Ternary Porphyrin Aggregates and Their Chiral Memory - The Journal

By contrast, the stable aggregates made up of an equimolar mixture of anionic and cationic porphyrins retain “memory” of the original matrix chira...
0 downloads 0 Views 116KB Size
Download PDF
10900

J. Phys. Chem. B 2000, 104, 10900-10908

Ternary Porphyrin Aggregates and Their Chiral Memory† R. Purrello,* A. Raudino,*,‡ L. Monsu` Scolaro,*,§ A. Loisi, E. Bellacchio, and R. Lauceri| Dipartimento di Scienze Chimiche, UniVersita` di Catania, Viale Andrea Doria 6, 95125 Catania, Italy ReceiVed: February 14, 2000; In Final Form: June 15, 2000

In this paper we investigate the self-aggregation of tetracationic porphyrin meso-tetrakis(N-methylpyridinium4-yl)porphinatocopper(II) (CuT4) onto a chiral polymeric matrix (polyglutamic acid, PG) as well as mixtures of tetracationic (CuT4) and tetraanionic, meso-tetrakis(4-sulfonatophenyl)porphine (H4TPPS), porphyrins onto the same PG matrix as a function of pH, temperature, ionic strength, and reactant concentration. The systems have been studied by light absorption, fluorescence, circular dichroism (CD), and resonance light scattering techniques. PG chains undergo a pH-dependent phase transition from a chiral R-helix conformation (low charge density) to an achiral random coil structure (high charge density). By contrast, a pH increase also enhances cationic porphyrin adsorption and self-aggregation onto the polymer matrix, the chirality of which induces strong dichroism in the adsorbed porphyrins. In the case of binary systems (ionic polymer and oppositely charged porphyrin) the competition between these opposite demands has been rationalized on the basis of a thermodynamic model for self-aggregation in a two-phase system (bulk solution and polymer adsorption sites) and gives rise to a maximum in the induced CD intensity and hypochromicity of the adsorbed porphyrins on varying the pH. An even more complex behavior has been observed in the case of ternary systems (ionic polymer interacting with mixtures of anionic and cationic porphyrins) because of the (pH-modulated) possibility of self-aggregation both onto the polymer matrix and in bulk solution. The studied systems also show interesting effects depending on the strength of self-association among porphyrins. In fact, weakly aggregated porphyrins (CuT4 onto PG) lose their chirality upon pH-induced disruption of the PG R-helix conformation. By contrast, the stable aggregates made up of an equimolar mixture of anionic and cationic porphyrins retain “memory” of the original matrix chirality even after months. This behavior is reminiscent of that of a ferromagnetic material when the strong magnetic field has been turned off: below a certain critical temperature the magnetization goes to zero, while above that critical value the magnetization slowly decreases, eventually reaching a constant nonzero value. Furthermore, tightly bound anionic-cationic porphyrins to the chiral PG template do not show any CD change upon addition of a large excess of template bearing opposite chirality, confirming a very slow exchange kinetics among the aggregates.

Introduction The ability of inducing,1-7 tuning,8 and memorizing9,10 a given “shape” at a supramolecular level allows the properties (absorption, emission, redox potentials, etc.) of the assemblies to be controlled and then is of particular interest for possible technological applications.11,12 Under these premises, aggregation of cationic or anionic achiral chromophores onto oppositely charged chiral polymeric templates proposes as an easy strategy to obtain supramolecular complexes molded by, and then reporting, the shape of the template.10,13-20 The resulting induced circular dichroism (ICD) has a conformational origin (arising from the self-organization of the achiral guest by a polymeric chiral template) and not a configurational one (due to the interaction of the achiral guest with the locally asymmetric environment). This is supported by the observation that the ICD modifies or disappears13,15,16 following matrix conformational transition. Owing to their peculiar and tunable spectroscopic and electronic properties, meso-substituted water-soluble porphyrins †

Part of the special issue “Thomas Spiro Festschrift”. E-mail: [email protected]. Fax: +39-095-580138. Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica, Universita` di Messina, ITCPN CNR Sezione di Messina, Messina, Italy. | Istituto per lo Studio delle Sostanze Naturali di Interesse Alimentare e Chimico Farmaceutico, CNR, Via del Santuario 110, 95028 Valverde (CT), Italy. ‡ §

are excellent building blocks for such supramolecular species.10,14-20 Interactions of achiral porphyrins onto chiral molds also present other advantages: (i) The appearance of an ICD band in the Soret region confirms complexation. (ii) The shape of the ICD reports the aggregation state of porphyrins (e.g., being split in the case of aggregation). The “binary” porphyrin-template supramolecular complexes are labile, and their existence can be modulated by different experimental conditions (pH, ionic strength, and temperature).15,17 This aspect can be usefully employed for sensing purposes,21,22 but in the meantime, it is also a limitation for possible technological applications11 for which quite robust species are needed. On the other hand, it is known that aggregation of oppositely charged porphyrins leads to quite “stable” species,23-26 but only a few attempts of shaping these assemblies have been published to date.10,20 We have very recently reported10 that the ternary aggregates of the achiral oppositely charged porphyrins meso-tetrakis(4sulfonatophenyl)porphine (H2TPPS; Figure 1) and meso-tetrakis(N-methylpyridinium-4-yl)porphinatocopper(II) (CuT4; Figure 1) onto R-helical polyglutamic acid led to induced optical activity of the Soret band. Surprisingly, the ICD remains unaltered even after the pH-induced conformational transition from the polyglutamic acid R-helix to the polyglutamate random coil. This finding, seemingly antithetical to the Stryer and Blout model,13 has been attributed to a remarkable inertness of the

10.1021/jp0005930 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/29/2000

Ternary Porphyrin Aggregates and Their Chiral Memory

J. Phys. Chem. B, Vol. 104, No. 46, 2000 10901 related to the adsorbed molecules depends both on the size of the aggregates and on their own chirality per unit size. The adsorption of molecules onto the polymer can be described by the variable θn defined as the volume fraction of binding sites occupied by an n-mer, while their spatial orientation is accounted for through the parameter ηn (ηn ) 0 means random orientation, while ηn ) (1 describes the orientations parallel or antiparallel to the polyelectrolyte helical axis of the adsorbed molecules). Adopting units where the Boltzmann constant is set equal to 1, the free energy per site of the bound molecules reads in a mean-field picture

FBOUND /T ) TOT 1

[

N

N

∑ Tn)1 Figure 1. Schematic structure of meso-tetrakis(4-sulfonatophenyl)porphine (H2TPPS) and meso-tetrakis(N-methylpyridium-4-yl)porphinatocopper(II) (CuT4).

supramolecular heteroaggregates, which permits them to retain the “memory” of the template even after its disruption.10 Here we present a more detailed characterization of these species, discussed in the frame of a theoretical model, aiming to understand the factors which govern these aggregation processes and render these species so stable. Experimental Section Polyglutamic acid (PG; polymerization degree ) 95) was obtained from Sigma Chemical Co. Its concentration is expressed as moles of glutamic acid residues per liter and was determined using 205 ) 3500 M-1 cm-1 in ultrapure water. H2TPPS and H2T4 (meso-tetrakis(N-methylpyridinium-4-yl)porphine) were obtained from Mid-Century. H2T4 metalation was performed by using a literature method.27 Unless otherwise specified, the same concentration (4 × 10-6 M) of both porphyrins has been used for all spectroscopic measurements. Absorption measurements were carried out on a HP8452A spectrometer. CD spectra were recorded on a Jasco J-600 spectropolarimeter. To allow for the formation of the ternary species (see the Results and Discussion), all the measurements have been carried out in the pH range 3.3-4.5 (citrate buffer 5 mM). In this pH range H2TPPS is protonated (H4TPPS, pKa ≈ 5). Theoretical Model The aim of this section is to develop an approximate picture for the spectroscopic variations (induced CD and hypochromicity) of molecules (e.g., porphyrins) adsorbed onto a chiral polymer matrix (e.g., a polyelectrolyte in its R-helix conformation). Consider a dilute solution of molecules that do not selfaggregate in solution. The solution also contains many mobile “binding sites” (the negatively charged PG residues in the present study), the number of which can be modulated by pH variations. Because of the increased local concentration of adsorbed molecules, these may self-aggregate onto the polymer matrix. An important difference between the R-helical and random coil structures of polyelectrolytes resides in the chiral nature of the helix. Therefore, the adsorbed molecules may arrange themselves either following the helicity of the polyelectrolyte (parallel to the helical axis orientation) or adopting a random adsorption (a mixture of parallel and antiparallel orientations). As a consequence, the resulting induced chirality

θn

θn

N

N

∑ log νn + (1 - n)1 ∑θn) log(1 - n)1 ∑θn) n)1νn

]

+

n-1 1 N (β(ηn) - TS(ηn))θn (1) J(ηn)θn + n Tn)1

∑

where ν is the molecule:solvent volume ratio. The first term in the square brackets describes the mixing entropy of the adsorbed n-mers, and the second one is the mixing entropy of the adsorbed solvent.28 The size n of the aggregates runs from 1 (monomers) to N, where N is the maximum number of contiguous adsorbing sites (proportional to the fraction of binding sites Φ) within a single chain. Clearly N ≡ N(Φ) ∝ Φ is strongly dependent on pH. N ((n - 1)/n)J(ηn)θn, is the self-aggregaThe third term, ∑n)1 tion energy gain inclusive of the unfavorable energy due to the two edges of the aggregate (scaling as29 (n - 1)/n). At variance of the standard models of self-aggregation, the interaggregate energy J(ηn) depends also on the relative orientation of the adsorbates. In the case of stronger interactions in the helical rather than random arrangement, we may write

J(ηn) ) -Jh - Bηn2

(2)

N (β(ηn) - TS(ηn))θn, Finally, the fourth term in eq 1, ∑n)1 describes the self-energy of the adsorbed molecule as described below. The self-energy depends on an interaction term with the polymer, β(ηn), plus an entropic term, -TS(ηn). The first one h + hηn) and behaves like a can be written as β(ηn) ) -(β magnetic or electric field acting upon a spin system: orientations parallel to the polymer helicity axis are favored, while antiparallel orientations are unfavored. The entropic term can be described by the usual combinatorial procedure for orderdisorder transitions, and the final expression for the total selfenergy reads

h + hηn) + FSELF(ηn) ) β(ηn) - TS(ηn) ) -(β 1 1 1 1 T (1 + ηn) log (1 + ηn) + (1 - ηn) log (1 - ηn) (3) 2 2 2 2

(

)

The energy minimum is attained when ∂FBOUND /∂ηn ) 0, and TOT utilizing eqs 1-3, after straightforward differentiation

1 + ηn n-1 T -2B η - h + log )0 n n 2 1 - ηn

(4)

from which ηn can be calculated. For small ηn values eq 4 can be recast in the standard Landau form for a spin system in the presence of a field h:

b(T)ηn3 + (T - Tc(n))ηn ) h

(5)

10902 J. Phys. Chem. B, Vol. 104, No. 46, 2000

Purrello et al.

(with b(T) ) T/3 and Tc(n) ) 2B(n - 1). As for a magnetic system where the magnetic coupling among the spins determines the critical temperature Tc, here the strength of the chiral interactions determines the value of Tc(n), the only difference being that now the critical temperature Tc(n) slowly depends on the aggregation number n. Asymptotic solutions in the case of large and small Tc(n) values can be easily calculated from eqs 4 and 5 both in the presence of the polymer “chiral field” h(R-helix) and upon its disruption (random coil)

0

(

Tc(n) e T

)

Tc(n) - T Tc(n) > T T 1 - 2 exp(-2Tc(n)/T) Tc(n) . T

ηn(h ) 0) ≈ 31/2

1/2

(6a)

h Tc(n) , T T - Tc(n) (6b) h ηn(h) ≈ Tc(n) ≈ T tgh T 1 - 2 exp(-2(Tc(n) + h)/T) Tc(n) . T

()

while η1 takes the exact expression η1 ) tgh(h/T). The calculation of the concentration of different aggregates can be easily accomplished as follows. Defining the chemical potential as µn ≡ ∂FTOT/∂θn, the chemical equilibrium condition requires the coincidence of the chemical potentials of each species in each phase (free and bound), namely

µFREE 1

)

µBOUND 1

(7a)

) µBOUND µBOUND 1 n

(7b)

where µFREE is the chemical potential of the isolated molecule 1 up to in bulk solution. The analytical expression of µFREE 1 linear terms in CFREE (high dilution limit) reads

1 1 /T ) µ(o) µFREE 1 1 /T + log(CFREE/ν) + - 1 + O(CFREE) (8) ν ν CFREE being the molecule concentration (expressed as volume fraction) in the bulk. Without loss of generality we may set µ(o) 1 ) 0; namely, the zero of the energy scale is the self-energy of the isolated solvated molecule in its monomeric state. Differentiating the free energy eq 1 with respect to θ1 and θn and inserting the result back into eqs 7a,b, we get after some rearranging

[

]

(9a)

) CFREEeβeff(η1)

(9b)

n θ1 θn ) nγ(ηn) eReff(ηn) e-Reff(ηn) ν ν

θ1 N

(1 -

∑θn)ν

n)1

with

ν Reff(ηn) ≡ 1 - (-Jh - Bηn2 + FSELF(ηn) - FSELF(η1)) (10a) T ν γ(ηn) ≡ exp 1 - (FSELF(ηn) - FSELF(η1)) T

(

ν βeff(η1) ≡ - FSELF(η1) T

)

(10b) (10c)

FSELF(ηn) being defined through eq 3. Equation 9a represents an extension of the well-known expression for self-aggregation in homogeneous solution,29 which includes the existence of different internal states for the aggregate molecules as described by the new variable ηn. In fact, in the limit ηn ) 0, we have Reff ) 1 + ν(Jh/T) and γ ) e, and relating the volume fraction θn with the mole fraction Xn by θn/ν ) Xn, one finds29 Xn ) ne(X1eReff)ne-Reff. Even in the case of ηn ) 0, eq 9a is coupled to eq 9b through the term θ1 (concentration of adsorbed monomers), which can be calculated by using eq 9b. Then, this latter equation represents a generalized Langmuir adsorption equation. It can be easily seen that eq 9b reduces to the well-known adsorption isotherm in the hypothesis that only monomer species are adsorbed N θn f θ1) and that the size difference between the solute (∑n)1 and solvent is negligible (ν f 1). Disregarding also the dependence of the binding on the internal states of the adsorbed molecule, one obtains βeff(η1) ≡ h/T, and eq 9b reduces to the classical expression θ1/(1 - θ1) ) CFREEeh/T. An additional equation follows from the mass conservation law, which imposes that the sum of the free and adsorbed molecules must be constant: N

(1 - Φ)CFREE + Φ

∑ θn ) C

(11)

n)1

where C is the total stoichiometric concentration of the considered molecule and Φ is the fraction of binding sites, Φ ) ΦMAXΘ, where ΦMAX is the polymer volume fraction and Θ is the fraction of charged polymer residues (0 < Θ < 1). Inserting eq 9 into eq 11 and disregarding the very weak n dependence of the Reff(n) and γ(ηn) terms, the sum over n appearing in eq 11 can be easily performed, yielding N

N

∑θn ) e-R n)1 ∑nXn ) e-R n)1 eff

eff

∂ N n X X ∂X n)1

∑

X ) e-Reff (1 + (N - 1)XN - NXN-1) ≈ 2 (1 - X)

{

e-Reff

X ≈ 1, N f ∞ (1 - X)2 (12) 1 -Reff N(N + 1) X ≈ 1, N small e 2

with X ≡ θ1eReff e 1. Combining the above result with eqs 9-11 yields a set of nonlinear algebraic equations from which the expressions for the concentrations θn of the aggregates can be obtained. Once the concentration θn of the various aggregates on the chiral polymer matrix has been calculated, we may easily estimate the variation of the induced chirality and hypochromicity as a function of different parameters (pH, temperature, ionic strength, concentration). Hypochromicity. The variation of hypochromicity as a result of porphyrin aggregation onto a polymer matrix is proportional to the number of porphyrin-porphyrin contacts. Since the number of contacts in a linear aggregate scales like 2(n - 1), N (n - 1)wn, where the the average number of contacts is 2∑n)1 weights wn are proportional to the aggregate concentration θn: wn ) Aθn, A being a normalization constant calculated from N N wn ) A∑n)1 θn ) 1. Hence the condition ∑n)1

Ternary Porphyrin Aggregates and Their Chiral Memory

J. Phys. Chem. B, Vol. 104, No. 46, 2000 10903

N

∑(n - 1)θn

n)1

ψHYP ∝

(13)

N

∑ θn

n)1

Inserting the analytical expression of θn into eq 13 and using N n2Xn ≈ 2/(1 - X)3 for X ≈ 1, N f ∞ and the relationships ∑n)1 N ∑n)1n2Xn ≈ (1/6)(2N + 1)(N + 1)N for X ≈ 1, N small, we may calculate the hypocromicity effect. Qualitative compact asymptotic expressions are easily obtained:30

0 Φ3c

ψHYP ∝ Φ -

( )( ) ( )( ) U(h) C T Φ

1/2

U(h ) 0) C T Φ

1/2

exp

exp

Φ2

Φ < Φ c , Φo

(14a)

Φ > Φc , Φ o

(14b)

Φ > Φ c < Φo

(14c)

Φ > Φ c > Φo

(14d)

where Φc is the critical concentration of polymer charged residues needed to form the smallest aggregates (dimers) of porphyrin and Φo is the critical charge concentration for the R-helix f random coil transition. The function U(h) ≡ U(ηn(h)) assumes the following approximate values:

≈ 1 for Φ < Φo and X(Φ) ≈ e-ζΦ for Φ ≈ Φo), we introduce a further but numerically small decrease of the hypochromic effect above the R-helix f random coil transition (eqs 14d and 15b). This decrease is related to the stronger binding occurring when both polymer and adsorbed molecules have the same chirality, an effect which disappears after the transition as clearly shown by eqs 15a,b. A more thorough comparison between theory prediction and experimental results will be discussed in the Results and Discussion. Induced Chirality. Although the studied porphyrins do not exhibit any CD in bulk solution, their adsorption onto a chiral polymer chain (R-helix polyglutamate) induces strong CD that disappears when the polymer chain assumes a random coil conformation (see the next section for details). We may guess that the CD comes from the self-aggregate porphyrins, which take the same helicity of the matrix. Hence, the unaggregated adsorbed monomers do not contribute to the CD signal, the induced ellipticity arising from aggregates with length comparable with or larger than the R-helix turn of the matrix (on the order of a few porphyrins31). The aggregates contribute to the CD signal depending on their length (or aggregation number n), concentration of aggregates θn, and their own chirality ηn. Therefore, a single aggregate of aggregation number n contributes to the ICD intensity as nθnηn. Averaging over all the aggregation numbers and recalling that ηn is practically independent of n (unless n is extremely small), it follows that the normalized induced CD intensity, ψCD, is proportional to Nc′

N

1 ν Tc . T 1 + Jh + Tc T 2 U(h) ≈ ν 1 Tc Tc , T 1 + Jh + h T 2 T - Tc

(

(

(

)

)

)

1 Tc - T T > T ν Jh + c T 2 T U(h ) 0) ≈ ν Tc < T 1 + Jh T 1+

ψCD ∝ η(h)

(15a)

∑nθn - n)1 ∑nθn

n)1

(16)

N

∑ θn

n)1

(15b)

To qualitatively understand the behavior of the hypochromism, it is convenient to separately discuss the different regimes. When the concentration Φ of polymer charged residues is smaller than the critical concentration Φc required to form the smallest aggregate (Φc is therefore on the order of the critical aggregation concentration), there is no hypochromic effect (eq 14a). On increasing Φ above the critical threshold Φc, small aggregates start to form. Since the fraction of binding sites is small near Φc as compared with the number of the free porphyrins in the bulk and, in addition, the charged polymerporphyrin and porphyrin-porphyrin energies of interaction are strong energies (i.e., eReff . 1 or equivalently X ≈ 1), it follows therefore that practically all the available binding sites N are occupied (with N ∝ Φ a small number). This leads, with the aid of eq 12, to an almost linear increase of the hypochromic effect with Φ as evidenced by eq 14b. On further increasing the number of binding sites Φ over the stoichiometric porphyrin N concentration (i.e., 1 - ∑n)1 θn ≈ 1), the dilute large aggregates are no longer stable. Their concentration can be easily estimated from eqs 9a,b in the limit of large stacking energies (X ≈ 1) by a perturbation expansion, obtaining with the aid of eq 12 that the hypochromic effect slowly decreases as Φ-1/2 (eqs 14c and 15a). By considering also the loss of helicity on approaching the charge-induced R-helix f random coil transition (the fraction of R-helical segments X(Φ) behaves as X(Φ)

where Nc′ is the aggregation number of the smallest chiral aggregate (on the order of a few porphyrins). Therefore, the charge density on the polymer matrix affects the ICD of the adsorbed molecule in two ways: (i) by modulating the aggregation number n and concentration θn of bound molecules which assume a chirality ηn proportional to that of the template and (ii) by varying the chirality of the polymer template (through the variation of the R-helix (pH < 5):random coil ratio (pH > 5)). This polymer chirality decrease has a small effect on θn but could have a dramatic influence on ηn. Inserting the analytical expressions for θn and ηn ≈ η(h) into eq 16, we may calculate the induced CD. Summarizing the main results, we obtain the following qualitative behavior:

Φ < Φc′ , Φo (17a)

0

(

η(h) Φ ψCD ≈

η(h) exp

)

Φc′3 Φ2

Φ > Φc′ , Φo

( )( ) ( )( ) U(h) C T Φ

η(h ) 0) exp

1/2

U(h ) 0) C T Φ

1/2

(17b) Φ > Φc′ < Φo (17c) Φ > Φc′ > Φo (17d)

where Φo is the critical charge concentration for the R-helix f random coil transition and Φc′ is the critical charge concentration

10904 J. Phys. Chem. B, Vol. 104, No. 46, 2000 needed to form the smallest chiral aggregate, while the explicit expressions for η(h) and U(h) are given by eqs 6 and 15, respectively. To better understand the behavior of the induced CD, it is convenient to investigate separately the different regimes. When the concentration Φ of polymer charged residues is smaller than the critical concentration Φc′ required to form the smallest chiral aggregate (Φc′ is therefore on the order of the critical aggregation concentration), the CD signal is zero (eq 17a). On increasing Φ above the critical threshold Φc′, small helicoidal aggregates start to form while the polymer still remains in its R-helical conformation. We assume that the ordering “chiral field” h of the polymer in its R-helical conformation is strong; therefore, η(h) ≈ 1 - O(exp(-2h/T) (see eq 6). Since the fraction of binding sites is small near Φc′ as compared with the number of unbound porphyrins and, in addition, the charged polymerporphyrin and porphyrin-porphyrin energies of interaction are strong, it follows that almost the available binding sites N are occupied (with N ∝ Φ a small number). This leads, with the aid of eq 12, to an almost linear increase of the CD signal with Φ (eq 17b). On further increasing the number of binding sites Φ over the stoichiometric porphyrin concentration (i.e., 1 N θn ≈ 1), the dilute large aggregates are no longer stable. ∑n)1 Their concentration can be easily estimated from eqs 9a,b in the limit of large stacking energies (X ≈ 1) by a perturbation expansion, obtaining that the CD signal slowly decreases as Φ-1/2 (eqs 17c and 15a). However, on approaching the chargeinduced R-helix f random coil transition, the chirality of the polymer matrix vanishes. The abrupt decrease of the polymer chirality has a small effect on the fraction θn of adsorbed molecules (ruled by the parameter U(h); see eq 15) but may have a sharp influence on their own chirality ηn. In fact, for temperatures much lower than the critical value Tc (i.e., for strong chiral interactions among the adsorbed porphyrins) ηn does not decrease appreciably (roughly from 1 - 2 exp(-2(h + Tc)/T) to 1 - 2 exp(-2Tc/T); see eqs 6a,b), while for T > Tc the polymer-induced chirality of the adsorbed molecules, ηn, jumps from 1 - 2 exp(-2(h + Tc)/T to zero! In that latter situation one observes a sharp decrease of the ICD, which eventually disappears after the completion of the polymer conformational transition (eqs 6a and 17d) with η(h ) 0) ) 0). Apart from the fact that the onset of the hypochromic effect occurs at smaller polymer charge density than that required for the onset of CD effects (i.e., Φc e Φc′ ), the behavior of the induced CD as a function of Φ is very similar to that of hypocromicity; great deviations, however, could be observed at high Φ values. In that regime the CD variation is given by eqs 17c,d, which for T > Tc considerably differ from those of the hypochromic effect (eqs 14c,d), where the decline of hypochromicity with polymer charge density is smoother. This is because the R-helix f random coil transition has a negligible effect on the binding and aggregation onto the polymer matrix, while the disruption of the helix also causes the disruption of the induced chiral aggregates. A more thorough comparison between theory prediction and experimental results will be discussed in the next section. Results and Discussion Binary Systems Formation. Before discussing the formation of the ternary species, we will briefly describe the two binary systems CuT4-polyglutamate and CuT4-H4TPPS.32 In our experimental conditions no complexation between H2TPPS and polyglutamate has been observed. CuT4-Polyglutamate System. The interaction of CuT4 with polyglutamic acid leads only to very subtle variations in the

Purrello et al.

Figure 2. Absorption spectra of CuT4 alone (4 µM, full line) and in the presence of polyglutamate (400 µM, dashed line) at pH 3.6.

Figure 3. CD spectra at pH 3.6 of (A) CuT4 (4 µM) in the presence of polyglutamate (200 µM) and (B) after the addition of a 4-fold excess of polyglutamate. Adapted from ref 10.

Figure 4. CD intensity and percentage of hypochromicity of CuT4 (4 µM) in the presence of polyglutamate (400 µM) vs pH.

absorption spectrum (Figure 2). The formation of the binary species is, however, confirmed by the appearance in the Soret region of an induced split CD signal, which also suggests the formation of porphyrin aggregates (Figure 3).14,33 pH exerts a central role in modulating the extent of complexation, CuT4 aggregation state, and chirality (both the intrinsic and induced one). Polyglutamic conformation is, in fact, pH-dependent, being R-helical for the protonated form (pKa ≈ 5) and random coil for the ionized one. Also, owing to the electrostatic nature of CuT4 interactions with polyglutamate, it is expected that both the type and extent of complexation are pH-dependent. Figure 4 indicates that the total CD intensity maximizes from pH ≈ 3.8 to pH ≈ 4.3 and then diminishes. However, the ICD (conservative) shape does not depend on pH, suggesting that porphyrins are still partially aggregated at pH close to the matrix pKa. In this pH range polyglutamate is R-helical;16 then, the CD intensity trend cannot be (simply) assigned to matrix

Ternary Porphyrin Aggregates and Their Chiral Memory

Figure 5. Salt effect on the CD intensity of the CuT4-polyglutamate complex at pH 3.6.

conformational changes. The rationale for these variations with pH have been analyzed in the previous section (eq 8). When the concentration Φ of polymer charged residues is smaller than the critical concentration Φc required to form the smallest chiral aggregate, the CD signal is zero (eq 17a). On increasing Φ above the critical threshold Φc, small helicoidal bound aggregates start to form, indicating a CD increase (eq 17b). On further increasing the number of binding sites Φ over the stoichiometric porphyrin concentration, the dilute large aggregates are no longer stable; consequently the CD signal slowly decreases as Φ-1/2 because of a dilution effect (eq 17c) and eventually disappears (eq 17d) because of the loss of helicity on approaching the charge-induced R-helix f random coil transition (pH ≈ 5). This means that η(h ) 0) ) 0, a result following from the assumption that the entropic forces overcome the chiral interactions (Tc < T). In that case the induced chirality disappears when the polymer chiral field h has been turned off, a result closely resembling that of a ferromagnetic material in a strong magnetic field. This explanation is supported by the analogous plot of Soret hypochromicity vs pH, which starts at a smaller threshold than that required for the CD signal, reaches a maximum, and then does not go to zero at pH 5, as for CD, but continues to decrease smoothly, indicating that porphyrins are still bound to the anionic template. All these findings together with the different behavior of ICD and hypochromicity are in qualitative agreement with the prediction of the theory (see eqs 14a-d). The spectroscopic variations recorded as a function of the ionic strength also confirm the importance of the cationic porphyrin-anionic matrix electrostatic interactions. Figure 5 shows, in fact, that these complexes do not form for sodium chloride concentrations ∼0.02 M. Even this result agrees with the theory which predicts a lowering of the CD signal on decreasing the porphyrin-polymer binding energy -β h .30 As already anticipated, the porphyrin binary aggregates are labile, and this is well evidenced in Figure 3, which shows that the ICD phase of the L-assemblies is reversed following the addition of an excess of D-polyglutamic acid. The resulting spectrum is not the perfect mirror image of the initial one but is averaged by the contemporary presence of the L- and D-aggregates. These species present a (pH-independent) peculiar behavior vs the temperature increase. In particular, the CD intensity decreases up to ∼55 °C and then reverts and increases up to ∼80 °C (Figure 6). This behavior, already observed for other porphyrin aggregates, agrees with the theory which predicts a lowering of the CD signal on increasing the temperature (through the terms η(h) and exp(U(h)/T in eqs 17c,d). At high T values, however, other effects may compete; for instance also

J. Phys. Chem. B, Vol. 104, No. 46, 2000 10905

Figure 6. Temperature effect on the CD intensity of the CuT4polyglutamate complex at pH 3.6 (no salt added).

Figure 7. Optical spectra at pH 3.6 of (a) H4TPPS (4 µM), (b) CuT4 (4 µM), and (c) their aggregate in a 1:1 mixing ratio.

the fraction of charged residues Φ may decrease, partially compensating the sharp decrease of the ICD observed at lower temperature. CuT4-H4TPPS System. Aggregation between CuT4 and H4TPPS is accompanied by a quite strong hypochromicity of the Soret band (Figure 7) together with ∼50% quenching of the H4TPPS emission (CuT4 does not fluoresce). Absorption and fluorescence Job’s plots indicate the formation of species in a 1:1 stoichiometric ratio.32 No induced optical activity is observed throughout the UV-vis region for these assemblies formed in the absence of chiral matrixes, and this holds also if the matrix is added as the last component (even after months), showing that these accordion-type aggregates are quite kinetically inert. Ternary Species Formation. The addition of H2TPPS (3.3 ≈ pH ≈ 4.5; see further discussion) to preformed CuT4-polyL-glutamate complexes leads to absorption and emission changes identical to those (already described) accompanying the formation of the CuT4-H4TPPS achiral aggregates. Also in this case the CD variations are very indicative and report the formation of ternary chiral species. The CD intensity, in fact, increases by 2 orders of magnitude with respect to that of the binary complex (Figure 8). The mirror image signals are obtained when the anionic porphyrins are added to preformed CuT4-poly-Dglutamate species, indicating that the chirality of the ternary assemblies is related to template chirality (Figure 8). To ascertain the cationic to anionic stoichiometric ratio in the ternary complex, we performed various CD experiments, adding increasing concentrations of H2TPPS to different CuT4polyglutamate solutions with fixed concentration. Figure 9 shows that the CD intensity maximizes for equimolar concentrations of the two porphyrins, which then aggregate in a 1:1 stoichiometric ratio. The pH exerts a relevant role in governing and tuning the bias toward the formation of chiral or achiral porphyrin

10906 J. Phys. Chem. B, Vol. 104, No. 46, 2000

Purrello et al.

Figure 8. CD spectra at pH 3.6 of CuT4-H4TPPS (4 µM each) in the presence of D-polyglutamate and L-polyglutamate (200 µM).

Figure 11. Disappearance of the CuT4-polyglutamate induced CD signal (A) after the addition at pH 4.5 of H2TPPS (4 µM) (B).

Figure 9. CD intensity dependence at pH 3.6 on the CuT4:H4TPPS ratio.

Figure 12. Kinetic profiles of CD intensity for the formation of (A) a CuT4 homoaggregate on poly-L-glutamate (the curve represents the best fitting to a first-order model with Ao ) 10.1 ( 0.1, Ainf ) 12.6 ( 0.1, and kobs ) 0.034 ( 0.002) and (B) the ternary assembly H2TPPSCuT4 on poly-L-glutamate. The inset reports an expansion of the kinetic run, showing the presence of an induction period. Experimental conditions: [porphyrin(s)] ) 3 µM, [PG] ) 300 µM, pH 3.3, T ) 298 K. Figure 10. CD intensity decrease (of the negative and positive bands at 565 and 396 nm, respectively) of the CuT4-H4TPPS-poly-Lglutamate species as a function of pH.

aggregates. Figure 10 shows that the formation of the chiral ternary supramolecular complex decreases with pH in the range 3.4-3.8. Addition of H2TPPS for pH values ∼4.0 causes the disappearance of the ICD in the Soret region, indicating that both the ternary and binary chiral species do not form anymore (Figure 11). Both absorption and fluorescence data, however, still indicate the formation of the achiral CuT4-H4TPPS complex in bulk solution (data not shown). A reasonable explanation of pH role in “tuning” the formation of the two aggregates can be found in the repulsion between the two anionic components. In fact, increasing the pH, the number of negative charges on polyglutamate augments and, because of the electrostatic repulsion, hinders anionic porphyrin approach and complexation. Then, anionic porphyrins strip CuT4 from the template surface and form the CuT4-H4TPPS achiral aggregates in bulk solution, indicating that in these experimental

conditions the porphyrin-porphyrin binary species is more favored than the CuT4-polyglutamate one. Increasing concentrations of salt strongly influence the formation of the ternary chiral species, and as observed for the CuT4-polyglutamate complex, already at 0.02 M sodium chloride the ternary species does not form anymore (Figure 5). Therefore, in a picture of a ternary complex model we should consider the charged H4TPPS as a building block involved in specific molecular recognition processes rather than as an electrolyte whose main effect is the electrostatic screening of charges. The differences between binary and ternary complexes are also evident when we investigate the kinetics of self-aggregation onto the polymer matrix. The binary system (PG + CuT4) follows simple first-order kinetics (Figure 12A), where the CD signal linearly increases on increasing the concentration of polyglutamate (data not shown). This result clearly indicates that the availability of negative charges on the supporting surface allows a large enough concentration of the oppositely charged CuT4 porphyrin to foster self-aggregation. The time traces of the ternary system are different in that they exhibit an initial

Ternary Porphyrin Aggregates and Their Chiral Memory

Figure 13. Melting behavior of the ternary complex, and (see the inset) its ionic strength dependence.

lag time followed by a sigmoidal profile (Figure 12B). This behavior is not unprecedented in the formation of large molecular assemblies and has been recently described in the interactions of cationic porphyrins with DNA.34 Considering that the kinetics process derives from a competition between association and dissociation processes, it follows that in the ternary system the smaller dissociation rate of the CuT4-H4TPPS (stabilized by the interaction of four positive net charges with four negative charges) is overwhelmed by a slower association kinetics because the system has to select an alternate binding of positive and negative porphyrins to obtain a stable supramolecular complex. This balance gives a more efficient formation of the aggregate on approaching thermodynamic equilibrium, but the growing rate in the early stages might be slower than that of the binary system and, apparently, not firstorder. However, the most interesting kinetic phenomena are observed when we consider effects related to the dissociation of the ternary aggregates instead of their association. A first effect is related to the temperature dependence of the ternary complexes. In Figure 13 are reported a series of CD spectra recorded at different temperatures in the range 25-80 °C. The CD intensity is reduced only by 50% in this range. Intriguingly, the addition of 0.3 M NaCl to preformed ternary complexes only slightly affects their thermal behavior (inset of Figure 13). This is in contrast with the previous findings where the salt was added just before porphyrin addition: the chiral porphyrin aggregates are easily disrupted on raising the temperature. This observation implies that, once formed, these ternary species are kinetically inert. As shown in the next section, this inertness enables these aggregates to retain the memory of template chirality. Ternary Species “Memory”. An even more striking evidence of the kinetic inertness of the chiral ternary aggregates derives from the experiment shown in Figure 14. Here a 5-fold excess of poly-D-glutamate has been added to a preformed L-ternary complex; however, differently from the binary species (Figure 3), the sign of the CD bands in the visible region does not invert (even after many days following the addition). Why do porphyrin aggregates on a polymeric matrix not easily dissociate? The answer can be easily found by considering the partition coefficient P between the bulk and adsorbed conformation: P ≈ exp(-〈n〉(νβ h /kT)), where -β h is the porphyrin-polymer binding energy and 〈n〉 ≈ 2((C/Φ))1/2 exp(U(h)/ T) (provided the concentration Φ of adsorbing sites is much greater than the concentration C of the porphyrins in the bulk). When the porphyrin-porphyrin stacking energy -U(h) is large (as for instance in the CuT4-H4TPPS, where the porphyrins carry four negative (H4TPPS) and four positive (CuT4) net

J. Phys. Chem. B, Vol. 104, No. 46, 2000 10907

Figure 14. CD spectra of (full line) a solution of CuT4-H2TPPS (4 µM each) in the presence of poly-L-glutamate (200 µM) and (dotted line) the same solution after the addition of a 5-fold excess of polyD-glutamate.

Figure 15. CD spectra of (full line) a solution of CuT4-H2TPPS (4 µM each) in the presence of poly-L-glutamate (400 µM) at pH 3.6 and (dotted line) the same solution at pH ≈ 12.

charges), 〈n〉 grows exponentially (in our experimental conditions (C/Φ)1/2 is on the order of 10-1). Therefore, even if the binding energy β h /T is relatively small, because of the factor 〈n〉, the product 〈n〉νβ h /T may be very large, making the dissociation of the aggregate from the polymer matrix difficult. It is worth mentioning that the lifetime of other self-aggregates in water solution is on the order of 10-3-10-1 s for micelles35 and 10-102 h for lipid bilayers,35 strongly depending on the hydrocarbon chain length and temperature. Therefore, aggregates made up of oppositely charged porphyrins are in the far edge of this stability scale, showing phenomena that cannot be adequately described in the framework of the classical thermodynamics (binding isotherms). Another strong evidence of the ternary assembly inertness derives from a pH-jump experiment (Figure 15). Here the ICD of aggregated porphyrins does not change on going from pH 3.6 to pH 12 despite the fact that the polyglutamate conformation goes from R-helix to random coil in this pH range (as testified by the CD changes in the UV region, Figure 15). These findings strongly indicate that these porphyrin assemblies retain their original chirality even when the chiral matrix loses it. We have also checked the time stability under such critical conditions (pH 12). It turns out that these complexes remain stable for several months as indicated by the CD intensity in the Soret region, which decreases only by less than 30% in about four weeks. Therefore, if the previous experiment shows that these aggregates do not easily dissociate from a polymeric matrix,

10908 J. Phys. Chem. B, Vol. 104, No. 46, 2000 the latter one clearly also testifies that these species retain the memory of the imprinted chirality (conformational inertness). This behavior strongly resembles that of a ferromagnetic substance as shown in the theoretical section. When a ferromagnetic material is submitted to an intense magnetic field, all the spins assume the same orientation of the field. In the present case the orienting field is the chiral field of the R-helix, which can be switched on and off by simple pH variations. When the field is turned off, two different scenarios arise: at low temperature (or strong spin-spin interactions) the spins basically retain the original ordered conformation. At high temperatures (or small spin-spin interactions) the entropic forces prevail and the magnetization decays to zero. In our studies we observed both regimes. In the case of binary systems (porphyrin + polymer) the pH-induced disruption of the chiral field leads to the disappearance of the porphyrin imprinted chirality (see the discussion concerning the binary systems). On the contrary, when ternary systems are considered (anionic porphyrin + cationic porphyrin + polymer), the strong interactions among oppositely charged porphyrins make the system more cooperative. In that case the chirality persists (even if to a less extent) after the disruption of the inducing field. This is just what we observe in Figure 15, where the chirality of the polymer matrix goes to zero (the band at ∼200 nm), while the CD features of the Soret band are essentially maintained. A further test of the above physical picture can be obtained by considering the ICD behavior of the ternary complexes after the pH-induced R-helix f random coil transition. Here the induced chirality should disappear when the critical temperature decreases (or, equivalently, the temperature increases above a critical threshold), and the CD decay should be larger than that observed in the presence of a chiral field (R-helix conformation). The ICD decay can be obtained either by reducing the porphyrin-porphyrin interactions through, for instance, salt addition to oppositely charged porphyrins (lowering Tc), or by raising the temperature. Our preliminary results confirm this theoretical trend, but as expected, the kinetics are very slow and much care has to be put into the experiments. A full analysis will be reported elsewhere. Interestingly, the ICD decay is not due to the disassembling of the adsorbed molecules, which are still in an aggregated form as shown by the hypochromicity effect and RLS data. All together, both theoretical and experimental findings show new effects consequent to the binding of achiral molecules onto a switchable chiral-nonchiral matrix. Both binding and induced CD are strongly coupled and appear to be very cooperative phenomena. Therefore, the present, or related, systems cannot be treated by the usual adsorption equations of the Langmuir type but require the use of different concepts borrowed from the physics of spin glasses and self-replicating structures. Acknowledgment. We thank CNR and MURST for partial financial support. References and Notes (1) Seto, C. T.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 905. (2) De Rossi, U.; Da¨hne, S.; Meskers, S. C. J.; Dekkers, H. O. J. M. Angew. Chem., Int. Ed. Engl. 1996, 35, 760.

Purrello et al. (3) Ferrarini, A.; Moro, G. J.; Nordio, P. L. Mol. Phys. 1996, 87, 495. (4) Atwood, J. L.; MacGillivray, L. R Nature 1997, 389, 469. (5) Saurez, M.; Branda, N.; Lehn, J.-M.; De Cian, A.; Fischer, J. HelV. Chim. Acta 1998, 81, 1. (6) Rowan, A.; Nolte, R. J. M. Angew. Chem., Int. Ed. Engl. 1998, 37, 63. (7) Prins, L. J.; Huskens, J.; de Jong, F.; Timmerman, P.; Reinhoudt, D. N. Nature 1999, 398, 498. (8) Oda, R.; Huc, I.; Schmutz, M.; Candau, S. J.; MacKintosh, F. C. Nature 1999, 399, 566. (9) Yashima, E.; Maeda, K.; Okamoto, Y. Nature 1999, 399, 449. (10) Bellacchio, E.; Lauceri, R.; Gurrieri, S.; Monsu` Scolaro, L.; Romeo, A.; Purrello, R. J. Am. Chem. Soc. 1998, 120, 12353. (11) Lehn, J.-M. Supramolecular Chemistry; VCH: Weinheim, 1995. (12) Ashwell, G. J.; Jefferies, G.; Hamilton, D. G.; Lynch, D. E.; Roberts, M. P. S.; Bahra, G. S.; Brown, C. R. Nature 1995, 375, 385. (13) Stryer, L.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 1411. (14) Gibbs, E. J.; Tinoco, I.; Maestre, M. F.; Ellinas, P. A.; Pasternack, R. F. Biochem. Biophys. Res. Commun. 1988, 157, 350. (15) Pasternack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, A.; Gibbs, E. J. J. Am. Chem. Soc. 1993, 115, 5393. (16) (a) Ikeda, S.; Nezu, T.; Ebert, G. Biopolymers 1991, 31 1257. (b) Nezu, T.; Ikeda, S. Bull. Chem. Soc. Jpn. 1993, 66, 25. (17) Pasternack, R. F.; Giannetto, A.; Pagano, P.; Gibbs, E. J. J. Am. Chem. Soc. 1991, 113, 7799. (18) Marzilli, L. G.; Gabor, P.; Mengfen, L.; Min Sook, K.; Dixon, D. W. J. Am. Chem. Soc. 1992, 114, 7575. (19) Fuhrhop, J.-H.; Demoulin, C.; Boettcher, C.; Ko¨ning, J.; Siggel, U. J. Am. Chem. Soc. 1992, 114, 4159. (20) Purrello, R.; Monsu` Scolaro, L.; Bellacchio, E.; Gurrieri, S.; Romeo, A. Inorg. Chem. 1998, 37, 3647. (21) Purrello, R.; Bellacchio, E.; Gurrieri, S.; Lauceri, R.; Raudino, A.; Monsu` Scolaro, L.; Santoro, A. M. J. Phys. Chem. B 1998, 102, 8852. (22) Purrello, R.; Gurrieri, S.; Lauceri, R. Coord. Chem. ReV. 1999, 190, 683. (23) Hofstra, U.; Koehorst, R. B. M.; Schaafsma, T. J. Chem. Phys. Lett. 1986, 130, 555. (24) Segawa, H.; Nishino, H.; Kamikawa, T.; Honda, K.; Shimidzu, T. Chem. Lett. 1989, 1917. (25) Hugerat, M.; van der Est, A.; Ojadi, E.; Biczok, L.; Linschitz, H.; Levanon, H.; Stehlik, D. J. Phys. Chem. 1996, 100, 495. (26) Endisch, C.; Fuhrhop, J.-H.; Buschmann, J.; Luger, P.; Siggel, U. J. Am. Chem. Soc. 1996, 118, 6671. (27) Herrmann, O.; Mehdi, S. H.; Corsini, A. Can. J. Chem. 1978, 56, 1084. (28) See e.g.: De Gennes, P. G. Scaling Concepts in Polymer Physics; Cornell University Press: Ithaca, NY, 1979. Extension to the case where monomer and solvent molecules have different sizes can be found, e.g., in Shibayama, M.; Young, H.; Stein, R. S.; Han, C. C. Macromolecules 1985, 18, 2179. (29) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: New York, 1995. (30) At first sight it seems strange that both hypochromicity (eqs 15ad) and induced CD (eqs 18a-d) do not depend on the adsorbed moleculepolymer binding energy β h . However, a more advanced series expansion procedure that includes higher order terms introduces in eqs 15c,d and 18c,d a multiplicative factor, the expression of which reads (1 - (1/C) exp(2U(h)/T) exp(-νβ h /T))1/2. This term does depend on the binding energy and, correctly, increases with β h , but it is small unless C goes to zero. (31) A small chiral signal from the porphyrin ring has been observed using ZnTPPS absorbed onto chiral polypeptides (Purrello, R.; Bellacchio, E.; Gurrieri, S.; Lauceri, R.; Raudino, A.; Monsu`-Scolaro, L.; Santoro, A. M. J. Phys. Chem. 1998, 102, 8852). ZnTPPS may form only dimers because of water molecule complexation with Zn. These results, however, do not change the physical picture we are proposing. (32) Lauceri, R.; Gurrieri, S.; Bellacchio, E.; Contino, A.; Monsu` Scolaro, L.; Romeo, A.; Toscano, A.; Purrello, R. Supramol. Chem., in press. (33) Kasha, M.; Rawls, H. R.; El-Bayoumi, M. A. Pure Appl. Chem. 1965, 11, 371. (34) Pasternack, R. F.; Gibbs, E. J.; Collings, P. J.; de Paula, C. C.; Turzo, L. C.; Terracina, A. J. Am. Chem. Soc. 1998, 120, 5873. (35) Jain, M. K. Introduction to Biological Membranes; Wiley: New York, 1988.