New BODIPY lipid probes for fluorescence studies of membranes

Ivan A. Boldyrev¹^,*, Xiuhong Zhai¹^,, Maureen M. Momsen, Howard L. Brockman, Rhoderick E. Brown²^, and Julian G. Molotkovsky²^,*

^* Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
Hormel Institute, University of Minnesota, Austin, MN 55912

Published, JLR Papers in Press, April 7, 2007.

^¹ I. A. Boldyrev and X. Zhai contributed equally to this work.

^² To whom correspondence should be addressed. e-mail: reb{at}umn.edu (R.E.B.); jgmol{at}ibch.ru (J.G.M.)

Many fluorescent lipid probes tend to loop back to the membraneinterface when attached to a lipid acyl chain rather than embeddingdeeply into the bilayer. To achieve maximum embedding of BODIPY(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore intothe bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholineswas synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl(Me₄-BODIPY-8) at the end of C₃-, C₅-, C₇-, or C₉-acyl. A strategywas used of symmetrically dispersing the methyl groups at BODIPYring positions 1, 3, 5, and 7 to decrease fluorophore polarity.Iodide quenching of the phosphatidylcholine probes in bilayervesicles confirmed that the Me₄-BODIPY-8 fluorophore was embeddedin the bilayer. Parallax analysis of Me₄-BODIPY-8 fluorescencequenching by phosphatidylcholines containing iodide at differentpositions along the sn-2 acyl chain indicated that the penetrationdepth of Me₄-BODIPY-8 into the bilayer was determined by thelength of the linking acyl chain. Evaluation using monolayersshowed minimal perturbation of <10 mol% probe in fluid-phaseand cholesterol-enriched phosphatidylcholine. Spectral characterizationin monolayers and bilayers confirmed the retention of many featuresof other BODIPY derivatives (i.e., absorption and emission wavelengthmaxima near 498 nm and $~$ 506–515 nm) but also showed theabsence of the 620–630 nm peak associated with BODIPYdimer fluorescence and the presence of a 570 nm emission shoulderat high Me₄-BODIPY-8 surface concentrations. We conclude thatthe new probes should have versatile utility in membrane studies,especially when precise location of the reporter group is needed.

Supplementary key words spectral properties • monolayers • lipid lateral packing • surface compressional modulus • lipid phase state • fluorophore position • fluorescence quenching • iodide • fluorophore location in bilayers • parallax analysis • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

Abbreviations: AV12-PC, 1-acyl-2-[12-(9-anthryl)-11E-dodecenoyl]-sn-glycero-3-phosphocholine; B3-, B5-, B7-, or B9-PC, phosphatidylcholine bearing at the sn-2 position ${omega}$ -Me₄-BODIPY-8-C₃-, -C₅-, -C₇-, or -C₉-fatty acid, respectively; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; I7- or I11-PC, phosphatidylcholine bearing 7-iodoheptanoic or 11-iodoundecanoic acid, respectively, at the sn-2 position; K_sv, Stern-Volmer quenching constant; Me₂-BODIPY-3, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl; Me₄-BODIPY-8, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl; PB-PC, 1-palmitoyl-2-Me₂-BODIPY-3-pentanoyl-sn-glycero-3-phosphocholine; T_m, phase transition temperature

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