New Synthetic Approaches to Small Molecules for Imaging

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Fluorophores in the NIR range enables in vivo optical imaging due to the significant tissue penetration of light in this range. Despite a central role in modern biology and medicine, the compounds employed in NIR fluorescence techniques have changed little in recent decades. Using molecular design concepts borrowed from related fields (e.g. medicinal chemistry and modern organic synthesis), we seek to develop new agents with improved utility for cancer-related imaging and microscopy. The long-term goal is to identify readily synthesized, stable, and bright fluorophores with optimal properties for biomedical imaging. The heptamethine cyanine class of near-IR fluorophores are used for many applications, with extensive recent progress in the context of fluorescence-guided surgery. We have developed a new rearrangement reaction that enables the synthesis of previously inaccessible variants. Compared to existing agents, the compounds we have prepared exhibit improved optical properties and significantly greater chemical stability to biological nucleophiles. Through an extensive optimization campaign, we have developed molecules that are exceptionally resistant to aggregation following labeling on both targeting antibodies and nanoparticles. These molecules exhibit reduced liver uptake and improved in vivo signal when compared to existing agents. In related work, we have shown that small changes in the polar functional groups appended to these fluorophores can have a dramatic impact on biodistribution and tumor accumulation when used without targeting motifs. Over the past year, we have continued to develop agents for use in labeled sensitive abdominal features such as bile duct anatomy and ureter structures. These agents have been tested in several settings, and we are carrying out additional preclinical studies. Towards the goal of improving the photon output of these agents, we have developed a chemical strategy to assemble polycyclic pentamethine cyanines through a cross metathesis/polycylcization strategy. When compared to conventional pentamethine cyanines, the resulting compounds exhibit significantly higher fluorescence quantum yield (4X) and, additionally, recover from sodium borohydride reduction with improved efficiency. These features allow these compounds to be used for super resolution microscopy and enable excellent photon counts without recourse to complex deoxygenation buffers. We have recently prepared molecules with improved antibody and nucleic acid labeling properties. Over the past year these molecules have been used to map cell surface receptors with unprecedented resolution using light-sheet microscopy. We have extended this approach to longer wavelength dyes, through the synthesis and testing of conformational restricted heptamethine cyanines dyes. Detailed studies found that, in contrast to prior reports, photoisomerization does not contribute significantly to the excited-state chemistry of these molecules. We also discovered that the fluorescence lifetime of the restrained heptamethine cyanine is temperature-insensitive and extended at moderately elevated temperatures. Fluorogenic probes in the near-infrared (NIR) region could provide stimuli-dependent information in living organisms. We recently developed the first class of NIR-responsive fluorogenic probes, which are based on the broadly used heptamethine cyanine scaffold. These compounds were created by modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. These cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (170X) due to dual requirements for NIR emission, carbamate cleavage through 1,6-elimination and chromophore protonation. We have applied these several settings, including the used a gamma-glutamate substituted CyBam which was applied to imaging gamma-glutamyl transpeptidase (GGT) activity in a metastatic model of ovarian cancer. By optimizing the cellular uptake and retention of these probes, we have been able to create mAb-targeted variants. Wavelengths between 1000 to 2000 nm, referred to as the shortwave-infrared (SWIR) or NIR-II range, can enable high-resolution in vivo imaging at depths not possible with conventional NIR wavelengths. There is a significant need for the type of bioconjugatable probes that have proven invaluable for multicolor imaging in the visible and NIR range. Enabled by a rational design process, we generated persulfonated nonamethine indocyanine dyes with absorbance/emission maxima beyond 800 nm.These compounds are ideally suited for targeted multiplexed imaging in combination with existing cyanines and have extended the range of wavelengths available for targeted multicolor in vivo imaging. Going forward, our plans are to optimize these probes for multicolor surgical applications and to develop responsive variants using strategies that build on our CyBam chemistry.