Investigation of the Complexation of Pyrene and Naphthalene with Hydroxypropyl-/%Cyclodextrin

J O N A T H A N B. Z U N G , T H I L I V H A L I T. N D O U , and I S I A H M. W A R N E R * Department of Chemistry, Emory University, Atlanta, Georgia 30322

The complexation of the now commercially available chemically modi- fied cyclodextrin hydroxypropyl-O-cyclodextrin with pyrene and naph- thalene is compared against the unmodified O-cyciodextrin with the use of fluorescence measurements. A pronounced difference in the complex- ation properties of the two cyclodextrins is observed for the interaction with both pyrene and naphthalene.

Index Headings: Inclusion complex; Hydroxypropyl-8-cyclodextrin; Py- rene; Naphthalene; Fluorescence enhancement.

INTRODUCTION

The inherent low aqueous solubility (1.80% w/v at 25°C) of beta-cyclodextrin (~-CD) can somewhat limit its practical utility. 1 To overcome the poor solubility of ~-CD, workers have synthesized numerous chemically modified CDs. 2-~ The two derivatives which have received most attention are heptakis (2,6-di-O-methyl)- and hep- takis (2,3,6-tri-O-methyl)-~-CD. Recently, however, much effort has been placed in designing nontoxic and rela- tively safe chemically modified CDs which provide in- creased aqueous solubility while retaining the inclusion complexing ability of the unmodified B-CD. In particular, great interest has been shown in hydroxypropyl-~-CD (HP-CD) due to its apparent high aqueous solubility (in excess of 50 % w/v) and favorable toxicity data, as com- pared with those from the methyl substituted deriva- tives. 4,5 The utility of these new substituted CDs is es- pecially important for improvement of pharmaceutical properties such as bioavailability, chemical stability, and solubility.

Although these chemically modifed CDs offer en- hanced aqueous solubility, they appear to have com- plexing properties different from those of the unmodified CDs. Using solubility measurements, Mueller and Brauns demonstrated that the average degree of substitution of the modified CD can affect the formation of the complex. 5 Their work suggested that, in most cases, CD derivatives with a lower degree of substitution are better solubilizing agents than the more substituted derivatives. The effect of the degree of substitution becomes more apparent when Corey-Pauling-Koltun (CPK) models, are exam- ined. From the space-filling models it is seen that as bulky groups (e.g., propyl and ethyl) are substituted for the hydroxyl groups on the CD, steric blocking of the cavity can result, which ultimately prevents some guests from entering the cavity. Thus, complexation between the guest and CD may or may not occur, depending upon the size of the guest and the size of the CD cavity. Ul- timately, this can prevent guests which normally form

Received 21 May 1990. * Author to whom correspondence should be sent.

complexes with the unmodified ~-CD from forming com- plexes with the modified ~-CD.

In this paper, we report on the complexing properties of now commercially available HP-CD as compared with unmodified ~-CD using steady-state fluorescent mea- surements. Fluorescence measurements are well suited for studying inclusion complexes due to the often-ob- served intensity enhancement phenomena2 Pyrene and naphthalene were used as probe molecules to investigate the complexing ability of the unmodified and modified ~-CD. In particular, these two guests were chosen in order to examine the inclusion complexes between a molecule such as naphthalene, which should readily fit into both the unmodified and modified CD cavity, and a molecule such as pyrene, which may be too large to enter the cavity of the modified CD. In addition, both molecules have been shown to demonstrate an intensity enhancement when complexed with ~-CD. 6-9 In addition to fluores- cence measurements, CPK models are used to help in- terpret the fluorescence data and demonstrate the effect of the substituted groups on the CD.

EXPERIMENTAL

All CDs used in this work were obtained from Amer- ican Maize Products (Hammond, IN). The ~-CD was recrystallized three times from water and the HP-CD was used as received. The molecular weight and degree of substitution (D.S.) of HP-CD were determined by use of negative-ion Fast Atom Bombardment Mass Spec- trometry (Fig. 1). The degree of substitution was cal- culated according to the formula D.S. = Z (peak height × S)/Z(peak height), where S = the number of substit- uents. The HP-CD shows a relatively symmetric peak distribution around the average molecular weight of the main product (approximately 1542) having a D.S. of ap- proximately 7. Unlike the unmodified/~-CD, which con- tains just one product, the modified CDs contain nu- merous homologs and isomers of the products that are symmetrically distributed around the average molecular weight.

Pyrene and naphthalene were used as received (Al- drich 99 + % purity). Potassium iodide was reagent grade and used as is (Merck). Stock solutions of pyrene were prepared in cyclohexane, and solutions of naphthalene in absolute ethanol. Aqueous pyrene solutions were pre- pared by pipetting a stock solution of pyrene into a 25- mL flask. The cyclohexane was then evaporated with the use of dry nitrogen and the flask was diluted with deion- ized water to give a 0.19 ~M solution. Naphthalene so- lutions were prepared by diluting the stock solution in deionized water to give a 1 ~M solution in 1% ethanol. Weighed amounts of solid CD were added to the re-

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9

I li~ Ill III i,h hlh ~, il I I i '1400 14S0 1500 "1550 1600 1650 1700 1750

MASS

FIG. 1. Negative-ion FAB mass spectrum of hydroxypropyl fl-CD in thioglycerol matrix.

spective solutions, and the resulting mixtures were me- chanically shaken for 2 h and allowed to equilibrate for 18 h before analysis. Steady-state fluorescence measure- ments were then acquired on a modified Perkin-Elmer 650-10S fluorometer. ~° The excitation wavelenghts used for pyrene and naphthalene were 337 and 280 nm, re- spectively. Excitation and emission bandwidths of 5 and 1.5 nm, respectively, were used. All measurements were acquired at (21 ± 0.1)°C.

RESULTS AND DISCUSSION

Pyrene is an especially suitable fluorescent probe since its emission spectrum contains fiv e vibronic bands which dramatically change in the presence of different solvents. The intensity ratio of the first and third bands (I/III) is well established as a diagnostic tool for the assessment of the molecular environment of the fluorophore. ~,~] For instance, in a polar solvent such as water, pyrene has a I/III ratio of approximately 1.89, while in a nonpolar solvent such as cyclohexane the ratio decreases to 0.60. Thomas and co-workers have used the I/III ratio to in- vestigate the environment inside the CD cavity, which they correlated with the hydrophobicity of the solvent. 7,s

Figure 2 shows the uncorrected fluorescence emission spectrum for pyrene in water and in ~-CD. It is clear that, upon addition of ~-CD, band III increases relative to band I, giving a smaller I/III ratio. For a I m M ~-CD solution, the ratio decreases from 1.89 in pure water to 1.35, indicating a change in the microenvironment or, more specifically, the formation of an inclusion complex. The I/III ratio continues to decrease as the concentration of the ~-CD is increased (Fig. 3). The effect is attributed to the transfer of pyrene from the hydrophilic aqueous environment into the nonpolar CD cavity. The band in- tensity ratio reaches a constant level at approximately 5.5 m M and at 10 m M has a value of 0.60, suggesting a substantially more nonpolar environment surrounding the pyrene than in just water. However, when HP-CD is added to the pyrene solution the vibronic band ratio is only slightly altered, as seen in Figs. 2 and 3. Figure 3 shows that, upon addition of HP-CD, the I/III ratio de- creases slightly and that, as the concentration of HP-CD is increased, little effect is seen on the I/III ratio. This slight change in the ratio of vibronic bands indicates that

z

360 I I ! I

380 400 420 440 WAVELENGTH (nm)

FIG. 2. Fluorescence spectra of 0.19 ~M pyrene in water ( CD ( - - - ) , and ~-CD ( ).

), HP-

the microenvironment around the pyrene is only slightly altered and suggests that not as much of the pyrene is getting into the substituted CD cavity as in the unmod- ified ~-CD. This result also suggests that the propyl groups on the CD play an important role in the formation of the inclusion complex. It would appear that the propyl groups are blocking the cavity, thus restricting the py- rene from fully entering the CD. Alternatively, the propyl groups could change the normal CD conformation, mak- ing interaction with pyrene less favorable. Our former hypothesis is more likely since space-filling CPK models suggest that only part of the pyrene molecule actually fits into the unmodified ~-CD cavity. Considering that ~-CD has an approximate internal cavity diameter of 0.78 nm and depth of 0.78 nm while pyrene has an approxi- mate width of 0.82 nm and length of 1.04 nm, only part of the pyrene actually enters the cavity. However, in the case of HP-CD the models suggest that the bulky propyl groups significantly hinder the pyrene from getting into the cavity since the bulky propyl groups effectively re- duce the entrance diameter.

To further investigate the complexing ability of HP- CD with pyrene, we made quenching measurements us- ing KI. We have previously shown that B-CD can effec- tively protect pyrene from a strong quencher such as KI. 12 Under normal conditions, the KI would signifi-

2.0"

1.6

1.2'

0.8'

0.4 4 8 12

HP-CD B-CD

CD CONCENTRATION (mM)

Fro. 3. Changes in the I/III ratio of 0.19 ~M pyrene in the presence of increasing concentrations of ~-CD ( , ) and HP-CD (B).

1 4 9 2 V o l u m e 44 , N u m b e r 9, 1 9 9 0

Z

fig

I I I I

360 380 400 4.20 44,0 WAVELENGTH ( n m )

Fro. 4. Fluorescence spectra of 0.19 #M pyrene in water ( ), 100 mM KI ( - - - ) , 100 mM KI + 5 mM ~-CD ( - - - ) , and 100 uM KI + 15 mM HP-CD ( ).

cantly quench the pyrene fluorescence; however, upon complexation with ~-CD the pyrene is protected from the KI. Figure 4 shows how the pyrene fluorescence is effectively quenched upon addition of 100 mM KI. Upon addition of 5 mM ~-CD to the pyrene-KI solution, the pyrene fluorescence intensity is increased above the ini- tial fluorescence and the I/III ratio dramatically changes, indicating that the pyrene is being protected by the ~-CD. When HP-CD is added to the pyrene-KI solution, only a slight change in the I/III ratio is observed, along with a small enhancement in the fluorescence intensity. In this case, the pyrene is more accessible to quenching from the KI. The quenching data suggest that the HP-CD does not offer as much protection to the pyrene as does the unmodified/~-CD. Partial blocking of the cavity by the propyl groups offers a better explanation of the ob- served fluorescence behavior with pyrene. This apparent blocking is in agreement with the prediction made with the CPK models for large guest molecules.

For smaller guest molecules such as naphthalene, size considerations for the guest are not as crucial in deter- mining whether the complex will or will not form, due to the small size of the guest relative to the CD. Figure 5 shows the fluorescence spectra for naphthalene in water and in the presence of equal amounts (1.1 w/v % ) of ~-CD and HP-CD. It is seen from the spectra that naphthalene forms a complex with ~-CD. This is evident by the en- hancement in fluorescence when naphthalene is in the presence of B-CD. In the presence of HP-CD, naphtha- lene, unlike pyrene, shows a greater fluorescence en- hancement as compared to fl-CD. Although there is ac- tually a higher concentration of ~-CD (10 mM) than HP-CD (7 mM), the HP-CD shows a larger enhancement in fluorescence. This greater enhancement with the HP-CD can be a t t r ibuted to the better protection that is achieved in the HP-CD cavity. It is also indicative of relatively strong interactions between naphthalene and HP-CD. In contrast, the results for pyrene imply weak interactions with HP-CD, suggesting that pyrene is par- tially in the cavity. In addition, the CPK models suggest that naphthalene should be able to completely fit into both the ~-CD and HP-CD due to its small size (width of 0.68 nm and length of 0.84 nm) relative to the CD.

310 323 335 348 360 WAVELENGTH (nm)

), FIG. 5. Fluorescence spectra of 1 #M naphthalene in water ( 1.1% w/v/~-CD ( - - - ) , and 1.1% w/v HP-CD ( ).

CONCLUSION

The present work suggests that, although the chemi- cally modified HP-CD offers enhanced solubility, it ap- pears to have significantly different complexing prop- erties than unmodified ~-CD. The most noticeable difference is that the substituted CD can prevent a bulky guest from completely entering the CD cavity, while of- feting better protection to smaller guests. Molecules which normally form complexes with ~-CD may or may not form complexes with the modified cyclodextrins, de- pending upon the size of the guest and the degree of substitution of the CD. Further work is currently un- derway with the use of computer modeling to better un- derstand the role of the substituted groups on the CD in the inclusion process and to examine the effect of the degree of substitution. In addition, individually isolated isomers from the HP-CD mixture are being examined in order to better evaluate their individual properties.

ACKNOWLEDGMENTS

This work was supported in part by grants from the National Science Foundation (CHE-8609372), the National Institutes of Health (GM 39844), and the Office of Naval Research. I.M.W. is also grateful for support from a Presidential Young Investigator Award (CHE-8351675). Recognition is made of an NIH shared instrument grant ($10-RR- 02478) for use of the VG 70-S mass spectrometer. The authors are also grateful to G. A. Reed of American Maize Products for providing the CDs used in this study.

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