19F-NMR, 1H-NMR, and Fluorescence Studies of Interaction between

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Article 19

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F-, H-NMR, and Fluorescence Studies of Interaction between 5-Fluorouracil and Polyglycerol Dendrimers Hejoo Lee, and Tooru Ooya J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 14 Sep 2012 Downloaded from http://pubs.acs.org on September 17, 2012

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The Journal of Physical Chemistry

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F-, 1H-NMR, and Fluorescence Studies of

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Interaction between 5-Fluorouracil and Polyglycerol

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Dendrimers

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Haejoo Lee† and Tooru Ooya*

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Graduate School of engineering, Kobe University, 1-1, Rokkoudai-cho, Nada-ku, Kobe 657-

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8501, Japan

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†

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Nishi-ku, Fukuoka 819-0395, Japan

Present Address: Department of Chemical Engineering, Kyushu University, 744 Motooka,

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*CORRESPONDING AUTHOR FOOTNOTE

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Dr. Tooru Ooya

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Graduate School of Engineering, Kobe University

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1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.

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TEL/FAX: +81-78-803-6255

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E-mail: [email protected]

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ACS Paragon Plus Environment

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ABSTRACT.

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Polyglycerol dendrimers (PGDs), which exhibit a well-defined structure consisting of only

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glycerol units, were examined as a host molecule of 5-fluorouracil (5-Fu) used as a model anti-

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cancer drug.

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estimate the molecular interaction between PGDs and 5-Fu in a buffer. Results of the NMR

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titrations revealed that PGD of generation 3 (PGD-G3) encapsulated 5-Fu in the buffer, whereas

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PGD-G2 and G1 partially incorporated 5-Fu molecule into the space. Fluorescent spectra of 5-

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Fu in the presence of PGD-G3 indicated that the diketo (lactam) form of 5-Fu changed to the

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enol-keto (lactim) form of 5-Fu, suggesting attraction of the imine proton of 5-Fu by ether

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oxygen of PGD-G3. Therefore, the encapsulation state of 5-Fu in PGDs at molecular level was

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modulated by the well defined branched structure depending on the generation of PGDs.

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F- and 1H-NMR titrations and fluorescence measurements were performed to

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KEYWORDS. Polyglycerol Dendrimers;

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Fluorescence; Encapsulation

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F-NMR; Molecular interaction; 5-Fluorouracil;

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1. INTRODUCTION

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The chemical and biochemical characteristics of dendrimers can be regulated through fine

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control of their molecular architecture and molecular weight.1-6 One of the unique characteristics

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of dendrimers is molecular encapsulation of guest molecules, where the inside region of the

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dendrimer has the ability to act as a site of molecular recognition.7-9 For example, a protected,

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amino acid-modified poly(propyleneimine) (PPI) dendrimer can encapsulate rose bengal due to

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steric effects of the dendritic periphery.10 PPI dendrimers can also encapsulate anionic dyes via

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electrostatic interactions in aqueous media, which is confirmed by UV/Vis titrations.11,12

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Poly(amidoamine) (PAMAM) dendrimer and its derivatives have also been studied for

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molecular encapsulation of 8-anilino-1-naphthalenesulfonic acid,13,14 based on electrostatic

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interactions in aqueous media. PAMAM dendrimers have been confirmed as drug carriers.15,16

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As PAMAM dendrimers contain surface primary amine and internal tertiary amine groups, these

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groups can be protonated below the pKa under aqueous conditions, which becomes a driving

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force to form complexes with oppositely charged drug molecules, including 5-fluorouacil (5-

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Fu).17-19 However, PAMAM dendrimers are toxic due to their high cationic charge density and

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this has limited their application in vitro and in vivo.20

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Although chemical modification of the peripheral amine group using poly(ethylene glycol)

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(PEG) is thought to be a good approach to overcome toxicity issues,15 nitrogen inclusion in the

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dendrimers is thought to be necessary. Recently, polyglycerol dendrimers (PGDs) and

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hyperbranched polyglycerols (HyPGs) have therefore been the focus of various biomedical

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applications.21 As PGDs and HyPGs do not contain nitrogen atoms and bear numerous hydroxyl

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groups on their peripheries, it is apparently possible to possess hydrophilic properties without

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containing nitrogen atoms and to exhibit good biocompatibility22-24 and hydrotropy25, similarly to


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PEG. In addition, we found that PGD of generation 2-4 (PGD-G2, G3 and G4) takes up

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fluorescent molecules into the nano-spaces, which was clarified by isothermal titration

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calorimetry and

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nanostructure with very narrow molecular weight distribution. Thus, PGDs may be good

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candidates as biocompatible and well-defined drug carriers capable of precisely defined analysis,

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as reported in the NMR analysis of PAMAM dendrimer-drug complexes.28-33

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H-NMR titration.26,27 Unlike HyPGs, PGDs exhibit a well-defined

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In this study, molecular interaction between 5-Fu (Figure 1(a)) and PGDs of generation 3, 2

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and 1 (Figure 1 (b-d)) was examined by 19F-, 1H-NMR titrations and fluorescence measurements.

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19

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of 5-Fu by observing the chemical shift changes. As 5-Fu has been previously studied as a

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photosensitizer capable of fluorescent change via tautomeric formation,34 fluorescence

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measurements in the presence of various concentration of PGDs were preformed to discuss the

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interaction-induced tautomeric change that was correlated with the data of the 19F- and 1H-NMR

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titrations.

F-NMR technique was employed to discuss how the generation of PGDs affects the interaction

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(Insert Figure 1)

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2. MATERIALS AND METHODS

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2.1 Materials

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5-Fu was purchased from Nacalai Tesque (Kyoto, Japan). Glycerol was purchased from Kanto

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Chemical Co., Inc. (Tokyo, Japan). PGD-G1, G2 and G3 were prepared according to the method

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of Haag et al.35 Deuterium oxide and acetic acid-d4 were purchased from Cambridge Isotope

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Laboratories, Inc. (Andover, MA). Sodium deuteroxide was purchased from Merck Chemicals

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(Darmstadt, Germany). Potassium fluoride was purchased from Wako Pure Chemical Industries

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(Osaka, Japan) and was used without further purification.


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2.2 1H- and 19F-NMR titration of 5-Fu with PGDs

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5-Fu was dissolved in a 10 mM acetate buffer prepared with D2O, and pD was adjusted to 5.0

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by adding NaOD and acetic acid-d4 (concentration of 5-Fu: 12.5 mM). Each PGD (PGD-G3, G2

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or G1) dissolved in buffer was added to 5-Fu solution to give concentrations of 2.5, 5.0 and 10.0

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mM (final concentration of 5-Fu: 2.5 mM). 1H-NMR and 19F-NMR spectra of each solution were

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obtained using a 500 MHz FT-NMR (Bruker Advanced 500, Bruker BioSpin K.K., Yokohama,

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Japan) and a 400 MHz FT-NMR apparatus (Varian Unity Inova-400, Agilent Technologies, Co.

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Ltd., Tokyo, Japan), respectively.

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2.3 Fluorescence measurements of 5-Fu in the presence of various concentrations of PGDs

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A stock solution of 5-Fu was prepared by dissolving 5-Fu in a 10 mM acetate buffer (pH 5) to

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be 12.5 mM. Separately, PGD-G3, G2 and G1 (6, 12.5, 25, 50 mmol) were dissolved in 800 µL

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of the same buffer. 5-Fu stock solution (200 µL) was added to each PGD solution (final

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concentrations: 5-Fu, 2.5 mM; PGD, 1.25, 2.5, 5.0 or 10.0 mM). The mixture of 5-Fu and PGDs

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was left to stand for 30 min at room temperature in a quartz cell, and then the emission spectra of

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the solution were measured (excitation wavelength: 267 nm) at room temperature using a

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spectrofluorometer (F-2500, 45 Hitachi, Ltd., Tokyo, Japan) with a 20-nm slit width.

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3. RESULTS AND DISCUSSION

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Our previous studies have suggested that higher generation PGDs have the potential to

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incorporate a guest molecule inside the PGDs.26 Especially, PGD-G3 and the guest molecule

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interacted attractively, showing a binding constant of 5.3 x 105 (M-1).27 Thus, PGD-G3 was


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subjected to analysis of its interaction with 5-Fu. Figure 2 shows the

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titrated with PGD-G3. The 19F chemical shift of 5-Fu itself was observed at 40.0 ppm, as a sharp

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doublet that means coupling with neighbor proton (Figure 2 (a)). When the concentration of

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PGD-G3 was the same as that of 5-Fu (2.5 mM), the 19F signal shifted downfield maintaining the

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same doublet peak (PGD-G3:5-Fu = 1:1, Figure 2(b)). The

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deshielding effect, hence, electron density around the 19F atom decreased, which might be related

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to deprotonation of the imino proton (i.e. N-H proton) by a proton acceptor.36 At a 1:1 molar

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ratio of 5-Fu and PGD-G3, the number of ether oxygen, which could act as a proton acceptor, is

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twenty-one; thus the densely packed ether oxygen of the PGD-G3 possibly formed hydrogen

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bonds with the imino proton of 5-Fu. When the ratio increased to 2:1 and 4:1, the 19F signal was

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difficult to find ((PGD-G3:5-Fu = 2:1 and 4:1, Figure 2(c) and (d)). The 5-Fu molecule is likely

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to be buried within the interior regions of PGD-G3, as the 19F signal was no longer detected, as

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seen in the case of encapsulated drugs in polymeric micelles.37 Thus, when the concentration of

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PGD-G3 was not less than 5.0 mM, 2.5 mM of free 5-Fu molecules are likely to be preferred to

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locate in the dendritic interiors. Since PGD-G3 molecules associates in aqueous solution with an

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average diameter of around 83 nm at a concentration of 0.2 mM,27 the PGD-G3 molecules at the

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concentration 5.0 mM also possibly make an association, in which 5-Fu molecues might be

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incorporated.

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F-NMR spectra of 5-Fu

F signal shift denotes enhanced

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(Insert Figure 2)

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H-NMR spectra were measured under the same conditions as 19F-NMR measurements. The Ha

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signal of 5-Fu itself appeared as a broad signal at around 7.55 ppm (Figure 3 (a)). Generally,

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peak broadening of 1H-NMR signals contains the meaning of slower mobility of protons than


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NMR time scale, which is correlated with increased viscosity, fast exchange of proton bound to

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hetero atoms such as –OH and –NH2, various conformations of hydrogen bonds, etc. One

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possible cause of this broad signal is the formation of the 5-Fu dimer, the type of which contains

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both a hydrogen-bonded conformations with both rings lying in the plane and a stacked structure

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that is constructed by strong hydrogen bonds with four water molecules.38 According to the

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results in the literature of molecular dynamics simulation for clustering made up of 5-Fu dimer

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embedded in water, several dimer conformations showing relevant structural and energetic data

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were suggested; thus, one can imagine that multiple Ha signals of 5-Fu on the NMR spectrum

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exist due to the various dimetrization in D2O leading to the broaden Ha signals. At a 1:1 molar

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ratio of PGD-G3 and 5-Fu, the Ha signals of 5-Fu became sharp. This is likely due to the

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dissociation of 5-Fu dimers, where PGD-G3 molecule might act as a water–disruptor to break the

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hydrogen-bond cluster of 5-Fu and water molecules. In addition, 1H signals attributed to PGD-G3

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became sharp (Figure 3 (b)) in comparison with the peaks of PGD-G3 in the absence of 5-Fu

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(Figure 3 (e)), suggesting that the molecular motion of PGD-G3 in the buffer was restricted by

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interaction with 5-Fu.

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When increasing the molar ratio from 2:1 to 4:1, the Ha signal of 5-Fu was deshielded (Figure

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3 (c, d)). In addition, 1H signals at around 4.0 ppm (CH2 protons adjacent to the terminal OH

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groups of PGD-G3: A in Figure 3) and 3.1 ppm (CH2 protons adjacent to the quaternary carbon

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in the core region of PGD-G3: B in Figure 3) became broad. The peak broadening of the

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position at A and B, accompanied with downfield shift of the Ha signal of 5-Fu, suggest hydrogen

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bonds between the Ha and ether oxygen adjacent to the CH2 at A and B region of PGD-G3.

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Taking the results of

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encapsulated within the internal region of PGD-G3 at over two molar amounts of PGD-G3

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against 5-Fu.

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F-NMR titration (Figure 2) into account, the 5-Fu molecule was


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(Insert Figure 3)

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The encapsulation of 5-Fu within the interior region of PGD-G3 was examined by fluorescence 19

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measurements under the same conditions as NMR measurements. Based on the results of

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and 1H-NMR titrations, we hypothesized that fluorescence spectra would also provide insight

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into the location of 5-Fu within the interior region of PGD-G3. The fluorescence spectrum of 5-

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Fu (i.e. concentration of PGD-G3 was 0 mM) showed excitation at 267 nm and exhibited bands

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between 300 and 600 nm (Figure 4). With increasing concentration of PGD-G3, the intensity

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around 342 and 462 nm increased markedly. The alpha-hydrogen on the imine (N3) of 5-Fu is a

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relatively acidic hydrogen (pKa = 7.26),39 so that the imine proton could be deprotonated in the

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presence of proton acceptors. If the ether oxygen of the PGD-G3 acts as the proton acceptors, the

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proton can be transferred to the carbonyl oxygen; the π-conjugated system of 5-Fu is extended by

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changing the diketo (lactam) form to the enol-keto (lactim) forms (Figure 5). This is consistent

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with previous report.40

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increased intensity at 462 nm suggests the encapsulation of 5-Fu within the interior region of

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PGD-G3, accompanied by the tautomeric change from the lactam to the lactim. The generation

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dependency of PGDs for the encapsulation of 5-Fu was demonstrated by the fluorescence change.

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The same experiments using PGD-G1 and G2 were performed by the fluorescence measurements,

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and the increased intensities at 462 nm were observed (data not shown). Figure 6 shows the

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fluorescence intensity change in 5-Fu at 462 nm (Ex: 267 nm) with increasing concentration

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PGD-G1, G2, and G3 in the acetate buffer. The intensity increase for PGD-G1 and G2 with

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increasing concentration was significantly smaller than with PGD-G3, indicating that the ability

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of attracting the imine proton within the interior of PGDs with low generations was poor. The

Taking the results in

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F-

F- and 1H-NMR titration into account, the


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binding constants were calculated using curve fitting software (OriginPro 8J) from the

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concentration-dependent fluorescence intensity change, according to the following equation, ∆I 11 2 K [G ]0

 2 2 1 + K [G ]0 + K [H ]0 − (1 + K [G ]0 + K [H ]0 ) − 4 K [G ]0 [H ]0 

{

}  1 2

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∆I = I − I 0 =

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where ∆I is the change of fluorescence intensity at 462 nm, I is the fluorescence intensity at

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462 nm in the presence of the host molecules (PGDs), I0 is the fluorescence intensity at 462 nm

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in the absence of the guests, ∆I11 is a difference in the fluorescence intensity at 462 nm between a

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host-guest complex formed at 1:1 molar ratio and the free host, K is a binding constant, [G]0 and

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[H]0 are initial concentrations of the guest (AHSA) and the host molecules. The obtained

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binding constants for PGD-G1, G2 and G3 were 7.7×103, 2.2×104, and 4.0×103 M-1, respectively.

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These results indicate that the generation dependency of PGDs for the equilibrium interaction

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with 5-Fu was small, but the magnitude for the transformation from the lactam to the lactim was

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in the order of PGD-G3 > G1 = G2.



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(Insert Figures 4, 5 and 6)

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As in the case of PGD-G3, 19F-NMR titration of 5-Fu against PGD-G2 and G1 were performed

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under the same conditions. With increasing concentration of PGD-G2 and G1, 19F signal of 5-Fu

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shifted downfield, and the signal broadening seen in the case of PGD-G3 (Figure 2) was not

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observed, even when the molar ratio of PGDs and 5-Fu was over 2:1 (Figure 7 (b)-(d), Figure 8

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(b)-(d)). These results suggest that 5-Fu actually interacted with PGD-G2 and G1, but was not

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fully incorporated into the interior part of PGD-G2 and G1. The results of 1H-NMR titration

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between 5-Fu and PGD-G2 or G1 supported partial incorporation, because the broadening of 1H


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signals attributed to both the branch and core region of PGD-G2 and G1 was not observed (see

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SI; Figures. S1 and S2).

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(insert Figures 7 and 8)

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4. CONCLUSIONS

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The polyglycerol dendrimer of generation 3 (PGD-G3) was able to encapsulate 5-Fu under

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aqueous conditions, as demonstrated by 19F- and 1H-NMR titration. The fluorescence spectra of

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5-Fu in the presence of PGD-G3 revealed that the π-conjugated system of 5-Fu was extended by

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attraction between the imine proton of 5-Fu and the ether oxygen in PGD-G3. Although such

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attraction was suggested by 19F-NMR and fluorescence measurements in the presence of PGDs

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with lower generation (PGD-G2 and G1), broadening of the 19F-NMR signals, as seen in the case

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of PGD-G3, was not observed. The binding constants for PGD-G3, G2 and G1 were calculated

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by the fluorescent intensity change at 462 nm and found to be 103-104 M-1 order. Therefore, the

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increased branch number of glycerol units contributed to the encapsulation of 5-Fu, where the

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ether oxygen plays an important role in the attraction of the imine proton of 5-Fu. When the

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generation of PGDs increased to four (PGD-G4), polarity of the PGD-G4-dissolved solution

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decreased, and hydrophobic interaction with a guest molecule might be included as the driving

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forces27; thus; we can predict that PGDs with much higher generations encapsulates 5-Fu by not

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only the hydrogen bonding but also hydrophobic interaction. In fact, a PGD of generation 5

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(PGD-G5) could dissolve a poorly water soluble drug, which is possibly related to hydrophobic

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interaction25. Encapsulation of 5-Fu and structurally resembled drugs such as pyrimidine analogs

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(e.g; cytarabine and gemcitabine) and the other metabolic antagonists (e.g.; mercaptopurine and

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pentostatin) in aqueous solution using biocompatible PGDs is expected to lead to their


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development as nano-sized drug carriers with well-defined size and structure, which are believed

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to be key factors for drug targeting in vivo.

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ASSOCIATED CONTENT

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Supporting Information Available. The 1H-NMR spectra upon titration between 5-Fu and

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PGD-G2 or G1 (Figures. S1 and S2). This material is available free of charge via the Internet at

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http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author

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* Tel.: +81 78 803 6025; Fax: +81 78 803 6025, E-mail address: [email protected]

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Present Address

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†Department of Chemical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka

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819-0395, Japan

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Funding Sources

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This work was partially supported by a Grant-in-Aid for Scientific Research B (No. 22300165)

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and for challenging Exploratory Research (No. 24650279) from JSPS.

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ACKNOWLEDGMENTS

We thank Prof. Toshifumi Takeuchi, Kobe University (Japan) for the fruitful discussions.


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2010, 392, 170-177. (38) Alagona, G.; Ghio, C.; Monti, S. International Journal of Quantum Chemistry, 2002, 88, 133–146. (39) Jang, Y. H.; Sowers, L. C.; Çagin, T.; Goddard, W. A. The Journal of Physical Chemistry A 2001, 105, 274-280. (40) Markova, N.; Enchev, V.; Timtcheva, I. The Journal of Physical Chemistry A 2005, 109, 1981-1988.

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FIGURE CAPTIONS

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Figure 1. Chemical structure of (a) 5-fluorouracil (5-Fu), (b) PGD-G3, (c) PGD-G2, and (d)

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PGD-G1

314

Figure 2. 19F-NMR titration of 5-Fu with PGD-G3. PGD-G3:5-Fu = (a) 0:1, (b) 1:1, (c) 2:1, and

315

(d) 4:1. Solvent: 10 mM acetate buffer (pD 5.0). Concentration of 5-Fu was 2.5 mM.

316

Figure 3. 1H-NMR titration of 5-Fu with PGD-G3. PGD-G3:5-Fu = (a) 0:1, (b) 1:1, (c) 2:1, (d)

317

4:1, and (e) 1:0. Solvent: 10 mM acetate buffer (pD 5.0). Concentration of 5-Fu in (a)-(d) was

318

2.5 mM.

319

Figure 4. Fluorescence spectral change (Ex: 267 nm) of 5-Fu (2.5 mM) on the addition of PGD-

320

G3 in 10 mM acetate buffer (pH 5) at room temperature: PGDs concentration = 0, 1.25, 2.5, 5.0

321

mM.

322

Figure 5. Proposed image of the extended π-conjugated system of 5-Fu by changing the diketo

323

(lactam) to the enol-keto (lactim) form.

324

Figure 6. Fluorescence intensity change of 5-Fu (2.5 mM) at 462 nm (Ex: 267 nm) with

325

increasing the concentration of PGD-G1, G2, and G3 (each concentration: 0, 1.25, 2.5, 5.0 mM)

326

in the acetate buffer (pH 5). Each plot was the average value of 4 times measurements (n=4).

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328


329


330



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Figure 1.

332

333 334 335 336


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Figure 2.

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339 340


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Figure 3.

342

343 344 345 346


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347

Figure 4.

348 12

5 mM 2.5

10

1.25

Intensity

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8

0

6 4 2 0 300

350

400

450

500

550

600

Wavelength (nm) 349 350


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Figure 5.

352

353

Diketo (lactam)

Enol-keto (lactim)

354 355


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Figure 6.

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358 359


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Figure 7.

361

(d)

(c)

(b)

(a) 362

40.8

40.6

40.4

40.2

40.0

39.8

39.6 PPM

363 364 365


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Figure 8.

367

(d)

(c)

(b)

(a) 368

40.8

40.6

40.4

40.2

40.0

39.8

39.6 PPM

369


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Table of Contents (TOC) Image

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19F-NMR, 1H-NMR, and Fluorescence Studies of Interaction between

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