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Reaction Mechanism of Covalent Modification of Phosphatidylethanolamine Lipids by Reactive Aldehydes 4-hydroxy-2-nonenal and 4-oxo-2-nonenal Katarina Vazdar, Danijela Vojta, Davor Margeti#, and Mario Vazdar Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00443 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Chemical Research in Toxicology

Reaction Mechanism of Covalent Modification of Phosphatidylethanolamine Lipids by Reactive Aldehydes 4-hydroxy-2-nonenal and 4-oxo-2-nonenal

Katarina Vazdar, Danijela Vojta, Davor Margetić, Mario Vazdar* Division of Organic Chemistry and Biochemistry, Rudjer Bošković Institute, Bijenička 54, HR-10000 Zagreb, Croatia Corresponding author: Dr. Mario Vazdar Rudjer Bošković Institute, Bijenička 54, HR-10000 Zagreb, Croatia Phone: +385-1-4571-382 Fax: +385-1-4560-195 E-mail: [email protected]

Abstract

4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) are biologically important reactive aldehydes formed during oxidative stress in phospholipid bilayers. They are highly reactive species due to presence of several reaction centers and can react with amino acids in peptides and proteins, as well as phosphoethanolamine (PE) lipids, thus modifying their biological activity. The aim of this work is to study in a molecular detail the reactivity of HNE and ONE towards PE lipids in a simplified system containing only lipids and reactive aldehydes in dichloromethane as an inert solvent. We use a combination of quantum chemical calculations,

1

H-NMR

measurements, FT-IR spectroscopy and mass spectrometry experiments and show that for both reactive aldehydes two types of chemical reactions are possible – formation of Michael adducts and Schiff bases. In the case of HNE, an initially formed Michael adduct can also undergo an additional cyclization step to a hemiacetal derivative, whereas no cyclization occurs in the case of ONE and a Michael adduct is identified. A Schiff base product initially formed when HNE is added to PE lipid can also further cyclize to a pyrrole derivative in contrast to ONE, where only a Schiff base product is isolated. The suggested reaction mechanism by quantum-chemical calculations is in a qualitative agreement with experimental yields of isolated products and is also additionally investigated by 1H-NMR measurements, FT-IR spectroscopy and mass spectrometry experiments.

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Introduction

In the process of oxidative phosphorylation which naturally occurs in mitochondria organelles, various reactive aldehydes (RAs), such as 4-hydroxy-2-nonenal (HNE)1 and 4-oxo-2-nonenal (ONE)2 (Figure 1) are formed during adenosine triphosphate generation (ATP) in the electron transport chain located in the inner mitochondrial membrane.3 In particular, RAs are formed in a cascade of peroxidation reactions between in situ generated reactive oxygen species (ROS)4 with polyunsaturated fatty acids located in phospholipid bilayers which are an integral part of mitochondrial membranes.5,6,7 It has been shown experimentally that RAs can readily react with different amino acids in membrane proteins thus severely modifying their function.8,9,10,11 Although RAs are primarily stabilized inside phospholipid bilayers due to the presence of alkyl hydrophobic chains, RAs can exit the bilayer and freely diffuse across cellular membranes12 thus reacting with proteins at other locations as well, including cytosol and cell nucleus.13,14

Figure 1. Schematic representations of HNE, ONE, POPE – model, POPE and various POPE adducts with HNE and ONE.

Specifically, due to the presence several electrophilic reaction sites, RAs are prone to chemical reactions with side chain heteroatoms of nucleophilic amino acids, such as cysteine, lysine, histidine and arginine.15 In addition to reactions with amino acids, some RAs (such as acrylamide16 and urethane17) can modify DNA bases in turn inducing DNA damage and 2 ACS Paragon Plus Environment

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carcinogenesis, whereas numerous adducts of acrolein, crotonaldehyde and HNE with DNA bases have been identified18 and also have been found in carcinogenic tissue.19 Moreover, RAs can also react with the amino group of phosphoethanolamine (PE) lipids20,21,22 which are making up to 30 % of all inner mitochondrial membrane lipids.23 For example, it has been previously found that RAs react with PE lipids which surround different membrane proton transporters (such as mitochondrial uncoupling protein 1), in turn indirectly affecting proton conductivity across cellular membranes, but differently for HNE and ONE.22 The mitochondrial dysfunction, as a result mitochondrial uncoupling protein modification, has profound implications on the bioenergetics in living organisms, especially relevant in diabetes 2.24,25 Although it is well established that upon reaction of amino group with HNE the most common products are Michael adducts and Schiff bases,26 together with their hemiacetal27 and pyrrole derivatives,20,28 the molecular details of the reaction mechanism are not yet described. Moreover, since addition of HNE and ONE result in covalent adducts which change biophysical properties of phospholipid bilayers,22 it is important to understand the difference in chemical reactivity towards PE lipids between HNE and ONE, which differ only slightly having either hydroxyl group (HNE) or ketone group (ONE), respectively, in the corresponding chemical structure (Figure 1). Herein, we present a detailed study on the reaction mechanism of covalent modification of 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) lipid by HNE and ONE, respectively, using combination of quantum chemical calculations supported with 1H-NMR measurements in neat dichloromethane solvent, transmission FT-IR spectroscopy and experimental mass spectrometry (MS) measurements in gas phase in turn showing important differences in chemical reactivity of studied RAs. In particular, the reaction between POPE and HNE yields the mixture of Michael adduct 1a and the corresponding hemiacetal derivative 1b together with the mixture of the Schiff base 2a and the corresponding pyrrole derivative 2b. On the contrary, the reaction between POPE and ONE yields the Michael adduct 3 and the Schiff base 4. (Figure 1). Although biologically relevant reactions of PE lipids with RAs take place in heterogeneous phospholipid bilayers where water plays a pivotal role, here we use the simplest environment composed of an inert aprotic dichloromethane solvent since both RAs and POPE are easily soluble in it, unlike polar protic solvents such as water where RAs are almost insoluble and detailed analysis of reaction mechanism is very difficult. 3 ACS Paragon Plus Environment


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Experimental Methods All reactions were performed under argon atmosphere. Commercially available reagents were used without further purification. HNE and ONE were prepared according to known literature procedures.29,15 The corresponding aldehyde (1.1 equiv) was dissolved in 1 mL of dry dichloromethane and POPE (Sigma Aldrich, purity ≥ 95%, tm=25 °C, 1.0 equiv) was added. The reaction mixture was stirred at 40 °C for 18 h until the corresponding aldehyde was consumed. The reaction was monitored using TLC on silica (mobile phase CHCl3:CH3OH:Et3N=65:5:1). The crude reaction mixture was purified by flash column chromatography on silica using CHCl3:CH3OH:Et3N=65:5:1 as an eluent. The isolated products were identified by mass spectrometry together with 1H-NMR and FT-IR spectroscopy (Supporting Information). Column chromatography on silica gel 60, 70-230 mesh, 60 Å (E. Merck, Darmstadt, Germany) was performed at RT. Thin layer chromatography was carried out on TLC aluminium sheets, 20x20 cm, silica gel 60 F254. Mass spectrometry (MS) analysis of was performed on an Agilent 1200 LC-MS System equipped with an Agilent Diode Array Detector and an Agilent 6410 Triple Quadrupole Mass Spectrometer Detector applying electrospray ionization (ESI) (Agilent Technologies Inc., Wilmington, DE, USA) in positive mode. The NMR spectra (in CDCl3 at RT) were measured on a Bruker AV 600 spectrometer (Bruker BioSpin GmbH., Rheinstetten, Germany). FT-IR spectra of products (viscous oils at RT) were collected on Bruker Alpha-T spectrometer (universal sampling module). Their transmission spectra were obtained from the empty KBr discs (Sigma Aldrich, 99 % purity, spectrometric grade), prepared by weighing approximately 120 mg of the inert salt, which were subsequently covered by investigated compounds. POPE was used for comparative analysis and prepared as a pellet by mixing and grinding 2 mg of POPE with about 110 mg of KBr. A spectrum of the atmosphere was used as the background spectrum. All spectra were collected at nominal resolution of 4 cm-1 and 16 scans at RT.

Computational Methods


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Instead of using a whole POPE lipid molecule in quantum chemical calculations, we used a simplified POPE – model where sn-1 and sn-2 acyl chains were cut out and replaced with a methyl group. In this way, the chemically relevant part of POPE lipid participating in the reaction is kept intact (Figure 1). This choice is reasonable since acyl chains have large conformational flexibility and also a considerable size which would make the calculations unfeasible. Geometry optimizations were performed at M06-2X/6-31G(d) level of theory and single point M06-2X/6311++G(2d,p)//M06-2X/6-31G(d) energy calculations were executed using optimized geometries with the smaller basis set.30,31,32 M06-2X functional has been shown to have a great reliability and accuracy for main group thermochemistry and kinetics.33 During geometry optimization and single point calculations, solvation effects were taken into account by the SMD solvation model34 with dichloromethane as a solvent at 298 K and 1 bar. NBO analysis35 was performed at the SMD/M06-2X/6-311++G(2d,p)//M06-2X/6-31G(d) level of theory. Minima and transition states on the potential energy surface were verified by vibrational analysis. In the case of transition states, a single imaginary frequency corresponding to bond breaking/forming process was obtained in all relevant cases and verified by IRC calculations.36 Gibbs free energy is calculated as a sum of single point electronic energy, thermal correction to Gibbs free energy and energy of solvation at 298 K and 1 bar. Free energy of solvation of proton in dichloromethane is taken to be -199.5 kcal mol-1 (for water this value is -264.6 kcal mol-1) and is included in the calculation of protonation/deprotonation processes in dichloromethane.37All quantum chemical calculations were performed with Gaussian09 suite of codes.38 All structures are visualized with CYLView.39

Results

Synthesis of POPE adducts with HNE and ONE POPE was treated with HNE or ONE in dry dichloromethane at 40 °C for 18 h. In the reaction of POPE with HNE two products were isolated, 1 and 2 in 45% and 24% yield (Table 1), respectively. Detailed mass spectrometry together with 1H-NMR and FT-IR spectroscopy (Supporting Information) suggests that 1 is actually a mixture of the Michael adduct 1a and the hemiacetal form of the Michael adduct 1b whereas product 2 is also a mixture of the Schiff base 2a and the pyrrole derivative of the Schiff base 2b (Figure 1). In the reaction of POPE with ONE two products were also isolated, 3 and 4 in 56% and 17% yield (Table 1), respectively. However, 5 ACS Paragon Plus Environment


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structural analysis of products by 1H-NMR and FT-IR spectroscopy and MS revealed that product 3 is the Michael adduct where no cyclic hemiacetal formation is observed, whereas product 4 is the Schiff base (Figure 1), unlike in the case of HNE where a subsequent cyclization to the pyrrole derivative takes place.

Table 1. Covalent adducts of POPE with HNE and ONE, respectively, with an indicated experimental yield. product

type

yield / %

1

Michael adduct + hemiacetal

45

2

Schiff base + pyrrole

24

3

Michael adduct

56

4

Schiff base

17

HNE

ONE

Michael addition of POPE lipid to HNE and ONE In Scheme 1, a schematic representation of Michael addition to HNE and ONE, respectively, is presented. First, we start with a detailed description of Michael addition to HNE which is shown in Scheme 1a. It has been suggested previously by mass spectrometry that POPE forms a Michael adduct with HNE without further cyclization to a hemiacetal derivative.20 However, since Michael adduct and hemiacetals have identical mass, it is difficult to distinguish whether a hemiacetal derivative has been formed too, which is a common reaction during protein modification.27 In order to check the reaction mechanism and shed more light on the reaction products, we calculated all steps of the reaction using the SMD/M06-2X/6-311++G(2d,p)//M062X/6-31G(d) theoretical approach. Table S1 shows calculated total energies and free energy differences along the proposed reaction path whereas Figure 2 shows schematic representation of energy diagrams. As we see from Table S1 and Figure 2, the free energy barrier for Michael addition of POPE to HNE by nucleophilic attack of amino group in POPE to the C=C double bond and via the transition state TS1-H in HNE is 16.8 kcal mol-1 (Figure 2). After the initial step, the unstable intermediate MH2 undergoes proton transfer via the transition state TS2-H with a barrier of 18.0 6 ACS Paragon Plus Environment

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kcal mol-1 yielding the Michael adduct MH3 which is by 7.5 kcal mol-1 more stable than the starting pre-reaction complex MH1. However, the reaction can still proceed further after formation of the Michael adduct MH3. After facile rotation around single C–C bond with a free energy barrier of 2.4 kcal mol-1 resulting in MH4, the additional nucleophilic attack of oxygen atom in the –OH group to the electrophilic carbon atom in the carbonyl C=O group can occur in two ways – the first possibility is a direct proton transfer coupled with formation of C–O bond through the transition state TS3-H and a free energy barrier of 25.0 kcal mol-1 resulting in the final cyclic hemiacetal derivative MH5 which is by 12.8 kcal mol-1 more stable than MH1 and by 5.3 kcal mol-1 than MH3. This suggests that hemiacetal derivative 1b is thermodynamically the most stable reaction product and should be predominantly isolated in thermodynamic equilibrium.

Scheme 1. Schematic representation of reaction mechanism of Michael addition of POPE lipid to a) HNE and b) ONE. In quantum chemical calculations, R is replaced by methyl –CH3 group. An alternative reaction mechanism of hemiacetal formation involving the protonation of MH4 is shown in red color.

The second possibility of nucleophilic attack of the –OH group towards the electrophilic carbon atom in the C=O group can be achieved far more easily if the carbonyl group is 7 ACS Paragon Plus Environment


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protonated. This greatly facilitates the reaction by increasing positive charge on carbon atom in the carbonyl group which is witnessed by the NBO analysis showing the increase of charge of the carbon atom from +0.48 to +0.63 therefore making it more electrophilic.40 Subsequently, a cyclization of the ring occurs via the transition state TSp-H with almost a negligible barrier of 1.9 kcal mol-1 leading to the protonated hemiacetal MH7 which after deprotonation provides the hemiacetal derivative MH5. However, protonation in this system is not very probable since the reaction occurs in dichloromethane where a source of proton does not exist. Therefore, we expect that the protonation route towards the hemiacetal derivate will be dominant in polar solvents, and especially in water where an unlimited source of protons exists which could explain why hemiacetal derivative formation was observed in earlier experiments.27,41,42

In the case of Michael addition of POPE lipid to ONE (Scheme 1b), calculated free energy barrier for the first step of the reaction going from MO1 to MO2 via the transition state TS1-O is somewhat lower as compared to HNE, being 14.2 kcal mol-1. A subsequent proton transfer via the transition state TS2-O and a free energy barrier of 33.9 kcal mol-1 yields the final Michael adduct MO3 which is 8.8 kcal mol-1 more stable than the initial pre-reaction complex MO1. However, in this case there is a major difference in the reaction mechanism – namely, further cyclization to hemiacetal derivative is not possible since oxygen atom in the C=O group is a poor nucleophile as compared to oxygen atom in the –OH group. This is also suggested by the NBO analysis which shows that charge on oxygen atom is -0.62, whereas the charge on oxygen atom in the –OH group is more negative, being -0.77 and in turn more nucleophilic. However, it is still theoretically possible for the carbonyl oxygen atom to act as a nucleophile in the enol form of MO3 (MO3-enol, Scheme 1), but this form of the hypothesized adduct is by 13.5 kcal mol-1 less stable than MO3 thus excluding the possibility of hemiacetal formation. We should mention that we calculated reaction path using implicit water model at the SMD/M06-2X/6-311++G(2d,p)//M06-2X/6-31G(d) level of theory using geometries optimized in dichloromethane solvent to capture the effect of the polar aqueous environment. Free energies, together with free energy differences are shown in Table S1 and the corresponding Figure S11. We see that inclusion of implicit water does not change qualitatively the reaction path, except for the slight stabilization of zwitterionic intermediates MH2 and MO2. Additionally, since free energy of solvation of proton in water37 is -264.6 kcal mol-1 (which is larger than free energy of 8 ACS Paragon Plus Environment

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protonation of MH6 being -258.8 kcal mol-1, Table S1), protonation process is not favorable and therefore not shown in Figure S11.

Figure 2. Schematic representation of reaction mechanism of Michael addition of POPE lipid to a) HNE and b) ONE. All free energy differences are given in kcal mol-1 and calculated at SMD/M06-2X/6311++G(2d,p)//M06-2X/6-31G(d) level of theory in dichloromethane. An alternative reaction mechanism of hemiacetal formation involving the protonation of MH4 is shown in red color.

Figure 3 shows optimized geometries of the transition structures TS1-H and TS1-O for the first step of Michael addition to HNE and ONE leading to intermediates MH2 and MO2, respectively, together with the second step involving a direct proton transfer during formation of Michael adducts MH3 and MO3, respectively. We see that bond lengths indicating formation of new C–N bonds in transition structures TS1-H and TS1-O are similar (Figure 3a and 3b), being 1.936 Å and 1.974 Å, respectively, which corresponds to similar free energy barriers for formation of the intermediates of 16.8 and 14.2 kcal mol-1, respectively. A comparable situation holds also for transition state structures TS2-H and TS2-O, where bond distances indicating formation of Michael adducts MH3 and MO3 are similar too – in TS2-H they are 1.290 and 1.481 Å (Figure 3c) and in TS2-O the corresponding values are 1.280 and 1.502 Å, respectively. 9 ACS Paragon Plus Environment


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Figure 3. Optimized transition state structures at the M06-2X/6-31G(d) level of theory – a) TS1-H, b) TS1O, c) TS2-H and d) TS2-O. Bond lengths corresponding to formation/cleavage of selected new bonds are shown in transparent color and are given in Å.

The present computational results are additionally investigated by the experiments described earlier. Namely, in the case of HNE adducts, MS analysis showed a product with the mass m/z of 874.8 which is an indication of either the Michael adduct 1a or the hemiacetal adduct 1b since they have an identical mass (Figure S1). Therefore, we performed complementary 1H-NMR measurements of the product 1 which actually indicated existence of a mixture of 1a and 1b. In particular, the indication of the HNE aldehyde proton peak in the Michael adduct 1a is present at the expected position of 9.59 ppm. Together with the spectral signature of Michael adduct 1a, a hydroxide proton peak assigned to the final hemiacetal adduct 1b is also found at around 5.15 – 5.26 ppm (Figure S2), thus indicating the possibility of the hemiacetal formation too as computationally suggested. Unfortunately, the resolution of aldehyde proton peak intensity in 1a is very weak due to strong broadening of 1H-NMR signals induced by dipolar interactions between lipid moieties.43,44 Therefore, we performed additional FT-IR measurements which suggest that Michael adduct 1a is indeed a dominant component of the product mixture 1. Specifically, a medium intensity band located at about 1650 cm-1 (Figure S10) is a result of superposition of carbonyl stretching of an aldehyde group (– CHO) in the adduct 1a and of C=C stretching in the oleyl chain of the lipid adduct. However, it is impossible to quantitatively determine the exact ratio of products 1a and 1b in the mixture using a combination of described experimental techniques, but we predict that both products exist in the reaction mixture 1. 10 ACS Paragon Plus Environment

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In the case of ONE adducts, the assignment of the final product of Michael addition is easier since there is no possibility of further cyclization to the hemiacetal derivative. Accordingly, mass spectrometry measurements showed a compound with the mass m/z of 872.4 which is assigned to the final Michael adduct 3 (Figure S5). Additional 1H-NMR measurements detected the aldehyde proton of ONE at the position of 9.71 ppm thus confirming existence of the Michael adduct 3 (Figure S6). Additionally, in the FT-IR spectrum of compound 3 a strong band with maximum at 1674 cm-1 (Figure S10) is very likely a result of convolution of the bands attributed to the stretching of ketone and aldehyde carbonyl groups of the Michael adduct 3.

Schiff base formation of POPE lipid with HNE and ONE Scheme 2 shows a schematic representation of Schiff base formation between the POPE model lipid and HNE and ONE, respectively. As indicated in the presented Scheme, we see that in the case of addition of the POPE model to HNE, a Schiff base is not the final product and the model pyrrole adduct SH8 could be formed by cyclization of the unsaturated HNE hydrocarbon tail towards nitrogen atom (Scheme 2a). This is in accordance to previous literature results for liquid chromatography-mass spectrometry study of addition of POPE lipids and HNE.20 On the other hand, a final product of covalent addition of the model POPE lipid to ONE yields the model Schiff base SO3 instead, since the cyclization to the pyrrole adduct is not possible in this case (Scheme 2b). We also look into the details of the energetics of the reaction mechanism employing SMD/M06-2X/6-311++G(2d,p)//M06-2X/6-31G(d) calculations and calculated free energies are presented in the Table S2 together with the schematic representation of the reaction mechanism shown in Figure 4.



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Scheme 2. Schematic representation of reaction mechanism of the Schiff base formation of POPE lipid to a) HNE and b) ONE. In quantum chemical calculations, R is replaced by methyl –CH3 group.

We will start first with the analysis of addition of the POPE model lipid to HNE. In a first step, carbinolamine intermediate SH2 is formed after nucleophilic attack of the POPE model lipid to HNE with a free energy barriers of 29.3 kcal mol-1. This is illustrated in Scheme 2 and Figure 4, whereas SMD/M06-2X/6-311++G(2d,p)//M06-2X/6-31G(d) calculations and calculated free energies are presented in Table S2.


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Figure 4. Schematic representation of reaction mechanism of Schiff base formation of POPE lipid with a) HNE and b) ONE (inset). All free energy differences are given in kcal mol-1 and calculated at SMD/M062X/6-311++G(2d,p)//M06-2X/6-31G(d) level of theory in dichloromethane

After carbinolamine formation, water elimination occurs in a second step45 where the model Schiff base complexed with water SH3 is formed with a high corresponding free energy barrier of 52.8 kcal mol-1. After water removal leading to SH4, three additional isomerization steps via three isomers SH5, SH6 and SH7 with the transition states TSisomerization1-H, TSisomerization2-H and TSisomerization3-H (with free energy barriers of 26.2, 7.3 and 45.1 kcal mol-1, respectively) result in the final isomer SH7 where a hydroxyl group –OH is in a vicinity of nitrogen atom. A direct water elimination where proton would be detached from carbon atom is not possible and in turn a proton transfer to nitrogen atom occurs via the transition state TSCH-NH-H with a free energy barrier of 25.6 kcal mol-1 resulting in SH8. Interestingly, water elimination from SH8 is again not possible and an intermediate step where a five-membered ring is formed (SH9) is taking place through the transition state TSclosure-H with a high free energy barrier of 51.2 kcal mol-1. Finally, water elimination by abstraction of proton from nitrogen atom and –OH group detachment via the transition state TSpyrrole-H and a free energy barrier of 15.2 kcal mol-1 result in the final product of the reaction SH10. Although the calculated free energy barriers are significantly higher than in 13 ACS Paragon Plus Environment


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the case of Michael addition (Figure 2), the driving force for Schiff base adduct and in turn pyrrole formation is strong thermodynamic stabilization of the products by 7.1 and 27.5 kcal mol1

(Figure 4) as compared with the pre-reactive model complex SH1 and the model Schiff base

SH4, respectively. In the case of addition of the POPE model lipid to ONE, a reaction mechanism is analogous to HNE (Scheme 2b). In a first step, the carbinolamine intermediate SO2 is formed after nucleophilic attack of the POPE model lipid to ONE with a free energy barriers of 28.2 kcal mol1

. This is followed by water elimination in a second reaction step where the Schiff base

complexed with water SO3 is formed with a corresponding high free energy barrier of 54.6 kcal mol-1. Similar to HNE, SO3 is more stable than the pre-reactive complex SO1 by 6.5 kcal mol-1. Finally, we also used implicit water and calculated the reaction path for Schiff base formation at the SMD/M06-2X/6-311++G(2d,p)//M06-2X/6-31G(d) level of theory using geometries optimized in dichloromethane solvent (Table S2 and Figure S12). Similar to Michael adduct formation, inclusion of implicit water does not change qualitatively the reaction path, except for the slight stabilization of transition states TScarbinolamine-H, TSwater-H, TScarbinolamine-O and TSwater-O. However, the free energy barrier heights for water elimination still remain high, being over 50 kcal mol-1. Figure 5 shows the optimized transition structures TScarbinolamine-H, TSwater-H, TScarbinolamineO and TSwater-O for the model Schiff base formation during nucleophilic attack of the POPE model lipid to HNE and ONE and subsequent water elimination, respectively. Additionally, Figure 5 also shows the transition structures for five-membered ring formation TSclosure-H and pyrrole formation TSpyrrole-H in the case of HNE. In the structures TScarbinolamine-H (Figure 5a), TSwater-H (Figure 5b), TScarbinolamine-O (Figure 5c) and TSwater-O (Figure 5d) the transition state is of four-membered ring shape and bond distances are very similar for both HNE and ONE, respectively. In particular, in the case of the carbinolamine formation (Figures 5a and 5c) we see a cleavage of N–H bond and a simultaneous formation of single O–H and C–N bonds in the carbinolamine adduct. In the case of water elimination step (Figures 5b and 5d), N–H single bond and double C=N bond are formed together with a simultaneous water elimination. Figure 5e shows the transition state TSclosure-H where C–N bond is formed as a part of a five-membered ring which is indicated by the bond length of 2.015 Å. Finally, Figure 5f shows the transition state TSpyrrole-H indicating water elimination and closure of the unsaturated pyrrole ring in SH10. 14 ACS Paragon Plus Environment

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Figure 5. Optimized transition state structures at the M06-2X/6-31G(d) level of theory – a) TScarbinolamineH, b) TSwater-H, c) TScarbinolamine-O, d) TSwater-O, e) TSclosure-H and f) TSpyrrole-H. Bond lengths corresponding to formation/cleavage of selected new bonds are shown in transparent color and are given in Å.

Computational results are again additionally investigated by experimental measurements. In particular, in the case of the product mixture 2, MS analysis shows a single product with the mass m/z of 838.7 which is an indication of the pyrrole derivative 2b formed after elimination of water from the Schiff base 2a (Figure S3).28 This is expected according to computational results indicating a high stability of the model pyrrole derivative (Figure 4). However, complementary 1

H-NMR experiments of the product mixture 2 show characteristic peaks corresponding also to

the Schiff base 2a (7.53 and 7.71 ppm, Figure S3) together with the pyrrole derivative 2b (5.80 – 5.83 ppm, 6.01 ppm and 6.67 ppm, Figure S4). Additionally, in FT-IR spectrum of 2 a very broad and medium weak band feature of rather poor defined band maximum (~1650 cm-1) is observed at low-frequency side of C=O stretching of acyl chains (1740 cm-1). Although the spectrum could correspond to a presumed pyrrole adduct 2b, this cannot be unambiguously confirmed because its characteristic signals are not fully resolved. Firstly, an eventual =C–H stretching of pyrrole moiety, expected between 3200 and 3100 cm-1, is covered by –OH stretching of water which is 15 ACS Paragon Plus Environment


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unavoidable when working with KBr pellets in the air atmosphere. Secondly, C=C stretching of pyrrole moiety, expected at about 1600 cm-1, at least partially coincides with C=C stretching of oleyl chain. Moreover, for the adduct 2a (Scheme 2a) with conjugated C=C and C=N functional groups is expected to absorb in this spectral range too. Although the change in dipole moment upon C=N stretching is greater than upon C=C stretching, this difference is not as high as when compared to C=C with C=O stretching in compound 1a. As the observed vibrational feature cannot discriminate between products 2a and 2b, this might indicate that 2 is actually a mixture of these two products (Figure S10) which is also suggested by 1H-NMR measurements. Just like in the case of Michael reaction, we cannot determine the exact ratio between the products 2a and 2b, but we suggest that both products are isolated in the mixture 2. Conversely, in the case of ONE adducts, MS measurements recorded a product with the mass m/z at 854.7 which corresponds to the Schiff base SO3 (Figure S7). 1H-NMR spectra also confirmed the existence of the Schiff base by identification of characteristic peaks at positions of 7.69 and 7.85 ppm and a lack of peaks which would correspond to pyrrole formation (Figure S8). Additional FT-IR spectroscopy measurements of 4 indicated that there is a lack of distinguished band maximum just like in the spectrum of compound 2, but with the exception that the intensity of the band is larger than in compound 2 (Figure S10). Therefore, as C=C, C=N and C=O groups absorb in investigated spectral range, it is very likely that this envelope is a result of their superposition and that presence of C=O groups is responsible for greater intensity than in 2 indicating that the compound is indeed the Schiff base adduct 4.

Discussion

A detailed analysis of the computational results reveals why product mixtures 1 and 2 are actually experimentally isolated. Thermodynamically, the model Michael adduct MH3 and the model hemiacetal derivative MH5 are stabilized by 7.5 and 12.8 kcal mol-1 (Table S1 and Figure 2) vs. model initial pre-reactive complex MH1. These values are comparable to the stabilization of the model Schiff base adduct SH3 which is stabilized by 6.5 kcal mol-1 vs. the model initial pre-reactive complex SH1. On the other hand, since there is water elimination occurring during formation of the model pyrrole derivative SH10, it is impossible to compare the free energy difference to the model initial pre-reactive complex SH1. However, the stabilization gained 16 ACS Paragon Plus Environment

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during pyrrole cyclization in the model pyrrole adduct SH10 is 27.5 kcal mol-1 which is significantly larger than stabilization of 6.5 kcal mol-1 achieved during formation of SH3. According to these values, one would expect that the hemiacetal adduct 1b and the pyrrole adduct 2b would be isolated as the final products of the reaction in the thermodynamic limit, instead of mixtures 1a + 1b and 2a + 2b, respectively. The reason why these adducts are not exclusively isolated is due to kinetic properties of the reaction. Namely, the formation of hemiacetal derivative 1b is hampered by higher free energy barrier of hemiacetal cyclization as compared to free energy barrier for formation of the Michael adduct 1a. This is reflected from calculated free energy barriers for hemiacetal cyclization which is 38.3 kcal mol-1 being 7.9 kcal mol-1 higher that cumulative barrier for formation of the model Michael adduct (Figure 4). Somewhat different situation holds for the formation of the pyrrole adduct 2b. Although it is strongly stabilized as compared to 2a, in order to obtain 2b it is necessary to proceed through a series of additional reaction steps possessing high free energy barriers, calculated to be as high as 51.5 kcal mol-1 in model systems, Figure 2. Although the largest calculated free energy barrier is not as high as free energy barrier for water elimination during formation of the Schiff base 2a (which is 52.8 kcal mol-1 in the model system, Figure 4), in order to proceed towards 2b it is necessary to adopt favorable configurations along a long reaction path which might be difficult in dichloromethane solution due to energy dissipation to the solvent46,47,48 in turn resulting in lowered yield of formation of the pyrrole adduct 2b as compared to the Schiff base 2a. Interestingly, mass spectrometry experiments in the gas phase indicate exclusive formation of the pyrrole adduct 2b (Figure S1) which can be explained due to high energy contained in the mass spectrometry apparatus and lack of solvent which restricts conformational freedom in the system leading to the thermodynamically most stable product. We can also briefly comment on the difference observed in the experimental yield of the reaction resulting in product mixtures 1 and 2. Namely, experimental yield of product mixture 1 is larger, being 45 % vs. yield of 2 which is 24 %. The reason behind a better experimental yield of the mixture 1 lies again in the calculated free energy barriers of the model reaction – namely, the highest free energy barrier along the reaction path of Michael addition is 38.0 kcal mol-1 for formation of the model hemiacetal derivative MH5. On the other hand, the highest free energy barrier for formation of the model Schiff base SH3 is 52.8 kcal mol-1 which is substantially higher than for formation of the model Michael adduct. Therefore, a higher experimental yield for 17 ACS Paragon Plus Environment


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formation of the mixture 1 is in accordance with computational results. In the case of ONE adducts, the calculated stabilization energy of the model Michael adduct MO3 vs. model initial pre-reactive complex MO1 is 8.8 kcal mol-1 (Figure 2) which is larger than stabilization of the model Schiff base adduct SO3 vs. the model initial pre-reactive complex SO1 being 6.5 kcal mol-1 (Figure 5). The calculated cumulative free energy barrier for formation of MO3 is 33.9 kcal mol-1 which is by 20.7 kcal mol-1 lower than the free energy barrier for water elimination during formation of the model Schiff base SO3 which is 54.6 kcal mol-1. Therefore, from both thermodynamic and kinetic point of view it is expected that the Michael adduct 3 will be preferred to the Schiff base 4, which is indeed experimentally observed (yields are 56 % for 2 vs. 17 % for 4, Table 1). Incidentally, there is also another experimental evidence connected with the present results. Namely, Petersen and coworkers have been incubating HNE and ONE with different peptides containing nucleophilic amino acids and have concluded that ONE is more reactive than HNE towards peptides producing exclusively Michael adducts.15 Our calculated free energy barriers for Michael addition in the case of ONE are somewhat larger than for HNE (33.9 vs. 30.4 kcal mol-1, Figure 2) thus being in a slight disagreement with the observed difference in reactivity between these two aldehydes towards peptides. The reasons for this discrepancy lie in the fact that reaction with peptides occur in water phase, where an additional protonation step is available as well as excess of water which implies different mechanism of the reaction which will be discussed in our future work. In line with that, formation of Schiff adducts was not observed in described experiments, but this may also be due to different reaction setup where HNE and ONE were added in a great excess in tricine buffer. The resulting excess of water is not thermodynamically favorable for formation of Schiff bases and in particular pyrrole derivatives since one of the reaction products upon Schiff base and pyrrole formation is water itself which pushes the equilibrium towards reactants. Taken together, the calculated differences in both thermodynamic and kinetic aspects of the reaction mechanism are in qualitative agreement with experimental results and experimental product yields. Since chemical reactions take place over 18 h at 40 °C, it is expected that Schiff base derivatives 1b and 2b can be formed in the end regardless of very high free energy barriers since they are strongly thermodynamically stabilized, which is especially valid for the pyrrole adduct 2b. However, calculated free energy barriers presented in this work which are larger than 18 ACS Paragon Plus Environment

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50 kcal mol-1 are probably overestimated since the reaction with so high barriers would occur extremely slowly according to transition state theory.49 Therefore, it is necessary to consider additional factors when determining free energy barriers in model systems. In a first place, model systems used in the present calculations are considerably truncated having no lipid tails (Figure 1). This may be important since a number of different conformations induced by lipid tail flexibility can result in a different reaction environment which cannot be appropriately described using a truncated lipid model. Additionally, a lack of explicit inclusion of solvent and in turn sufficiently accurate descriptions of entropic effects,50 and/or proton tunneling during proton transfer reactions,51 may also be responsible for too high free energy barriers. Since the high energy free energy barriers are involved mostly in reactions where proton transfer occurs via four-membered transition state, especially pronounced during Schiff base formation where free energy barriers are larger than 50 kcal mol-1 (TSwater-H and TSwater-O, Figures 5b and 5d), it is plausible to assume that additional water molecule(s) will catalyze the proton transfer reaction and significantly lower the free energy barrier as computationally predicted.40 Therefore, water assistance will be important in biological membranes where phospholipid bilayers are immersed in water which can diffuse to phosphoethanolamine headgroups and catalyze the reaction. However, since phosphoethanolamine headgroups are zwitterionic in water, it is necessary first to deprotonate –NH3+ group which is very facile in water. Unfortunately, it is not possible to fully capture this effect using simple implicit water model as shown earlier (Tables S1 and S2, Figures S11 and S12) where free energy barrier heights were almost unchanged as compared to implicit dichloromethane solvent. Consequently, more demanding calculations including explicitly included water molecules and realistic membrane environment are needed to fully account for its influence on the reaction pathway which is out of the scope of the present work and will be addressed in the future.

Conclusions In the present paper, we show by a combination of quantum chemical calculations, 1H-NMR measurements, FT-IR spectroscopy and mass spectrometry experiments the detailed reaction mechanism of covalent modification of POPE lipid by reactive aldehydes HNE and ONE, which is the onset of protein and lipid modification in living organisms during oxidative stress. Using a 19 ACS Paragon Plus Environment


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simplified reaction system with dichloromethane as an inert solvent, we identified two types of chemical reactions – Michael addition and Schiff base formation which is operative for both HNE and ONE. In the case of Michael addition, we isolated the product mixture in the case of HNE consisting of the Michael adduct 1a and the hemiacetal derivative 1b in contrast to ONE where the Michael adduct 3 is the only product. We identified that in the case of HNE a cyclization of an initially formed Michael adduct to hemiacetal follows, which is not the case with ONE where no hemiacetal derivative is formed. In the case of Schiff base products, we again isolated the product mixture of the Schiff base 2a and the pyrrole derivative 2b in the case of HNE and only Schiff base 4 was isolated. A five-membered ring closure and the formation of the pyrrole derivative 2b of an initially formed Schiff base 2a is strongly thermodynamically favored in the case of HNE whereas this is not the case with ONE where pyrrole cyclization does not follow and a Schiff base 4 is formed instead. Detailed calculations of thermodynamic stability of final products, as well as a kinetic aspect assessed by determining free energy barriers during the course of investigated reactions, are in qualitative agreement with experimental yields of reaction showing prevalence of mixtures of Michael adducts 1 and 2. In addition, calculated free energy barriers are significantly lowered by tens of kcal mol-1 in the case for formation Michael adducts as compared to Schiff base adducts, which also explains why product mixtures 1 and 2 are formed in a greater extent. This study sets and analyzes the general reaction mechanism of biologically important reaction of POPE lipid with reactive aldehydes HNE and ONE in the simplest system containing only dichloromethane as an inert solvent in a full molecular detail. In the future, we will use the general reaction mechanism set up here to expand the research including reactions of peptides and proteins in more realistic biological environments, such as phospholipid bilayers immersed in water.

Supporting Information 1

H-NMR, mass spectrometry and FT-IR spectroscopy analysis of compounds 1 – 4.

Computational results (free energies, energy differences and energy diagrams in implicit water), Cartesian coordinates and vibrational frequencies for all compounds. This material is available free of charge via the Internet at http://pubs.acs.org. 20 ACS Paragon Plus Environment

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Funding Sources

This study was supported by Croatian Science Foundation, project no. UIP-2014-09-6090.

Acknowledgments

We also thank Dr. Srećko I. Kirin and Dr. Ivanka Jerić for the instrumental support.

Abbreviations

ATP, adenosine triphosphate; ESI, electron spray ionization; FT-IR, Fourier transform infrared; HNE, 4-hydroxy-2-nonenal; MS, mass spectrometry; NMR, nuclear magnetic resonance; ONE, 4-oxo-2-nonenal;

PE,

phosphoethanolamine;

POPE,

1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphoethanolamine; RA, reactive aldehyde; ROS, reactive oxygen species; TLC, thin layer chromatography

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Scheme 1. Schematic representation of reaction mechanism of Michael addition of POPE lipid to a) HNE and b) ONE. In quantum chemical calculations, R is replaced by methyl –CH3 group. An alternative reaction mechanism of hemiacetal formation involving the protonation of MH4 is shown in red color. 357x207mm (150 x 150 DPI)

ACS Paragon Plus Environment


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Scheme 2. Schematic representation of reaction mechanism of the Schiff base formation of POPE lipid to a) HNE and b) ONE. In quantum chemical calculations, R is replaced by methyl –CH3 group. 284x273mm (150 x 150 DPI)


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Figure 1. Schematic representations of HNE, ONE, POPE – model, POPE and various POPE adducts with HNE and ONE. 279x132mm (150 x 150 DPI)



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Figure 2. Schematic representation of reaction mechanism of Michael addition of POPE lipid to a) HNE and b) ONE. All free energy differences are given in kcal mol-1 and calculated at SMD/M06-2X/6311++G(2d,p)//M06-2X/6-31G(d) level of theory in dichloromethane. An alternative reaction mechanism of hemiacetal formation involving the protonation of MH4 is shown in red color. 332x172mm (150 x 150 DPI)


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Figure 3. Optimized transition state structures at the M06-2X/6-31G(d) level of theory – a) TS1-H, b) TS1O, c) TS2-H and d) TS2-O. Bond lengths corresponding to formation/cleavage of selected new bonds are shown in transparent color and are given in Å. 190x120mm (150 x 150 DPI)



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Figure 4. Schematic representation of reaction mechanism of Schiff base formation of POPE lipid with a) HNE and b) ONE (inset). All free energy differences are given in kcal mol-1 and calculated at SMD/M06-2X/6311++G(2d,p)//M06-2X/6-31G(d) level of theory in dichloromethane. 335x176mm (150 x 150 DPI)


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Figure 5. Optimized transition state structures at the M06-2X/6-31G(d) level of theory – a) TScarbinolamine-H, b) TSwater-H, c) TScarbinolamine-O, d) TSwater-O, e) TSclosure-H and f) TSpyrrole-H. Bond lengths corresponding to formation/cleavage of selected new bonds are shown in transparent color and are given in Å. 174x172mm (150 x 150 DPI)



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85x37mm (150 x 150 DPI)


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