ORIGINAL PAPER

Theoretical studies on models of lysine-arginine cross-linksderived from α-oxoaldehydes: a new mechanismfor glucosepane formation

Rasoul Nasiri & Mansour Zahedi & Hélène Jamet &Ali Akbar Moosavi-Movahedi

Received: 28 April 2011 /Accepted: 21 June 2011 /Published online: 3 August 2011# Springer-Verlag 2011

Abstract Availability and high reactivity of α-oxoaldehydeshave been approved by experimental techniques not only invivo systems but also in foodstuffs. In this article we re-examine the mechanism of glucosepane formation by usingcomputational model chemistry. Density functional theory hasbeen applied to propose a new mechanism for glucosepaneformation through reaction of α-oxoaldehydes with methylamine (MA) and methyl guanidine (MGU) models of lysineand arginine residues respectively. This non enzymatic processcan be described in three main steps: (1) Schiff base formationfrom methyl amine, methyl glyoxal (MGO) (2) addition ofmethyl guanidine and (3) addition of glyceraldehyde. Weshow that this process is thermodynamically possible andpresents a rate-determining step with a reasonable free energybarrier equal to 37.8 kcal mol-1 in water solvent. Comparisonswere done with the mechanism formation of GODIC(glyoxal-derived imidazolium cross-link) and MODIC

(methyl glyoxal-derived imidazolium cross-link), two otherimportant cross-links in vivo.

Keywords α-oxoaldehydes . Cross-link . Densityfunctional theory . Glucosepane . GODIC .MODIC .

Physiological glucose levels

Introduction

The chemical modification and cross-linking of proteins bythe Maillard reaction is the results of a cascade of reactions.Condensations, rearrangements, fragmentation and oxida-tion processes of reducing carbohydrates (glucose, ribose,fructose) or α-oxoaldehydes (methyl glyoxal (MGO),glyoxal (GO), 3-deoxyglucosone (3-DG) and glyceralde-hyde (GLA)) occur with amino groups in lysine residuesand guanidine groups in arginine residues [5, 7, 9, 25, 29,33, 34, 46]. The mechanism of the glycation reaction wasfirstly tested by Maillard at the beginning of the twentiethcentury by incubation of amino acids with different sugars[37] and can be described at three stages. The early stage ofreaction involves initiation of glycation through amino-carbonyl interaction leading to early glycation products,which contain an intact sugar moiety. In the intermediatestage, a variety of carbonyl compounds, which aregenerally more reactive than the original amino acids, areformed. Finally, successive rearrangements lead irreversiblyto the formation of a variety of heterogeneous structures,known as the Advanced Glycation End-products (AGEs).

AGEs accumulate in body and have been implicated inthe development of many pathophysiologies associatedwith ageing in general as well as with complications ofdiabetes, atherosclerosis and renal insufficiency [8, 56].

Electronic supplementary material The online version of this article(doi:10.1007/s00894-011-1161-x) contains supplementary material,which is available to authorized users.

R. Nasiri :M. Zahedi (*)Department of Chemistry, Faculty of Sciences,Shahid Beheshti University,G.C., Evin,19839-63113, Tehran, Irane-mail: mansourzahedi@yahoo.come-mail: m-zahedi@sbu.ac.ir

R. Nasiri :H. JametDépartement de Chimie Moléculaire, Chimie Théorique,UMR-5250, Université Joseph Fourier,BP 53, 38041, Grenoble Codex 9, France

A. A. Moosavi-MovahediInstitute of Biochemistry and Biophysics, University of Tehran,Tehran, Iran

J Mol Model (2012) 18:1645–1659DOI 10.1007/s00894-011-1161-x

They are implicated in pathogenesis of various devastatingdisorders, such as coronary heart diseases [26], cancergrowth [11] and metastasis, insulin resistance [56] and nonalcoholic fatty liver disease [36]. They have also beenshown to have a role in neuro degenerative disease such asAlzheimer's and Parkinson's diseases [22, 48].

According to the type of carbonyl compounds that canbe a reducing agent in glycation reaction, six distinctclasses of AGEs have been found in serum. They areglucose-derived AGEs (GLC-AGEs), MGO-derived AGEs(MGO-AGEs), GO-derived AGEs (GO-AGEs), 3-DG-derived AGEs (3-DG-AGEs), glyceraldehyde-derivedAGEs (Glycer-AGEs) and glycolaldehyde-derived AGEs(Glycol-AGEs) [42, 49]. On the other hand, Ahmed et al.classified AGEs to the first four in aforementioned ones [1,2]. Interestingly, clinical studies have shown that Glycer-AGEs are more important than GLC-AGEs in cardiovas-cular disease (CVD) and nonalcoholic steatohepatitis(NASH) in diabetes [31, 32].

An extense variety of AGEs has been discovered in vitroand in vivo systems; for example, glucosepane, DODIC,GOLD, MOLD, MODIC and GODIC, etc. These cross-links have also been identified in foodstuffs [4]. It is thecase of the glucosepane, recognized as the major cross-linkin vivo under non oxidative conditions [6, 46]. To explainits formation, Biemel et al. proposed a mechanism withlysine linked α-dicarbonyl compound as intermediate [7].This mechanism involves formation of a dideoxyosonefrom glucose and lysine; dideoxyosone then undergoescyclization into aldimine, which then reacts with the sidechain of arginine to yield glucosepane (see Scheme 1).

Experimental studies have previously suggested thatAGEs can arise not only from reducing sugars, but alsofrom α-oxoaldehyde compounds obtained from the degra-dation of sugars [53–55, 58], lipids [3], Schiff baseintermediate or Amadori product [38] in buffered solutionat 37 °C. Decomposition of amadori products has beenillustrated in Scheme 2. The α-oxoaldehyde compoundsfound in vivo may have a dietary origin [27, 35]. In theglycation of human serum albumin (HSA) under physico-logical conditions, separation of glucose from HSA [43]1

and its degradation to the α–oxoaldehydes (such as MGO,GO, 3-DG and GLA) [51], imply another possibility forglucosepane formation. It has been found that there arethree pathways for the in vivo formation of glyceralde-

hydes: namely, glycolytic pathway, polyol pathway, andfructose metabolic pathway [42] and on the other hand, itplays a key role in Maillard cross linking of food proteins[17–19, 57].

According to these results which show availability of theα–oxoaldehydes, a new mechanism for the formation ofglucosepane is re-examined by using computational modelchemistry. This article is devoted to describe a novelmechanism for glucosepane in vivo and food systems.Computational model chemistry will be used to understandand establish proper reaction mechanism. It can givemolecular level insights into a reaction through determina-tion of intermediates and transition states structures andenergies. The mechanism considers reactions between theα-oxoaldehydes MGO and GLA with lysine and arginineamino acids.

We have investigated mechanisms of formation of thecross-links with three main aims: first, to provide insightinto the formation mechanism of glucosepane consistentwith experimental results found from glycation of humanserum albumin under physiological conditions (Scheme 3);second, to study and compare the reaction mechanisms ofthe glucosepane, GODIC, and MODIC; third, to analyzethe changes in the thermodynamic and kinetic propertiescaused by the solvent.

Computational details

The geometries of reactants, products, intermediates, andtransition states involved in the proposed mechanismhave been fully optimized with using the wB97XDfunctional [13]. This functional includes empirical disper-sion and can better treat hydrogen bonding and van derWaals interactions than conventional DFT. The 6-31+G*basis set with both polarized and diffuse function is aneffective choice for more accurate description of hydrogentransfer reactions and hydrogen-bonded species of thisstudy.

For validation of method and basis set, all optimizedstructures in the first step of glucosepane formation processfrom wB97XD level were reoptimized with frozen coremoller-plesset perturbation theory, MP2(FC)/6-31+G*,computational level. Moreover, CCSD(T)/6-31+G* singlepoint calculations have been carried out for the samecompounds to evaluate the reliability of barrier calculated atwB97XD and B3LYP/6-31+G* level. Extension of thebasis set to 6-311++G (3df, 3p) in DFT calculations werealso examined to compare to the same calculations with6-31+G* basis set (see Table 1). This strategy showed thatfirstly, extension of basis set did not have significant effectson free energy diagram while obtained results fromwB97XD/6-31+G* are more reliable than B3LYP/6-31+G

1 In this stage of the reaction, there will be a competition betweenglucose and α-oxoaldehydes in the nucleophilic attack on aminogroups in the proteins. Glucose, because of high stability in its cyclicform, will be less subject to the nucleophilic attacks compared to theα-oxoaldehydes and since α-oxoaldehydes are up to 20,000-fold morereactive than glucose [50]. Therefore, glucosepane can likely bederived through reaction of the α-oxoaldehydes with lysine andarginine amino acids

1646 J Mol Model (2012) 18:1645–1659

ones since former considers non-bonded interactions alongwith long rang correlations better than latter one (comparerelevant CCSD(T) values in Table 1).

The intrinsic reaction calculations (IRC) [20, 21] in thegas phase confirmed that all TSs were in the right reactionpathways unambiguously.

To evaluate the effects of water as a high-permittivitysolvent, on the cross-linking mechanisms we employedpolarizable continuum model (PCM) calculations [52]based on solvent reaction field method (SCRF) to furtheroptimize the geometries of the stationary points in water.Vibrational analyses were performed on all optimizedstructures with the same functional and basis set. The

zero-point energy (ZPE) corrections were also included inthe reported free energies with a scale factor of 0.89 attemperature 298.15 K and 1 atm pressure in water. Allcalculations were performed using the Gaussian 09 soft-ware [16] and the Gauss View program [15] by employingthe software and hardware facilities of the theoretical groupof the DCM, University of Grenoble I Codex, France.

Results and discussion

Density functional theory (DFT) calculations were per-formed on methyl amine (MA) and methyl guanidine

C

C OH

C

C

C

C

OHH

HHO

OHH

OHH

OHH

H

C

C NHH

C

C

C

C

OHH

HHO

OHH

OHH

OHH

H

R1

OH

C

C NH

C

C

C

C

OHH

HHO

OHH

OHH

OHH

H

R1

-H2O

C

CHNH

C

C

C

C

OH

HHO

OHH

OHH

OHH

H

R1

enolization+R1-NH2

CH

HNH

C

C

C

C

HHO

OHH

OHH

OHH

H

R1

C O

a

CH

HNH

C

C

C

C

HHO

OHH

OHH

OHH

H

R1

C O

CH

HNH

C

C

C

C

HO

OHH

OHH

H

R1CH

HN

H

C

C

C

C

HO

OH

OHH

H

R1

C OH

CH

HNH

C

C

C

C

HO

H

OHH

OH

R1

HC OH

-H2O

C O

H H

1,5 H+ shift

tautomerization

CH

HNH

C

C

C

C

HO

OH

OH

R1

HC OH

H

H

keto-enol tautomerization

1,3 H+ shift

tautomerization

CH

HNH

CH

C

C

C

HO

H

OH

OH

R1

HC OH

CH

HNH

CH

CH

C

C

HO

H

O

OH

R1

HC OHketo-enol

tautomerization

enolization

b

CH

HNH

CH

CH

C

C

HO

H

O

OH

R1

HC OH

cyclization

+H+

-H2O

R2N C

NH2

NH -H+

lysine linked alpha-dicarbonyl (dideoxyosone)

R1 = CH2CH2CH2CH2CHNH2COOH

R2 = CH2CH2CH2CHNH2COOH

NH+

R1

HO

OH

O

HON+

R1

HO

OHO

N

R1

OH

OH

H N

N

H

N

R2

H

N

R1

OH

OH

N

NN

R2

H

H

c

aldimine

Scheme 1 Scheme of proposedmechanism for glucosepaneby Biemel, (a) Scheme ofamadori formation step, (b)Scheme of lysine linked alpha-dicarbonyl formation step, (c)Scheme of addition of argininestep (R1 and R2 stand forCH2CH2CH2CH2CHNH2COOH and CH2CH2CH2CHNH2COOH, respectively)

J Mol Model (2012) 18:1645–1659 1647

(MGU) which are models for the lysine and arginine aminoacids with α-oxoaldehydes of MGO, GO and GLA todescribe the mechanism of the formation of glucosepane,MODIC and GODIC. Water molecules are added in themechanism, not to simulate the solvent but as reactivespecies. The proposed mechanism (Scheme 3) involvesthree main steps, each one of them involves severalintermediates and transition state structures: (1) Schiff baseformation (Scheme 3a), (2) addition of methyl guanidine(Scheme 3b) (only structures of Int3 to Int7) and (3)addition of glyceraldehyde (Scheme 3c). Because ofsimilarity in new mechanism of Glucosepane with eachone of MODIC and GODIC, we have put their mechanismsin the same schemes (Schemes 3a and b) but note that theyare different-on the two last TS in Schemes 3b.

This section is divided as follows: First, the results anddiscussion of different steps of the glucosepane formationare presented (section A). Then formation mechanism ofGODIC and MODIC is compared with glucosepane insection B. Finally, we discuss the solvent effects overthe reaction mechanisms (section C).

A. Formation mechanism of glucosepane

Schiff base formation

A molecular complex consisting of MGO, MA and onewater molecule was chosen as model compound to studythe initial step of the cross linking process. The free energyprofile in gas phase and solution for the process is depictedin Fig. 1. The electronic energy of the reactant complex isstabilized relative to each of the isolated species while itsfree energy is destabilized. The difference of values of theelectronic and free energies are −11.9 and +9.5 kcal mol-1

respectively. Hydrogen bond interactions between themolecules explain the stabilization of the electronic energyand the destabilization of the free energy reflects thenegative entropic contribution associated to the transforma-tion of three molecules to one specie. Because of instabilityof pre reactive complex, we do not include it in thediscussion.

The process starts with carbinolamine formation bynucleophilic attack of the methyl amine molecule to the

CH

HNH

C

C

C

C

HHO

OHH

OHH

OHH

H

R1

C OCH

HNH

C

C

C

C

OH

OHH

OHH

OHH

H

R1

C OH

-R1-NH2

CH2

C

C

C

C

O

OHH

OHH

OHH

H

C OH

-H2Oenolization

CH3

C

C

C

C

O

OHH

OHH

OHH

H

C O

Amadori Product 1-DG

enolization

C

HNH

C

C

C

C

HHO

OHH

OHH

OHH

H

R1

C OH-R1-NH2

HC

CH2

C

C

C

OHH

OHH

OHH

H

C OCH3

HC

C

C

C

O

OH

OHH

OHH

H

C O

+H+

-H2O

C

HNH

C

C

C

C

H

OHH

OHH

OHH

H

R1

C OH

+H2O

O

retro-aldolMGO

GLA

3-DG

C

C OH

C

C

C

C

OHH

HHO

OHH

OHH

OHH

H

C

C NHH

C

C

C

C

OHH

HHO

OHH

OHH

OHH

H

R1

OH

C

C NH

C

C

C

C

OHH

HHO

OHH

OHH

OHH

H

R1

+H2O

C

CHNH

C

C

C

C

OH

HHO

OHH

OHH

OHH

H

R1

enolization

-R1-NH2Schiff base

Glucose

C

C HO

C

C

C

C

OHH

HHO

HHO

OHH

OHH

HMannose

Scheme 2 The pathways ofamadori products degradation

1648 J Mol Model (2012) 18:1645–1659

R

HC

O

CO

HO

H

H

N

CH3

HC

Int 1 Int 2 Int 3

O

N

CR

TS1 TS2

Re

R

H

CO

CO

HO

H

H

N

CH3

H

R

H

C

O

CO

H

O

H

H

NCH3

H

H

CH3HO

H

H

OH

a

RC

CH

NCH3

O

HO

H

HOH

Int 4 Int 5Int 6

NC

NN

HR

C

C

N

CH3

H

HOH

OH

HO

H

NC

NN

H

H

H

H

H3CN

CN

N

H RC

CH

N

CH3

H

HO

H

HO

H

H3CH3CO

H

H

H

TS3 TS4

NC

NN

H RC

C

NCH3

H

HOH

OH

H OH

H3C

H

H

HC

Int 3

O

N

CR

+MGUH

CH3HO

H

H

OH

Int7 Int 8'

NC

NN

HRC

C

NCH3

H

HOH

OH H

O H

H3C

HPr1 / Pr2

TS6' TS7'

H

NC

NN

HRC

C

NCH3

H

HOH

OH H

OH

H3C

H

H

TS5

NC

N

NH

CH3

C

CHCH3

CH2

COH H

O

Int 9-10 Int 11

+GLATS6

H

NC

NN

HCH3

C

C

N

CH3H

CH3 H

Int7 Int8

N

CH3

CH

OH

NC

N

NH

CH3

C

CHCH3

CH2

COH H

O

H

N

CH3

CH

OH

NC

N

NH

CH3

C

CHCH3

CH2

COH H

O

H

N

CH3

CHTS7

Int 12-13 Int 14-15

TS 8

TS9

N

C

N

N

H

CH3

C

CHCH3

CH2

COH

O

H

N

CH3

CH

TS10

N

C

N

N

H

CH3

C

CHCH3

CH2

C

O

H

N

CH3

CH2

O

b

Int 18-19

TS12 TS13

Int 16-17

N

C

N

N

H

CH3

C

CHCH3

CH

C

OH

N

CH3

CH2

OH

TS11

N

C

N

N

H

CH3

C

CHCH3

CH

CH

O

N

CH3

CH2

OH

Int 22 Pr3

NC

NN

H

CH2

C

CCH3

CH

CH

OH

N

CH3

CH2

OH

N

OH

OH

CH3

N

NCN

CH3

H

H

H

Int 20-21

NC

NN

H

CH2

C

CHCH3

CH

CH

O

N

CH3

CH2

OHH

TS14

c

Scheme 3 Novel pathway for glucosepane formation proposed in thiswork, (a) Scheme of Schiff base formation step, (b) Scheme ofaddition of methylguanidine step (R for Pr1 and Pr2 is hydrogen and

methyl respectively), (c) Scheme of addition glyceraldehyde step (Allwater molecules have been omitted for the sake of clarity)

J Mol Model (2012) 18:1645–1659 1649

carbonyl carbon atom labeled as C4 in Fig. 2a. This attackis performed via a proton transfer from N1 to O15 with afree energy barrier equals to 15.7 kcal mol-1 (see Fig. 1). Inthis six-membered-ring TS called TS1 in Fig. 2a, the C4-N1

bond is almost formed (1.53 A) and the C4-O7 bond has alength close to that of a single bond (1.35 A). The Schiffbase formation is accomplished with the dehydration ofcarbinolamine to yield a ketoimine. As Int1 cannot bedehydrated directly, it isomerizes to Int2 through two singlebond rotations. The free energy barrier for this transforma-tion is less than 4 kcal without participation of watermolecule and can be done easily. Then Int2 evolves to theSchiff base as Hall and Smith reported this stage viareaction of MA and formaldehyde [24]. The reactionfollows through a concerted proton transfer in which H11

is transferred from N1 to O15 while H10 from O15 to O7 TS2in Fig. 2a illustrates these proton transfers. In this transitionstate, the double bond between N1 and C4 is formed.Finally, a ketoimine compound is created that has twoelectrophilic centers reacts with MGU. As can be seen fromfree energy profile (Fig. 1), the activation free energybarrier is high for the dehydration step and has a valueequal to 33.5 kcal mol-1 in the gas phase which is in goodagreement with high-level ab initio results (33.2 kcal mol-1)on similar reaction [24]. It is worth mentioning here thatthis barrier decreases significantly when more watermolecules are mediated around the reactive center ofreaction, but since water assisted effects are not the goalof this study we do not discuss it. In comparing to otherbarriers of the Schiff base formation (see Fig. 1) the last oneis the rate-limiting step of this reaction in agreement withexperimental kinetic studies [44, 45].

Addition of methyl guanidine (MGU)

In the second step, methyl guanidine was chosen asmolecular model of arginine residue and added to theSchiff base obtained from the first step. Because ofexistence five acidic protons on the guanidino group (seeScheme 4); there are five different neutral methylguanidine tautomers. The guanidino function is usually

assumed to be in the protonated form with an effectivepKa of about 12.0 in aqueous solution at 298 K [40]. Sothe first calculations were performed with this form.However our calculations show the protonated formmoving away from the Schiff base and we did not succeedto optimize an adduct from them. Hence, for this reasonone of the neutral forms was taken into account and couldexplain why rate of the glycation reaction increases withthe basicity [30]. Moreover, the pKa s values in biologicalsystems are not the same as in the solution. A previoustheoretical study shows that all the neutral forms of MGUare in equilibrium to each other [39]. Therefore, weconsidered only one of these tautomers2. Addition ofMGU to the Schiff base leads to Int4 in which guanidinogroup is perpendicular to the ketoimine plan. Two watermolecules assist this step, the water molecule was addedin the Schiff base formation step, and the water moleculeproduced from the last dehydration. Since the NH functionis more nucleophilic than the NH2 one, first N2 attack toC7 of the Schiff base (see Fig. 2b for the notation). Thefirst proton transfer in this step occurs through TS3 with afree energy barrier less than 8.4 kcal mol-1. In thistransition state, a twisted six-membered ring is formed inwhich the proton from NH is being transferred to the watermolecule which in turn has already started to transfer itsproton to O8. The completion of this reaction leads to acarbinolamine (Int5) whose free energy is stabilized about11.1 kcal mol-1 relative to Int4. This stability is due toformation of bond N2-C7 completely in Int5 while distanceof N2-C7 in Int4 is 1.54 Åand N2 has also positive charge. Anew step of dehydration occurs to give Int6. The calculatedfree energy barrier of this dehydration reaction which passesby TS4 (Fig. 1) is also high but 7.5 kcal mol-1 less than

Table 1 Changes of electronic energy for the first step of proposed mechanism in the gas phase (in kcal mol-1)

WB97XDa WB97XDb B3LYPc MP2d CCSD (T)e CCSD (T)f

Re——TS1 5.4 4.2 4.5 5.7 7.2 7.3

TS1——Int1 16.5 17.4 15.1 16.2 18.4 18.7

Int1——Int2 4.8 4.4 4.8 4.7 3.5 4.3

Int2——TS2 32.6 31.7 26.9 28.8 31.0 33.7

TS2———Int3 24.7 26.4 23.9 25.5 26.3 28.7

Superscripts a, b, c, d, e and f indicate calculations at the WB97XD/6-31+G*, WB97XD/6-311++G (3df, 3p), B3LYP/6-31+G*, MP2 (FC)/6-31+G*,CCSD (T)/6-31+G*//WB97XD/6-31+G* and CCSD (T)/6-31+G*//B3LYP/6-31+G* levels, respectively

2 Following the comment of a reviewer, we tested the influence of twodifferent isomers of MGU (see tautomers C and E in Scheme 4) on thereaction profile in second step of cross-linking process. We found thatactivation barriers are to be in different values when tautomers areinvolved in the process (see Figure S1). It is worth noting that isomerF cannot be involved in the process actively since Lederer and Klaiberdefinitely excluded formation of MODIC or GODIC via endocyclicnitrogens of arginine residues (see 34 for more detail)

1650 J Mol Model (2012) 18:1645–1659

similar one in the first step (TS2 in Fig. 1). The NH2 group ofthe MGU acts as a proton transferring carrier, therebylowering the barrier of this second dehydration reactionrelative to the first dehydration reaction. After downhill fromTS4, Int6 can produce a five-membered heterocyclic structurethrough TS5, which involves the concurrent transfer of twoprotons from N3 to N1. The free energy barrier for thisreaction is calculated to be 8.5 kcal mol-1 relative to Int6.From TS5 to Int7, distance of N3-C4 decreases from 1.51 to1.46 Å and the free energy decreases to 35.3 kcal mol-1 whichcould explain the irreversibility of the cross-linking process invivo (see Fig. 1).

Addition of glyceraldehyde (GLA)

Finally, we consider the formation of glucosepane by additionof glyceraldehyde to Int8 obtained from the previous step.Glyceraldehyde (GLA), the simplest reducing sugar, has beenrecognized as one of the most reactive species in the Maillardreaction in vivo and in vitro under physiological conditions[12]. Int8 is the reactant complex due to the addition of theGLA to Int7. N1 in Int8 must be dehydrogenated before itsaddition to the carbonyl function. It is done with the help ofthe water molecule. Thanks to hydrogen bonds with O19 andH11, it acts as a bridge to facilitate the proton transfer througha concerted TS (TS6 in Fig. 2c). This proton transfer is alsosubject to a relatively small barrier (13.5 kcal mol-1, Fig. 1).

In this step, because of the carbonyl mobility alongthe entire GLA backbone and the limited number ofwater molecules available, we had to change theposition of water molecules relative to reactive center

The tautomeric transformation of Int17 to Int18 issimilar to the tautomerization of Int13 to Int14 andtheir free energy barriers are equal to 18.7 and21.6 kcal mol-1 respectively. An enamine (Int19) is

Fig. 1 Free energy profile for the whole cross-linking process of glucosepane analog in gas phase and solvent (in parentheses) calculated at thewb97xd/6-31+G*level

J Mol Model (2012) 18:1645–1659 1651

manually. This explains the apparition of new stationarypoints after intermediates 9, 12, 14, 16, 18, 20 (seeFig. 1). As the free energy difference between thesestationary points is small, we can use this protocol todescribe the mechanism. After formation of anothercarbinolamine via the reaction of GLA and the five-membered heterocyclic amine (Int7), dehydration occursthrough two stepwise reactions. Firstly, dehydroxylationoccurs that leads to the formation of the iminium (Int11)with a high barrier of 38.7 kcal/ mol (TS7). Secondly, ahydrogen abstraction is done with a very small free energybarrier.With incorporation of twowater molecules, Int13 can beconverted to a keto amine (Int14). This tautomerization reactionoccurs through TS9 which involves three proton transferreactions from O16 to C20 concurrently within the eight-membered ring (Fig. 2c). As it can be seen from Fig. 1, thebarrier of this conversion is 21.6 kcal mol-1 and the keto form(Int14) is stabilized about 16.1 kcal mol-1 compared to thestabilization in the enol form (Int13). Conversion of this ketoform to a new enol form (Int16) is performed through TS10which has a high barrier of 40.8 kcal mol-1 consistent with thefact that the pKa of Cα in GLA is about 10.3 [23]. Our DFTcalculations show that the proton-transfer reaction in thistautomerisation is the rate-limiting step. This kind oftautomerization has also been reported by Biemel et al. [5,7, 33] in the backbone of sugars (see Scheme 1 forcomparison).

Re TS1 TS2

TS3 TS4

TS5

C4

N1

H11

O7

H16 O15

H10

H11C4

N1

H10O15

H16

O7 H11

N1

O15H10H16

O7

C4

C7

O8

H11

H12O15

O7

H10O8

C7

C4

N2 H17N3

O7

O8 H10H2N3

N2

H13

O15

H11H12 N1

N3

N2

C4

a

b

Fig. 2 (a) Optimized (wb97xd/6-31+G*) geometries of the initialreactant complex and the transition states for Schiff base formationstep. (b) Optimized (wb97xd/6-31+G*) geometries of the transition

states for addition methyl guanidine (MGU) step, (c) Optimized(wb97xd/6-31+G*) geometries of the transition states and the productfor addition of the glyceraldehyde step

1652 J Mol Model (2012) 18:1645–1659

formed after passing of TS12 which its formation barrieris 33.1 kcal mol-1. In this step of reaction H32, which is anacidic proton, is transferred to N2 (Fig. 2c). The lastcyclization is done through addition of enamine toaldehyde in backbone of glyceraldehyde which is a kindof cross-aldol reaction.

This kind of phenomena has also been found in Morita–Baylis–Hillman reaction and mechanism of direct aldolreactions catalyzed by amino amide derivatives in otherstudies [14, 41]. In this step, H16 is shuttled to O17 throughforming a less strained ten-membered-ring. Four watermolecules assist this cyclization; two of these molecules

serve as a bridge to help the proton transfer and the otherones make the cooperation effect to stabilize the structure viathe formation of a hydrogen bond (Fig. 2c). As can be seenfrom Fig. 1, activation free energy for last cyclization (TS13)is about 18 kcal mol-1. Distance between C23-C33 in TS13reach to 1.7 and it shows that second cyclization throughcarbon-carbon bond formation mechanism can be done.Addition reactions of enamines through new C-C bondformation have been observed experimentally [10]. Thereaction proceeds downhill from TS13 to form a seven-membered heterocyclic ring attached to a five-membered one(Int22) whose stability is 17.2 kcal mol-1 more than Int21.

TS6 TS7

TS8 TS9

TS10 TS12

N1

N2

N3

O8

O7

H11

O15

H13

H12

O19

C20

O7

O8

N2

N3H12

O19 H11H13

O15

C20

N1

O7

O8

N2 N1

N3

H13O15

H22

GLA

O17

O16C21

H26

O16

O17

O15O19

H11

C20N1

H22

H26

O17

O16

H27H12

O19 H26

O15

H30C21 C23

C33H32

O8

H10

O7

H16

N2

c

Fig. 2 (continued)

J Mol Model (2012) 18:1645–1659 1653

The last proton transfer in this process is performed from C8

to C7 (see Fig. 2c TS14) and leads to the final product.

B. Comparison of cross-links

Formation mechanism of GODIC and MODIC(Scheme 3) proceeds through two main steps namely: (1)Schiff base formation (Scheme 3a), (2) addition of methylguanidine (Scheme 3b). All optimized structures for these

cross-links are given in the Supporting information. Theirfree energy surfaces in gas and solvent phases are depictedin Figs. 3 and 4 respectively. When comparing these cross-links (see Scheme 3 along with Figs. 3 and 4), we find thatin the first TS corresponding to the carbinolamineformation, relative free energies as compared to separatedreactants are the same in both cross-links. Moreover,barriers of the first dehydration reaction (TS2) in theformation of GODIC and MODIC are equal to 32.3 and33.5 kcal mol-1 respectively. These barriers have highest

TS11

TS13 TS14

Pr

O15

O19

O17

H27

H13

H22

C21

C23

C33C23

O17

H13

O19O8

H16

N2

H32

C8 C7

H34

d

Fig. 2 (continued)

1654 J Mol Model (2012) 18:1645–1659

values in free energy profile and are predicted to be the rate-limiting steps. There is a significant difference of3.7 kcal mol-1 between the free energy barriers of thesecond dehydration reaction (TS4) in GODIC and MODIC.This is due to the induction effect of methyl group on the C7

in MODIC (see Fig. 2b for notation) which would lowerpositive charge from 0.337 in C7 of GODIC to 0.001 in C7of MODIC. Thus TS4 in GODIC is more advanced and thefree energy barrier of this step is higher than that in MODICformation.

N

N

C

H

HH

H

H

H

H

H

N

N

C

H

HH

H

H

H

H

N

N

C

H

HH

H

H

H

H N

N

C

H

HH

HH

H

H

N C

N

N

C

H

HH

HH

H

H

N CHN C

N C

N C

N C

N

N

C

HH

H

H

H

H

HA

B

C

D

E

F

Scheme 4 Structures of the protonated (A) and neutral forms of methyl guanidine (B-F)

Fig. 3 Free energy profilefor the whole cross-linkingprocess of GODIC andMODIC (in parentheses)analog in gas phase calculatedat the wb97xd/6-31+G*level

J Mol Model (2012) 18:1645–1659 1655

When we compare Figs. 1 and 3, it is clear that thesecross-links share steps of Schiff base formation andaddition of MGU with glucosepane. As it can be seen fromthe figures, from a thermodynamics point of view, the threeprocesses are exothermic and the glucosepane formation ismore favorable than each of GODIC and MODIC (by ca.4.1 and 12.0 kcal mol-1 respectively). however, the freeenergy barrier of the rate-limiting step in the glucosepaneformation is higher than those in the GODIC and MODICformation (40.8 kcal mol-1 against 32.3 and 33.5 kcal mol-1

respectively). So its formation is slower than others.Table 2 summarizes levels of cross-links in vivo. In the

different matrices of human, concentration of glucosepaneis more important than each of MODIC and GODIC, whichcorroborates with the fact that its formation is highlyexothermic. Moreover experimental results [6, 46] haveconfirmed that time incubation for its formation is higherthan the two others cross-links. So our results are in goodagreement with experimental evidence in vivo.

C. Solvent effects

To estimate the solvent effect, the structures of all stationarypoints were reoptimized at the wb97xd/6-31+G* level with

the PCM model. Cartesian coordinates optimized in thewater are given in the Supporting information. The freeenergy profiles in solution are represented in Figs. 1 and 4for glucosepane and GODIC (MODIC) respectively. Thefree energy barriers for each step of cross-linking processesin the gas and water phase for comparison are summarizedin Table 3.

It is noteworthy that entropic contributions in aqueoussolution can be somewhat overestimated since calculated inthe ideal gas model3 [47].

The presence of the polarizable dielectric of solventincreases the barriers when the dipole moment of the TSstructures is smaller than that in their intermediates insolvent phase. For example, in glucosepane formation,relative free energy of TS1 with respect to separated

3 Following the comment of a reviewer, we computed entropiccontributions on free energy profile in the gas phase and solvent forglucosepan. Our calculations revealed that this contribution hasmaximum values at the beginning of any step; (i.e, in addition ofMA, MGU and GLA on GO/MGO, Int4 and Int8, respectively)whereas in any other steps enthalpic contributions overcome onentropy penalty (see Table S1). Therefore, it can be concluded thatentropic contribution in most cases can not affect the reaction profile(exception in beginning of each steps) while that enthalpic terms aremore effective and determining on the proceeding of reaction

Fig. 4 Free energy profilefor the whole cross-linkingprocess of GODIC andMODIC (in parentheses) analogin solvent phase calculated atthe wb97xd/6-31+G*level

1656 J Mol Model (2012) 18:1645–1659

reactants increases by 1.5 kcal mol-1 in solution. It can beattributed to that the dipole moment of TS1 (5.1 Debye) issmaller than that of MA+MGO+H2O separated reactant(5.8 Debye). So the transition state is destabilized in solvent

water by solvation. On the contrary, the dipole moment ofTS7 (13.9 Debye) is larger than that of Int10 (6.2 Debye),and the solvation effect on TS7 is stronger than that onInt11. Therefore, the solvent water stabilizes the transitionstate TS11, leading to a decrease of free energy barrier by10.4 kcal mol-1. It can be conclude that by increasing ofdipole moment of TSs relative to their intermediates(positive values of Δμ (sol) in Table 3), the free energybarriers in solution decrease and inverse too.

Thermodynamics of the whole cross-linking processesfor glucosepane, GODIC and MODIC are disfavored by12.2, 10.4 and 10.8 kcal mol-1 when solvent is taken intoaccount. However these reactions are still favorable (by ca.18.1, 15.8 and 7.5 respectively for mentioned cross-links)(see Table 3 for comparison).

The rate-limiting steps stay the same for all three cross-links formation and the first dehydration reaction (TS2) for theformation of GODIC and MODIC and the last keto-enolconversion on the backbone of glyceraldehyde in the third

Table 2 Levels of the cross-links in different matrices of humana

Glucosepane GODIC MODIC

Human skin collagen 2000 (5000)b 15 (<60) 30 (<60)

Glomerular basementmembrane (GBM)

500 (900) 80 60

Human lens proteins 185.1 21.1 65.5

Human serum albumin(HSA)

17.1 (29.2) N.Dc 4.1 (3.9)

a Data obtained from references 5 and 17b All values are expressed as mmol/mg of protein and in parenthesesare amount of cross-links in diabetic subjectsc N.D stands for "not detected"

Mechanism of GODIC (MODIC)

ΔGgas≠ ΔGsol

≠ ΔGsol≠ - ΔGgas

≠ ΔμSOLa

Re1 0.0 0.0 0.0 0.0

Re1 –– TS1 15.8 (15.7b) 16.3 (17.2) 0.5 (1.5) −1.0 (−0.6)INT2 –– TS2 32.3 (33.5) 30.2 (32.2) −2.1 (−1.2) 2.3 (3.4)

INT4 –– TS3 8.8 (8.4) 13.3 (11.8) 4.5 (3.4) −2.5 (−2.0)INT5 –– TS4 29.7 (26.0) 27.6 (25.2) −2.1 (−0.8) 2.4 (2.1)

INT7 –– TS5 0.4 (0.3) −0.8 (0.9) −1.2 (−1.1) 0.5 (0.7)

INT8 –– TS6 22.0 (25.0) 22.0 (24.4) 0.1 (−0.6) −0.1 (0.1)

INT9 –– TS7 18.3 (18.1) 17.2 (16.7) −1.1 (−1.4) 1.0 (0.7)

Mechanism of glucosepane

Re2 0.0 0.0 0.0 0.0

Re2 –— TS1 15.7 17.2 1.5 −0.6INT2 –— TS2 33.5 32.2 −1.2 3.4

INT4 –— TS3 8.4 11.8 3.4 −2.0INT5 –— TS4 26.0 25.2 −0.8 2.1

INT7 –— TS5 0.3 −0.9 −1.1 0.7

INT9 –— TS6 13.5 13.0 −0.5 1.5

INT11——— TS7 38.8 28.3 −10.4 7.8

INT12——— TS8 0.2 2.3 2.1 −1.3INT14——— TS9 21.5 19.2 −2.4 13.0

INT16——— TS10 40.8 37.8 −3.0 0.2

INT18——— TS11 18.7 15.2 −3.5 0.2

INT20——— TS12 33.1 31.1 −1.9 0.7

INT22——— TS13 18.4 18.8 0.4 −2.4INT23——— TS14 13.8 15.5 1.7 −3.3Thermodynamics of reactions

ΔGgasr ΔGsol

r ΔGsolr - ΔGgas

r ΔμSOLc

Re3 –— Pr1 (Pr2) −26.2 (−18.3) −15.8 (−7.5) 10.4 (10.8) −7.8 (−6.4)Re4 –— Pr3 −30.3 −18.1 12.2 −6.5

Table 3 The reaction barriersand relative free energies re-spect to separated reactantsexpressed in kcal mol-1 for eachreaction step of GODIC(MODIC) and Glucosepanemodels in the gas and solventphase at wb97xd/6-31+G* levelof theory

aΔμSOL=μTS - μINT

b Value in parentheses are forMODIC modelcΔμSOL=μ

Pr - μRe

Re1 is MA+GO (MGO)+H2Oas separated

R2 is MA+MGO+H2O asseparated

Re3 is R1+MGU as separated

Re4 is R2+MGU+GLA asseparated

J Mol Model (2012) 18:1645–1659 1657

step of process for the Glucosepane are still rate-determiningsteps.

Conclusions

In summary, our studies focused upon the cross linkingreactions between α-oxoaldehydes and models of lysine andarginine residues in order to better understand cross-linkingchemistry in vivo and food chemistry. Our results show thatthese reactions are thermodynamically and kinetically favor-able in gas phase and solution with a high stabilization of thefree energy of the glucosepane in respect to the separatedreactants and values of free energy barriers are reasonable.Comparisons with the formation of two other important cross-links, GODIC and MODIC, show that the glucosepaneformation is more exothermic than others while its formationkinetics is slower. These results could illuminate mechanismsof AGE formation in diabetes and aging as well as suggestpotential therapeutic interventions in good agreement withexperimental results in glycation of human serum albuminunder physiological conditions.

Acknowledgments The financial support of Research Council ofShahid Beheshti University is gratefully acknowledged. Rasoul Nasiriwould like to thank the Ministry of Science, Research, andTechnology of Iran for financial support. Many thanks also go toMartin Field and Andrew Greene for their helpful discussions.

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