Encapsulation of vanillylamine by native and modified cyclodextrins: Spectral and computational studies

Journal of Molecular Structure 1028 (2012) 57–67

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Encapsulation of vanillylamine by native and modified cyclodextrins: Spectral and computational studies T. Sivasankar, A. Antony Muthu Prabhu, M. Karthick, N. Rajendiran ⇑ Department of Chemistry, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India

h i g h l i g h t s " HMBA forms 1:1 complex with

a-CD, b-CD, HPa-CD and HPb-CD.

" CH2NH2 group present in the inner part of the CD nano-cavity. " Biexponential decay was observed in the nanosecond time-resolved fluorescence. " DG, DH, DS, and HOMO–LUMO orbital calculations confirm the better stability of the inclusion complex. " Nanomaterial structure of the inclusion complex was different from guest and host.

a r t i c l e

i n f o

Article history: Received 19 April 2012 Received in revised form 9 June 2012 Accepted 11 June 2012 Available online 18 June 2012 Keywords: Vanillylamine Cyclodextrin Inclusion complex Molecular modeling

a b s t r a c t Inclusion complex formation of vanillylamine (HMBA) with a-, b-, hydroxyl propyl a- and hydroxyl propyl b-cyclodextrins were studied by absorption, steady state fluorescence, time resolved fluorescence, FT-IR, 1H NMR, molecular modeling methods. The study revealed that HMBA formed 1:1 complex with all the four CDs. Nanosecond time-resolved studies indicated that HMBA show single exponential decay in water whereas biexponential decay in CDs. Thermodynamic parameters and binding affinity of complex formation of all the CDs were determined and discussed. It was found that van der Waals interactions are mainly responsible for enthalpy-driven complex formation of HMBA with CDs. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Cyclodextrins (CD) are cyclic oligosaccharides composed of six, seven or eight a-1, 4-linked glucose residues and are characterized by a truncated cone shape. In their cavity, the CD can accommodate a wide class of organic molecules leading to inclusion complexes [1]. CD chemistry has caused much interest, not only due to its applications to pharmaceutical science and technology but also because the inclusion represents an ideal model mimicking enzyme–substrate interactions [2]. In addition, the study of host: guest interaction may lead to a better understanding of some fundamental topics (i.e. the nature of the hydrophobic and hydrophilic interactions). In a more general picture, the driving forces involved in the complexation have been attributed to factors including van der Waals force [3], dipole–dipole interactions [4] hydrophobic effect [5] or charge transfer [6]. van der Waals is the long range forces ⇑ Corresponding author. Tel.: +91 94866 28800; fax: +91 4144 238080. E-mail address: [email protected] (N. Rajendiran). 0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molstruc.2012.06.025

arising between two molecular clusters; the dipolar interaction arises when the two molecules have a dipole moment. The hydrophobic interaction arises between two non-polar molecules; they are widely believed to play a dominant role in the formation of large biological molecules. Nevertheless the mechanism of hydrophobic interaction is still under debate [7,8]. In the ‘‘classical’’ theory, the distribution of water molecules around the solute is a key feature of the phenomenon. The enthalpy and entropy changes of the process are both positive; the association is said to be ‘‘entropy driven’’. The theories suggested that the hydrophobic interaction arises from electrostatic fluctuations, changes in water structure or the interplay of density fluctuations at both small and large length scales [8]. Our earlier studies on vanillin (4-hydroxy-3-methoxy benzaldehyde, HMB) [9,10] suggested that the specific hydrogen bonding between the solvents and carbonyl group also plays a major role in the formation of the ICT state in the ground state. We reported the polarity of the medium, the specific interactions of the solvent molecules with electron donor and acceptor group can facilitate the formation of the ICT state. In continuation of our work, in this

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paper we have studied vanillylamine (4-hydroxy-3-methoxy benzylamine (or) 4-(amino methyl)-2-methoxyphenol, HMBA) in a-CD, b-CD, hydroxyl propyl a-CD and hydroxyl propyl b-CD, different polarity of solvents, pH and computational method. 2. Experimental 2.1. Materials and methods Vanillylamine (HMBA), spectrograde solvents, a-CD, b-CD, hydroxypropyl a-CD and hydroxypropyl b-CD were obtained from Sigma–Aldrich and used as such. Triply distilled water was used for the preparation of aqueous CD solutions. A stock solution of HMBA was prepared in methanol and 0.2 ml aliquots of this solution were added to aqueous solutions of CD. The concentrations of CDs were varied from 1  103 to 10  103 M and HMBA was 2  105 M. Solutions in the pH range 2.0–12.0 were prepared by adding appropriate amount of phosphate buffer (NaOH and H3PO4). The experiments were carried out at room temperature (303 K). Absorption spectra were measured with a Shimadzu UV 1601 PC model UV–Visible spectrophotometer. Steady state fluorescence spectra were recorded with a Shimadzu spectrofluorimeter model RF-5301. The pH values in the range 2.5–12.0 were measured on an Elico pH meter (model Li-120). FT-IR spectra were measured on Avatar-330 FT-IR spectroscopy using KBr pelleting in the range 500–4000 cm1. Bruker Advance DRX 400 MHz super conducting NMR spectrophotometer was used to record 1H NMR spectra. The fluorescence lifetime measurements were performed using a picoseconds laser and single photon counting setup from JobinVyon IBH. A diode pumped Millena CW laser (Spectra Analysis) 532 nm was used to pump the Ti-Sapphire rod in Tsunami picosecond mode locked laser system (Spectra physics Model No. 4690 M3S). The Ti-Sapphire rod is oriented at Brewster’s angle to the laser beam. The wavelength turning range is 720–850 nm, i.e., standard pico configuration. The fluorescence decay of the sample is further analyzed using IBH data analysis software. The fluorescence decay profiles were fitted to the expression:

IðtÞ ¼ A1 exp

IðtÞ ¼ A1 exp

  t

s1   t

s1

þ A2 exp

þ A2 exp

  t

ð1Þ

s2   t

s2

þ A3 exp

  t

s3

ð2Þ

where s1, s2 and s3 are lifetimes of the three components, A1, A2 and A3 are the pre-exponential factors of the same and t is time. The average fluorescence lifetime is calculated by using the equation:

hsi ¼

X

si ai

ð3Þ

2.2. Synthesis of inclusion complex Methanol solution of vanillylamine (1 m mol, 10 ml) was added drop wise to an aqueous solution of CD (1 m mol, 40 ml), and the reaction mixture was stirred at 50 °C for 6 h. After cooling to room temperature, the precipitate (white powder) was collected by filtration. The crude product was dissolved in hot water to make a saturated solution. After removing the insoluble substances by filtration, a small amount of water was added to the filtrate. The resultant solution was kept at room temperature for several days, and the white powder was collected for FT-IR and 1H NMR analysis.

HMBA and CD molecules were constructed using the Spartan 08 program. The initial molecular geometries of guest molecules, bCD and inclusion complexes were fully optimized using the PM3 (Parametric Method 3) method. The corresponding frequencies were calculated to ensure that the obtained stationary points were true minima. These semiempirical methods are very convenient for the modeling of large molecular systems, such as cyclodextrin inclusion complexes [11,12]. For, inclusion process, considering large number of atoms in the studied system, a compromise between the available computer power and the desired level of calculation was particularly important for full geometry optimization calculations, since these tasks are computationally expensive at the ab initio level for such large system. We used PM3 calculations for the inclusion complexes as implemented in Gaussian 03W. In our case for large molecules, semiempirical methods have been used which ignore or approximate some of the integrals used in ab initio methods. To compensate for neglecting integrals, the semiempirical methods introduce parameters based on molecules data. The parameter for PM3 was derived by comparing a much larger number and wider variety of experimental versus computed molecular properties. So, the most stable complexes were optimized by PM3 semiempirical methods under no constraints. Because of the computations Density Functional Theory (DFT) levels are prohibitively expensive (cost and takes long time) in treating such large molecular systems, We proceeded with single point energy calculations to the PM3 optimized geometries using Hartree–Fock (HF) method as implemented in Gaussian 03W [11–15]. 3. Results and discussion 3.1. Effect of cyclodextrins with HMBA Figs. 1 and 2 depicts the absorption and emission spectra of HMBA (2  105 M) in pH 7 solutions containing different concentrations of a-CD, b-CD, HP-a-CD and HP-b-CD. In the above four CDs, the absorption maxima of HMBA appeared at 277, 228 nm. A small but clearly observable effect appeared below a CD concentration of 0.01 M. In CD solutions, the absorbance slightly increased at the same wavelength. The inset Figs. 1 and 2 depicts the changes in the absorbance and fluorescence intensity was observed as a function of the concentration of CD added. The above results were due to the transfer of guest from more protic environments (bulk aqueous phases) to less protic CD nano-cavity environments [16–24]. Fig. 2 depicts the emission spectra for HMBA (excited at 280 nm) with varying concentration of a-CD, b-CD, HP-a-CD and HP-b-CD. In aqueous and CD solutions, HMBA gave one emission maximum. The emission maximum slightly red shifted from 312 to 316 nm and the fluorescence intensity was regularly increased from water to CD solutions. Interestingly, in HMBA, a similar absorption and emission spectral maxima were observed as in all the CD solutions indicating that HMBA form a similar type of inclusion complexes. Further the absorption and emission spectral behavior of HMBA is different from HMB [9] indicating the inclusion process is different from the latter molecule. The presence of isosbestic point in the absorption spectra, in the above CDs suggested HMBA form 1:1 inclusion complex with the CDs. The K value which is the association constant for the formation of 1:1 inclusion complexes can be evaluated from double reciprocal plot concerning the absorption and fluorescence intensity change [25]:

2.3. Molecular modeling

1=If  I0 ¼ 1=a þ 1=aK½CD0

The theoretical calculations were performed using the Gaussian 03W, Gaussian view 5.0 software package. The initial structures of

where If, I0, a are the fluorescence intensities in the presence and absence of CD and the proportionality constant respectively. Fig. 3

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T. Sivasankar et al. / Journal of Molecular Structure 1028 (2012) 57–67

1

0

5

Absorbance

0.09 0.06

0.14

10

15 [α-CD] × 10-3 M

0.07 0 200

275

0.16

Absorbance

Abs

Absorbance

0.12 0.09

350

0.22

7

0.15

0

5

10

15

[HP α-CD] × 10-3 M

0.10

0 200

275

0.36

0.18

1

0 5 10 15 [β-CD] × 10-3 M

Wavelength (nm)

0.30

0.20

0.09

0.08

Wavelength (nm)

7

0.15 0.12 0.06

1

0 200

350

Abs

7

0.12

Abs

Absorbance

0.24

0.15

7

0.14

0.24

0.10

1

0 5 10 15 [HP β-CD] × 10-3 M

0.12

0 200

350

275

0.18

Abs

0.21

Wavelength (nm)

350

275

Wavelength (nm)

Fig. 1. Absorbance spectra of HMBA in different a-CD, b-CD, HP a-CD and HP b-CD concentrations (M): (1) water, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008, (7) 0.01; Inset figure: abs versus [CD] 103 M.

1000

Fluorescence intensity

Fluorescence intensity

500 500

400

7

If

300

400 300 200

1

0

5

10

15

[α-CD] × 10-3 M

200 100

900

7

800

700 500 300

600

1

400

0

5 10 15 [β-CD] × 10-3

200 0

0 300

400

350

450

300

500

Wavelength (nm)

350

400

1000

750 600

7

If

750 600 450 300

450

0

5 10 15 [HP α-CD] × 10-3 M

1

300 150 0 300

350

400

450

500

Wavelength (nm)

450

500

Wavelength (nm)

Fluorescence intensity

Fluorescence intensity

If

900

7

800

If

700 500

600

300

0 5 10 15 [HP β-CD] × 10-3 M

1 400 200 0 300

350

400

450

500

Wavelength (nm)

Fig. 2. Fluorescence spectra of HMBA in different a-CD, b-CD, HP a-CD and HP b-CD concentrations (M): (1) 0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.006, (6) 0.008, (7) 0.01; (kexci  280 nm); Inset figure: IF versus [CD] 103 M.

illustrates the double reciprocal plot for the drugs containing the four CDs. The straight line in the Fig. 3 conforms the 1:1 stoichiometry of the CD–drug inclusion complex. From the plot, the K value is estimated. The stoichiometry of the inclusion complexes of the HMBA with the CDs are studied by using Jobs method [26,27]. A plot of (A  Ao)/Ao (or) (I  Io)/Io versus [CD]/([CD] + [HMBA]) gave a very good linear line as shown in Fig. 4. This analysis showed HMBA molecule formed 1:1 inclusion complex with CD. Further the Benesi–Hildebrand plot for 1:2 inclusion complex (HMBA:CD) showed

upward curve which confirmed the 1:2 complex was not formed (figures not shown). The similar slopes in Figs. 3 and 4 for all the inclusion complexes indicate that the interactions of hydrogen atoms are approximately same in both the ground and excited states. The hydrophobic contact with the guest is some what weak, since the polar substituents are far from the internal surface of the CD nano-cavity and the CH2NH2 group encapsulated in the interior part of the CD cavity. Further, the increased absorbance and emission intensities along with CD concentrations revealed that

T. Sivasankar et al. / Journal of Molecular Structure 1028 (2012) 57–67

(a)

300

1/A-Ao

60

200

(a)

α-CD β-CD HP-α-CD HP-β-CD

0.34

0.30 f

100 α-CD β-CD HP-α-CD HP-β-CD

0.26

0 0

200

400

600

1/[CD] M

800

1000

1200

0.22

-1

0

2

4

6

8

10

12

-3

[CD] × 10 M

1/I-Io

(b)

0.09

α-CD β-CD HP-α-CD HP-β-CD

0.06

(b)

4.50

4.00

0.03

1/ f 3.50

0

0

200

400

600

800

1000

1200

1/[CD] M-1

3.00 0

Fig. 3. Benesi–Hildebrand plot for the complexation of HMBA with CDs. Plot of 1/ DA versus 1/[CDs] and Plot of 1/(I  I0) versus 1/[CDs].

200

400

600

1/[CD] M

800

1000

1200

-1

Fig. 5. Plot of (a) quantum yield (Uf) versus concentration of CD; (b) Plot of 1/Uf versus 1/CD.

(a)

0.8

(A-A0)/A0

abs  215 M1, flu  365 M1; HP-a-CD: abs  232 M1, flu  297 M1; HP-b-CD: abs  258 M1, flu  440 M1). The small formation constant implies that the guest was not tightly embedded in the CD cavity. In order to ascertain the stochiometry and binding constants of the 1:1 complexes with the CDs, the plot of the fluorescence quantum yield (uf) against the CD concentration, has been fitted to the equation:

α-CD β-CD HP-α-CD HP-β-CD

0.6

0.4

0.2

uf ¼ ðu0 þ u1 þ K 1 ½CDÞ=ð1 þ K 1 ½CDÞ

0 0

0.2

0.4

0.6

0.8

1.0

1.2

1.0

1.2

[CD]/([CD]+[HMBA])

(b)

0.8 α-CD β-CD HP-α-CD HP-β-CD

(I-I0)/I0

0.6

0.4

0.2

0 0

0.2

0.4

0.6

0.8

[CD]/([CD]+[HMBA]) Fig. 4. Plot of (A  A0)/A0 and (I  I0)/I0 versus mole fraction of CD using (a) Absorption and (b) fluorescence intensity.

aromatic ring was encapsulated in the non-polar CD nano-cavity. Thus, for these reasons, the association constants for the guest is almost equal (a-CD: abs  161 M1, flu  235 M1; b-CD:

where u0 and u1 are the fluorescence quantum yields of the free HMBA and 1:1 complex respectively [28,29]. The values of u0 and K1 from Benesi–Hildebrand analysis are used as constants in the equation. The binding constants and the quantum yields are then calculated by iterative non-linear least squares regression (Fig. 5). The fluorescence quantum yield (uf) is found to be significantly higher in the 1:1 complexes with b-CD than in any of the other complexes. This is explained further using the time resolved fluorescence studies. Since the CD nano-cavities restrict the free rotation of the guest, the emission intensity and quantum yield were increased in all the CD solutions. The presence of broad emission in CD aqueous solution suggested viscosity plays major role in the inclusion process. The increase in full width at half maximum (FWHM) of emission spectra with increasing CD concentration further supported the above prediction. Further it is well known that, the cavity size of the CD is also responsible for the different association constants. The small association constant implied that the guest was not tightly encapsulated in the CD cavity. In other words, the CH2NH2 is more deeply entrapped in the non-polar CD cavity than the aromatic OH groups. The gradual enhancement of emission with increasing CD concentration is consistent with this speculation. Thus, the enhancement of the emission in CD indicated that the energy barrier is not affected by the entrapment of the guest in the

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T. Sivasankar et al. / Journal of Molecular Structure 1028 (2012) 57–67

Table 1 Absorption, fluorescence spectral data (nm) and Stokes shifts (cm1) of 4-hydroxy-3-methoxy benzylamine (HMBA) and 4-hydroxy-3-methoxy benzaldehyde (HMB) in selected solvents. No.

1 2 3 4 5 6 7 8 9

Solvent

Cyclohexane 1,4-Dioxane Ethyl acetate Acetonitrile t-Butyl alcohol 2-Butanol 2-Propanol Methanol Water

HMBA

HMB

kabs

log e

kflu

mss cm1

kabs

270 272 276 276 276 276 278 278 277

2.87 3.45 3.39 3.66 3.60 3.55 3.51 3.57 3.63

308 308 312 312 312 312 312 312 312

4570 4297 4180 4180 4180 4180 3920 3920 3972

295, 304, 305, 304, 311, 311, 311, 308, 289,

non-polar CD cavity. Further, the presence of CH2NH2 does not significantly change the inclusion process. The above findings confirmed that the hydroxyl group is present in the wider rim part and CH2NH2 is entrapped in the interior part of the CD cavity. As discussed above the orientation of the guest is same in the all the four CD inclusion complex. 3.2. Effect of solvents To understand the hydrophobic and hydrophilic interactions between the guest and CDs the solvent dependent changes in the absorption and emission spectra for HMBA in polar and non-polar solvents were recorded. Table 1 summarizes the spectral properties of HMBA and HMB [9] in different solvents. In any solvent, compared to that of HMB [9] (cyclohexane: kabs 295, 270, 232 nm, kflu 323, 348 nm and methanol: kabs 308, 279, 234 nm, kflu 327, 393 nm) and 4-hydroxybenzaldehyde (HB: cyclohexane 288, 281, 274, 265 nm and methanol 294, 221 nm), the absorption and emission spectra of HMBA (cyclohexane: kabs 278 nm, kflu 308 nm; methanol: kabs 278 nm, kflu 312 nm) was blue shifted. Both the absorption and emission spectra of HMBA was different from that of HMB. In all the solvents HMB exhibited a dual fluorescence (cyclohexane: kflu 348, 323 nm, methanol: kflu 327, 393 nm) whereas HMBA gave one fluorescence maximum in all solvent (cyclohexane 303 nm, methanol 312 nm). Among the two bands one occurred in shorter wavelength region (SW 325 nm) and the other in longer wavelength region (LW or ICT 350–425 nm). As the solvent polarity was increased, the emission maxima of HMB bands (both SW and ICT) shifted to red, the shift being greater for the ICT band. The absorption and fluorescence spectra of HMB were found to be solvent dependent, while no significant shift was noticed in HMBA. The red shifts of HMB with respect to HMBA in all solvents are due to the resonance interaction between the aldehyde group and phenyl ring. The large interaction in HMB is revealed by the maximum red shift in fluorescence observed for this molecule. The blue shift of the absorption and emission spectrum of HMBA are due to the weak interactions of the hydroxyl group with the p-cloud of the benzene ring [22–24]. The absorption maxima of HMBA was blue shifted than HMB indicating replacement of aldehyde group by CH2NH2 group in HB molecule which decreased interaction between hydroxy and CH2NH2 groups. 3.3. Effect of acid/base concentrations To know the effect of CD on the prototropic equilibrium between monocation, neutral and monoanion, the pH dependent changes in the absorption and emission spectra of HMBA in aqueous and CD medium were recorded. In aqueous medium, the

log e 270, 273, 273, 272, 281, 281, 281, 279, 256,

232 230 235 231 235 235 235 234 218

3.50, 3.94, 3.89, 4.02, 4.07, 4.06, 4.06, 3.97, 3.76,

mss cm1

kflu 3.88, 4.01, 3.99, 4.08, 4.04, 4.04, 4.04, 3.99, 4.01,

3.99 3.99 3.99 4.12 4.01 4.04 4.04 4.00 4.11

348, 357, 358, 359, 382, 382, 384, 393, 425,

323 328 326 323 326 326 327 327 327

5110, 2927 4865, 2342 4816, 2112 4871, 2308 5914, 1436 5997, 1567 5914, 1436 7001, 1639 10843, 3699

absorption and fluorescence spectra were studied in the H0/pH/ H range of 10 to 16.0. The results are summarized as below: With the decrease on pH from 7, the absorption spectrum of HMBA was unaffected up to H0 10. When the pH was increased from 5 to 10, the absorption maximum was moved to longer wavelength (HMBA: 277–294 nm). This is due to the formation of the monoanion obtained by deprotonation of the hydroxyl group. No further change was noticed in absorption spectrum even at H_16. The above behavior resembled to that of the aromatic compounds containing the hydroxyl group [16–19]. The fluorescence characteristic of the HMBA is similar to its absorption characteristics. When the acidity was increased, the emission intensity of the fluorescent band decreased considerably as the consequence of monocation formation. With an increase in the pH from 6, the emission intensity of the fluorescent band was quenched at the same wavelength (312 nm) suggesting monoanion was formed [9]. The formation of monoanion was completed in the pH range 10–11. The effect of CD on the prototropic equilibrium between monocation, neutral, monoanion on the pH dependent changes in the absorption and emission spectra of the HMBA in various CD solutions were measured. The absorption and emission maxima of HMBA in CD solutions were studied in 6  103 M in the pH range 0.1–11. No marginal spectral shift was observed in the absorption and emission maxima of HMBA in CD medium and aqueous medium. The pKa (pK a ) values of the neutral-monoanion equilibrium was also similar to aqueous medium.

3.4. Excited singlet state life time The formation of inclusion complex was also studied by the fluorescence decay curves obtained for all the four CD. The fluorescence lifetimes of the HMBA molecule in aqueous and CD medium were determined from the decay curves and given in Table 2 and Fig. 6. Single exponential decay was observed with HMBA in

Table 2 Time resolved fluorescence spectral data of HMBA (excitation wavelength = 280 nm; emission wavelength = 320 nm). Medium

Lifetime (ns)

s1 Water a-CD b-CD HP-aCD HP-bCD

1.93 1.59 1.63 1.99 1.95

Standard deviation

s2 2.18 2.26 6.62

4.84  1012 1.11  1010 1.17  1010 1.59  1011

7.19

1.31  1011

Relative amplitude

v2

A1

A2

2.65  1010 4.32  1011 2.74  1010

100 46.65 43.77 90.66

53.35 56.26 9.34

1.17 0.97 1.09 0.99

8.40  1011

76.32

23.68

1.04

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T. Sivasankar et al. / Journal of Molecular Structure 1028 (2012) 57–67

Fig. 6. Fluorescence decay curve of HMBA in different cyclodextrin medium (Excitation wavelength = 280 nm; emission wavelength = 320 nm).

Type I

Type II

Fig. 7. The proposed inclusion complex structure of HMBA with CDs cavity.

aqueous solution (1.93 ns). By the addition of the CDs, single exponential curve becomes biexponential. This showed HMBA encapsulated in the CD cavity. The life time of the inclusion complexes were higher than the isolated molecule (a-CD  2.18 ns, b-CD  2.26 ns, HP-a-CD  6.62 ns and HP-b-CD  7.19 ns). The life time of HMBA increased by the following order: water < a-CD < b-CD < HP-a-CD < HP-b-CD. The above order indicated the tendency of complexation ability of CD, in other words HP-b-CD has given the privileged encapsulation. The amplitude of the complexed component also increased due to increase in the complex formation and that of the decreased free species. The increase in the value of s1 and s2 with increase in the CD concentration is due to the encapsulation of HMBA in the CD cavity. The s1 and s2 values depend on the type of CD and the nature of the process with regard to short-lived species. The decay of the HMBA is dependent of the CD. This may be due to the vibrational restriction of HMBA in the excited state. With native CD, the lifetimes do not change considerably, but the lifetime is high in HP-CDs (Table 2, Fig. 6). A similar variation of amplitudes, with large change in lifetimes is observed with HP-CDs. The increase in the lifetime of the HMBA with longer lifetime in HP-CDs is explained by confinement effect experienced by the fluorophore within the cavities of these CDs. The decrease in the amplitude of the HMBA signifies a weaker interaction with the CD. The greater degree of encapsulation in HP-CDs than in aCD/b-CD is rationalized by the presence of hydroxyl propyl-CD, which provides a stronger interaction than native CDs. The fact that, the fluorescence decays become single-exponential low in native CDs. This has been explained in the light of the difference in the orientation of the HMBA molecule in the complexes with the

HP-CDs and native CDs, which results from a difference in the size of their cavities. The longer lifetime is ascribed to a deep encapsulation (inside the cavity), and short life time is due to a loosely associated form. The difference in the extents of enhancement of fluorescence quantum yield of HMBA induced by the two HP-CDs may be explained similarly. A deep encapsulation with the smaller HP-CD may be expected, compared to the loose complex with native CDs. This may be expected to cause a greater enhancement of fluorescence in the former than in the latter. 3.5. Inclusion complexation mechanism In HMBA, two different types of inclusion complex formation are possible i.e., (i) hydroxyl group captured or (ii) CH2NH2 group deeply present in the inner nano-cavity (Fig. 7). As per type I arrangement, if hydroxyl group deeply entrapped in the CD nano-cavity, the monoanion absorption and emission maxima in CD medium should be blue shifted than aqueous medium. In CD medium [9,10] the monoanion of HMBA is not blue shifted indicating OH group is not present in the hydrophobic part of the CD. However, the monoanion absorption and emission maximum in CD were similar to aqueous medium indicating OH group is not encapsulated in the inner CD cavity. This is possible because, in CD medium the hydroxyl anion is similar to aqueous medium (312 nm). The absorption and emission spectral maximum for HMBA is similar to HMB [9] and dihydroxy phenols [16,17] suggesting the CH2NH2 group is present in the narrow part of the nano-cavity. Moreover, in the aqueous and CD medium, no significant absorption and emission spectral shifts are noticed for the hydroxyl

T. Sivasankar et al. / Journal of Molecular Structure 1028 (2012) 57–67

Upper view

63

Side view

(a)

(b)

(c)

(d)

Fig. 8. Axial and equatorial views of low energy structures calculated for the complexes. between HMBA and (a) a-CD, (b) b-CD, (c) HP-a-CD and (d) HP-b-CD.

monoanion indicating that the hydroxyl group is present in the hydrophilic part of the CD cavity. It is already reported when the OH group is entrapped in the CD cavity, the hydroxyl anion should be blue shifted in CD than aqueous medium [16,17]. This because of the large rim of CD containing many secondary hydroxyl groups provides an environment qualitatively similar to poly hydroxy alcohols [30–33]. It is well known that CD is a very good proton donor [30–33] and this may provide proton to the guest hydroxyl groups. Moreover, the amino and hydroxyl groups in the aromatic rings are capable of forming hydrogen bonding with the CD hydroxyl groups. From the above discussion, it is clear that, in CD solution the CH2NH2 group of HMBA goes inside the nano-cavity and hydroxyl group is present in the upper part of the b-CD nano-cavity. The above results suggested that, the structural geometry of HMBA with all the four CD inclusion complexes were same in terms of orientation of guest molecules. We found that earlier in HMB with aqueous CD solution, hydrophobicity is the driving force for encapsulation of the molecule inside the cavity and naturally the hydrophobic part like to go inside the deep core of the non-polar cavity and the polar group will be projected in the hydrophilic part of the CD nano-cavity [9]. The increase in the FWHM and emission intensity along with CD concentration confirmed CH2NH2 group is present in the interior part of the CD cavity. On the basis of the above discussions, it is clear that as long as the CH2NH2 group

is present in the CD cavity, the free rotation of the guest is reduced in the CD nano-cavity. 3.6. Molecular modeling As described above, interactions between the four CDs and HMBA were studied experimentally which provided evidence for the formation of inclusion complexes [11–15]. For further evidence, the geometries and stabilities of the complexes, as well as the inclusion energetic for formation of the complexes were studied by theoretical method. The theoretical calculations were carried out using Spartan 08 and Gaussian 03W version 6.0 packages for molecular and quantum mechanics method respectively. The initial structure of HMBA, a-CD, b-CD, HP-a-CD and HP-b-CD were constructed by module builder of Spartan the optimized with PM3 method without imposing any symmetrical restrictions. By using this models as shown in Fig. 8, the binding energy change curves for HMBA passing through all the CD nanocavities are shown in Fig. 8 (for Head-up and for Head-down) respectively. For the construction of guest/host inclusion complex, the glycosidic oxygen atoms of b-CD were placed onto the XY plane: their center defined as the origin of the coordinate system. The primary hydroxyl groups were oriented pointing towards the positive Z axis.

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Table 3 Energies, thermodynamic characteristics and HOMO–LUMO calculations using PM3 method for HMBA, a-CD, b-CD, HP-a-CD and HP-b-CD, its inclusion complexes calculated by the PM3 method. Properties EHOMO (eV) ELUMO (eV) EHOMO  ELUMO (eV) l (eV) g (eV) S (eV) x (eV) Dipole (D) 

EHF (kcal mol1) DE° H° DH° G° DG° S° (kcal/mol Kelvin) DS° Zero-point energy (kcal mol1) Mullikan charges

a-CD

b-CD

HP-a-CD

HP-b-CD

HMBA: aCD

HMBA: bCD

HMBA:HP-aCD

HMBA: HP-bCD

9.04 0.14 9.19 4.45 4.59 0.22 2.15 0.97 57.93

10.38 1.26 11.64 4.56 5.82 0.17 1.78 11.34 1247.52

10.35 1.23 11.58 4.56 5.79 0.17 1.79 12.29 1457.63

10.20 1.24 11.44 4.48 5.72 0.17 1.75 10.38 1533.51

10.18 1.21 11.40 4.47 5.70 0.17 1.75 10.34 1683.18

9.11 0.01 9.11 4.55 4.56 0.22 2.27 10.14 1318.78

8.94 0.14 9.09 4.40 4.54 0.22 2.13 11.06 1538.94

8.98 0.04 8.94 4.51 4.47 0.22 2.27 8.44 1603.95

8.82 0.12 8.70 4.37 4.45 0.22 2.13 8.95 1750.79

121.14

676.73

789.52

1026.70

1087.02

89.07

571.21

667.55

882.52

973.95

12.51 1149.69 18.36 991.52 19.92 0.530 0.060 1082.94

8.98 1228.09 19.93 1044.62 18.40 0.572 0.050 1152.98

HMBA

0.107

0.353

0.409

0.483

0.515

113.31

634.47

740.56

967.28

1040.17

0.00

0.00

0.00

0.00

0.00

The guest molecule was placed on the Z axis and allowed to approach the CD cavity from the large rim at a distance of 8 Å which separates the CD equatorial plane and the reference atom (C6) in HMBA. The inclusion process emulation was then achieved along the Z axis is – 8 Å at a step of 1 Å. The generated structures at every step were minimized keeping the movement of the reference atom (C6) and the CD structure totally restricted. To obtain optical complex geometry, each complex was completely optimized without any restriction. The most stable inclusion complexes were optimized by PM3 semiempirical method under no constraints. The energy minimized structures of HMBA with a-CD, b-CD, HP-a-CD and HP-b-CD inclusion complex obtained from PM3 calculations are illustrated in Fig. 8. The geometries of the studied complexes by PM3 method showed that HMBA partially included in all the CDs. The energetic features, thermodynamic characteristics and electronic properties of these structures are summarized in Table 3. The first remark is that all binding energies are negative which demonstrate that the inclusion process of HMBA in all CDs were thermodynamically favorable. Compared to other CDs, b-CD-HMBA complex binding energy was more negative which indicated b-CD formed more stable complex than other CDs. Mulliken charges for the inclusion complex was zero suggesting no charge transfer interaction between the guest and host. From the semiempirical study, we noticed that the dipole moment values of CDs, free HMBA and the complexes are formed which is an indication of the augmentation of the polarity.

13.33 809.56 11.69 679.34 19.06 0.403 0.058 750.09 0.00

23.38 929.95 19.28 789.19 32.56 0.472 0.044 863.79 0.00

0.00

0.00

effect. Unfortunately, because of limitations in the calculation ability of our computer and the large molecular size of CD calculations for these systems could not be performed for aqueous solutions. However, it is observed that the solvent effect on the host–guest interactions easily changes the inclusion reaction from a nonspontaneous process in the gas phase to a spontaneous one in the aqueous phase. The host–guest interaction causes an enthalpy–entropy compensating process in the gas phase whereas the same interaction causes an enthalpy–entropy co-driven process in aqueous solution, because inclusion complexation releases a number of water molecules from the cavity of CDs. To quantity the interaction between host and guest in the optimized geometries, we have evaluated binding (DE) energy, which was calculated by subtracting the sum of the energies of individual free host and guest molecules from the energy of the inclusion complex using the following formula

DEcomplexation ¼ Ecomplexation  ðEbCD þ EHMBA Þ

ð4Þ

HOMO as Ionization energy (IE) and LUMO as electron affinity (EA) are used for calculating the electronic chemical potential (l) which is half of the energy of the HOMO and LUMO:

3.7. Thermodynamics parameters To investigate the thermodynamics of the inclusion process, the binding energies (DE), Gibbs energy changes (DG), enthalpy changes (DH) and entropy changes (DS) for the most stable HMBA:CD complexes (both Head-up and Head-down configurations) were calculated and are summarized in Table 3. The experimental free energy change values of the inclusion complexes (a-CD abs: 3.05, flu: 3.29; b-CD abs: 3.23, flu: 3.55; HP-a-CD abs: 3.28, flu: 3.43; HP-b-CD abs: 3.34, flu: 3.67) were calculated from the formation constant (unit: kcal mole1). The negative DG values suggest that the inclusion proceeded spontaneously at 303 K. The experimental results indicated that the inclusion reactions of the CDs with HMBA are an exothermic process. The experimental DG values are different from theoretical values (Table 3). This can be explained by the solvent

HOMO

LUMO

Fig. 9. The optimized structure and HOMO, LUMO energy structure of HMBA.

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T. Sivasankar et al. / Journal of Molecular Structure 1028 (2012) 57–67

l ¼ ðEHOMO þ ELUMO Þ=2

ð5Þ

Calculations showed that the HOMO and LUMO energy levels for the complexes are lower than those for HMBA (Fig. 9). HOMO–LUMO values of all the complexes are almost same. The HF calculations expressed that energies of the complexation are lower than those for the isolated host and guest molecules. The HF calculations indicated that, HP-b-CD/HMBA is more stable than the other CD/HMBA. The hardness (g) as half of the gap energy between HOMO and LUMO was calculated using the following expression:

Gap ¼ EHOMO  ELUMO

ð6Þ

g ¼ ELUMO  EHOMO =2

ð7Þ

The electrophilicity of the components were calculated by HF method using the following equation:

x ¼ l2 =2g

ð8Þ

These structural parameters calculated by the HF method for the isolated HMBA and its inclusion complex are summarized in Table 3. As it clear from Table 3 that complex formation of HMBA with all CDs under study were exothermic and most enthalpy-driven DH > DS. It should be noted that DH and DS values contain contributions from (i) release of cavity found water, (ii) partial destruction of hydration shells of the reagents, (iii) non-covalent interactions (van der Waals, hydrophobic and electrostatic interactions as well as hydrogen bonding, and (iv) hydration of the complexes. All these process should be taken into account while discussing thermodynamic parameters of complex formation. HMBA is bounds to b-CD with more negative DH and high binding constants. Probably geometric factor plays a considerable role in complexation process. The observed small negative DS is a confirmation of restriction of freedom of the molecule and formation of more compact structures. As it is evident from Table 3, hydroxyl propyl groups reduce binding affinity of HP-a-CD to HMBA, making the complexation process less enthalpy and more entropy favorable. It is assumed that the cavity size and substituents surrounding the a-CD cavity serve as a steric hindrance for HMBA inclusion. Binding of HMBA with CDs are enthalpy–entropy favorable showing negative DH and DS values. Penetration of HMBA into CD cavities can be deeper and HMBA molecules can be located inside the cavity. The observed small negative DS values are

assumed to be due to enhancement of disorder in the system. Moreover hydrophobic interactions, which are long range interactions, can be important in the CD complex formation. The inclusion complex Fig. 8 also suggested that benzene ring was completely inside the cavity and interacts with it through hydrophobic interactions. Further the small DH values can be explained by the prevalence of hydrophobic interactions. Furthermore the optimized inclusion structure bond length also suggests hydrogen binding interactions was not present between HMBA and the CDs. With regard to a-CD/HP-a-CD, b-CD/HP-b-CD forms more stable complexes with HMBA. The DG and DH values for a-CDs, b-CDs further confirmed this speculation. Comparison of DH and DS for both a-CDs and b-CDs showed that enthalpy changes are higher and entropy changes are lower for b-CDs complexation. Therefore complexes of both b-CDs are more enthalpy stabilized. A slight increase in exothermicity of HP-b-CD binding and as a consequence, the enhancement of complex stability could be possible by additional interactions between HMBA-OH group and flexible hydroxypropyl substituents. Upon deep inclusion into b-CD, OH group can protrude outside the cavity and form H-bonds with functional groups on the HP-b-CD rim. But the optimized structure in Fig. 8 shows, no hydrogen bond is formed between the HMBA and the CDs. This suggested that in all the inclusion complexes hydrophobic and electrostatic interactions played a major role. The bond distances, bond angles and the most interesting dihedral angles of HMBA before and after complexation in CDs obtained from PM3 calculations for the most stable structure (Fig. 8) are presented in Table 4. It is evident that in CDs the geometry of HMBA is slightly altered. The alterations are significant in dihedral angles, which indicated that HMBA must adopt a specific conformation to form a stable complex. The internal diameters of the CDs are approximately 5.6 Å for a-CD 6.5 Å or b-CD and the height of all the CDs are 7.8 Å. Considering the shape and dimensions of CD, the guest cannot be completely embedded in the CD nano-cavity. The vertical distance and length of HMBA (CH2NH2–OH) is (7.66 Å) is greater than the CD cavity and upper/lower rim of CD. Hence, the CH2NH2 and hydroxyl groups attached to benzene ring cannot be fully present inside of the CD nano-cavity. Further, the optimized theoretical structure of HMBA–CD inclusion complex by Gauss view method also confirmed the CH2NH2 attached to phenyl ring is present in the interior part of the CD and hydroxyl group is present in the hydrophilic part of the CD nano-cavity (Fig. 8). These finding confirmed HMBA is partially embedded in the CD cavity.

Table 4 Geometrical parameters of HMBA before and after inclusion in a-CD, b-CD, HP-a-CD and HP-b-CD bond distances (Å), angle (°) and dihedral angles (°) calculated by PM3 method. Parameters Bond lengths (Å) H1–H11 C6–C8 O1–N1 C7–O1 H3–H6 H4–H6 C8–N1 O1–O2 Bond angles (°) O1–C1–C2 C4–C7–N1 C2–O2–C8 Dihedral angle (°) C3–C4–C7–N1 C5–C4–C7–N1 C3–C2–O2–C8 O1–C1–C2–O2 O1–C1–C2–C3

Free HMBA 7.666 4.444 6.332 5.661 7.049 6.200 6.116 2.788

a-CD: HMBA

b-CD: HMBA

7.691 4.489 6.354 5.666 6.975 6.331 5.744 2.874

7.693 4.493 6.323 5.662 7.037 6.225 5.873 2.873

121.92 110.33 113.67

124.16 110.34 112.85

96.46 83.53 102.70 6.13 179.72

81.11 99.12 85.95 3.64 179.17

124.34 109.84 113.26 89.25 90.81 88.62 5.049 179.81

HP-a-CD: HMBA 7.761 4.486 6.385 5.636 6.989 6.317 5.964 2.876 124.54 110.57 112.53 99.61 29.40 83.67 1.51 177.77

HP-b-CD: HMBA 7.764 4.489 6.356 5.631 7.052 6.311 5.995 2.877 124.66 110.97 113.12 100.12 83.18 86.25 1.151 176.36

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It is well known that the strength of interaction is dependent on the size of the CD cavity and size of the substituent in the complex. This means that the interaction is more sensitive to the size and nature (polar and non-polar) of substituents and the CD in the complexation. The CDs are truncated, right-cylindrical, coneshaped molecules, 7.8 Å heights with a central cavity. The diameters of the wider and narrower rim of the cavity for b-CD are 6.5 Å and 5.8 Å, respectively. It is well known that the van der Waals forces including the dipole–induced dipole interactions are proportional to the distance between guest, the wall of the CD cavity and to the polarizabilities of the two components. The interaction of the phenyl ring with CD would play an important role because the phenyl moiety may achieve a maximum contact area with the internal surface of the cavity of the CD. Therefore, the guest is partially embedded in the CD cavity. Further, the increased absorbance in the CD solutions suggested that the aromatic ring is encapsulated in the non-polar part of the CD cavity. The above results suggest inclusion of HMBA molecule with CD nano-cavity is affected by hydrophobic and electronic interactions. Since CD has a permanent dipole the primary hydroxyl end is positive and the secondary hydroxyl end is negative in the glucose units of CD. The stability of binding by hydrophobic interaction is partly the result of van der Waals force but is mainly due to the effects of entropy produced on the water molecules. In the aqueous solution, a hydrophobic guest compound is restricted by the water shell formed by the hydrogen bonding network. It has a strong tendency to break down the water cluster and penetrate the non-polar cavity of the CD. This process is exothermic due to entropic gain. The association constants for the inclusion of CD with guest compounds were observed to be proportional to the substituent hydrophobic constant of the guest. From Fig. 8 it can be observed that the HMBA molecule is partially encapsulated in the CD nano-cavity. Further, no intermolecular H-bond is noticed in the structures. Here, the H-bond is defined as C–H  O or O–H  O with the distance between O and H being greater than 2.5 Å [34]. The minimum H-bond length is 2.89 Å in this structure, which is not within the reported length range for H-bonds. These data indicate that intermolecular Hbonds is not present in the inclusion complexes. The data in Table 3 also indicate that the inclusion complex bCDHMBA is more stable than the others, which can be explained by (i) b-CD has the optimal internal cavity (7.8 Å) to encase the HMBA molecule, (ii) The standard formation enthalpies (DH°) of the complexes a-CDHMBA, b-CDHMBA, HP-a-CDHMBA and HPb-CDHMBA are 11.69, 19.28, 18.36 and 19.93 kcal mol1 respectively, which indicated that the formation reactions for these complexes are weak exothermic processes, (iii) Negative values for the standard Gibbs energy changes (DG°) indicate that the formations of all the complexes are spontaneous processes and (iv) The entropy effects (DS°) for formation of the inclusion complexes were small negative than standard Gibbs energy and the heat effect. Finally, the computational results indicate the formation of all the inclusion complexes were enthalpy driven process. 4. Solid inclusion complex Further to confirm the HMBA:CD interactions, we also prepared the solid inclusion complexes and characterized by FTIR, and 1 HNMR methods. 4.1. Infrared spectral studies The OH stretching vibrations in HMBA produce a strong band in the 3178 cm1 region was largely red shifted in the inclusion complex (3351 cm1). The OH bending (1377 cm1) and out of plant

deformation (790 cm1) frequencies also blue shifted in the inclusion complex to 1368 and 757 cm1 respectively. The C–O–CH3 symmetric stretching (1032 cm1) frequency was slightly blue shifted in the complex. Further aromatic ring stretching (1611 cm1) was red shifted in the complex (1639 cm1). Further, the absorption intensity of the inclusion complex was also significantly varied from HMBA molecule. The above results confirm HMBA molecule was included into the b-CD cavity.

4.2. 1H NMR spectral studies Proton NMR (400 MHz in D2O-DMSO 5%) was used to compare the environments of the pure and complexed molecules and in particular to confirm interactions of the guest molecules. Proton nuclear magnetic resonance (1H NMR) spectroscopy has proved to be a powerful tool in the study of inclusion complexes [35,36]. The basis of information gained from NMR spectroscopy is located in this shifts, loss of resolution and broadening of signals observed for the host and guest protons. Although, only limited information can be obtained from the 1H NMR data, the observation of slight upfield shifts of the guest protons in the presence of b-CD is consistent with the inclusion of each guest into the cavity. The resonance assignment of the protons of b-CD are well established [35,36] and consists of six types of protons. The chemical shift of b-CD protons reported by different authors are very close to those reported in this work. The H-3 and H-5 protons are located in the interior of the b-CDs cavity, and it is, therefore likely that the interaction of the host with the b-CD inside the cavity will affect the chemical shifts of the H-3 and H-5 protons. A minor shift is observed for the resonance of H-1, H-2 and H-4 located on the exterior of b-CD. Owing to the poor solubility of the guest toward D2O, we are forced to employ at least 5% volume of DMSO-d6 as a co-solvent, which made the use of a buffered solution difficult. We define the change in chemical shift (Dd ppm) as the difference a chemical shifts between proton signals of the guest in the presence and absence of b-CD. Unfortunately, the addition of 5% volume of DMSO-d6 to a D2O solution of b-CD caused relatively large upfield shifts of the H-3 (Dd = +0.05) and H-5 (Dd = +0.18) signals, allowing us to expect that the presence of this co solvent lowers the equilibrium constant for the complexation with b-CD and hence, the guest is merely able to induce the small chemical shift of each proton signals for the host. Significant chemical shifts changes Dd was observed with equimolar mixer of guest and host molecules. Values of pure HMBA (inclusion complex) were as follows: OH = 9.26 (9.262), NH2 = 8.512 (8.500), OCH3 = 3.777 (3.773), CH2 = 3.878 (3.869), 2H = 7.223 (7.215), 5H = 6.823 (6.820), 6H = 6.874 (6.867).

5. Conclusions The above study reveals that: (i) CD studies confirm that, with all the four CDs HMBA form 1:1 inclusion complex, (ii) pH studies reveals that the deprotonation reactions in aqueous and CD medium follows the same trend, (iii) absorption and fluorescence spectral data provide OH group of the HMBA molecule was present in the hydrophilic part and CH2NH2 group present in the inner part of the CD nano-cavity, (iv) analysis of the thermodynamic data indicates that the stoichiometry of all of the CD complexes with HMBA are 1:1 and that formation of the inclusion complexes is driven by both enthalpy process and (v) theoretical studies suggest that hydrophobic interaction plays an important role in determining the stability of the inclusion complexes and that the HMBA into the cavity of the CDs is favored from the wide side rather than the narrow side.

T. Sivasankar et al. / Journal of Molecular Structure 1028 (2012) 57–67

Acknowledgements This work is supported by the CSIR (No. 01(2549)/12/EMR-II), UGC (No. F-351-98/2011 (SR) and DST, New Delhi (No. SR/FTP/ CS-14/2005). The author A. Antony Muthu Prabhu is thankful to CSIR, New Delhi for the award of Senior Research fellowship (ACK. No. 123029/2K9/1). The authors thank to Dr. P. Ramamurthy, Director, National centre for ultrafast processes, Madras University for allowing the fluorescence lifetime measurements available for this work.

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molstruc.2012. 06.025.

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