LETTER pubs.acs.org/Langmuir
Fluorescence Turn On by Cholate Aggregates Anthony Baldridge, Adrian Amador, and Laren M. Tolbert* School of Chemistry and Biochemistry, 901 Atlantic Drive, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, United States
bS Supporting Information ABSTRACT: Bile salts, including sodium cholate (NaCh), are amphiphilic molecules with a concave hydrophilic side and a convex hydrophobic side. By forming aggregates in aqueous solution, these natural surfactants fulfill vital biological roles in the solubilization of cholesterol, lipids, and fat-soluble vitamins and thus are involved in the transport and absorption of important biological molecules. Following our success with the encapsulation of fluorescent protein chromophore (FP) analogs by synthetic hydrophobic and hydrophilic hosts, based upon substitution patterns, we now report the binding and turn on of other analogs by bile salt aggregates, observations which may lead to new tools for studying trafficking in these important systems.
B
ile salts, including sodium cholate (NaCh), are amphiphilic molecules with a concave hydrophilic side and a convex hydrophobic side.1 By forming aggregates in aqueous solution, these natural surfactants fulfill vital biological roles in the solubilization of cholesterol, lipids, and fat-soluble vitamins and thus are involved in the transport and absorption of important biological molecules.2 The aggregation of bile salts has been the focus of a literature review.3 A widely adopted model argues that at low concentrations, bile salt monomers aggregate to form single primary binding sites whereas at high concentrations further aggregation results in the formation of secondary binding sites (Figure 1).4 Additional models argue the formation of hydrogen bonds between monomer units,5 and a third model argues that the interactions are not defined in that multiple interactions may interplay within the overall system.6 A general classification of the binding sites is marked by their encapsulation of both hydrophobic molecules (hydrophobic site) and hydrophilic molecules (hydrophilic site).7 Following our success with the encapsulation of fluorescent protein chromophore (FP) analogs by synthetic hydrophobic and hydrophilic hosts, based upon substitution patterns,8 we now report the binding and turn on of other analogs by bile salt aggregates, observations which may lead to new tools for studying trafficking in these important systems. Fluorescent protein chromophores exhibit diminished emission quantum yields (EQY) outside the protective β barrel.9 Sequestration within the β barrel is believed to inhibit twisting about the double bond (OBF represents the one-bond flip in Figure 2), leading to cis/trans isomerization as well as rotation about the formal single aromatic bond; both processes are believed to lead to the ultrafast nonradiative decay of the excited state. A combination of these two twisting mechanisms, a “hula twist”, has also been proposed.10 Our efforts have concentrated upon using the recovery of the high FP fluorescence through sequestration to develop structureactivity relationships that r 2011 American Chemical Society
will further enhance our understanding of these excited-state processes. Arylmethyleneimidazolinones (AMIs) were synthesized using previously described methods (Table 1).11 Substituents for the FP analogs were chosen upon the basis of their hydrophobic/ hydrophilic nature and their steric bulk. For all studies, 0.2 M NaCl aqueous solutions were used and were allowed to equilibrate 24 h prior to measurement. Encapsulation of the chromophores was marked by a shift in the absorbance maximum compared to that of free chromophore in solution (Supporting Information Figure S1). The effect of inclusion in bile salts is highlighted in Table 1, where turn-on ratios (F/Fo) of 50 are achieved for hydrophobic chromophores (for general reference, in the case of 11, Φ = 0.05). This is readily rationalized by the inclusion of hydrophobic chromophores into the hydrophobic restrictive aggregates, which inhibit nonradiative torsional motions. The hydrophilic chromophores are included in the hydrophilic sites whose interaction with the chromophores is starkly different, thus allowing for significantly more nonradiative processes and lower quantum yields. Additionally, a wide variation in turn-on ratios is noted, with the hydrophobic chromophores most sensitive to structural changes in the bile salt aggregation site structure upon inclusion. This phenomenon has been noted for bile salts.4,13 The titration of AMIs with NaCh shows an emission enhancement with increasing concentration, owing to the continuing formation of hydrophobic binding sites.14 Figure 3 shows the fluorescence response with 11 where peak enhancement is achieved with increasing cholate concentration up to 75 mM. Using the response of 11 as a standard, F/Fo ratios were Received: January 25, 2011 Revised: March 6, 2011 Published: March 11, 2011 3271
dx.doi.org/10.1021/la2003244 | Langmuir 2011, 27, 3271–3274
Langmuir
LETTER
Table 1. FP Analogs and Spectroscopic Characteristics
no.
Figure 1. Idealized bile salt aggregate showing hydrophobic (yellow) and hydrophilic (orange) binding sites.
R1
R2
F/Foa
λabsb
log P12 1.25
1
H
Me
19
347/352
2
4-Br
Me
96
353/360
2.06
3
2-CF3
Me
157
340/354
2.10
4 5
3-CF3 4-CF3
Me Me
75 122
346/357 348/359
2.12 2.15
6
4-OH
Me
2
372/383
0.77
7
2-Br
Me
57
348/359
2.01
8
2-Cl
Me
93
348/359
1.88
9
3-OH
Me
5
354/358
0.75
10
H
n-Pr
44
340/355
2.13
11
2-CF3
n-Pr
212
340/355
2.98
12 13
4-OH 2-Cl
n-Pr n-Pr
5 167
371/379 350/361
1.65 2.76
Intensity (AMI þ NaCh)/intensity(AMI) at λem. AMI [105 M] and NaCh [75 mM] were used. b λabs 0.2 M NaCl/λabs 75 mM NaCh. a
Figure 2. Torsional Modes of the GFP Chromophore.
calculated using 75 mM NaCh for all experiments. Support for the inclusion of hydrophobic chromophores into the hydrophobic sites is also shown through spectroscopic shifts in cyclohexane versus water. Table 1 and SI show a distinct red shift in absorbance upon inclusion due to the change in polarity. An additional bathochromic shift of 7 nm is observed compared to cyclohexane, perhaps due to the planarization of the chromophore. This result is also rationalized through the higher F/Fo ratios achieved by the hydrophobic chromophores in 75 mM NaCh solution. The data in Figure 3 were fit to a two-site cooperative binding model15 with two dissociation constants (Kd) of 74 and 2.0 mM and a saturation ratio of 73. In solution, the rapid deactivation (
