A chemically stable fluorescent marker of the ureter

Abstract

Surgical methods guided by exogenous fluorescent markers have the potential to define tissue types in real time. Small molecule dyes with efficient and selective renal clearance could enable visualization of the ureter during surgical procedures involving the abdomen and pelvis. These studies report the design and synthesis of a water soluble, net neutral C4'-O-alkyl heptamethine cyanine, Ureter-Label (UL)-766, with excellent properties for ureter visualization. This compound is accessed through a concise synthetic sequence involving an N- to O-transposition reaction that provides other inaccessible C4'-O-alkyl heptamethine cyanines. Unlike molecules containing a C4'-O-aryl substituent, which have also been used for ureter visualization, UL-766 is not reactive towards glutathione and the cellular proteome. In addition, rat models of abdominal surgery reveal that UL-766 undergoes efficient and nearly exclusive renal clearance in vivo. In total, this molecule represents a promising candidate for visualizing the ureter during a variety of surgical interventions.

Keywords: Fluorescence-guided surgery; Fluorophore synthesis; Near-IR fluorescence.

Fig 1.

Key prior cyanines, IRDye-800CW and FNIR-774, and the compound reported here, UL-766.

Figure 2:

(A) General reaction manifold for cyanine-thiol adduct formation. (B) Glutathione stability determine by fraction remaining of starting cyanine (10 μM) in 1 mM GSH in pH 7.4 PBS. (C) Proteome-wide reactivity of cyanines to HEK-293 cells (HEK-293 cell lysate incubated with 10 μM dye for 24 h and run on an SDS-PAGE gel). The green signal is emission using 800 nm excitation and the blue signal is Coomassie staining to confirm protein loading.

Fig 3.

NIR fluorescence-guided intraoperative identification of the ureter. Shown are the color video (left column), NIR fluorescence (middle column), and a pseudo-colored (Cyan) merged image of the 2 (right column). Exposure time was 33 ms for all NIR fluorescence images. Li:liver, Du:Duodenum, Ki:kidney, Ur: ureter, Ut: uterine, Bl: Bladder.

Fig 4.

Contrast-to-background ratio of kidney in rats (n = 4). Bars represent mean +/− SEM.

Scheme 1.

Synthesis of 8 and 9.