DOI: 10.1002/chem.201100313

Intramolecular Weak Interactions in the Thermodynamic Stereoselectivity ofCopper(II) Complexes with Carnosine–Trehalose Conjugates

Giuseppa I. Grasso,[a] Giuseppe Arena,[a] Francesco Bellia,[a] Giuseppe Maccarrone,[a]

Michele Parrinello,*[b] Adriana Pietropaolo,[a, b] Graziella Vecchio,*[a] andEnrico Rizzarelli[a]

Introduction

Sugar–peptide interactions play a subtle role in a wide rangeof fundamental biological processes: metabolic regulation,growth, embryogenesis, and apoptosis among many others.Understanding the mechanisms by which carbohydrates rec-ognize proteins,[1] as well as the specific structural determi-nants which cause the natural stereoselectivity[2–5] (d-sugarsversus l-amino acids) remains a fundamental question inbiochemistry.

Interest is growing in a specific class of peptides of thecarnosine family,[6] as witnessed by the growing number ofpatents.

Carnosine (b-alanyl-l-histidine) is a naturally occurringdipeptide present in the muscle and brain tissues of humans,and other vertebrates in relatively high concentrations (1–20 mm). Carnosine is synthesized by carnosine synthetase

and hydrolyzed in blood plasma by carnosinases.[7,8] Not sur-prisingly, there has been a considerable interest[9–14] in thedevelopment of carnosine-related structures with increasedmetabolic stability in view of potential therapeutic applica-tions,[15] owing to the role of carnosine in Alzheimer’s dis-ease,[15] nitrosative[16,17] and oxidative stress,[15] and to itsability to complex zinc and copper ions.[18] In particular, l-carnosine (lCar) conjugated to d-trehalose (Tr) resists car-nosinase hydrolysis and possesses an antioxidant activityhigher than that of l-carnosine.[14–19] The carbohydratemoiety has also been shown to be able to perform severalfunctions.[20, 21]

d-Trehalose has also been reported to in-crease human neuroblastoma cell viability in the presence ofAb aggregates[22] and alleviate the polyglutamine-inducedsymptoms in a mouse model of Huntington’s disease.[23]

The d enantiomer of l-carnosine, dCar, is not hydrolyzedby carnosinase and maintains the same quenching activity ofl-carnosine in vitro,[24] though does not show the beneficialeffects of the l enantiomer in cultured human fibroblasts.[25]

In view of its potential therapeutic applications, we per-formed an extensive combined experimental and theoreticalinvestigation of the dimeric copper(II) complexes with d-trehalose–l-carnosine (TrlCar) and d-trehalose–d-carnosine(TrdCar) (Scheme 1). Surprisingly, the diastereoisomersshow significantly different affinity for copper(II) mainlywith respect to the metal dimeric species(log bl

22�2�log bd

22�2 = 3.6). The potentiometric, spectroscopic,and isothermal titration calorimetry (ITC) measurementsand free-energy calculations allowed us to probe differentaspects of the copper–dipeptide interaction. A complete pic-

[a] Dr. G. I. Grasso, Prof. G. Arena, Dr. F. Bellia, Prof. G. Maccarrone,Dr. A. Pietropaolo, Prof. G. Vecchio, Prof. E. RizzarelliDipartimento di Scienze ChimicheUniversit� degli Studi di Catania6 Viale A. Doria, 95125 Catania (Italy)Fax: (+39) 095-337678E-mail : gr.vecchio@unict.it

[b] Prof. M. Parrinello, Dr. A. PietropaoloComputational Science Department of Chemistry andApplied Biosciences, ETH ZurichUSI-Campus, 6900 Lugano (Switzerland)E-mail : parrinello@phys.chem.ethz.ch

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201100313.

Abstract: The interactions of metalions with chiral molecules are of partic-ular interest for relevant biochemicalprocesses, as many of them are madepossible only with a selected chiralityof the stereocenters. In this work wereport a study of the stereoselectivityof copper(II) complexes with d-treha-lose–l-carnosine and d-trehalose–d-carnosine as a prototypical case of nat-ural chirality selection. The interest inl-carnosine dipeptide is compoundedby its antioxidant and antitumor prop-

erties, which are further enhancedwhen combined with d-trehalose. Po-tentiometric, calorimetric, and UV/cir-cular dichroism (CD) spectroscopicmeasurements show that the copper(II)dimer of d-trehalose–l-carnosine ismore stable than the d-trehalose–d-car-nosine diastereoisomeric copper(II)

dimer (log bl

22�2�log bd

22�2 = 3.6). Free-energy calculations highlight that thecause of this different behavior lieswith different intramolecular weak in-teractions between the diastereoiso-mers. The different pattern of hydro-gen bonds and the different CH–p in-teractions between the p-electron-richimidazole and the a-glucose rings aremore favorable by 5 kcal mol�1 in the l

dimer.Keywords: carbohydrates ·chirality · copper · peptides ·thermodynamics

� 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2011, 17, 9448 – 94559448

ture of all the factors determining the different stabilityemerges from the combination of different approaches.

Results and Discussion

Protonation equilibria and the relative enthalpy/entropycontributions : Protonation constant values for both TrlCarand TrdCar are reported in Table 1. The values for the threeprotonation steps involving amino, imidazole, and carboxyl-ate groups of TrlCar and TrdCar are not significantly differ-ent. However, compared with unfunctionalized carnosine,the derivatization of both lCar and dCar with trehalosecauses a drop in the protonation values of the amino groupof more than one logarithmic unit while small or no effectswere observed on protonation of the imidazole and carbox-ylate moieties (Table 1). Interestingly, this trend is reportedto be a leitmotif for carnosine derivatization.[10, 26] To haveinformation on this phenomenon we have split the DG8term into its enthalpic and entropic contribution by meansof calorimetry.

In particular, the enthalpy trend found is the one expect-ed for this class of peptides.[27–29] The protonation of theamino group usually shows an exothermic contribution,

since the formation of the H�N bond outweighs the endo-thermic effect resulting from the desolvation of both theproton and the amino group; the resulting charged particle(R1-NH2

+-R2) orders the water molecules and this accountsfor the relatively small entropy contribution. On the contra-ry, the protonation of a carboxylate group is usually associ-ated with a large entropy gain due to charge neutralizationwhile the process is slightly endothermic. In the present casethe protonation of the amino group is accompanied by un-usually large entropic values; in fact, for both systems theseare larger (TrlCar) or comparable (TrdCar) to those of thecarboxylate groups. It is noteworthy that the solvation pro-cess related to the protonation of the amino group usuallyoccurs more easily than that of the carboxylate and this ac-counts for its less favorable entropy contribution.[30] Thedrop in the basicity of the amino group after trehalose deri-vatization results from a less favorable enthalpy contributionto its protonation (�7.7 vs. �11.7 kcal mol�1 for TrlCar andlCar, respectively). A similar trend has been reported forthe amine protonation of functionalized cyclodextrins.[31–33]

The unusual enthalpy and entropy changes pertaining to thesecond protonation step of imidazole may be explained in asimilar manner. In addition, such a trend turns out to beeven more pronounced for the TrlCar diastereoisomer. Theprotonation process would involve the breaking of hydrogenbonds and, in turn, a lesser exothermic change and morepositive entropy contribution caused by the increase of thedegrees of freedom of the molecule. Contrary to TrlCar, forTrdCar the protonation of imidazole is more exothermic,which suggests a noncovalent interaction involving the het-erocyclic ring; the consequent stiffening of the molecule ac-counts for the less favorable entropy change. The third pro-tonation step shows thermodynamic parameters similar tothose found for lCar and the protonation process of the d

diastereoisomer is again enthalpically more favored than theanalogous step of the l diastereoisomer.

NMR spectroscopy measurements (Figures S1–S5 in theSupporting Information) indicate that trehalose interactswith the alanine residue and the protonation process de-stroys this interaction since trehalose moves away from theamine group. If compared to the analogous underivatizedamines,[30] the diminished exothermic contribution ofTrlCarH and TrdCarH (Table 1) has to be ascribed to the

cleavage of hydrogen bonds and it is responsible ofthe more positive contribution. Moreover, NMRspectroscopy data show that in the protonatedamine nitrogen species, the imidazole of the l-histi-dine bends toward the protonated amino group,thereby suggesting an interaction between the het-erocyclic ring and the alanine moiety, as supportedby recent findings.[34]

The rationalization of the thermodynamic data isfurther supported by molecular dynamics carriedout at different pH (see the Supporting Informa-tion), which show a dependence on pH of the twodiastereoisomer conformations. In particular, vary-ing from acidic to basic pH, the number of confor-

Scheme 1. Structure of TrlCar (*=l) and TrdCar (*=d).

Table 1. Log b, pKa, DG8, DH8, and DS8 values (3s in parentheses) for the protonationof lCar, TrlCar, and TrdCar at 25 8C and 0.1 mol dm�3 (KNO3).

Equilibrium Log b pKa DG8[kcal mol�1]

DH8[kcal mol�1]

DS8[calmol�1 K]

lCar+HQlCarH 9.40[a] 9.40 �12.78[b] �11.7[b] 3.6[b]

lCarH +HQlCarH2 16.18[a] 6.78 �9.24[b] �7.7[b] 5.2[b]

lCarH2 +HQlCarH3 18.75[a] 2.57 �3.54[b] �0.2[b] 11.2[b]

TrlCar+HQTrlCarH 8.21(3) 8.21 �11.19(4) �7.70(4) 11.7(1)TrlCarH +HQTrlCarH2 14.90(3) 6.69 �9.12(4) �4.9(4) 14(1)TrlCarH2 +HQTrlCarH3 17.46(6) 2.56 �3.49(8) �0.6(4) 10(1)TrdCar+HQTrdCarH 8.17(3) 8.17 �11.14(4) �8.7(4) 8(1)TrdCarH +HQTrdCarH2 14.90(3) 6.73 �9.18(4) �6.4(4) 9(1)TrdCarH2 +HQTrdCarH3 17.50(6) 2.60 �3.55(8) �1.2(5) 8(2)

[a] Reference [10]. [b] Reference [29].

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FULL PAPER

mations with a higher number of hydrogen bonds increases,owing to more available acceptor groups interacting withthe side chains (see Figures S6–S8 in the Supporting Infor-mation).

Copper coordination equilibria in solution

Potentiometry : As seen for the protonation, the introductionof trehalose modifies the species distribution of the copper-(II) complexes (Figure 1) of both the lCar and dCar deriva-tives. The refined equilibria for both TrlCar and TrdCarwith bpqr values (see the Experimental Section) are listed inTable 2.

A striking difference is immediately evident: the two dia-stereoisomers form species of comparable stability with cop-per(II), except for the dimeric species (see Figure 1 andTable 2 for the complete list of species). The formation ofthe l dimer [Cu2ACHTUNGTRENNUNG(TrlCar)2H�2]is favored over the analogousspecies with TrdCar and the dif-ference between the stabilityconstant values amounts to 3.6logarithmic units. In addition,[Cu2ACHTUNGTRENNUNG(TrlCar)2H�2] shows alarger stability if compared tothe analogous species with lCar(Table 3). To verify if these dif-

ferences between the stability constant values might resultfrom different coordination environments of the metal ionin the diastereoisomer dimeric complexes, the copper(II)complex systems with TrlCar and TrdCar were investigatedby UV-visible and CD spectroscopy.

UV-visible and CD spectroscopy : The copper(II)–lCarsystem has been previously investigated by different tech-niques.[35] For comparison we report here the results for thecopper(II)–dCar system. As expected, the UV-visible aswell as the CD spectra of the enantiomeric systems are thesame.

The addition of trehalose to the solution containing cop-per(II)–lCar binary complexes does not modify the CDspectra, thus suggesting that free trehalose does not interactwith the carnosine–copper(II) complexes. The presence of achiral moiety such as trehalose, covalently bound to carno-sine, induces some differences in the coordinating featuresof the peptide.

UV-visible spectra of the copper(II)–TrlCar and –TrdCarsystems were carried out at different pH values. Visiblespectra (pH 7.2) show a redshift of the d–d band (Table S2in the Supporting Information) and a higher e value in com-parison to the copper(II)–lCar system. In particular, theTrdCar and the TrlCar systems show bands centered at 645(e= 120) and 656 nm (e=139), respectively. CD spectra(Figure S9 in the Supporting Information) of the copper(II)complexes of TrlCar and TrdCar are quasi-enantiomeric inthe visible region at 600 nm (pH 7.2), but the jDe j valuesare larger for the TrlCar system. Both the CD spectra ofthe copper(II) complex with TrlCar (l=270 nm) and withTrdCar (l= 290 nm) show a positive Cotton effect. This

Figure 1. Species distribution diagrams for the copper(II)/L systems(1:1.2), cL =3.5� 10�3 mol dm�3. 1) Free Cu; 2) [CuLH]; 3) [CuL];4) [Cu2L2H�2]; 5) [CuLH�2]. L is for TrlCar in (a) and TrdCar in (b).

Table 2. Log b values for the complexes of copper(II) with lCar, TrlCar,and TrdCar at 25 8C and 0.1 mol dm�3 (KNO3).

Equilibrium bpqr Log bpqr[a]

Cu2+ +lCar�+H+Q[CuACHTUNGTRENNUNG(lCar)H]2+ b111 13.54[b]

Cu2+ +lCar�Q[Cu ACHTUNGTRENNUNG(lCar)]+ b110 8.49[b]

Cu2+ +lCar�Q[Cu ACHTUNGTRENNUNG(lCar)H�1] +H+ b11�1 2.98[b]

2Cu2+ + 2lCar�Q ACHTUNGTRENNUNG[Cu2 ACHTUNGTRENNUNG(lCar)2H�2]+2 H+ b22�2 8.06[b]

2Cu2+ + lCar�QACHTUNGTRENNUNG[Cu2 ACHTUNGTRENNUNG(lCar)H�1]2+ +H+ b21�1 5.35[b]

Cu2+ +TrlCar�+H+Q[CuACHTUNGTRENNUNG(TrlCar)H]2+ b111 12.1(1)Cu2+ +TrlCar�Q[Cu ACHTUNGTRENNUNG(TrlCar)]+ b110 7.27(1)2Cu2+ + 2TrlCar�Q ACHTUNGTRENNUNG[Cu2ACHTUNGTRENNUNG(TrlCar)2H�2]+2 H+ b22�2 8.83(4)Cu2+ +TrlCar�Q[Cu ACHTUNGTRENNUNG(TrlCar)H-2]

�+2 H+ b11�2 �7.52(4)Cu2+ +TrdCar�+H+Q[CuACHTUNGTRENNUNG(TrdCar)H]2+ b111 11.78(4)Cu2+ +TrdCar�Q[Cu ACHTUNGTRENNUNG(TrdCar)]+ b110 7.25(2)2Cu2+ + 2TrdCar�QACHTUNGTRENNUNG[Cu2ACHTUNGTRENNUNG(TrdCar)2H�2] +2H+ b22�2 5.23(3)Cu2+ +TrdCar�Q[Cu ACHTUNGTRENNUNG(TrdCar)H�2]

�+2 H+ b11�2 �7.43(3)

[a] 3s in parentheses. [b] Ref. [10].

Table 3. Log b, DG8, DH8, and DS8 values (3s in parentheses) for the dimeric copper(II) complex species withlCar, TrlCar, and TrdCar at 25 8C and 0.1 mol dm�3 KNO3.

Equilibrium Log b22�2 DG8[kcal mol�1]

DH8[kcal mol�1]

DS8[calmol�1 K]

2Cu2+ + 2lCar�Q ACHTUNGTRENNUNG[Cu2 ACHTUNGTRENNUNG(lCar)2H�2]+2 H+ 8.06[a] �11.39[b] �12.4[b] �3[b]

2Cu2+ + 2TrlCar�Q ACHTUNGTRENNUNG[Cu2ACHTUNGTRENNUNG(TrlCar)2H�2]+2 H+ 8.83(4) �12.04(5) �6.0(4) 20(1)2Cu2+ + 2TrdCar�QACHTUNGTRENNUNG[Cu2ACHTUNGTRENNUNG(TrdCar)2H�2] +2H+ 5.23(3) �7.13(4) �3.36(4) 12.6(1)

[a] Ref. [10]. [b] Ref. [29].

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G. Vecchio, M. Parrinello et al.

broad band is characteristic of charge transfer N-3d fromthe amino and amido groups that are overlapped in the tre-halose derivatives. The copper(II)–TrlCar system showsspectroscopic characteristics that are similar to those ofother carbohydrate derivatives of lCar.[10, 36,37] The function-alization of carnosine produces a larger Cotton effect and aredshift in the visible region. In the b-cyclodextrin derivativeof lCar, a distortion of the square-planar geometry of thecomplex was hypothesized.[10] Similarly, we can assume thatthe derivatization with the trehalose unit produces a distor-tion of the coordination plane of the metal ion, as also indi-cated by the lmax and the e values observed in UV-visiblespectra. However, the same atom binding in the coordina-tion sphere of carnosine can be suggested (NH, N�, Im,COO�). The magnitude of the Cotton effect depends on thedistortion and probably relates to the pendant attached tob-alanine. The interaction of the trehalose chain with thecarnosine–copper(II) complex moiety is correlated with his-tidine chirality, as indicated by the dichroic spectra. CDspectra of the TrlCar system do not change significantly atpH 10, in keeping with the distribution diagram (Figure 1),whereas the visible spectra of the TrdCar system show ablueshift of the band due to the d–d transition at 580 nm. Inthe CD spectra, the De of the band at 290 nm increases andtwo shoulders are detected at 250 and 500 nm, which sug-gests different coordination features in keeping with the for-mation of a new complex species. These data are consistentwith the involvement of a deprotonated hydroxyl group inthe coordination of the copper(II) in addition to imidazole,amino, and N� amido nitrogen atoms, as reported for similaraminoglycoside complexes.[38]

The spectroscopic parameters indicate that 1) in the di-meric species of the two diastereoisomeric complexes thedonor atoms bound to the metal ion are the same; and2) the derivatization with trehalose causes a distortion of thecoordination geometry observed in the analogous complexwith the natural dipeptide. These results do not provide evi-dence to explain the significant differences of the stabilityconstants, and consequently we went in depth to explain theorigin of this particular thermodynamic stereoselectivity.

Calorimetry : To further explore the origin and the extent ofthe interactions due to the presence of trehalose in the car-nosine backbone, we resorted to isothermal titration calo-rimetry (ITC) as a valuable approach for measuring theheat energy associated with a molecular interaction at agiven constant temperature[39,40] as well as in providing aprobe to determine the extent of interaction over the courseof the experiment.[41,42] The DG8, DH8, and DS8 values forthe formation of the main species for the two diastereoiso-mers are given in Table 3.

The larger DH8 value of the dimeric species formed byTrlCar with respect to that of TrdCar indicates that thehigher stability (��5 kcal mol�1) results (at least partly)from a more favorable arrangement of the two ligand moiet-ies, that, in turn, leads to different noncovalent bonds. In ad-dition, the D ACHTUNGTRENNUNG(DH8) may also reflect the difference between

the respective initial state of the two species of the two dia-stereoisomers (TrlCarH and TrdCarH) at this pH. Themore favorable enthalpic contribution, observed for the mo-noprotonation of the d diastereoisomer, would no longeroccur in the copper(II) dimeric species and, on the whole,would determine its smaller DH contribution. TrdCar con-formation is driven by the presence of a favorable networkof noncovalent interactions; these interactions would be lostin the dimeric complex species. Since the core of the binu-clear species is practically the same for the two diastereoiso-mers and the CD experiments show that the addition of un-bound trehalose to copper(II) complexes with lCar or dCarsolutions does not cause any change, the differences ob-served should result from noncovalent intramolecular inter-actions, invoked for binary and ternary metal complexes.[43]

However, structural information is needed to prove (or dis-prove) the above-mentioned conclusion. To this end wehave combined the spectroscopic and calorimetric resultswith computational free-energy estimates (see below).

Free-energy determination : For simplicity let us considerthe case of a single collective variable, s. The extension tomany variables is straightforward.

The free energy F(s) as a function of this variable is givenby Equation (1):

FðsÞ ¼ �1b

ln

Rdðs� sðrÞÞe�bVðrÞdrR

e�bVðrÞdrð1Þ

in which V(r) is the interaction potential and r the atomiccoordinates.

We now add to V(r) a harmonic restraining potential thatdepends only on the collective coordinates, 1

2 kðsðrÞ � s0Þ2.We define FB(s) as the free-energy surface for the modi-

fied potential VðrÞ þ 12 kðsðrÞ � s0Þ2 given by Equation (2):

FBðsÞ ¼ � 1b

ln

Rdðs� sðrÞÞe�bðVðrÞþ1

2kðsðrÞ�s0Þ2ÞdrR

e�b12kðsðrÞ�s0Þ2 e�bVðrÞdr

ð2Þ

It is then straightforward to show that FB(s) is related toF(s) by Equation (3):

FBðsÞ ¼ 12

kðs� s0Þ2 þ FðsÞ þ c ð3Þ

in which c is � 1b lnR

dse�bFðsÞe�b12kðsðrÞ�s0 Þ2

Rdse�bFðsÞ

In our case we have two collective variables, namely, thelength of the two trehalose–carnosine bonds and this rela-tion will be generalized in Equation (4):

FLl ðs1; s2Þ ¼

l

2Kðs1 � s0Þ2 þ

l

2Kðs2 � s0Þ2 þ FL

0 ðs1; s2Þ þ cL0 þ cL

l

FDl ðs1; s2Þ ¼

l

2Kðs1 � s0Þ2 þ

l

2Kðs2 � s0Þ2 þ FD

0 ðs1; s2Þ þ cD0 þ cD

l

ð4Þ

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FULL PAPERThermodynamic Stereoselectivity of Copper Complexes

in which cl =

� 1b lnR

ds1ds2e�bFL0ðs1 ;s2 Þe�b

l2Kðs1�s0 Þ2 e�b

l2Kðs2�s0 Þ2

Rds1ds2e�bFL

0ðs1 ;s2 Þ

The K is chosen to be theharmonic constant of the treha-lose–carnosine bond and for l =

1 it is in the bonded situation(Cu2ACHTUNGTRENNUNG(TrxCar)2H�2, in which x isl or d), whereas for l=0 it is inthe dissociated case. The re-markable property is that, intheory, with a single calculationone can obtain simultaneouslythe free energy of these twostates. In practice, to evaluateF(s) we chose an intermediatevalue of l such that the re-straining force is strong enoughto bring back the trehalose tothe carnosine, yet weak enoughfor the trehalose to explore re-gions of configuration space inwhich the system is fully disso-ciated. We have found thatthese conditions are satisfiedfor l= 10�3 (lK= 0.32 kcal mo-l�1 ��2), which allows s to reachdistances of 6 � where the freeenergy becomes flat, thereby in-dicating complete dissociation.Since at this limit any depend-ence on the trehalose chiralityis lost, we can equate the quan-tities according to Equation (5):

FL01ðs1; s2Þ þ cL

0 ¼ FD01ðs1; s2Þ þ cD

0

Dðc0Þ ¼ cL0 � cD

0 ¼ FD01ðs1; s2Þ � FL

01ðs1; s2Þð5Þ

thus aligning the two surfaces. Once the free-energy surfacesat this value of l are obtained (Figure S10 in the SupportingInformation) we can determine the free-energy difference atl=1, Fl

1�Fd

1, which is reported in Figure 2).The experimentally observed quantity is the formation

energy in the process DFx. This can be calculated as a resultof the cycle in Figure 3, where we first separate the treha-lose from the carnosine, which results in the free energy offormation DF ligand

x . The two free carnosines are then assem-bled to form the core [Cu2ACHTUNGTRENNUNG(lCar)2]; this step is clearly inde-pendent of the chirality and leads to the free energy, DFcore.We shall assume that within the error of calculation DF ligand

x

is negligible, as shown in Figure S11 of the Supporting Infor-mation and focus on the evaluation of DFassociation

x , where thecomplex is reassembled by adding the trehalose to the cen-tral core, DFassociation. This last step is clearly chirality-depen-dent.

Free-energy landscape of copper dimers : When looking atthe free-energy difference of the l and d diastereoisomersurface the result is striking: the l dimer is more stable by5 kcal mol�1 than the d one. We tried to clarify the chemicalorigin of the stereoselectivity by exploring the conforma-tional space with two metadynamics for l and d dimers atstandard force constant (l=1, K=320 kcal mol�1 ��2), usingas collective variables the two torsions involving the rotationof the trehalose chain around the binding distance.

It is evident from Figure S12 in the Supporting Informa-tion that the l copper dimer shows a higher percentage ofhydrogen bonds than the d one and, interestingly, in bothcases two conformations are more likely. In addition, thechemical features of both dimers suggest another possibleinteraction at the interface of the central core and the car-bohydrate chain. We analyzed the CH–p interactions, whichinvolve the p-rich imidazole and the a-glucose rings, bymonitoring the w angle formed by the C1–H1 bond vector ofthe a-glucose ring with the plane of the imidazole ring, andby following the distance between the H1 hydrogen and thecenter of mass of the imidazole ring.

The map for the d dimer appears diffuse (Figure 4),whereas a strong sharp minimum is present in the l dimer,revealing an antiparallel conformation. The formation of an

Figure 2. a) Representation of the collective coordinates for the binding free-energy calculation. b) Free-energy difference surface Fl

1�Fd

1, highlighting the �5 kcal mol�1 free-energy difference in the minimum. Theisolines were drawn using a 0.5 kcal mol�1 spacing and the energy scale is in kcal mol�1.

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G. Vecchio, M. Parrinello et al.

intrachain hydrogen bond between the hydroxyl groups oftwo different chains (Figure S13 in the Supporting Informa-tion) is responsible for a parallel orientation of the chains,despite being less populated than the antiparallel conforma-tion (Figure S14 in the Supporting Information). The twostates deriving from the hydrogen-bond distributions (Fig-ure S12) are therefore connected to the antiparallel (highestpeak) and the parallel (lowest peak) conformations, thesnapshots of which are reported in Figure 4.

Conclusion

Understanding the structural and thermodynamic phenom-ena that control the manner in which a carbohydrate inter-

acts with the peptide moiety is crucial for studying the com-plex mechanisms that lead to protein–sugar recognition andtheir interaction with metal ions.

To the best of our knowledge, these are the first data everreported on diastereoisomer copper(II) complexes based ondisaccharide–peptide conjugates. The presence of the metalion is very relevant for exploiting enantiospecific interac-tions. The dinuclear species formation is favored by the en-thalpy and entropy contributions in a different manner forthe two diastereoisomers.

The experimental approach, based on calorimetric andspectroscopic data, was supported by metadynamics-basedfree-energy estimates. The key is to study how d-trehaloseinteracts with the dinuclear l- and d-carnosine cores of cop-per(II). Since chirality centers of d-trehalose are fixed as d,being a natural carbohydrate, different interactions are thengenerated. The number of hydrogen bonds is larger in the l

diastereoisomer and moreover, an intrachain hydrogen bondwas found in the l dimer, which causes a parallel orienta-tion. In addition, CH–p interactions between the p-rich imi-dazole and a-glucose rings are found to be more stable inthe l dimer. This is an example of chirality selection basedon free-energy difference.

Experimental Section

Synthesis and 1H NMR spectroscopy measurements of the ligands are re-ported in the Supporting Information (Figure S1–S5).

Spectroscopic measurements : UV/Vis spectra of the copper(II) com-plexes were recorded on a Cary 500 spectrophotometer (Varian).

CD spectra of the ligands and their copper(II) complexes were recordedon a JASCO 810 spectropolarimeter at a scan rate of 50 nm min�1 and aresolution of 0.1 nm. Calibration of the CD instrument was performedwith a 0.06 % solution of ammonium camphor sulfonate in water (De=

2.40 mol�1 dm3 cm�1 at 290.5 nm). The 200–800 nm spectral range was cov-ered by using quartz cells of various path lengths. Results are reported ase (molar adsorption coefficient) and De (molar CD coefficient) inmol�1 dm3 cm�1.

Metadynamics (MD): All MD calculations were performed by usingNAMD 2.7[44] with the Charm22 forcefield.[45] To reconstruct the free-energy surface of the two copper dimers, we used well-tempered (WT)metadynamics, proved to be effective in previous studies,[46–49] within theframework of PLUMED.[50] Gaussians of 0.2 kcal mol�1 in height weredeposited at one picosecond time interval, with a bias factor of 10. Atemperature of 300 K was enforced using a Langevin thermostat. Thequantum mechanics/molecular mechanics (QM/MM) run that was usedto select the starting configuration of the copper core as well the copperpotential parameters are described in the Supporting Information andwere used throughout the simulation. To this copper core, with histidine,carboxylate, and amino groups deprotonated, we bound the trehalosechains, picking representative conformations from MD runs and equili-brated starting from a distance of 10 � for 5 ns, before running the meta-dynamics simulations at low force-constant value (lK=0.32 kcalmol�1 ��2),for 100 ns. In addition, two metadynamics simulations of 40 ns were car-ried out at standard force constant (320 kcal mol�1 ��2) for l and d

copper dimers.

Two metadynamics simulations of 100 ns were also run at low force-con-stant value (lK= 0.32 kcal mol�1 ��2), for trehalose–l-carnosine and tre-halose–d-carnosine to evaluate the free-energy difference estimate con-sidering the bond formation between trehalose and l-/d-carnosine. The

Figure 3. Top: Schematic representation of trehalose–l-/d-carnosine–cop-per(II) dimers. The two molecules may be seen as two enantiomeric cop-per(II) units (shown in black), bound to the trehalose chiral chains(shown in grey), which makes the energy split in two different levels.Bottom: Thermodynamic cycle for the free-energy difference calculationof the copper(II) dimers, [Cu2 ACHTUNGTRENNUNG(TrlCar)2H�2] and [Cu2 ACHTUNGTRENNUNG(TrdCar)2H�2]which involves a chirality-independent step (DFx core).

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FULL PAPERThermodynamic Stereoselectivity of Copper Complexes

relative free-energy difference is reported in Figure S11 of the SupportingInformation.

The evolution of the binding distances during metadynamics simulationsand the free-energy surfaces obtained for the copper complexes are re-ported in Figures S15–S16 and S10 in the Supporting Information.

EMF measurements : Potentiometric titrations were performed with twohome-assembled fully automated apparatus sets (Metrohm components:E654 meter, E665 dispenser; combined micro pH glass electrode,ORION 9103SC) controlled by appropriate software set up in our labora-tory. Further details are reported in the Supporting Information.

ITC measurements : The isothermal titrations were carried out by using aTA ITC 2G nanocalorimeter equipped with a 250 mL injection syringe.All titrations were run in overfilled mode, and thus the heat generatedrequires no correction, neither for liquid evaporation nor for the pres-ence of the vapor phase. The power curve was integrated using NanoA-nalyze (TA Instruments) to obtain the gross heat evolved in the reaction.The apparatus was calibrated before and after each run by introducingknown power values through built-in precision resistors. Solutions con-taining nitric acid (0.1–2.6� 10�2 mol dm�3) were added to a solution ofthe ligand (0.5–2 � 10�3 mol dm�3) or the ligand (5–9 � 10�4 mol dm�3) andthe metal ion (Cu2+/L ranged from 0.8 to 0.9). The net heat of the reac-tion was calculated by subtracting the heat evolved/absorbed duringblank experiments, in which the titrant was added to a solution contain-ing the same chemicals as the reaction run, except for the ligand. Thetotal number of data points used was 400 and 350 for the protonationand Cu2+ complexation with TrdCar, respectively, whereas 550 and 570data points were used for the analogous systems with the l derivative.

Chemical equilibria : All the equilibrium constants reported in the pres-ent work are overall association constants (b) and refer to the generalequilibrium given in Equation (6):

pCuþ qLþ rHÐ ½CupLqHr � ð6Þ

in which L is the most deprotonatedform of each ligand; in Equation (6),charges are omitted for simplicity. Theoverall stability constant (b), associat-ed with the equilibrium given in Equa-tion (6), is defined by Equation (7):

bpqr ¼½CupLqHr �½Cu�p½L�q½H�r ð7Þ

Acknowledgements

We thank Flamma S.p.A. (Bergamo,IT) for kindly supplying d-carnosineand MIUR (2008R23Z7K), PRIN2008F5A3AF_005 for financial sup-port. The computational work was per-formed on the Cineca supercomputercentre. ETHZ Brutus cluster is alsokindly acknowledged for providing ad-ditional computational resources.

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Received: January 28, 2011Published online: July 5, 2011

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FULL PAPERThermodynamic Stereoselectivity of Copper Complexes