Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions

Rachel Soon, Tony Velkov, Francis Chiu, Philip Thompson, Rashmi Kancharla, Kade Roberts, Ian Larson, Roger Nation, Jian Li

Research output: Contribution to journalArticleResearchpeer-review

29 Citations (Scopus)

Abstract

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary I?-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (Kd <1 I?M) than for whole cells (Kd 6a??12 I?M). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxin B3a??LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3a??LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC 16 I?g/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development.
Original languageEnglish
Pages (from-to)273 - 283
Number of pages11
JournalAnalytical Biochemistry
Volume409
Issue number2
DOIs
Publication statusPublished - 2011

Cite this

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title = "Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions",
abstract = "Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary I?-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (Kd <1 I?M) than for whole cells (Kd 6a??12 I?M). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxin B3a??LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3a??LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC 16 I?g/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development.",
author = "Rachel Soon and Tony Velkov and Francis Chiu and Philip Thompson and Rashmi Kancharla and Kade Roberts and Ian Larson and Roger Nation and Jian Li",
year = "2011",
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language = "English",
volume = "409",
pages = "273 -- 283",
journal = "Analytical Biochemistry",
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Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions. / Soon, Rachel; Velkov, Tony; Chiu, Francis; Thompson, Philip; Kancharla, Rashmi; Roberts, Kade; Larson, Ian; Nation, Roger; Li, Jian.

In: Analytical Biochemistry, Vol. 409, No. 2, 2011, p. 273 - 283.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions

AU - Soon, Rachel

AU - Velkov, Tony

AU - Chiu, Francis

AU - Thompson, Philip

AU - Kancharla, Rashmi

AU - Roberts, Kade

AU - Larson, Ian

AU - Nation, Roger

AU - Li, Jian

PY - 2011

Y1 - 2011

N2 - Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary I?-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (Kd <1 I?M) than for whole cells (Kd 6a??12 I?M). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxin B3a??LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3a??LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC 16 I?g/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development.

AB - Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary I?-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (Kd <1 I?M) than for whole cells (Kd 6a??12 I?M). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxin B3a??LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3a??LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC 16 I?g/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development.

U2 - 10.1016/j.ab.2010.10.033

DO - 10.1016/j.ab.2010.10.033

M3 - Article

C2 - 21050838

VL - 409

SP - 273

EP - 283

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -