Accepted Manuscript

Effect of Encapsulation in the Anion Receptor Pocket of Sub-domain IIA ofHuman Serum Albumin on the Modulation of pKa of Warfarin and StructurallySimilar Acidic Guests: A Possible Implication on Biological Activity

Shubhashis Datta, Mintu Halder

PII: S1011-1344(13)00238-8DOI: http://dx.doi.org/10.1016/j.jphotobiol.2013.10.013Reference: JPB 9594

To appear in: Journal of Photochemistry and Photobiology B: Bi-ology

Received Date: 2 August 2013Revised Date: 3 October 2013Accepted Date: 19 October 2013

Please cite this article as: S. Datta, M. Halder, Effect of Encapsulation in the Anion Receptor Pocket of Sub-domainIIA of Human Serum Albumin on the Modulation of pKa of Warfarin and Structurally Similar Acidic Guests: APossible Implication on Biological Activity, Journal of Photochemistry and Photobiology B: Biology (2013), doi:http://dx.doi.org/10.1016/j.jphotobiol.2013.10.013

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1

Effect of Encapsulation in the Anion Receptor Pocket of 1

Sub-domain IIA of Human Serum Albumin on the 2

Modulation of pKa of Warfarin and Structurally Similar 3

Acidic Guests: A Possible Implication on Biological 4

Activity 5

Shubhashis Datta, and Mintu Halder* 6

Department of Chemistry, Indian Institute of Technology Kharagpur 7

Kharagpur-721302, India 8

*Corresponding author: mintu@chem.iitkgp.ernet.in 9

Phone: +91-3222-283314, FAX: +91-3222-282252 10

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Abstract 22

Supramolecular and bio-supramolecular host assisted pKa shift of biologically relevant 23

acidic guests, warfarin and coumarin 343, has been monitored using both steady-state and 24

time resolved fluorescence spectroscopy. The anion receptors present in sub-domain IIA 25

of human serum albumin (HSA) stabilize the anionic form of the guest and thereby shift 26

pKa towards acidic range. On the other hand, the preferential binding of the neutral form 27

of guests in the non-polar hydrophobic cavity of β-cyclodextrin results in up-shifted pKa. 28

This shifting of pKa of drugs like warfarin, etc., whose therapeutic activity depends on 29

the position of the acid-base equilibrium in human system, is of great importance in 30

pharmacokinetics. The release of the active form of such drugs from macrocyclic carrier 31

and subsequent distribution through the carrier protein should depend on the modulation 32

of the overall pKa window brought about by the encapsulation in these hosts. Present 33

work also suggests that properly optimized encapsulation in appropriate receptor pocket 34

can enhance the bioavailability of drugs. This work also opens up the possibility to use 35

HSA as encapsulator, instead of traditional cyclodextrins or other polymeric hosts, since 36

such systems may overcome toxicity as well as biocompatibility issues. 37

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Keywords: host-guest interaction; acid-base equilibrium; bio-supramolecular host; 46

cationic side-chain; hydrophobic cavity; drug delivery. 47

48

49

3

Introduction 50

Modulation of physicochemical properties of guest, encapsulated in 51

supramolecular host cavity, has opened up its enormous uses in the areas of 52

supramolecular catalysis [1], chemosensing [2], photo-switchable devices [3], drug 53

delivery [4, 5], and so on. Dsouza et al. [6] have summarized the changes in 54

physicochemical properties of fluorescent dyes due to their supramolecular complexation 55

with macrocycles in aqueous media. The role of non-covalent interactions such as 56

electrostatic (ion-ion, ion-dipole, dipole-dipole, dipole-induced dipole and higher order 57

interactions), hydrogen bonding, -stacking, CH- interactions, and hydrophobic effect 58

between host-guest pairs have been reported to tune the physicochemical properties of 59

guests [7, 8]. Among various physicochemical properties, modulation of acid-base 60

equilibrium (pKa shift) of the guest is of great relevance. Various research groups have 61

reported and applied host assisted pKa shift in different contexts [9-11]. Pischel et al. and 62

Nau et al. applied pKa shift for the designing of supramolecular logic by complexation 63

with cucurbit-7-uril (CB7) as a supramolecular input [12]. Pal et al. also have applied 64

pKa shift for the designing of logic gates using the same macrocyclic hosts [13]. More 65

interestingly, Pluth et al. have shown that the possibility of acid catalysis in some 66

supramolecular host under alkaline conditions is due to its ability to protonate the 67

incoming guest [14]. Such shift in pKa of biologically important guest molecules are also 68

relevant in pharmacokinetics and drug delivery [15, 16]. Saleh et al. have shown that the 69

activity of proton-pump inhibitor drug, lansoprazole, can be improved by increasing the 70

basicity of the benzimidazole group by encapsulating with CB7 [15] and thereby 71

reducing the production of gastric acid. Sanguinarine, an anticancer, antimicrobial and 72

antifungal drug has two different forms namely, iminium and alkanolamine. The 73

alkanolamine form predominates in basic pH. The active form of the drug, the iminium 74

form, predominates in acidic pH. Miskolczy et al. have shown that the pH window of the 75

active form of this drug can be extended by encapsulating with CB7 and this 76

encapsulation also improves the resistance towards photooxidation [17]. The solubility is 77

another important factor in drug delivery because the drug should be soluble in 78

physiological intestinal fluids to perform its pharmacological activity. Day et al. and 79

4

Koner et al. have shown that the encapsulation of drug molecules of benzimidazole 80

family within CB7 cavity increases their pKa values and thus stabilizing the water soluble 81

ionic form [18]. 82

A recent review by Ghosh et al. have extensively summarized the use of 83

supramolecular pKa shift to enhance the bioavailabilty of drugs [19]. This review briefly 84

describes the pKa shift of basic guests (drugs) containing amine group or nitrogen 85

containing heterocycles and reports their bioavailability. Recently, Mohanty et al. have 86

reported that guest molecules can be actively released from their complexes with CB7 87

(having cation receptor) in presence of inorganic cations and relocate to hydrophobic 88

pocket of bovine serum albumin (BSA) [20]. Pischel et al. have shown that some guests 89

can be released from CB7 cavity by a phototriggered pH jump [21]. Although the above 90

two release mechanisms look to be fair enough, but Ghosh et al. have doubted in their 91

review [19] about the applicability of these release mechanisms to real biological systems 92

(in vivo) and commented that these mechanisms may not be straightforward particularly 93

due to their natural buffering action and pH jumps which are already observed in the 94

living cells. Now most of the drugs are found to reversibly bind with plasma proteins, 95

e.g., albumins, because these are the major protein components of blood plasma [22]. 96

Human serum albumin (HSA) is the major component present in the blood plasma of 97

human. Among the several binding sites of HSA, sub-domain IIA is one of the important 98

sites where majority of drugs bind [23]. The extent of binding depends on the affinity of a 99

particular drug for some binding site(s) and ranges from less than 10% to as high as 99% 100

of the plasma concentration. As the free drug (unbound) diffuses into intestinal fluid and 101

cells, drug molecules dissociate from plasma proteins to maintain the equilibrium 102

between free drug and bound drug. So serum protein has the key role in the distribution 103

of the active form of drug to organs and tissues via circulation. Because of the availability 104

of charge receptors and hydrophobic cavity in the binding pocket, serum proteins may 105

function like macrocyclic host and can shift the pKa of guest in either direction (likewise 106

the case with cyclodextrin, cucurbiturils and sulfonatocalixarene), depending on the 107

nature of the charge receptors and hydrogen bonding groups. Recently Pal et al. have 108

reported negative pKa shift of basic guest neutral red in presence of BSA [20]. Therefore, 109

5

it is highly probable that the active form of the drug complexed initially with macrocycles 110

(like cyclodextrins) may be converted into inactive form due to binding with serum 111

proteins. Also the switching of the encapsulated drug from cyclodextrin cavity to serum 112

protein depends on the magnitude of partition coefficient of the drug between two 113

different host cavities. So before choosing a molecule as drug we should have knowledge 114

about their pKa values within supramolecular and biosupramolecular host and also the 115

relative binding strengths between the hosts. Ghosh et al. have also concluded that the 116

study of pKa of acidic guest like phenols and carboxylic acids in presence of macrocyclic 117

host, especially with negative receptor groups, is really challenging as till now no such 118

report is available in the literature [19]. Therefore, we are curious to explore the possible 119

effect of pocket specific binding on the pKa of prototropic guests with serum proteins as 120

model system. 121

We have chosen structurally similar warfarin (WF) and coumarin 343 (C343) as 122

guests where the former possesses hydroxyl group and the latter possesses carboxylic 123

acid function. Both of them contain standard coumarin moiety. WF has been used as an 124

oral anticoagulant for more than 20 years for the prevention of cardio-vascular diseases 125

[24, 25]. It possesses several structural isomers [26, 27]. Most importantly it has been 126

reported that deprotonation of hydroxyl group of WF is one of the important steps of the 127

reaction related to anticoagulation activity [28]. 128

Coumarin-3-carboxylic acids constitute another important class of compounds for 129

their wide range of applications. These are the relevant intermediates for the synthesis of 130

natural products with diverse biological activities. Further these 3-carboxycoumarins 131

have been used for the synthesis of modified cephaloporins [29], penicillins [30], isoureas 132

[31] and oxygen-bridged tetrahydropyridones [32] with specific inhibition activity of α-133

chymotrypsin and human leukocyte elastase [33]. Recently, coumarin-3-carboxylic acid 134

derivatives have been found to be potent and selective inhibitors to monoamine oxidase 135

and showed marked potency to inhibit cancer cell invasion in vitro and tumor growth in 136

vivo [34]. Their metal complexes have also been found to exhibit good biological 137

properties [35]. The bioavailability of these molecules in human system is dependent on 138

the associated acid-base equilibrium. To explore such equilibrium, which can be relevant 139

6

in blood plasma, we have chosen C343 as a good mimic of these 3-carboxycoumarins. 140

The choice of C343 is perfect in the present context because the change in prototropic 141

equilibrium is sharply reflected through modulation of its photophysical properties [36-142

39] and therefore can be readily monitored spectroscopically. The pKa of C343 is also 143

reported to be shifted in presence of ionic surfactants due to possible coulombic 144

interaction with different prototropic forms of the probe [36]. C343 has also been used in 145

different solvatochromic studies [40], determination of microenvironment of 146

heterogeneous systems [36] and solvent relaxation process [41, 42]. Here we have studied 147

acid-base equilibrium of WF and C343 in presence of HSA. The results are also 148

compared with that in two cyclodextrins namely, β-cyclodextrin (β-CD) and γ- 149

cyclodextrin (γ-CD) to throw light on the behavior of such guest in the 150

biomacromolecular host. 151

152

2. EXPERIMENTAL SECTION 153

2.1. Materials 154

HSA (>98%), hemin (>98%, HPLC), ibuprofen (>98%, GC), γ- cyclodextrin (γ-155

CD) and β-cyclodextrin (β-CD) are purchased from Sigma Aldrich and warfarin 156

(analytical grade) is purchased from TCI chemicals, Japan. Coumarin 343 (laser grade, 157

Exciton) is also used as received. All other chemicals are of analytical reagent (AR) 158

grade and ultra pure water is used throughout the study. 159

160

2.2. Sample Preparation 161

The probe (C343 and WF) solutions are prepared in ethanol. The appropriate 162

volume of probe solution has been taken in a volumetric flask and then ethanol is 163

evaporated, and to this ethanol-evaporated probe the required volume of the chosen 164

buffer solution is added and stirred to get the required concentration. HCl or HClO4 and 165

NaOH solutions are used for pH adjustment. The pH is measured with a pre-calibrated 166

EUTECH pH 510 ion pH-meter. 167

2.3. Instrumentation and methods 168

7

The UV-VIS absorption spectra are recorded by scanning the solution on a 169

Shimadzu UV-2450 absorption spectrophotometer against solvent reference. 170

The steady state fluorescence spectra are recorded on a Hitachi (Model F-7000) 171

spectrofluorimeter equipped with temperature controlled water cooled cuvette holder and 172

quartz cuvette of 1-cm path length is used. The steady-state fluorescence measurements 173

have been performed at 298 K. The excitation and emission slits are kept at 5 nm and 2 174

nm, respectively, in all steady state fluorescence measurements. 175

Fluorescence lifetimes are measured by means of a single photon counting 176

apparatus where the samples are excited at 408 nm using a picosecond laser diode (IBH, 177

Nanoled), and the signals are collected at the magic angle 54.7° using a Hamamatsu 178

microchannel plate photomultiplier tube (R3809U), instrument response is 100 ps. The 179

decay analyses are done with IBH DAS, version 6 software. The perfectness of fitting is 180

judged in terms of a χ2 value and weighted residuals. 181

For measuring time resolved anisotropy [r(t)] (Eq.(1)) the samples have been 182

excited at 408 nm using a picosecond laser diode (IBH, Nanoled). We have used a 183

motorized polarizer in the emission side. The emission intensities at parallel and 184

perpendicular polarizations are collected alternately, until a certain peak difference has 185

been reached between them. The peak differences depend on the tail matching of the 186

parallel and perpendicular components of decays. The analysis of the data has also been 187

done with above mentioned software. 188

……………………… (1) 189

190

Visualization of all the crystal structures is made using the Discovery Studio Visualizer 191

2.5 (Accelrys, Inc., San Diego, CA). 192

193

3. Results and Discussion 194

3.1. Modulation of photophysical properties of WF and C343 in presence of HSA 195

and cyclodextrins 196

In aqueous solution, depending upon pH, two prototropic forms of C343 (scheme 197

1) can exists, namely the neutral and anionic forms, both showing their characteristic 198

( ) . ( )( )

( ) 2. . ( )

I t G I tr t

I t G I t

8

structured absorption bands at 454 and 427 nm, respectively, and characteristic emission 199

peaks at 495 and 490 nm, respectively [36, 40, 43]. The ground state pKa of C343 is 200

reported to be 4.6 [36, 37] in aqueous solution. 201

Scheme-1 202

203 204

WF is a biologically and medicinally important fluorescent molecule. Like C343, 205

in aqueous solution two different forms (scheme 2) can exist for WF, namely neutral and 206

anionic, where the fluorescence quantum yield of the former is low [44] and that of the 207

latter is more [27]. The absorption spectra of WF in acidic pH show maxima at 270, 280 208

and 308 nm [45]. With increase of pH the absorbance at 270 and 280 nm bands decreases 209

and that at 308 nm increases, and in pH above its pKa (the ground state pKa is reported to 210

be 5.1[46, 47]) the former two bands disappear [45]. 211

Scheme 2 212

213 214

Petitpas et al. and Wilting et al. have reported that in physiological pH the 215

binding constant of WF with HSA is reasonably high and also the binding constant is pH 216

dependent especially above pH 8 where neutral conformation of serum albumin (N-form) 217

9

is converted into basic conformation (B-form) [48, 49]. Importantly, the study shows that 218

the anionic form of WF undergoes interaction with HSA [48]. Also till now no literature 219

report is available on the exploration of binding interaction of C343 with HSA. So 220

keeping these facts in mind, we plan to explore the possible effects of biosupramolecular 221

encapsulation on the acid-base equilibrium of these guests, and hence pH is so chosen 222

(pH 3.5 for C343 and 4.0 for WF) that the deprotonation of the neutral form of probe due 223

to interaction with HSA can be monitored. 224

<Fig. 1> 225

<Fig. 2> 226

In presence of HSA the absorption maxima of C343 is shifted towards shorter 227

wavelength by about 27 nm (454 to 427 nm) along with a concomitant decrease in 228

absorbance (Fig. 1). An isosbest at 422 nm indicates prototropic equilibrium in the 229

protein-bound state. Also the emission maxima (Fig. 2) are blue shifted (from 495 to 484 230

nm, in presence of HSA) and the corresponding fluorescence intensity is also quenched. 231

The absorbance of WF at 308 nm is increased (Fig. S1, supporting information) in 232

presence of HSA, but the absorbance at two other wavelengths (270 and 280 nm) could 233

not be monitored because of the overlap of strong absorption centered at 278 nm due to 234

added protein. Also the low fluorescence quantum yield of the neutral form of WF is 235

largely increased in presence of HSA (Fig. S2, supporting information). These results 236

indicate that neutral probe molecule gets converted to anionic species in the presence of 237

HSA. 238

Warfarin is already known to bind in sub-domain IIA of HSA [45, 48]. In order to 239

locate the binding site of C343, we have performed the competitive binding study with 240

HSA in presence of site markers like warfarin, ibuprofen, and hemin. The fluorescence 241

intensity of C343 bound with HSA at pH 7.5 is increased with the addition of warfarin 242

(Fig. S3, supporting information) indicating the displacement of the former by the latter, 243

whereas with ibuprofen and hemin no competition has been observed. Thus the binding 244

site of C343 is in the sub-domain IIA of HSA, which is also known as the warfarin 245

binding site. 246

10

3-Carboxycoumarins and WF are also known to form inclusion complex with 247

cyclodextrins [50-54]. The binding constants of WF with three different cyclodextrins are 248

in the order [53]. The binding constant of WF with β-CD varies in the range 249

99-633 M-1

[46, 52]. Zingone et al. have reported that the interaction of β-CD with WF, 250

measured by phase solubility method, depends on pH of the solutions [54]. The neutral 251

form of WF shows much stronger interaction with β-CD than the anionic form. But all 252

other studies related to inclusion complex of WF and 3-carboxycoumarins with β-CD, 253

using spectroscopic technique, are performed at pH much above pKa of the guest [46, 254

53]. Here we have explored the interaction of WF and C343 with β-CD and γ-CD at two 255

different pH, namely, pH 3.5 and 7.5 which are well below and above of their respective 256

pKa values in water, to look into both the forms individually. 257

In presence of β-CD at pH 3.5 the absorbance of C343 increases with a little red 258

shift in the maxima, and at the same time the fluorescence intensity is found to increase 259

sharply (Fig. S4, supporting information). Because of the limited solubility of β-CD (16 260

mM) [55] in aqueous solutions the fluorescence changes of C343 could only be 261

monitored upto 15 mM of host. Similar measurements at pH 7.5 show no observable 262

changes in absorbance spectra and emission intensity (Fig. S5, supporting information). 263

The plot of change in fluorescence intensity, IF vs [β-CD], (Fig. S6, supporting 264

information) at both the pH shows that β-CD strongly interacts with the neutral form of 265

the fluorophore. In presence of γ-CD, under similar conditions, the absorption maxima of 266

C343 is little blue shifted with a small increase in the absorbance, but the corresponding 267

fluorescence intensity increases very little without any significant change of emission 268

maximum (Fig. S7, supporting information). These results indicate that γ-CD does not 269

have any preference for either of the forms of C343 and interaction is indeed very weak. 270

At pH 3.5, the weak fluorescence at 360 nm due to the neutral form of WF is not 271

found to be increased significantly in presence of either β-CD or γ-CD (Fig. S8, 272

supporting information). But under the same condition, the absorbance of WF at 308 nm 273

decreases, whereas the absorbance at 270 nm and 280 nm increase (Fig. S9, supporting 274

information) which indicates shifting of the acid-base equilibrium of the fluorophore 275

11

towards the neutral form. At pH 7.5 the fluorescence intensity of WF is significantly 276

enhanced in presence of β-CD (Fig. S10, supporting information) than with γ-CD. 277

The enhancement of fluorescence intensity of WF in presence of HSA and 278

cyclodextrin is simply due to encapsulation of fluorescent guest within host cavity which 279

results in increase of emission of the former [6, 56, 57], as the rotational and vibrational 280

motions of the bound guest are retarded by the geometrical confinement within the cavity 281

[58, 59]. This reduces the rate of non-radiative deactivation of the excited guest molecule. 282

Therefore, our observation of fluorescence quenching of C343 with HSA is apparently 283

unusual. But there are few reports on fluorescence quenching of emitting guests due to 284

their interaction with host and has been attributed to a number of specific interactions, 285

like hydrogen bonding, charge transfer, etc [6, 60-62]. 286

Results of absorbance data and fluorescence titration indicate shifting of acid-287

base equilibrium towards the anionic form of WF and C343 in the biosupramolecular 288

host cavity of HSA. On the other hand, β-CD tub prefers the neutral form of both WF and 289

C343 and shifts the prototropic equilibrium to the other side. 290

291

3.2 Comparison of pKa values of WF and C343 in absence and presence of various 292

hosts 293

In order to find out the preferred protomer within the host (HSA, β-CD and γ-294

CD), pKa of WF and C343 are calculated by measuring the absorbance spectra at 295

different pH. The physiological ionic strength (~150 mM NaCl [63]) condition has been 296

maintained during the measurements in presence of HSA. 297

The plot of absorbance as a function of pH in the absence and presence of serum 298

proteins are shown in the following Fig. 3(a), Fig. 3(b) and Fig. 4. The 454 nm band 299

corresponding to the neutral form of C343 and the 308 nm band for the open-chain 300

deprotonated form of WF are monitored here. 301

<Fig. 3(a), Fig. 3(b) and Fig. 4> 302

pKa of C343 and WF in aqueous solution, calculated from the Fig. 3(a) and Fig. 4, 303

are found to be (4.5 0.06) and (4.9 0.07), respectively, which is similar to the value 304

reported in the literature [36, 37, 46, 47]. Interestingly, for both the guests, the calculated 305

12

pKa are found to be shifted in presence of HSA. The pKa of WF and C343 is decreased by 306

about 1.2 and 1.6 units in presence of 8M and 15M HSA, respectively. So it can be 307

concluded that acid-base equilibrium of these two guests is shifted towards the basic form 308

(anionic form) in presence of HSA. Since HSA favors anionic form, it is expected that 309

higher concentration of HSA should shift pKa towards more acidic region, and actually in 310

presence of saturating concentration of HSA (15M in case of WF and 50M in case of 311

C343) pKa decrease is saturated by 1.6 and 1.9 units, respectively, with respect to water 312

medium. Now this concentration dependent pKa shift can also be explained with the help 313

of partition coefficient of guest molecules between aqueous phases to receptor cavity of 314

protein. Using the procedure as described elsewhere [64], the partition coefficient 315

(protein: water) of C343 in presence of 15M HSA is calculated to be 2.6103 and is 316

increased to a saturation value of 5.6104 at 50M concentration of HSA. Likewise, the 317

calculated value of partition coefficient of WF has been found to increase from 1.5103 318

(with 8M HSA) and 8.610

3 (with 15M HSA). The increase of partition coefficient 319

indicates that in the protein-bound state the overall concentration of the anionic form of 320

the guest becomes more compared to neutral species and hence higher pKa shift is 321

observed. Now the efficacy of a drug is related to the serum level in blood plasma, and 322

therefore, such concentration dependent pKa shift of guest can be very important. 323

Furthermore, it may be noted that by monitoring the pKa shift one can determine the 324

serum protein level in human blood plasma also. 325

<Fig. 5> 326

It is interesting to note that in presence of γ-CD, pKa of C343 remains almost 327

unperturbed (Fig. 5) whereas in presence of β-CD the value increases from 4.6 to 5.2. 328

Due to the limited solubility of β-CD, the pKa value of C343 determined in presence of 329

15 mM β-CD is not the true value but the apparent one. 330

<Fig. 6> 331

From Fig. 6 it is found that pKa of WF is increased by only about 0.5 units in 332

presence of β-CD and remains almost unchanged in presence of γ-CD. Therefore, the pKa 333

calculation also indicates that the neutral form of WF is the preferred protomer in the β-334

CD tub. 335

13

Ghosh et al. also have correlated pKa shift of basic guests with the nature of the 336

host in an excellent way [19]. They have interpreted that positive pKa shift of basic guest 337

is generally observed in presence of cation receptors or hydrogen bond-acceptor host 338

(like cucurbiturils and sulfonatocalixarene). Negative pKa shift is observed in presence of 339

anion receptors (calixpyridine, C4P) or hydrogen bond–donor host molecules or 340

molecules which offer non-polar cavities (e.g., cyclodextrins). Due to lack of 341

experimental data at hand, they have to presume that for acidic guests (with hydroxyl and 342

carboxylic acid functions) similar but opposite effect should apply. This means that 343

positive pKa shift with these acidic guests are expected in presence of hydrogen bond–344

acceptor host or molecules with non-polar cavities or cation receptors, and negative pKa 345

shift is expected in presence of anion receptors or hydrogen bond-donor host. Our results 346

seem to conform to these aforementioned predictions. We have observed a negative pKa 347

shift for both WF and C343 in HSA which indicates the presence of either anion receptor 348

or hydrogen bond donor host. 349

We have visualized the crystal structure of HSA (PDB ID: 1AO6) [65,] which 350

shows that the amino acid residues present in the sub-domain IIA are mainly Tryptophan, 351

Arginine, Histidine, Glutamic acid, Glutamine, Alaline, Tyrosine and Leucine (Fig. S11, 352

supporting information). Among these residues, Arginine and Histidine with cationic side 353

chain can function as anion receptors; Glutamic acid with anionic side chain can function 354

as cation receptor, but other amino acids either with polar uncharged side chain (like 355

Glutamine) or hydrophobic side chain (like Tryptophan, Alaline, Tyrosine and Leucine) 356

can only act as hydrogen bond acceptor but not as donor host. It is also important to 357

mention here that the significantly larger shift in pKa (pKa 1.2 units) of guests reported 358

in the literature is generally observed in presence of ion receptors [19]. Ghosh et al also 359

have commented that groups with either hydrogen bond donating or accepting ability 360

cannot shift pKa to such significant amount. Consequently our observed HSA-induced 361

change in pKa by more than 1.5 units for both WF and C343 indicates that the anion 362

receptors (availability of positively charged amino acid residues) present in sub-domain 363

IIA pocket of HSA should be responsible for the negative pKa shift (lowering of pKa). 364

We have also visualized the crystal structure of HSA-WF complex [48], obtained from 365

14

the protein data bank (PDB ID: 1H9Z), which shows that the active surface around the 366

bound WF is composed of positive charge density (Fig. 7, shown in blue) arising out of 367

Arg and His with positive side-chains. Since C343 is also a WF-site binder ligand, the 368

positive charge density due to those cationic side chains should be responsible for 369

stabilizing the C343 anion. 370

<Fig. 7> 371

The hydrophobic cavity of β-CD only stabilizes the neutral form of guest and 372

thereby shifts the acid-base equilibrium towards neutral form [6, 19]. Therefore positive 373

pKa shift of WF and C343 in presence of β-CD is also in accordance with the above 374

mentioned facts. Although γ-CD also possesses non- polar cavity but it is not able to shift 375

pKa due to very weak interaction with both WF and C343. There are several reports 376

available in the literature related to the study on pKa of basic guest in presence of β-CD 377

and γ-CD which shows that due to the large cavity size γ-CD is unable to bind the guest 378

and hence pKa shift is not observed [6, 19]. 379

3.3. Binding constant and Stoichiometry of complexation 380

pKa of WF and C343, as determined above in presence of HSA, are found to be 381

on the strongly acidic side. In order to determine the binding strength of HSA with 382

neutral form of probe, pH should be kept much below its protein-modified pKa. But in 383

such highly acidic conditions, the conformation of HSA is significantly affected [66]. 384

Therefore, the determination of strength of binding of HSA with neutral form of probe 385

has not been attempted. Also the binding constant data at pH much lower than the 386

physiological conditions may not be very useful. The binding constant (KBH) for the 387

complexation of WF and C343 with supramolecular host has been calculated using the 388

Benesi-Hildebrand (B-H) equation (Eqs. (2) and (3)) [67 68]. 389

max max

1 1 1 1

[ ]n

BHI I K Host I

…………….. (2) 390

or, 0

0

( ) 11

( ) ( [ ] )n

t BH

I I

I I K Host

…………………… (3) 391

Here ∆I= It-I0 and ∆Imax= I∞-I0 where I0, It and I∞ are the emission intensities of guest in 392

the absence of the host, at any intermediate concentration of host and at the level of 393

15

saturation of interaction, respectively, and n is the number of binding sites. The plot of 394

0

0

( )

( )t

I I

I I

vs

1

[ ]protein at pH 7.5 at 298K using the Benesi-Hildebrand equation is shown 395

below. 396

<Fig. 8> 397

<Fig. 9> 398

The linear nature of plots (Fig. 8 and Fig. 9) indicates a 1:1 stoichiometry of HSA 399

[68] and C343 or WF. The magnitude of KBH has been calculated from the slope. The 400

values of KBH for HSA-C343 and HSA-WF complexes are found to be (4.95 ± 0.03) ×105 401

M-1

and (8.5 ± 0.02) ×105 M

-1, respectively. As mentioned in the previous section that no 402

reasonable fluorescence titration curves could be obtained for the interaction of C343 403

with β-CD at pH 7.5 and with γ-CD at both the pH and also for the interaction of 404

cyclodextrin with WF at pH 3.5, and hence binding constants could not be estimated for 405

these systems. The B-H plot for the interaction of β-CD with C343 at pH 3.5 and with 406

WF at pH 7.5 are shown in the following figures. 407

<Fig. 10> 408

<Fig. 11> 409

The plots (Fig. 10 and Fig. 11) indicate that cyclodextrins also form 1:1 inclusion 410

complex with these guests. The values of binding constant at pH 3.5 and pH 7.5 in 411

presence of cyclodextrin are listed in Table 1 which shows that β-CD binds stronger with 412

the neutral form of both the guests. On the other hand, γ-CD binds the guest weakly 413

without preference for any of the prototropic forms. 414

<Table 1> 415

We have also determined the stoichiometry alternatively by Job’s method of 416

continuous variation using absorption spectroscopy [69]. A modified version of Job’s plot 417

has been utilized and the details of this method can be found elsewhere [70, 71]. The 418

Job’s plot for complexation of C343 with HSA at pH 7.5 is shown below. 419

<Fig. 12> 420

16

From Fig. 12 the intersection of two lines is found to be around 0.5 for HSA-421

C343 complex which show 1:1 stoichiometry. We have determined stoichiometry in 422

presence of both the cyclodextrins which also indicates 1:1 composition. 423

3.4 Time resolved measurements 424

Time resolved fluorescence decay 425

Time resolved fluorescence study is an important and very sensitive technique to 426

monitor the prototropic changes of fluorescent probes [72, 73]. We have measured the 427

fluorescence lifetime of C343 in the absence and presence of host at different pH 428

conditions. A representative decay plot is shown below. 429

<Fig. 13> 430

Analysis shows that the fluorescence decay profile (Fig. 13) of C343 in absence 431

of any host at pH 3.5 and 7.5 can be fitted to single exponential and the corresponding 432

lifetime values are (4.18±.05) ns and (4.48±.04) ns, respectively. These two values should 433

correspond to the lifetime of neutral and anionic forms of C343, respectively [37, 74]. In 434

the HSA-bound state (at pH 3.5) the fluorescence decay becomes double exponential 435

with lifetimes (4.24±.04) ns (~93%) and (1.80±.02) ns (~7%). Here the lifetime of the 436

major decay component is found to be lower than that of the unbound anionic form 437

(4.48±.04 ns) of C343 at pH 7.5 but is higher than the lifetime of its unbound neutral 438

form (4.18±.05 ns) at pH 3.5. Therefore, the major decay component in presence of HSA 439

at pH 3.5 corresponds to the lifetime of the anionic form of C343 and its lifetime 440

decreases due to binding and consequent quenching by the protein [60]. The steady-state 441

fluorescence quenching data is also supportive to this. Thus encapsulation of C343 within 442

the receptor cavity of HSA results in shifting of the acid-base equilibrium, and thereby 443

C343 gets deprotonated. The biexponential nature of fluorescence decay of similar probes 444

is a common observation in presence of serum proteins [75]. The other shorter lifetime 445

component in presence of HSA may also correspond to another kind of bound C343 446

anion [75]. It may also correspond to bound neutral form of C343. But importantly its 447

percentage contribution is significantly less. Average lifetimes of C343 at pH 3.5 and 7.5 448

in the absence and presence of β-CD and γ-CD have been calculated using the Eq. (4) 449

[76] and are shown below in the Table 2. 450

17

………………. (4) 451

<Table 2> 452

Here τ and α is the time constant and relative amplitude, respectively, obtained 453

from the fitted fluorescence decay. Since the fluorescence intensity of C343 is increased 454

in presence of β-CD, the average lifetimes are also found to be increased, as expected. 455

From the Table 2, it is noticeable that the change in average lifetime of C343 in presence 456

of β-CD is higher at pH 3.5 than at pH 7.5 which indicates stronger interaction between 457

the host and the neutral form of C343. With γ-CD the fluorescence decay profile for 458

C343 at both the pH is single exponential and the decrease in fluorescence lifetime is 459

insignificant which indicates very weak interaction with both the neutral and anionic 460

form of the guest. We have not been able to monitor fluorescence lifetime of WF because 461

of unavailability of excitation source between 295 and 375 nm. 462

Time resolved fluorescence anisotropy 463

Time resolved fluorescence anisotropy is also another important technique to 464

gather information about the microenvironment of the guest in different micro-465

heterogeneous systems like protein, molecular aggregates [77, 78]. We have measured 466

the time resolved fluorescence anisotropy decay of C343 in the absence and presence of 467

different concentration of HSA at pH 7.5 and it is shown below. 468

<Fig. 14> 469

Uncomplexed C343 exhibits single exponential anisotropy decay in pH 7.5 (Fig. 470

14). In presence of HSA the anisotropy decay profile becomes bi-exponential with two 471

distinct correlation times (one fast and another slow). The bi-exponential anisotropy 472

decay in HSA can be represented as follows (Eq. (5)) [79]. 473

0 1 2

1 2

( ) exp expr r

r r

t tr t r

………… (5) 474

Here r0 is the limiting anisotropy that corresponds to the inherent depolarization of the 475

probe. α and τ are the pre-exponential factors and rotational correlation time constants, 476

respectively, as obtained from the fitting of anisotropy decay. We have also measured 477

time resolved fluorescence anisotropy decay of C343 in presence of both β-CD and γ-CD 478

1 1 2 2avg

18

at pH 3.5 and 7.5. The average rotational correlation time constants are calculated 479

applying the following Eq. (6) [78] and are shown in Table 3. 480

481

1 1 2 2rot r r r r ………………………… (6) 482

483

<Table 3> 484

From Table 3 it is observed that the value of average rotational correlation time 485

constant increases significantly in presence of HSA. This also indicates a strong 486

interaction and hence C343 experiences rotationally restricted environment in the binding 487

pocket of HSA [77]. It is interesting to note that the change in the average rotational time 488

constant of C343 in presence of β-CD is higher at pH 3.5 than at pH 7.5. This result also 489

supports our earlier observation in fluorescence titration that the binding constant of β-490

CD with C343 is higher at pH 3.5 than at pH 7.5, that is, the neutral form binds stronger. 491

The average rotational time constant of C343 is not significantly affected in presence of 492

γ-CD at both the pH. 493

Therefore, the results of fluorescence titration and pKa measurements provide the 494

same insight as obtained from time resolved fluorescence decay and anisotropy 495

measurements. All theses experimental results indicate negative pKa shift of C343 and 496

WF is due to interaction with anion receptors in the pocket of HSA and positive pKa shift 497

due to encapsulation in the non-polar cavity of β-CD by hydrophobic interactions and 498

hydrogen bonding. On the other hand, pKa value is not affected significantly due to very 499

weak binding with γ-CD. 500

501

CONCLUSION 502

The present work shows that pKa of acidic guests, warfarin (WF) and coumarin 503

343 (C343) are shifted towards lower value in presence of serum proteins like HSA and 504

up-shifted in presence of β-CD, a cyclic oligosaccharide. This indicates that the anionic 505

form is the preferred isomer (protomer) of acidic guest when encapsulated in the 506

biosupramolecular cavity (protein pocket), which happens to be the anion receptor. It is 507

19

also interesting to note that the negative pKa shift for WF in presence of HSA favors the 508

formation of active component responsible for anticoagulation activity. 509

Our results are important especially for the family of acidic drugs whose activity 510

depends on the position of their prototropic equilibrium in human body system. Present 511

study also shows that the release of the active form of the acidic guests from the host 512

cavity to blood serum is not the last thing to talk about but the survival of its active form 513

is rather more challenging. With the knowledge of pKa shift due to supramolecular and 514

biosupramolecular encapsulation, one can optimize the relevant species through inclusion 515

in macrocyclic systems to enhance the bioavailability of such drugs. Moreover, fine 516

tuning the host-assisted pKa shift by proper fictionalization of the guest and choice of the 517

receptor cavity can indeed open up the vast possibility to design more effective drugs. 518

Sub-domain IIA is known as drug site 1. In the present study WF and C343 are 519

acting as reporter of pocket environment in sub-domain IIA of HSA through modulation 520

of their prototropic activity. Sub-domain IIIA is another binding site of HSA where few 521

other drugs like ibuprofen, camptothecin etc bind and this is known as drug site 2. 522

Similar studies with sub-domain IIIA binder guests could also be important in 523

pharmacokinetics. 524

Finally this study raises the possibility of using HSA as encapsulator of 525

prototropic drugs instead of conventional polymeric hosts. Drugs pre-equilibrated with 526

HSA should be more biocompatible and may avoid toxicity issues. 527

528

Abbreviations 529

HSA, Human serum albumin; C343, Coumarin 343; WF, Warafrin; 530

531

Acknowledgments 532

We thank DST-India (Fund no. SR/FTP/CS-97/2006), CSIR-India (Fund no. 533

01/(2177)/07 EMR-II, dated 24/10/2007) and IIT-Kharagpur (ISIRD-EEM grant) for 534

financial support. SD thanks IIT Kharagpur for a fellowship. We thank Prof. N. Sarkar of 535

IIT Kharagpur for his help in measuring time resolved data. SD thanks Mr. N. Mahapatra 536

20

and Dr. P. Bolel for help in various instances. We would also like to thank the 537

anonymous reviewers for their critical comments and important suggestions. 538

539

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Tartrazine/Cationic Surfactant Interaction, J. Phys. Chem. B, 115 (2011) 14435-14444. 732

[71] S. Datta, N. Mahapatra, M. Halder, pH-insensitive electrostatic interaction of 733

carmoisine with two serum proteins: A possible caution on its uses in food and 734

pharmaceutical industry, J. Photochem. Photobiol. B-Biol, 124 (2013) 50-62. 735

[72] N. Barooah, J. Mohanty, H. Pal, A.C. Bhasikuttan, Stimulus-Responsive 736

Supramolecular pK(a) Tuning of Cucurbit[7]uril Encapsulated Coumarin 6 Dye, J. Phys. 737

Chem. B, 116 (2012) 3683-3689. 738

[73] M. Shaikh, Y.M. Swamy, H. Pal, Supramolecular host-guest interaction of acridine 739

dye with cyclodextrin macrocycles: Photophysical, pK(a) shift and quenching study, J. 740

Photochem. Photobiol a, 258 (2013) 41-50. 741

27

[74] H.N. Ghosh, Charge transfer emission in coumarin 343 sensitized TiO2 742

nanoparticle: A direct measurement of back electron transfer, J. Phys. Chem B, 103 743

(1999) 10382-10387. 744

[75] J.R. Lakowicz, Priniples of Fluorescence Spectroscopy, 3rd ed., Plenum, New York, 745

2006. 746

[76] B. Sengupta, P.K. Sengupta, Binding of quercetin with human serum albumin: A 747

critical spectroscopic study, Biopolymers, 72 (2003) 427-434. 748

[77] B. Bhattacharya, S. Nakka, L. Guruprasad, A. Samanta, Interaction of Bovine Serum 749

Albumin with Dipolar Molecules: Fluorescence and Molecular Docking Studies, J. Phys. 750

Chem. B, 113 (2009) 2143-2150. 751

[78] J.R. Lakowicz, Principles of fluorescence spectroscopy, springer, New York, 2006. 752

[79] R.D. Ludescher, L. Peting, S. Hudson, B. Hudson, Time-Resolved Fluorescence 753

Anisotropy for Systems with Lifetime and Dynamic Heterogeneity, Biophys. Chem, 28 754

(1987) 59-75. 755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

28

Table 1: Binding constant (KBH) of C343 and WF in presence of -CD and -CD at 772

298K 773

774

775

776

777

778

779

780

aChange in fluorescence intensity is too small to calculate KBH accurately. 781

782

Table 2: Average lifetime of C343 at different pH at 298K in absence and presence 783

of -CD and -CD 784

785

786

System Binding constant (KBH) (M-1

)

pH 3.5 pH 7.5

C343-CD complex (13011) a

C343-CD complex (2505) a

WF- CD complex a (28003)

WF-CD complex a (7506)

System Average

lifetime

(ns)

Change in

average

lifetime (ns)

χ2

C343 in absence of host, pH3.5 (4.18±.05) - 1.01

C343 in presence of 15 mM -CD, pH3.5 (4.50±.03) 0.32 0.99

C343 in presence of 6 mM γ-CD, pH3.5 (4.23±.02) 0.05 1.10

C343 in absence of any host, pH7.5 (4.48± .04) - 0.99

C343 in presence of 15 mM -CD, pH7.5 (4.51±0.03) 0.03 1.05

C343 in presence of 6 mM γ-CD, pH7.5 (4.49±0.02) 0.01 0.98

29

787

788

789

Table 3: Average fluorescence anisotropy decay constants of C343 with different 790

hosts at 298K 791

792

793

794

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

810

811

812

813

814

815

816

817

818

819

820

System

<τrot>

(ns) χ

2

C343 without host, pH 7.5 (0.20±.02) 0.99

C343+HSA (15μM), pH 7.5 (3.63±0.03) 0.99

C343+-CD, pH 3.5 (0.95±0.05) 1.03

C343+-CD, pH 7.5 (0.32±0.02) 0.99

C343+-CD, pH 3.5 (0.41±0.04) 1.05

C343+-CD, pH 7.5 (0.30±0.01) 1.10

30

Figure Captions 821

822

Fig. 1: Absorption spectra of C343 in the absence (blue line) and presence (black 823

lines) of different concentrations of HSA at pH 3.5 at 298K. [C343] = 2µM and 824

[HSA] is varied from 0-50µM. 825

Fig. 2: Emission spectra of C343 in the absence and presence of increasing 826

concentrations of HSA at pH 3.5 at 298K. [C343] = 1.5µM and [HSA] is varied from 827

0-22.5µM. 828

Fig. 3: Determination of pKa: Absorbance at 454 nm of C343 as a function of pH in 829

the absence and presence of 15µM (a) and 50µM (b) HSA, respectively, at 298K. 830

Fig. 4: Determination of pKa: Absorbance at 308 nm of warfarin as a function of pH 831

in the absence and presence of HSA (15µM) at 298K. 832

Fig. 5: Determination of pKa: Absorbance at 454 nm of C343 as a function of pH in 833

the absence and presence of -CD (15 mM) and -CD (6 mM) at 298K. 834

Fig. 6: Determination of pKa: Absorbance at 308 nm of warfarin as a function of pH 835

in the absence and presence of -CD (15 mM) and -CD (10 mM) at 298K. 836

Fig. 7: Active surface around bound warfarin in sub-domain IIA of HSA. Blue color 837

corresponds to positive charge density due to the cationic side chains of amino acids 838

residues. 839

Fig. 8: Benesi-Hildebrand plot for the complexation of C343 with HSA in pH 7.5 at 840

298K. 841

Fig. 9: Benesi-Hildebrand plot for the complexation of WF with HSA in pH 7.5 at 842

298K. 843

31

Fig. 10: Benesi-Hildebrand plot for the complexation of C343 with -CD in pH 3.5 844

at 298K. 845

Fig. 11: Benesi-Hildebrand plot for the complexation of WF with -CD in pH 7.5 at 846

298K. 847

Fig. 12: Job’s plot for C343-HSA complex in pH 7.5 at 298K. The total 848

concentration of C343 and protein is 6µM. 849

Fig. 13: Fluorescence decay profiles of C343 at pH 7.5 and in the absence and 850

presence of HSA at pH 3.5. ex = 408 nm and monitored at the emission maxima. 851

[C343] = 1.5μM. 852

Fig. 14: Time resolved fluorescence anisotropy decay of C343 in the absence and 853

presence of HSA at pH 7.5 at 298K. [C343]=1.5μM. 854

855

856

857

858

859

860

861

862

863

864

865

866

32

400 440 4800.00

0.02

0.04

0.06

0.08

0.10 27 nm

Isosbest

Ab

sorb

ance

Wavelength (nm)

[HSA] increasing

867

Fig. 1 868

869

870

871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

Fig. 2 886

887

475 500 525 550 5750

400

800

1200

1600

[HSA] increasing

Flu

ore

scen

ce I

nte

nsi

ty (

a.u

.)

Wavelength (nm)

11nm

33

888

889

890

891

892

893

894

895

896

897

898

899

900

901

902

903

904

905

906

907

908

909

910

911

Fig. 3 912

913

2 3 4 5 60.04

0.08

0.12

0.16

0.20 HSA-C343 C343

pKa>1.3

A

bso

rban

ce (

= 4

54

nm

)

pH

(a)

2 3 4 5 6

0.05

0.10

0.15

0.20

Ab

sorb

ance

(

= 4

54

nm

)

pH

HSA-C343 C343

pKa>1.8

(b)

34

914

915

916

917

918

919

920

921

Fig. 4 922

923

3 4 5 6

0.1

0.2

0.2

pKa>0.5

Ab

sorb

ance

(=

45

4n

m)

pH

C343

(-CD)-C343

(-CD)-C343

924 Fig. 5 925

926

927

928

929

2 3 4 5 6

0.16

0.18

0.20

pH

Ab

sorb

ance

(

=3

08

nm

)

WF HSA-WF

pKa>1.6

35

4 5 6 7

0.16

0.18

0.20

0.22

pKa>0.4

Ab

sorb

ance

(

= 3

08

nm

)

pH

WF

(-CD)-WF

(-CD)-WF

930 Fig. 6 931

932

933

934

935

936

937

938

939

940

941

942

943

944

945

946

947

948

949

950

951

Fig. 7 952

953

36

954

955

956

957

958

959

960

961

962

963

964

965

966

967

968

969

Fig. 8 970

5.0x104

1.0x105

1.5x105

2.0x105

1.0

1.1

1.2

1.3

(I-I

0)/

(It-I

0)

[Protein]-1(M-1)

HSA-WF

971 Fig. 9 972

973

974

975

976

977

2x105

4x105

6x105

8x105

1x106

1

2

3

(I-I

0)/

(It-I

0)

[Protein] -1

(M-1)

HSA-C343

37

978

979

980

981

982

983

984

985

986

987

988

989

990

991

992

993

994

995

Fig. 10 996

997

998

999

1000

1001

1002

1003

1004

1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015

Fig. 11 1016

100 150 200 250 300

1.0

1.5

2.0

2.5

3.0

3.5

(I-I

0)/

(It-I

0)

[-CD]-1(M

-1)

100 150 200 250

1.00

1.04

1.08

1.12

1.16

(I-I

0)/

(It-I

0)

[-CD]-1(M

-1)

38

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

Fig. 12 1035

1036

1037

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

Fig. 13 1052

1053

5 6 7 8 9 100

1k

2k

3k

4k

5k

Co

un

ts

Time (ns)

IRF

C343 pH 3.5

C343 pH 7.5

C343-HSA pH 3.5

0.00 0.25 0.50 0.75 1.00

-0.018

-0.009

0.000

HSA-C343

A

XC343

39

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

1066

1067

1068

1069

1070

1071

1072

1073

1074

1075

1076

1077

1078

Fig. 14 1079

1080

1081

1082

1083

1084

1085

1086

1087

1088

1089

1090

1091

1092

4 8 120.0

0.1

0.2

0.3

C343 pH 7.5C343-HSA pH 7.5

An

iso

tro

py

(r)

Time (ns)

Graphical Abstract

Modulation of pKa of prototropic drugs due to encapsulation in the host pocket

Research Highlights

● Supramolecular and bio-supramolecular host induced pKa shift of WF and C343.

● Positive pKa shift with β-CD and the reverse with human serum albumin.

● Activity of prototropic-equilibrium-based drugs may be lost or enhanced with HSA.

● Properly functionalized drug with appropriate receptor pocket is very important.

● Encapsulation in HSA receptor pocket can lead to efficient drug delivery system.