Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy

The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable twocarbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygeninitiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool

Tetrazine-mediated bioorthogonal prodrug–prodrug activation

The selective and biocompatible activation of prodrugs within complex biological systems remains a key challenge in medical chemistry and chemical biology. Herein we report, for the first time, a dual prodrug activation strategy that fully satisfies the principle of bioorthogonality by the symbiotic formation of two active drugs. This dual and traceless prodrug activation strategy takes advantage of the INVDA chemistry of tetrazines (here a prodrug), generating a pyridazine-based miR21 inhibitor and the anti-cancer drug camptothecin and offers a new concept in prodrug activation.ISSN:2041-6520ISSN:2041-653

Development and Application of Strategies in Prodrug Chemistry

A novel strain-promoted 1,3-dipolarcycloaddition between trans-cyclooctene and a masked p-azidobenzyloxycarbonyl (PABC) pro-probe is introduced and its applications in a click-to-release strategy is discussed in Chapter 2. The reaction of trans-cyclooctenol (TCO-OH, 59) with a caged 7-hydroxycoumarin pro-probe 56 resulted in a rapid 1,3-dipolar cycloaddition with second-order rates of approx. 0.027 M-1s-1. 1H NMR studies indicated that activation proceeded via a triazoline and imine intermediate, both of which upon rapid hydrolysis released the uncaged probe. Doxorubicin prodrug 58 was found to be non-toxic to cells, however, the cytotoxicity was restored in vitro upon activation with 59. While the rates of the developed click reaction were comparable to some examples of the strained azide-alkyne cycloaddition reactions, it was still too slow to achieve low dosing in vivo. To improve the kinetics of bioorthogonal prodrug activation, a series of five fluorine substituted-azido-PABC pro-moieties 79-88 (masking 7-hydroxycoumarin and doxorubicin) are reported (Chapter 3). The rates of cycloaddition (with TCO-OH 59) escalated as the number of fluorine substituents on the PABC linker increase, with a tetra-fluoro-substituted pro-probe 81 exhibiting a 10-fold increase in reaction rate over the non-substituted PABC pro-probe 56. Additionally, the number of fluorine substituents determined how fast the drug released from the triazoline and imine intermediates that are formed in situ. Experimental results and ab initio calculations demonstrated that increasing the fluorine substituents lowered the transition-state energy for the conversion of the triazoline to the imine intermediate and also indicated that the rate-determining step could be the hydrolysis of the imine intermediate, potentially due to the drop in the predicted pKa values from 8.5 (for the non-substituted analogue) to 5.1 (tetrafluoro-substituted analogue). Modified PABC linkers used in Chapter 2 and Chapter 3 are ineffective at caging phenols as the linker is attached to the probe via a carbonate ester group, which is susceptible to chemical and enzymatic hydrolysis. To overcome these shortcomings, three cinnamyl ether linkers that are stable under physiological conditions and have varying rates of release (seconds-to-hours) are reported in Chapter 4. The -methyl-linker 134 can release phenols 7-hydroxycoumarin and etoposide (pKa 7.8 and 9.8 respectively) with a t1/2 < 2.5 min in a 1:1 aqueous-organic solvent. The slower releasing linker (-methyl) was found to release 7-hydroxycoumarin with a t1/2 = 54 min. Additionally, the activation studies for the -methyl linker 134 suggest the generation of a highly stable aza-cinnamyl-methide, which is significantly more stable than aza-PABC-methides generated. The (aza)-quinone-methides 131a-131c and 163, are the major by-products of the strategies involving PABC-self-eliminating linkers (Chapter 2 – Chapter 4). They have been widely underestimated in regards to their toxicity and are assumed to interact with the proximal water molecules to form p-aminobenzylalcohol 60. However, the poor nucleophilicity of water (under physiological conditions) can lead to the alkylation of a wide range of advantageous nucleophiles (e.g. DNA) leading to unwanted side effects (e.g. cancer). Chapter 5 reports the design and synthesis of a nucleophile appended PABA-based self-eliminating linker 167. Upon activation, the attached model-probe is released by a 1,6-elimination. The nitrogen nucleophile that is appended to the linker trapped the generated azaquinone-methide, forming a tetrahydroisoquinolone that could be isolated by HPLC and detected using MS. The proof-of-principle studies indicate that the nucleophile trap is capable of mopping-up the (aza)quinone-methide. In summary, this thesis describes the development of a number of novel self-immolative strategies that can be triggered by biorthogonal or other reactions for use in produg science

Development and Application of Strategies in Prodrug Chemistry

A novel strain-promoted 1,3-dipolarcycloaddition between trans-cyclooctene and a masked p-azidobenzyloxycarbonyl (PABC) pro-probe is introduced and its applications in a click-to-release strategy is discussed in Chapter 2. The reaction of trans-cyclooctenol (TCO-OH, 59) with a caged 7-hydroxycoumarin pro-probe 56 resulted in a rapid 1,3-dipolar cycloaddition with second-order rates of approx. 0.027 M-1s-1. 1H NMR studies indicated that activation proceeded via a triazoline and imine intermediate, both of which upon rapid hydrolysis released the uncaged probe. Doxorubicin prodrug 58 was found to be non-toxic to cells, however, the cytotoxicity was restored in vitro upon activation with 59. While the rates of the developed click reaction were comparable to some examples of the strained azide-alkyne cycloaddition reactions, it was still too slow to achieve low dosing in vivo. To improve the kinetics of bioorthogonal prodrug activation, a series of five fluorine substituted-azido-PABC pro-moieties 79-88 (masking 7-hydroxycoumarin and doxorubicin) are reported (Chapter 3). The rates of cycloaddition (with TCO-OH 59) escalated as the number of fluorine substituents on the PABC linker increase, with a tetra-fluoro-substituted pro-probe 81 exhibiting a 10-fold increase in reaction rate over the non-substituted PABC pro-probe 56. Additionally, the number of fluorine substituents determined how fast the drug released from the triazoline and imine intermediates that are formed in situ. Experimental results and ab initio calculations demonstrated that increasing the fluorine substituents lowered the transition-state energy for the conversion of the triazoline to the imine intermediate and also indicated that the rate-determining step could be the hydrolysis of the imine intermediate, potentially due to the drop in the predicted pKa values from 8.5 (for the non-substituted analogue) to 5.1 (tetrafluoro-substituted analogue). Modified PABC linkers used in Chapter 2 and Chapter 3 are ineffective at caging phenols as the linker is attached to the probe via a carbonate ester group, which is susceptible to chemical and enzymatic hydrolysis. To overcome these shortcomings, three cinnamyl ether linkers that are stable under physiological conditions and have varying rates of release (seconds-to-hours) are reported in Chapter 4. The -methyl-linker 134 can release phenols 7-hydroxycoumarin and etoposide (pKa 7.8 and 9.8 respectively) with a t1/2 < 2.5 min in a 1:1 aqueous-organic solvent. The slower releasing linker (-methyl) was found to release 7-hydroxycoumarin with a t1/2 = 54 min. Additionally, the activation studies for the -methyl linker 134 suggest the generation of a highly stable aza-cinnamyl-methide, which is significantly more stable than aza-PABC-methides generated. The (aza)-quinone-methides 131a-131c and 163, are the major by-products of the strategies involving PABC-self-eliminating linkers (Chapter 2 – Chapter 4). They have been widely underestimated in regards to their toxicity and are assumed to interact with the proximal water molecules to form p-aminobenzylalcohol 60. However, the poor nucleophilicity of water (under physiological conditions) can lead to the alkylation of a wide range of advantageous nucleophiles (e.g. DNA) leading to unwanted side effects (e.g. cancer). Chapter 5 reports the design and synthesis of a nucleophile appended PABA-based self-eliminating linker 167. Upon activation, the attached model-probe is released by a 1,6-elimination. The nitrogen nucleophile that is appended to the linker trapped the generated azaquinone-methide, forming a tetrahydroisoquinolone that could be isolated by HPLC and detected using MS. The proof-of-principle studies indicate that the nucleophile trap is capable of mopping-up the (aza)quinone-methide. In summary, this thesis describes the development of a number of novel self-immolative strategies that can be triggered by biorthogonal or other reactions for use in produg science

Structure-Activity Relationships of Antibody-Drug Conjugates: A Systematic Review of Chemistry on the Trastuzumab Scaffold

Antibody-drug conjugates (ADCs) are a rapidly growing class of cancer therapeutics. The goal of ADCs is to overcome the low therapeutic index of conventional cytotoxic agents. However, realizing this goal has been a significant challenge. Consisting of an antibody linked to a therapeutic payload, ADCs comprise many components which can be modified, including the target, payload, linker, and bioconjugation method. Many approaches have been developed to improve the physical properties, potency, and selectivity of ADCs. The anti-HER-2 antibody trastuzumab, first approved in 1998, has emerged as an exceptional targeting agent for ADCs, as well as a broadly used platform for testing new technologies, The extensive work in this area enables the comparison of various linker strategies, payloads, drug-to-antibody ratios (DAR), and mode of attachment. In this review, these conjugates, ranging from the first clinically approved trastuzumab ADC, Kadcyla (T-DM1) to the latest variants, are described with the goal of providing a broad overview and comparison of existing and emerging conjugate technologies.</jats:p

Structure-Activity Relationships of Antibody-Drug Conjugates: A Systematic Review of Chemistry on the Trastuzumab Scaffold

Antibody-drug conjugates (ADCs) are a rapidly growing class of cancer therapeutics. The goal of ADCs is to overcome the low therapeutic index of conventional cytotoxic agents. However, realizing this goal has been a significant challenge. Consisting of an antibody linked to a therapeutic payload, ADCs comprise many components which can be modified, including the target, payload, linker, and bioconjugation method. Many approaches have been developed to improve the physical properties, potency, and selectivity of ADCs. The anti-HER-2 antibody trastuzumab, first approved in 1998, has emerged as an exceptional targeting agent for ADCs, as well as a broadly used platform for testing new technologies, The extensive work in this area enables the comparison of various linker strategies, payloads, drug-to-antibody ratios (DAR), and mode of attachment. In this review, these conjugates, ranging from the first clinically approved trastuzumab ADC, Kadcyla (T-DM1) to the latest variants, are described with the goal of providing a broad overview and comparison of existing and emerging conjugate technologies

Core remodeling leads to long wavelength fluoro-coumarins

Fluoro-Coumarins are a novel class of far-red and near-infrared solvent sensitive dyes of exceptionally low molecular weight.</p

Preparation of Fluoroalkenes via the Shapiro Reaction: Direct Access to Fluorinated Peptidomimetics

Fluoroalkenes represent a useful class of peptidomimetics with distinct biophysical properties. Current preparations of this functional group commonly provide mixtures of E- or Z-fluoroalkene diastereomers, and/or mixtures of nonfluorinated products. To directly access fluoroalkenes in good stereoselectivity, a Shapiro fluorination reaction was developed. Fluoroalkene products were accessed in one- or two-step sequences from widely available ketones. This strategy should be useful for the preparation of fluorinated analogs of peptide-based therapeutics, many of which would be challenging to prepare by alternate strategies