Photostable, Amino Reactive and Water-Soluble Fluorescent Labels Based on Sulfonated Rhodamine with a Rigidized Xanthene Fragment

Abstract

Highly water soluble fluorescentdyes were synthesized and transformedinto new amino reactive fluorescentlabels for biological microscopy.To this end,rhodamine 8 (preparedfrom 7-hydroxy-1,2,3,4-tetrahydroquinoline(7) and phthalic anhydride in85% aq. H3PO4) was sulfonated with30% SO3 in H2SO4 and afforded thewater soluble disulfonic acid 3a (64%).Amidation of the carboxy group in 3awith 2-(methylamino)ethanol in thepresence of O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium·PF6(HATU) led to alcohol 3b (66%),which was transformed into the aminoreactive mixed carbonate 3d with di(Nsuccinimidyl)carbonate and Et3N. Reactionof the carboxy group in 3a withMeNHACHTUNGTRENUNG(CH2)2CO2Me and N,N,N’,N’-tetramethyl-O-(N-succinimidyl)-uronium·BF4 (TSTU) yielded methylester 13. After saponification of the aliphaticcarboxy group in 13,the compoundwas converted into NHS-ester3e (using HATU and Et3N). Heatingof 7 with trimellitic anhydride inH3PO4 gave a mixture of dicarboxylicacids 14 and 15 (1:1). Regioisomer 15was isolated,sulfonat ed with 30% SO3in H2SO4,and disulfonic acid 3 f wasused for the synthesis of the monoNHS-ester 3g,in which the stericallyunhindered carboxy group was selectivelyactivated (with N-hydroxysuccinimide,HA TU,and Et3N). The sulfonatedrhodamines 3b, c and f are solublein water (up to 0.1m),have excellentphotostabilities and large fluorescencequantum yields. Subdiffractionresolution images of tubulin filamentsof mammalian cells stained with thesedyes illustrate their applicability aslabels for stimulated emission depletionmicroscopy and other fluorescencetechniques.