Absorbance spectra of both Ab-conjugates for additional QC

Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect posttranslational modifications with single-molecule sensitivity. Here, we demonstrate single-molecule protein sequencing using a dynamic approach in which single peptides are probed in real time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. We annotate amino acids and identify the peptide sequence by measuring fluorescence intensity, lifetime, and binding kinetics on an integrated semiconductor chip. Our results demonstrate the kinetic principles that allow recognizers to identify multiple amino acids in an information-rich manner that enables discrimination of single amino acid substitutions and posttranslational modifications. With further development, we anticipate that this approach will offer a sensitive, scalable, and accessible platform for single-molecule proteomic studies and applications.

Optical methods offer the potential to manipulate living biological systems with exceptional spatial and temporal control. Caging bioactive molecules with photocleavable functional groups is an important strategy that could be applied to a range of problems, including the targeted delivery of otherwise toxic therapeutics. However existing approaches that require UV or blue light are difficult to apply in organismal settings due to issues of tissue penetration and light toxicity. Photocaging groups built on the heptamethine cyanine scaffold enable the targeted delivery of bioactive molecules using near-IR light (up to 780 nm) in live animal settings. Here we provide a detailed procedure demonstrating the utility of the heptamethine cyanine caging group to create a light-cleavable linker between an antibody, panitumumab, and a therapeutic small molecule in the duocarmycin class of natural products. Descriptions of the design and synthesis of the small molecule component, assembly of the antibody conjugate, in vitro analysis of uncaging, in vivo imaging, and impact on tumor progression are provided.

Heptamethine cyanines are broadly used for a range of near-infrared imaging applications. As with many fluorophores, these molecules are prone to forming nonemissive aggregates upon biomolecule conjugation. Prior work has focused on persulfonation strategies, which only partially address these issues. Here, we report a new set of peripheral substituents, short polyethylene glycol chains on the indolenine nitrogens and a substituted alkyl ether at the C4′ position, that provide exceptionally aggregation-resistant fluorophores. These symmetrical molecules are net-neutral, can be prepared in a concise sequence, and exhibit no evidence of H-aggregation even at high labeling density when appended to monoclonal antibodies or virus-like particles. The resulting fluorophore–biomolecule conjugates exhibit exceptionally bright in vitro and in vivo signals when compared to a conventional persulfonated heptamethine cyanine. Overall, these efforts provide a new class of heptamethine cyanines with significant utility for complex labeling applications.

Despite advances, visual inspection, palpation, and intraoperative ultrasound remain the most utilized tools during surgery today. A particularly challenging issue is the identification of the biliary system due to its complex architecture partially embedded within the liver. Fluorescence guided surgical interventions, particularly using near-infrared (NIR) wavelengths, are an emerging approach for the real-time assessment of the hepatobiliary system. However, existing fluorophores, such as the FDA-approved indocyanine green (ICG), have significant limitations for rapid and selective visualization of bile duct anatomy. Here we report a novel NIR fluorophore, BL (Bile Label)-760, which is exclusively metabolized by the liver providing high signal in the biliary system shortly after intravenous administration. This molecule was identified by first screening a small set of known heptamethine cyanines including clinically utilized agents. After finding that none of these were well-suited, we then designed and tested a small series of novel dyes within a prescribed polarity range. We validated the molecule that emerged from these efforts, BL-760, through animal studies using both rodent and swine models employing a clinically applicable imaging system. In contrast to ICG, BL-760 fluorescence revealed a high target-to-background ratio (TBR) of the cystic duct relative to liver parenchyma 5 min after intravenous injection. During hepatic resection surgery, intrahepatic ducts were clearly highlighted, and bile leakage was easily detected. In conclusion, BL-760 has highly promising properties for intraoperative navigation during hepatobiliary surgery.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death in the world. Therapeutic outcomes of HCC remain unsatisfactory, and novel treatments are urgently needed. GPC3 (glypican-3) is an emerging target for HCC, given the findings that 1) GPC3 is highly expressed in more than 70% of HCC; (2) elevated GPC3 expression is linked with poor HCC prognosis; and (3) GPC3-specific therapeutics, including immunotoxin, bispecific antibody and chimeric antigen receptor T cells. have shown promising results. Here, we postulate that GPC3 is a potential target of antibody-drug conjugates (ADCs) for treating liver cancer. To determine the payload for ADCs against liver cancer, we screened three large drug libraries (> 9,000 compounds) against HCC cell lines and found that the most potent drugs are DNA-damaging agents. Duocarmycin SA and pyrrolobenzodiazepine dimer were chosen as the payloads to construct two GPC3-specific ADCs: hYP7-DC and hYP7-PC. Both ADCs showed potency at picomolar concentrations against a panel of GPC3-positive cancer cell lines, but not GPC3 negative cell lines. To improve potency, we investigated the synergetic effect of hYP7-DC with approved drugs. Gemcitabine showed a synergetic effect with hYP7-DC in vitro and in vivo. Furthermore, single treatment of hYP7-PC induced tumor regression in multiple mouse models. Conclusion: We provide an example of an ADC targeting GPC3, suggesting a strategy for liver cancer therapy.

Optical approaches that visualize and manipulate biological processes have transformed modern biomedical research. An enduring challenge is to translate these powerful methods into increasingly complex physiological settings. Longer wavelengths, typically in the near-infrared (NIR) range (similar to 650-900 nm), can enable advances in both fundamental and clinical settings; however, suitable probe molecules are needed. The pentamethine and heptamethine cyanines, led by prototypes Cy5 and Cy7, are among the most useful compounds for fluorescence-based applications, finding broad use in a range of contexts. The defining chemical feature of these molecules, and the key chromophoric element, is an odd-numbered polymethine that links two nitrogen atoms. Not only a light-harvesting functional group, the cyanine chromophore is subject to thermal and photochemical reactions that dramatically alter many properties of these molecules. This Account describes our recent studies to define and use intrinsic cyanine chromophore reactivity. The hypothesis driving this research is that novel chemistries that manipulate the cyanine chromophore can be used to address challenging problems in the areas of imaging and drug delivery. We first review reaction discovery efforts that seek to address two limitations of long wavelength fluorophores: undesired thiol reactivity and modest fluorescence quantum yield. Heptamethine cyanines with an O-alkyl substituent at the central C4 ' carbon were prepared through a novel N- to O-transposition reaction. Unlike commonly used C4 '-phenol variants, this new class of fluorophores is resistant to thiol modification and exhibits improved in vivo imaging properties when used as antibody tags. We have also developed a chemical strategy to enhance the quantum yield of far-red pentamethine cyanines. Using a synthetic strategy involving a cross metathesis/tetracyclization sequence, this approach conformationally restrains the pentamethine cyanine scaffold. The resulting molecules exhibit enhanced quantum yield (Phi(F) = 0.69 vs Phi(F) = 0.15). Furthermore, conformational restraint improves interconversion between reduced hydrocyanine and intact cyanine forms, which enables super resolution microscopy. This Account then highlights efforts to use cyanine photochemical reactivity for NIR photocaging. Our approach involves the deliberate use of cyanine photooxidation, a reaction previously only associated with photodegradation. The uncaging reaction sequence is initiated by photooxidative chromophore cleavage (using wavelengths of up to 780 nm), which prompts a C-N bond hydrolysis/cyclization sequence resulting in phenol liberation. This approach has been applied to generate the first NIRactivated antibody-drug conjugates. Tumor uptake can be monitored in vivo using NIR fluorescence, prior to uncaging with an external irradiation source. This NIR uncaging strategy can slow tumor progression and increase survival in a MDA-MB-468-luc mouse model. Broadly, the vantage point of cyanine reactivity is providing novel probe molecules with auspicious features for use in complex imaging and drug delivery settings.

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.

Abstract Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody–photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab–IR700 (pan-IR700) and the NIR-releasing compound, CyEt–panitumumab–duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line in vitro. In in vivo studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor–background ratio. To evaluate the effects of combination therapy in vivo, tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.05), and significantly prolonged survival was achieved (P < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. Mol Cancer Ther; 17(3); 661–70. ©2017 AACR.

Near-IR photocaging groups based on the heptamethine cyanine scaffold present the opportunity to visualize and then treat diseased tissue with potent bioactive molecules. Here we describe fundamental chemical studies that enable biological validation of this approach. Guided by rational design, including computational analysis, we characterize the impact of structural alterations on the cyanine uncaging reaction. A modest change to the ethylenediamine linker (N,N'-dimethyl to N,N'-diethyl) leads to a bathochromic shift in the absorbance maxima, while decreasing background hydrolysis. Building on these structure function relationship studies, we prepare antibody conjugates that uncage a derivative of duocarmycin, a potent cytotoxic natural product. The optimal conjugate, CyEt-Pan-Duo, undergoes small molecule release with 780 nm light, exhibits activity in the picomolar range, and demonstrates excellent light-to-dark selectivity. Mouse xenograft studies illustrate that the construct can be imaged in vivo prior to uncaging with an external laser source. Significant reduction in tumor burden is observed following a single dose of conjugate and near-IR light. These studies define key chemical principles that enable the identification of cyanine-based photocages with enhanced properties for in vivo drug delivery.

An extract of Eudistoma sp. provided eudistidine C (1), a heterocyclic alkaloid with a novel molecular framework. Eudistidine C (1) is a racemic natural product composed of a tetracyclic core structure further elaborated with a p-methoxyphenyl group and a phenol-substituted aminoimidazole moiety. This compound presented significant structure elucidation challenges due to the large number of heteroatoms and fully substituted carbons. These issues were mitigated by application of a new NMR pulse sequence (LR-HSQMBC) optimized to detect four- and five-bond heteronuclear correlations and the use of computer-assisted structure elucidation software. Synthesis of eudistidine C (1) was accomplished in high yield by treating eudistidine A (2) with 4(2-amino-1H-imidazol-5-yl)phenol (4) in DMSO. Synthesis of eudistidine C (1) confirmed the proposed structure and provided material for further biological characterization. Treatment of 2 with various nitrogen heterocycles and electron-rich arenes provided a series of analogues (5-10) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided (+)-(R)-eudistidine C (1a) and (-)-(S)-eudistidine C (1b). The absolute configuration of these enantiomers was assigned by ECD analysis. (-)-(S)-Eudistidine C (1b) modestly inhibited interaction between the protein binding domains of HIF-1α and p300. Compounds 1, 2, and 6-10 exhibited significant antimalarial activity against Plasmodium falciparum.

Herein we report the syntheses and comparative photophysical, electrochemical, in vitro, and in vivo biological efficacy of 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-cyanine dye (HPPH-CD) and the corresponding indium (In), gallium (Ga), and palladium (Pd) conjugates. The insertion of a heavy metal in the HPPH moiety makes a significant difference in FRET (Forster resonance energy transfer) and electrochemical properties, which correlates with singlet oxygen production [a key cytotoxic agent for photodynamic therapy (PDT)] and long-term in vivo PDT efficacy. Among the metalated analogs, the In(III) HPPH-CD showed the best cancer imaging and PDT efficacy. Interestingly, in contrast to free base HPPH-CD, which requires a significantly higher therapeutic dose (2.5 ymol/kg) than imaging dose (0.3 ymol/kg), the corresponding In(III) HPPH-CD showed excellent imaging and therapeutic potential at a remarkably low dose (0.3 itmol/kg) in BALB/c mice bearing Colon26 tumors. A comparative study of metalated and corresponding nonmetalated conjugates further confirmed that STAT-3 dimerization can be used as a biomarker for determining the level of photoreaction and tumor response.

Near-infrared (NIR) fluorophores have several advantages over visible-light fluorophores, including superior light penetration in tissue and lower autofluorescence. We recently demonstrated that a new class of NIR cyanine dyes containing a novel C4'-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4'-O-aryl variants. We synthesized two NIR cyanine dyes with the same core structure but different indolenine substituents: FNIR-774 bearing four sulfonate groups and FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, resulting in an anionic (-3) or monocationic (+1) charge, respectively. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-774 and FNIR-Z-759 with panitumumab (pan) at antibody-to-dye ratios of 1:2 or 1:5. Conjugates of both dyes demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower background in mice, resulting in higher tumor-to-background ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-774 conjugates, especially in the abdominal region, regardless of the dye-mAb ratio. These results suggest that zwitterionic cyanine dyes are a promising class of fluorophores for improving in vivo optical imaging with antibody-NIR dye conjugates.

Despite significant progress in the clinical application of antibody drug conjugates (ADCs), novel cleavage strategies that provide improved selectivity are still needed. Herein is reported the first approach that uses near-IR light to cleave a small molecule from a biomacromolecule, and its application to the problem of ADC linkage. The preparation of cyanine antibody conjugates, drug cleavage mediated by 690nm light, and initial in vitro and in vivo evaluation is described. These studies provide the critical chemical underpinning from which to develop this near-IR light cleavable linker strategy.

New synthetic methods to rapidly access useful fluorophores are needed to advance modern molecular imaging techniques. A new variant of the classical Smiles rearrangement is reported that enables the efficient synthesis of previously inaccessible C4'-O-alkyl heptamethine cyanines. The key reaction involves N- to O-transposition with selective electrophile incorporation on nitrogen. A representative fluorophore exhibits excellent resistance to thiol nucleophiles, undergoes productive bioconjugation, and can be used in near-IR fluorescence imaging applications.

In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.

Cyanines are indispensable fluorophores that form the chemical basis of many fluorescence-based applications. A feature that distinguishes cyanines from other common fluorophores is an exposed polyene linker that is both crucial to absorption and emission and subject to covalent reactions that dramatically alter these optical properties. Over the past decade, reactions involving the cyanine polyene have been used as foundational elements for a range of biomedical techniques. These include the optical sensing of biological analytes, super-resolution imaging, and near-IR light-initiated uncaging. This review surveys the chemical reactivity of the cyanine polyene and the biomedical methods enabled by these reactions. The overarching goal is to highlight the multifaceted nature of cyanine chemistry and biology, as well as to point out the key role of reactivity-based insights in this promising area.

Experimental and computational studies indicate that the photodegradation of heptamethine cyanines proceeds via a regioselective photooxidative C–C cleavage reaction involving singlet oxygen and dioxetane intermediates.