Formation of inclusion complexes between dimers of (R)-3-hydroxybutanoic acid and β-cyclodextrin: thermodynamic study of the complexation and conformational analysis of the complexed dimers

Abstract

The formation of inclusion complexes of di[(R)-3-hydroxybutanoate] (Dimer 1) and the methyl ester of di[(R)-3-hydroxybutanoate] (Dimer 2) with β-cyclodextrin (β-CD) was studied by NMR spectroscopy. The stoichiometry of the complexation was 1 ∶ 1 (host ∶ guest). Thermodynamic analysis revealed that the complex formation is enthalpically favorable, but entropically unfavorable. Dimer 1 forms hydrogen bonds with β-CD more frequently than Dimer 2 because 1 has two hydroxy groups. Conformational analysis of the 3HB (3-hydroxybutanoate) dimers in the complexes indicates that they have extended (trans) forms. In contrast, in solution without β-CD, the end of both Dimers 1 and 2 takes a sickle (gauche) shape due to formation of intermolecular hydrogen bonds.

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Introduction

Poly[(R)-3-hydroxybutanoate] [P(3HB)] is an optically active biopolyester synthesized as a storage material for carbon and energy in many prokaryotic microorganisms.^1,2 Besides storage properties, a trace of narrow-disperse low-molecular-weight P(3HB) of physiological significance was found in a variety of prokaryotic and eukaryotic organisms,^3,4 and even in human blood plasma.⁵ Low-molecular-weight P(3HB) always appears to be complexed with other specific large molecules.⁶ Although the mechanism is still not clear, there is no doubt that molecular recognition plays an important role in the physiological processes involving P(3HB).^3c

Oligomers of 3HB have attracted much attention in terms of their synthesis, structures, and functions.^7–9 We recently studied conformational structures of dimers and some oligomers of 3HB in solution, and showed that the monomer unit adjacent to the terminal hydroxy group has a different conformational distribution due to intramolecular hydrogen bonding between the hydroxy and carbonyl groups.¹⁰

Cyclodextrins (CDs) are a series of doughnut-shaped cyclic oligosaccharides composed of 6, 7, and 8 D-(+)-glucose units linked by α-1,4-linkages, and named α-, β-, and γ-CD, respectively. Their hydrophobic cavities are able to bind various molecules to form inclusion complexes.¹¹ They have been extensively studied as models to mimic enzyme activity and to provide understanding of the mechanism of molecular recognition.^11,12 In a previous paper,¹³ we reported that di[(R)-3-hydroxybutanoate] (Dimer 1) selectively forms an inclusion complex with β-CD in aqueous solution, and carried out a thermodynamic analysis of the complex formation. The methyl ester of di[(R)-3-hydroxybutanoate] (Dimer 2) was synthesized as another model compound of P(3HB). We have also found an inclusion complex between Dimer 2 and β-CD in aqueous solution. It is of great interest to learn how the complexation affects the conformations of the guest molecules. In this paper, we report the conformational analysis of Dimer 1 and Dimer 2 in the β-CD complexes, and compare the conformational structures of the 3HB dimers in the complexes with those of free 3HB dimers in solution. The thermodynamic analysis of Dimer 2–β-CD complexation is also reported and compared with that of complexation of Dimer 1. Fig. 1 shows the chemical structures of β-CD, P(3HB), Dimer 1, and Dimer 2.

Fig. 1 The chemical structures of β-CD, P(3HB), Dimer 1, and Dimer 2.

Results and discussion

Complex formation between Dimer 2 and β-CD

Upon addition of β-CD to a D₂O solution of Dimer 2, ¹H and ¹³C NMR spectra of Dimer 2 were found to show changes in chemical shift and/or in resonance pattern. Fig. 2 shows ¹H NMR spectra of Dimer 2 in the absence and the presence of various amounts of β-CD in D₂O solution. Upon addition of β-CD, resonances of H2, H3, and H3′ of Dimer 2 show a marked shift to higher magnetic fields, while those of H4 and –OCH₃ shift downfield, and resonance patterns of H2 and H2′ show marked changes. On the other hand, upon addition of α-CD or γ-CD to a D₂O solution of Dimer 2, both ¹H and ¹³C NMR spectra of Dimer 2 were found not to show marked changes in chemical shift or in resonance pattern. The results indicate that Dimer 2 selectively forms a complex with β-CD in aqueous solution, the cavity size of which is a fit for the dimers of 3HB. As we reported in a previous paper,¹³ monomeric 3HB does not form inclusion complexes with α-, β-, or γ-CD at all. Therefore, the complex formation between Dimer 2 and β-CD is of high selectivity, and the size fitting between the dimers of 3HB and β-CD plays an important role in the inclusion complexation.

Fig. 2 500-MHz ¹H NMR spectra of Dimer 2 in the absence and the presence of β-CD in D₂O at 27 °C. The concentrations for β-CD–Dimer 2 are (from top to bottom) 0 ∶ 20.0, 5.0 ∶ 15.0, 6.7 ∶ 13.3, 10.0 ∶ 10.0, 13.3 ∶ 6.7, 15.0 ∶ 5.0 mmol dm⁻³.

Fig. 3 shows the ¹H NMR spectra of β-CD in the absence and the presence of Dimer 2 in D₂O solution. Upon addition of Dimer 2 to a D₂O solution of β-CD, the spectra show marked changes in chemical shift of proton resonances of H-3 and H-5 of β-CD, which are located in the interior of the cavity,¹¹ while the resonances of H-1, H-2, H-4, and H-6, which are located in the exterior of the cavity,¹¹ remain unchanged. The results reveal that Dimer 2 is included in the interior of the β-CD cavity to form an inclusion complex. The ¹H NMR spectra of α-CD and γ-CD in the absence and the presence of Dimer 2 were recorded too. Fig. 4 shows a comparison of changes in chemical shift of protons of α-, β-, and γ-CD upon addition of Dimer 2 in D₂O solution. Only H-3 and H-5 of β-CD shift markedly upon complexation with Dimer 2. For all protons of α-CD and γ-CD, no marked changes in chemical shift were observed, because Dimer 2 does not form inclusion complexes with α-CD or γ-CD.

Fig. 3 500-MHz ¹H NMR spectra of β-CD in the absence and the presence of Dimer 2 in D₂O at 27 °C. The concentrations for Dimer 2–β-CD are (from top to bottom) 0 ∶ 20.0, 5.0 ∶ 15.0, 6.7 ∶ 13.3, 10.0 ∶ 10.0, 13.3 ∶ 6.7, 15.0 ∶ 5.0 mmol dm⁻³.

Fig. 4 Changes in ¹H NMR chemical shift for protons of α-, β-, and γ-CD induced by Dimer 2 in D₂O at 27 °C. The concentration for Dimer 2–CD is 15.0 ∶ 5.0 mmol dm⁻³.

The stoichiometry of the inclusion complex formation between Dimer 2 and β-CD may be obtained by continuous variation experiments, i.e., recording ¹H NMR spectra for solutions where the molar ratio of Dimer 2 and β-CD is varied but the overall molar concentration of the two components is kept constant. Fig. 5 shows the continuous variation plots (Job plots¹⁴) for changes in chemical shift of protons of Dimer 2 (a) and β-CD (b). Plots for all protons in Fig. 5 show a maximum at a molar fraction of Dimer 2 or β-CD equal to 0.5, indicating the formation of a 1 ∶ 1 inclusion complex. The stoichiometry is the same as for the complex formation between Dimer 1 and β-CD as we previously reported.¹³

Fig. 5 Continuous variation plots for Dimer 2–β-CD system in D₂O at 27 °C. (a) Job plots for changes in chemical shift of H3 (●), –OCH₃ (■), H4 (▲), H2 (◆), and H3′ (▼) of Dimer 2; (b) Job plots for changes in chemical shift of H-5 (●) and H-3 (■) of β-CD.

Dissociation constant and thermodynamic parameters

Since Dimer 2 and β-CD form a 1 ∶ 1 inclusion complex, the equilibrium of the complex may be expressed by: Then, the dissociation constant K_d for the complex formation is given by:

K_d = [D][CD]/[D·CD] (2)

where [D] and [CD] are the equilibrium concentrations of Dimer 2 and β-CD, respectively, and [D·CD] is the equilibrium concentration of the complex. For a proton of Dimer 2, the observed chemical shift δ = δ_D + ([D·CD]/[D]₀)(δ_D·CD − δ_D), where [D]₀ is the initial concentration of Dimer 2, and δ_D and δ_D·CD are the chemical shifts of the proton in pure Dimer 2 and in pure complex, respectively. Therefore, the observed induced chemical shift Δδ is:

Δδ = δ − δ_D = ([D·CD]/[D]₀)Δδ_D·CD (3)

where Δδ_D·CD = δ_D·CD − δ_D. The following equation then is applicable from eqns. (2) and (3):

1/Δδ = 1/Δδ_D·CD + (K_d/Δδ_D·CD)(1/[CD]) (4)

If one is working under a condition of initial β-CD concentration [CD]₀ in large excess over [D]₀, then eqn. (4) becomes:

1/Δδ = 1/Δδ_D·CD + (K_d/Δδ_D·CD)(1/[CD]₀) (5)

This is an analogue of the Benesi–Hildebrand equation.¹⁵ For a series of solutions, a plot of 1/Δδ against 1/[CD]₀ should be linear. The intercept with the ordinate yields 1/Δδ_D·CD and from the gradient K_d/Δδ_D·CD may be obtained.

However, our conditions were only [CD]₀ > 5. So we first obtained approximate values of Δδ_D·CD and K_d by using eqn. (5). The values were taken to calculate [CD] in the given solutions. Then we estimated new values of Δδ_D·CD and K_d by using eqn. (4). These procedures were repeated until new values of Δδ_D·CD and K_d were identical to previous ones.¹⁶ For our data, the identical values were reached after three repetitions. The dissociation constants K_d at various temperatures between 17 and 45 °C were determined. The temperature dependence of the dissociation constants yields an enthalpy ΔH° of −6.5 kcal mol^−l for the complex formation. The free energy ΔG° and the entropy ΔS° were also calculated. Table 1 summarizes all of the thermodynamic parameters for the complex formation between Dimer 2 and β-CD. For comparison, thermodynamic parameters for complex formation between Dimer 1 and β-CD are also listed in the table. It is clear that the driving force for formation of both complexes is the large favorable enthalpy. It seems that the large favorable enthalpy is due to the contribution of van der Waals forces and hydrogen bonding between the host and the guest molecules, while the unfavorable entropy is due to the loss of conformational and rotational freedom of the dimers and/or conformational freedom of β-CD.¹⁷ The changes both in enthalpy and in entropy reveal a tight inclusion and a deep penetration of the guest molecules within the cavity of β-CD. A key factor which affects the complex formation is the size correspondence between the dimers and the β-CD cavity. Therefore, we can understand why the 3HB dimers do not form complexes with α-CD and γ-CD, whose cavities are too small or too large for the 3HB dimers. Another factor is the capability to form hydrogen bonding between the 3HB dimers and β-CD. The hydroxy end group of both Dimer 1 and Dimer 2 can form hydrogen bonding with β-CD. However, Dimer 1 has another free carboxy end group capable of forming hydrogen bonding with β-CD more efficiently than Dimer 2, whose corresponding end group is a methyl ester. This may be a primary reason that the enthalpy for Dimer 1–β-CD is larger than that for Dimer 2–β-CD. The decrease in entropy for Dimer 1–β-CD is larger than that for Dimer 2–β-CD, which may result from loss of more rotational freedom of Dimer 1 due to formation of more hydrogen bonding between the guest molecule and β-CD.

Table 1 Thermodynamic parameters for complex formation of 3HB dimers with β-CD in D₂O solution at various temperatures^a

System	Temperature/°C	K d/mol dm^−3	ΔG°/kcal mol^−1	ΔH°/kcal mol^−1	ΔS°/cal mol^−1 K^−1
a Parameters were determined from ^1H NMR titration. b This work. Chemical shift changes of H4 were used for the calculations. c Data from ref. 13.
Dimer 2–β-CD^b	17.0	1.07 × 10^−2	−2.61	−6.5	−13.0
	27.0	1.58 × 10^−2	−2.46
	35.0	2.49 × 10^−2	−2.25
	45.0	3.27 × 10^−2	−2.15
Dimer 1–β-CD^c	15.0	6.59 × 10^−3	−2.87	−10.4	−25.6
	27.0	1.06 × 10^−2	−2.71
	37.0	2.56 × 10^−2	−2.21
	47.0	3.69 × 10^−2	−2.10

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Conformational analysis and structures of complexes

For each monomer unit of a 3HB dimer, there are three possible conformers: trans (T), plus gauche (G), and minus gauche (Ḡ), as shown in Fig. 6. The two monomer units of a 3HB dimer show different conformational behavior due to formation of intramolecular hydrogen bonding and the different polarities of the solvents.¹⁰ It is of great interest to analyze the conformations of the 3HB dimers in the complexes and make a comparison of the conformational structures between the complexed and uncomplexed dimers.

Fig. 6 Newman projections of possible conformers of 3HB dimers for C²–C³ bond (a) and C^2′–C^3′ bond (b).

As described in previous papers,^10,18 the distribution of conformers around the CH₂–CH bonds of a 3HB unit in solution can be determined by means of ¹H NMR spectroscopy. The methylene proton resonances are associated with the methine proton (H_X) and are analyzed as an ABX three-spin system with a vicinal coupling of H_A and H_B protons. Then, the coupling constants J_AX and J_BX are presented by average values of the component coupling constants in the three conformers weighted by their fractional populations P_T, P_G, and P_Ḡ, as follows:

J_AX = P_TJ_t + P_GJ_g + P_ḠJ_g (6)

J_BX = P_TJ_g + P_GJ_t + P_ḠJ_g (7)

1 = P_T + P_G + P_Ḡ (8)

where J_g and J_t are the gauche and trans vicinal coupling constants, respectively. Assuming the reasonable values of J_g = 2.1 Hz and J_t = 11.0 Hz,¹⁹ we can calculate the fractional populations P_T, P_G, and P_Ḡ for the CH₂–CH bonds under various conditions.

Table 2 summarizes the coupling constants of methylene protons and conformer distributions of CH₂–CH bonds in 3HB dimers in D₂O solution obtained from the ¹H NMR spectra of 3HB dimers in solution and in β-CD complexes. For the C²–C³ bond of both free 3HB dimers in D₂O solution, predominant conformers are trans and plus gauche, while the minus gauche conformer is strongly disfavored. This is in accordance with the steric energies of the trans and plus gauche conformers being much lower than that of the minus gauche conformer for both 3HB dimers, because the carbonyl oxygen receives van der Waals repulsion from only one of either –CH₃ or –OR groups in the trans and plus gauche conformers, but from both –CH₃ and –OR groups in the minus gauche conformer. For the C²–C³ bond of both 3HB dimers in β-CD complexes, the most preferred conformer becomes trans, while the plus gauche conformer becomes the next preferred. The conformational distribution is more like the case in a non-polar organic solvent.¹⁰ The results indicate that the complexed 3HB dimers are in a different environment from that formed by D₂O solvent, where the dimers have less chance to interact with the aqueous molecules.

Table 2 Coupling constants of methylene protons (H2 and H2′) in 500-MHz ¹H NMR spectra and conformer distributions of CH₂–CH bonds of 3HB dimers in D₂O solution in the absence and the presence of β-CD at 27 °C

			Coupling constant/Hz		Conformer fraction
System	Bond	Probe H	J AX	J BX	P T	P G	P Ḡ
a [Dimer 1] = 5.0 mmol dm^−3. b [Dimer 1] = 5.0 mmol dm^−3; [β-CD] = 15.0 mmol dm^−3. c [Dimer 2] = 5.0 mmol dm^−3. d [Dimer 2] = 5.0 mmol dm^−3; [β-CD] = 15.0 mmol dm^−3.
Dimer 1^a	C^2–C^3	H2	6.6	6.6	0.50	0.50	0.00
	C^2′–C^3′	H2′	5.5	7.3	0.38	0.58	0.04
Dimer 1–β-CD^b	C^2–C^3	H2	7.4	6.0	0.58	0.43	0.00
	C^2′–C^3′	H2′	7.4	6.0	0.58	0.43	0.00
Dimer 2^c	C^2–C^3	H2	6.4	6.4	0.49	0.49	0.02
	C^2′–C^3′	H2′	5.2	7.8	0.35	0.64	0.00
Dimer 2–β-CD^d	C^2–C^3	H2	7.3	6.1	0.57	0.44	0.00
	C^2′–C^3′	H2′	7.3	5.8	0.57	0.41	0.02

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For the C^2′–C^3′ bond of both free 3HB dimers in D₂O solution, the predominant conformer is plus gauche, and the next preferred conformer is trans, while the minus gauche conformer is strongly disfavored (Table 2). As described in our previous papers,¹⁰ the plus gauche conformer of the C^2′–C^3′ bond of 3HB dimers in D₂O solution is stabilized by the formation of an intramolecular hydrogen bond between the hydroxy end group and the vicinal carbonyl group. However, for the C^2′–C^3′ bond of both 3HB dimers in β-CD complexes, the most preferred conformer becomes trans, while the plus gauche conformer becomes the next preferred, indicating that the intramolecular hydrogen bond is broken. It seems that the hydroxy end group of the 3HB dimers tends to form hydrogen bonding with β-CD, which contributes to the favorable enthalpy for the complexation, as discussed in a previous section of this paper.

Table 3 summarizes ¹H NMR chemical shifts of Dimer 2 in the absence and the presence of β-CD and the chemical shifts for protons of Dimer 2 induced by β-CD in D₂O. The induced chemical shifts for H2, H3, and H4 are more marked than those for H2′, H3′, and H4′, respectively, indicating that the 3HB unit with a –COOCH₃ end group is more subject to the effect of the inner wall of the cavity of β-CD. A probable explanation of the results is that the 3HB unit with a –COOCH₃ end group is included in the narrow side of the β-CD cavity. Such geometry makes the 3HB unit with a –COOCH₃ end group closer to the inner wall of the β-CD cavity, and the chemical shifts of protons of the 3HB unit with a –COOCH₃ end group are more subject to the effect of β-CD. Based on the data of ¹H NMR induced chemical shifts, and the thermodynamic and conformational analyses, a schematic illustration of a proposed structure of the Dimer 2–β-CD complex is shown in Fig. 7. In the complex, the hydroxy end of Dimer 2 forms hydrogen bonding with one of the secondary hydroxy groups of β-CD.

Table 3 ¹H NMR chemical shifts of Dimer 2 in the absence and the presence of β-CD and the chemical shifts of Dimer 2 (Δδ) induced by β-CD in D₂O at 27 °C

	Chemical shift of protons of Dimer 2 (ppm)
	–OCH3	H2	H2′	H3	H3′	H4	H4′
a [Dimer 2] = 5.0 mmol dm^−3. b [Dimer 2] = 5.0 mmol dm^−3; [β-CD] = 15.0 mmol dm^−3.
Dimer 2^a	3.690	2.717	2.507	5.281	4.196	1.299	1.209
Dimer 2–β-CD^b	3.722	2.691	2.495	5.243	4.172	1.328	1.217
Δδ	0.032	−0.026	−0.012	−0.038	−0.025	0.029	0.008

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Fig. 7 Proposed structure of Dimer 2–β-CD complex (left, side view; right, top view).

Conclusions

Two 3HB dimers, Dimer 1 and Dimer 2, selectively form inclusion complexes with β-CD in D₂O solution, while 3HB monomer does not form any complex with cyclodextrins. The thermodynamic analysis reveals that the complex formation is enthalpically favorable but entropically unfavorable, indicating that the driving forces for the complex formation are van der Waals forces and hydrogen bonding. A comparison of the thermodynamic parameters for complex formation of the two 3HB dimers with β-CD reveals that Dimer 1 may form hydrogen bonding more efficiently than Dimer 2, while the decrease in entropy for Dimer 1–β-CD is larger than that for Dimer 2–β-CD, which may result from loss of more rotational freedom of Dimer 1 due to formation of more hydrogen bonding between the guest molecule and β-CD. Both the selectivity and the thermodynamic analysis imply that the size correspondence between the guest and host molecules plays an important role in the complex formation.

Conformational analysis of the 3HB dimers in solution and in the complexes indicates that both C²–C³ and C^2′–C^3′ bonds of the two 3HB dimers in β-CD complexes mainly take trans conformations, which is different from the case of the free 3HB dimers in D₂O solution. A 3HB dimer complexed in the cavity of β-CD is in a different environment from that formed by D₂O solvent, where the dimer has less chance to interact with the solvent molecules. In D₂O solution, an intramolecular hydrogen bond between the hydroxy end group and the oxygen of the adjacent carbonyl group is formed and the C^2′–C^3′ bond takes mainly the plus gauche conformation for the two 3HB dimers. In the complexes, instead of the intramolecular hydrogen bonding, the hydroxy end group forms hydrogen bonding with β-CD, and the C^2′–C^3′ bond takes mainly the trans conformation for the two 3HB dimers. It is also concluded that the 3HB unit with a –COOCH₃ or –COOH end group is included in the narrow side of the β-CD cavity from an analysis of the ¹H NMR induced chemical shifts for respective protons of the 3HB dimers.

Experimental

Materials

α-CD, β-CD, and γ-CD were purchased from Wako Pure Chemical Ind. Ltd. Other chemicals for synthesis were supplied by Aldrich. Deuterium oxide (D₂O, 99.95%) was obtained from Merck. Dimer 1 and Dimer 2 were prepared according to the procedures described in our previous papers.¹⁰

Dimer 1: ¹H NMR δ_H(500 MHz; CDCl₃; DSS) 5.34 (m, 1H, C(3)H), 4.21 (m, 1H, C(3′)H), 2.55–2.69 (m, 2H, C(2)H₂), 2.39–2.48 (m, 2H, C(2′)H₂), 1.33 (d, J = 6.4 Hz, 3H, C(4)H₃), 1.23 (d, J = 6.4 Hz, 3H, C(4′)H₃). FAB-MS m/z 191.3 (M⁺ + l; 100%), 105.6 (57%). Elemental Anal. Found: C, 49.80; H, 7.44%. Calc. for C₈H₁₄O₅: C 50.52; H, 7.42%.

Dimer 2: ¹H NMR δ_H(500 MHz; CDCl₃; DSS) 5.29–5.36 (m, 1H, C(3)H), 4.16–4.22 (m, 1H, C(3′)H), 3.69 (s, 3H, OCH₃), 3.05 (s, broad, 1H, OH), 2.51–2.67 (m, 2H, C(2)H₂), 2.37–2.48 (m, 2H, C(2′)H₂), 1.32 (d, J = 6.1 Hz, 3H, C(4)H₃), 1.23 (d, J = 6.4 Hz, 3H, C(4′)H₃). FAB-MS m/z 205.0 (M⁺ + 1; 100%), 119.2 (44%). Elemental Anal. Found: C, 50.83; H, 7.68%. Calc. for C₉H₁₆O₅·0.4H₂O: C, 51.13; H, 8.01%.

Measurements

The ¹H and ¹³C NMR spectra were recorded on a JEOL GX-500 NMR spectrometer at 500 and 125 MHz, respectively. The ¹H NMR measurements were carried out with 5.3-s pulse repetition, 5000-Hz spectral width, and 32768 data points. Chemical shifts were referred to DSS (δ = 0.00 ppm) as external reference in a D₂O solution. Mass spectra were obtained on a JEOL JMS-HX 110 mass spectrometer by the fast atom bombardment (FAB) method in positive ion mode with a glycerol matrix, 5.0–10.0 kV acceleration voltage, and 1000 resolution. Mass calibration was carried out with a CsI spectrum.

Continuous variation experiments

The total concentrations of 3HB dimer and β-CD were maintained at 20.0 mmol dm⁻³ in D₂O, while the molar ratios of 3HB dimer and β-CD were varied to be 1 ∶ 0, 3 ∶ 1, 2 ∶ 1, 1 ∶ 1, 1 ∶ 2, 1 ∶ 3, and 0 ∶ 1. The ¹H NMR spectra of the samples were recorded at 27 °C. The Job plots¹⁴ were drawn based on the induced chemical shifts of protons of 3HB dimer and β-CD, respectively.

Determination of dissociation constant

A series of D₂O solutions which include 1.5 mmol dm⁻³ of 3HB dimer and 0.0, 8.0, 10.0, 12.0, 14.0, and 16.0 mmol dm⁻³ of β-CD were prepared. The ¹H NMR spectra of the solutions were recorded at different temperatures. The dissociation constants and other thermodynamic parameters were estimated from the induced chemical shifts of H4 of Dimer 2. The average chemical shift values for the double peaks of H4 were used.

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