎧⎛ ⎪ [H3O+] [H O+]2 ⎞⎛ [H3O+] ⎟⎟⎜⎜1 + + 3 /⎨⎜⎜1 + ⎪ K2 K2K1 ⎠⎝ K2P ⎩⎝ +

⎪ [H3O+]2 [H3O+]3 ⎞⎫ ⎟⎟⎬ + ⎪ K2PK1P K2PK1PK 0P ⎠⎭

(2)

+ +2 ⎧ ⎪ 2 [H3O ] 4 3 [H3O ] + + k−1 = ⎨ k k k 1 − 1 − 1 − ⎪ K3SB K3SBK2SB ⎩ 1 + k− 1

⎫ ⎪ [H3O+]3 [H3O+]4 ⎬ + k−0 1 ⎪ K3SBK2SBK1SB K3SBK2SBK1SBK 0SB ⎭

+ ⎧ ⎪ [H O ] [H3O+]2 [H3O+]3 3 + + /⎨ ⎪ K3SBK2SB K3SBK2SBK1SB ⎩ K3SB

+

⎫ ⎪ [H3O+]4 ⎬ ⎪ K3SBK2SBK1SBK 0SB ⎭

(3)

k1i

where denotes the formation constant for the Schiff base of each ionic species, K1 and K2 are the ionization equilibrium constants for the aminophosphates, and KiP the ionization equilibrium constants for PLP. Based on this scheme, PLP2 can form two differently protonated Schiff bases. Since SB4 and SB3 differed in the protonation status of the phosphate group in the amino compound, we assumed the formation constants for the two i bases to be identical. k−1 and KiSB in eq 3 represent the hydrolysis and ionization constant, respectively, of each Schiff base. Scheme 2 also allowed the following equation relating the equilibrium formation constant of the Schiff base (KpH) to the ionization constants for the Schiff bases, PLP and aminophosphates

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RESULTS AND DISCUSSION Formation of PLP−Aminophosphate Schiff Bases. Figure 1 shows the variation of log k1 for the Schiff bases of

⎧ ⎛ ⎪ [H3O+] [H3O+]2 [H3O+]3 + + K pH = ⎨⎜⎜1 + ⎪ K3SB K3SBK2SB K3SBK2SBK1SB ⎩⎝ +

⎞ ⎫ ⎪ [H3O+]4 ⎟⎟KM ⎬ ⎪ K3SBK2SBK1SBK 0SB ⎠ ⎭

⎧ ⎛ ⎪ [H3O+] [H O+]2 [H3O+]3 ⎞ ⎟ + 3 + /⎨⎜⎜1 + ⎪ K2P K2PK1P K2PK1PK 0P ⎟⎠ ⎩⎝ ⎛ ⎪ [H3O+] [H O+]2 ⎞⎫ ⎟⎟⎬ × ⎜⎜1 + + 3 ⎪ K K K 2 2 1 ⎠⎭ ⎝

(4)

where KM is the equilibrium formation constant for the Schiff 4 base at a very high pH (= k14/k−1 , see Scheme 2). The experimental data for k1, k−1, and KpH were fitted to eqs 2−4 by using the software Sigma Plot.33 Determination of Equilibrium Ionization Constants. The ionization constants for PEA and PSer were determined by potentiometric titration on a Metrohm Titrino 718 pH-Stat,

Figure 1. Variation of log k1 as a function of pH for the Schiff bases of PLP with (●) O-phosphorylethanolamine and (▲) O-phospho-D,Lserine. Points are experimental values and solid lines represent the theoretical fitting to eq 2.

PLP with O-phosphorylethanolamine (PEA) and O-phosphoD,L-serine (PSer) as a function of pH. The variation was similar 1900



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to that previously observed in Schiff bases of PLP with aminoacids and primary amines.34,43,44 However, the change over the pH range 5.0−10.0 was less than 1 unit for the PLP− PSer system but amounted to almost two units for the PLP− PEA system. The individual kinetic formation constants of the Schiff bases of the different ionic species in Scheme 2 were obtained by fitting k1 to eq 2. Figure 1 shows the best fit of the experimental data and Table 1 the values of the calculated constants in Table 1. Best Kinetic Constants and pK Values (Scheme 2) Obtained by Fitting of Experimental Values of k1, k−1, and KpH to eqs 2−4

log k14 log k13 log k12 log k11 pK2P pK1P pK0P 4 log k−1 3 log k−1 2 log k−1 1 log k−1 0 log k−1 pK3SB pK2SB pK1SB pK0SB log kM pK2 pK1 a

PLP−PEA

PLP−PSer

2.74 ± 0.04 3.65 ± 0.09 5.60 ± 0.09 8.30 ± 0.15 8.33c 5.90c 3.60c 1.20 ± 0.05 −0.60 ± 0.10 0.90 ± 0.10 −0.02 ± 0.03 −0.06 ± 0.03 11.1 ± 0.3 6.9 ± 0.2 6.0 ± 0.20 5.4 ± 0.20 1.5 ± 0.2 10.2 ± 0.2 5.6 ± 0.2

1.63 ± 0.08 2.90 ± 0.26 5.35 ± 0.22 8.40 ± 0.30 8.33c 5.90c 3.60c 0.80 ± 0.05 −0.60 ± 0.09 0.77 ± 0.15 0.40 ± 0.20 0.30 ± 0.20 11.1 ± 0.3 7.5 ± 0.1 5.9 ± 0.2 5.5 ± 0.2 0.60 ± 0.2 10.2 ± 0.2 5.7 ± 0.2

PLP−ε-amino caproic acida

PLP− serineb

3.16 4.15 5.52 7.78 8.37 5.90 3.58 1.11 −1.28

2.37 3.20 4.77 6.65 8.36 5.91 3.62 1.05 −0.83

−0.41 −0.27 11.70

−0.05 0.13 11.04

6.42 5.69 2.07 10.45 4.07

6.58 5.58 1.31 9.15 2.21

Figure 2. Variation of log k−1 as a function of pH for the Schiff bases of PLP with (●) O-phosphorylethanolamine and (▲) O-phospho-D,Lserine. Points are experimental values and solid lines represent the theoretical fitting to eq 3.

the Schiff bases of PLP with the aminophosphates with pH. The points in the graph represent experimental data and the lines their best fit to eq 3. The pH-dependence of k−1 for the PLP−aminophosphate Schiff bases was similar to that for bases of PLP with amines and aminoacids.34,43 Fitting the experimental data to eq 3 allowed us to determine the ionization and hydrolysis constants of the Schiff bases (Table 1). Species SB5 was the fully deprotonated Schiff base (see Scheme 2), with a net charge of −5 in PLP−PEA and −6 in PLP−PSer. The incorporation of a proton produced species SB4, which corresponded to the protonation of the imine nitrogen at 4′ in the aqueous medium. The pK3SB value for PLP−PEA and PLP−PSer, 11.1, was similar to those for the Schiff bases of PLP with primary amines and aminoacids, which ranged from 11.0 to 11.7.43 15N NMR measurements allowed Sharif et al.48 to calculate pKa for the imine nitrogen in the N-(pyridoxyl-5′-phosphate-15N-idine)−methylamine Schiff base 3 , in water: 11.4 ± 0.2. The hydrolysis constant for species SB4, k−1 4 was smaller than that for SB5 (k−1 ) (see Table 1), which suggests that the former is more resistant to water hydrolysis than the latter. A similar result was previously obtained for Schiff bases of PLP with amines, aminoacids, and hydrazines.34,43,47 The incorporation of a second proton into SB4 produced species SB3, the ionization constant for which, pK2SB, was 6.9 in PLP−PEA and 7.5 in PLP−PSer, and corresponded to the protonation of the phosphate group in the two model compounds. By using 31P NMR spectroscopy, Szpoganicz and Martell37,49 determined the pKa values for the Schiff bases of PLP with 2-aminoethylphosphonic acid and 2-amino-3phosphonopropionic acid, two analogues of our model compounds, in D2O at 35 °C. Correcting their values for the aqueous medium led to a pKa value for the phosphonate group of 6.85 in PLP−PEA and 7.54 in PLP−PSer, both of which are consistent with the results of fitting our experimental data to 2 = eq 3. The hydrolysis constant for SB3 in PLP−PEA (log k−1 0.90) exceeded that for SB4; therefore, the incorporation of a second proton by protonation of the phosphate group in the aminophosphate instabilizes the Schiff base. It has been suggested on a similar system37 that the phosphate group in the aminophosphate lateral chain in species SB3 may form

From ref 43. bFrom ref 45. cThese values were fixed.

addition to those reported for ε-aminocaproic acid43 and serine.45 The fitted pKa values for the two aminophosphates are consistent with those obtained by potentiometric titration. As can be seen from Table 1, k1i decreased with increasing pH, consistent with previous results for PLP−aminoacid Schiff bases irrespective of their chemical structure.43 This was a result of acid catalysis in the dehydration of the corresponding carbinolamine, which involved all protonable groups in PLP.45,46 The linear Brönsted plots of k1i for the PLP−PEA and PLP− PSer Schiff bases confirm the presence of intramolecular acid catalysis; in fact, α (0.93−1.00) was slightly higher than it was for the Schiff bases of PLP with amines, aminoacids and hydrazines, which fall in the range 0.65−0.80.34,45−47 The k1i values obtained reveal the influence of the carboxyl group in α and the phosphate group on the formation of the Schiff base. The k14 and k13 values for the Schiff bases of PLP with PEA and PSer differed by about 1 order of magnitude. This is consistent with previous reports for Schiff bases of PLP with aminoacids (see Table 1) and suggests that the carboxyl group in α causes a decrease in the formation constants of the Schiff bases of PLP. The phosphate group present in the aminophosphates additionally reduced k14 and k13 with respect to the bases of the aminoacids. Hydrolysis of PLP−Aminophosphate Schiff Bases. Figure 2 shows the variation of the hydrolysis constants of 1901



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calculated as the ratio k1/k−1. As can be seen, the highest stability as determined from KpH was reached at pH 9.0, which is in between pKa for the amine and the highest pKa value for the aldehyde and consistent with previous results of Metzler for similar systems.50 The highest KpH value was 180 M−1 for PLP−PEA and 40 M−1 for PLP−PSer. Martell and co-workers obtained a peak KpH value of 14.5 and 16 M−1 for the Schiff base of pyridoxal with 2-aminoethylphosphonic acid51 and 2amino-3-phosphonopropionic acid,37 respectively. These values are smaller than those for Schiff bases of PLP with aminoacids, which fall in the range 300−1200 M−1.43 These results suggests that the negative charge of the phosphate group in a PLP− aminophosphate Schiff base decreases its equilibrium constant relative to PLP−aminoacid adducts. In a recent study, Barannikov et al.52 examined the thermodynamic properties of Schiff bases of PLP with various aminoacids and also found excess negative charge in the aminoacids to instabilize the Schiff bases −and to decrease their equilibrium constants as a result. Fitting the experimental data to eq 4 provided the equilibrium constant at alkaline pH, KM. The fact that KM value for PLP−PEA was nearly 8-fold that for PLP−PSer suggests that PLP forms more stable Schiff bases with PEA than it does with PSer owing to the instabilizing effect of the carboxyl group in α in the latter compound. Also, KM for the PLP−aminophosphate systems was smaller than the values for the Schiff bases of PLP with amines and aminoacids.43 Modeling the Formation of Schiff Bases of PLP with Model Compounds. Our experimental study of the formation of Schiff bases between PLP and the two model aminophospholipids was completed with theoretical calculations performed with the DFT. The reaction of a primary amine with a carbonyl group involves the formation of a carbinolamine intermediate that undergoes dehydration to the final Schiff base. Based on experimental data, the carbinolamine dehydration is the rate-determining step of the reaction,53 as previously shown in theoretical studies by our group.54−56 This led us to focus the present study on such a step. The compounds used in the theoretical study included methylamine, alanine and PEA. The former two were used to examine the influence of the carboxyl group in α with the amino group, and the latter that of the phosphate group in the nucleophile. The study focused on the dehydration of the most basic carbinolamine (pH > 10). Figure 4A shows the structures of the carbinolamine (CA), transition state (TS), and Schiff bases of PLP with methylamine and alanine. The carbinolamine formed between PLP and alanine (CAPLP−Ala) exhibited two successive hydrogen bonds (phenol−hydroxyl−carboxyl) less than 1.71 Å in length that stabilized the starting structure. The distance between the phenol group at 3 and the oxygen atom at 4′, 1.708 Å, being smaller than that in the carbinolamine of PLP with methylamine (CAPLP−methylamine, 1.821 Å). Figure 5 shows the energies of the carbinolamine, the transition state of its dehydration and the resulting Schiff base. The activation energy for the dehydration of CAPLP−Ala (step CA → TS) was 19.9 kcal/mol and that for CAPLP−methylamine 15.4 kcal/mol. This energy difference accounts for the fact that nucleophiles containing no carboxyl group in α have a decreased activation energy for the dehydration step and hence a greater kinetic formation constant for the Schiff base, consistent with the experimental results at alkaline pH for PLP−aminophosphate (log k14 = 2.74 for PEA and 1.63 for PSer) and PLP−amine bases (log k14 = 3.16 for ε-aminocaproic acid and 2.37 for serine).

hydrogen bonds with the phosphate group at position 5′; by contrast, SB4 might simultaneously form a hydrogen bond between the imino group, the phosphate group in the aminophosphate and the phenolate group at 3, thereby stabilizing the Schiff base (see Scheme 3). Scheme 3

The incorporation of a third proton into SB3 produced species SB2, the pK1SB value for which was 6.0 in PLP−PEA and 5.9 in PLP−PSer. Protonation occurred in the phosphate group at 5′ in the Schiff base. Szpoganicz and Martell37,49 reported pKa values of 6.2−6.3 for similar compounds. As can be inferred 1 from the k−1 values of Table 1, protonation of the phosphate group at 5′ stabilizes the Schiff base. The addition of a fourth proton to the Schiff base produced SB1, with a protonated pyridine nitrogen. The pK0SB values for the studied compounds were 5.4−5.5. It has been reported a value of 5.5 for the pyridine nitrogen in a PLP−PSer analogue,37,49 and one of 5.8 ± 0.2 for the N(pyridoxyl-5′-phosphate-15N0 idine)−methylamine Schiff base.48 A comparison of the k−1 1 and k−1 values in Table 1 reveals that protonation of the pyridine nitrogen had no effect on the stability of the Schiff base. Studies on the Schiff bases of PLP and aminoacids have suggested the presence of ionic groups around the imine nitrogen to decrease their stability and increase k−1.43 Based on our results, the Schiff bases of PLP with aminoacids and primary amines are more stable than those with aminophosphates, consistent with the above-described effects of ionizable groups. Equilibrium formation constants for the Schiff bases of PLP with PEA and PSer. Figure 3 shows the variation of log KpH (viz. the formation equilibrium constant of the Schiff bases of PLP with PEA and PSer) with pH. The constant was

Figure 3. Variation of log KpH as a function of pH for the Schiff bases of PLP with (●) O-phosphorylethanolamine and (▲) O-phospho-D,Lserine. Points are experimental values and solid lines represent the theoretical fitting to eq 4. 1902



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Figure 4. Mechanism of carbinolamine dehydration in the Schiff bases of PLP with methylamine and alanine (A) and of PLP with Ophosphorylethanolamine (B).

The experimental values of k14 and k13 for the Schiff bases of PLP with the aminophosphates were slightly smaller than those for the bases with amines (see Table 1). A DFT study of the carbinolamine formed by PLP and PEA (CAPLP−PEA) was conducted in order to assess the significance of the phosphate group. As can be seen in Figure 4B, CAPLP−PEA exhibits a strong hydrogen bond between the phenol group at 3 and the amino group at 4′ (d = 1.704 Å). The activation energy for the dehydration of this compound, 16.6 kcal/mol, is slightly higher than the calculated value for CAPLP−methylamine (15.4 kcal/mol). This result suggests that an increase in negative charge in the carbinolamine by effect of the presence of a phosphate group in the aminophosphate raises the energy barrier of the

dehydration step and leads to a decreased formation constant for the Schiff base (see Table 1). We also studied the hydrolysis of the Schiff bases of PLP with methylamine, alanine and PEA via DFT calculations. As can be seen from Figures 4A and 4B, the bond distance between the phenol and hydroxyl groups in the transition state, and that between the imine nitrogen and phenolate group in the Schiff base, were similar. The activation energy for the hydrolysis of the Schiff base in Figure 5 corresponds to the step SB → TS. The energy barriers for the hydrolysis of the three bases differed by less than 0.5 kcal/mol, which is within the error range of the method; therefore, the corresponding 4 constants should be similar as well. The experimental k−1 and 1903



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of a research fellowship by the Regional Government of the Balearic Islands and Fundación Carolina, respectively.

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Figure 5. Free energy profiles for carbinolamine dehydration in the Schiff bases of PLP with methylamine (blue triangle), alanine (red square), and PEA (green circle) Theoretical profile as calculated at the SMD/M062x/6-31+G(d,p) level. 3 k−1 values for the PLP−PEA and PLP−PSer systems were slightly different, consistent with the theoretical results.

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CONCLUSIONS In this work, we studied the formation of Schiff bases between PLP, an inhibitor of biomolecular glycation, and two compounds mimicking the polar head of natural aminophospholipids (viz. O-phosphorylethanolamine and Ophospho-D,L-serine). Based on the results, the pH-dependence of the microscopic constants (log k1, log k−1, and log KpH) of a PLP−aminophosphate Schiff base is similar to that of a PLP− aminoacid base. However, the presence of a carboxyl or phosphate group in the nucleophile influences some kinetic or equilibrium constants. Thus, the formation constants k14 and k13 for a PLP−aminophosphate system are smaller than those for a PLP−aminoacid system. Also, the presence of a carboxyl group in α with the amino group decreases k1. DFT calculations revealed that the activation energy for the dehydration of carbinolamine, which is the rate-determining step in the formation of Schiff bases, is raised by the presence of increased negative charge in a PLP−aminophosphate system relative to a PLP−aminoacid system. Also, consistent with the experimental results, the theoretical calculations suggest that the activation energy for the hydrolysis of Schiff bases is not altered by the presence of ionic groups in the nucleophile.

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ASSOCIATED CONTENT

S Supporting Information *

All atom coordinates for the minimum and the transition states and IRC curves. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was funded by the Spanish Government (Project CTQ-2008-02207/BQU). C.C. and J.M.G. acknowledge award 1904



Article

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Formation of Schiff Bases of O-Phosphorylethanolamine and O

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