table # in ‘section’ Table [2.2.0].1 Major CA cysteine protease inhibitors, categorized by their mechanism of attack ...12 Table [2.2.0].2 Comparison of the inhibition constants of di
About the Investigation
Significance of the Investigation
Cysteine proteases of the CA clan, a 13-member family that includes calpains and papain-like enzymes, support normal cellular function and structure by driving lysosomal hydrolysis and tissue homeostasis They have unique active-site properties and tissue-specific expression patterns that shape their roles in health and disease Of these enzymes, cathepsins K, B, and S are especially critical, directly mediating osteoclastic bone resorption and macrophage invasion When CA proteases are imbalanced or mislocalized, their dysregulated activity can disrupt normal cellular processes and tissue integrity, contributing to the progression of osteoporosis, atherosclerosis, and various cancers of the lung, breast, and prostate, among other systemic diseases For this reason, CA clan cysteine proteases hold high medical significance.
Cathepsin K (CST-K) plays a pivotal role in bone remodeling and function, and its upregulation and mislocalization drive osteoporosis and bone metastases in lung, breast, and prostate cancers CST-K is more highly expressed in bone marrow macrophages and stromal cells within the metastatic microenvironment than in the primary tumor, making metastatic bone cells an attractive target for tumor reduction Studies show that growth and progression of intratibial prostate carcinoma cells (PC3) are significantly reduced in cathepsin K knockout mice compared with wild-type controls, confirming its importance in bone metastasis.
Cancer is a malignant tumor that results from uncontrolled division of genetically mutated cells and, worldwide, it claims more than 17,000 lives daily, making it the focus of extensive research by teams and pharmaceutical companies Clinically, there are many strategies to combat the 100-plus cancer types, including surgery, immunotherapy, laser therapy, stem cell transplant, hyperthermia, photodynamic therapy (PDT), and blood transfusion; among the most common are orally administered chemotherapy and radiation therapy However, oral treatments often cannot achieve location-specific inhibition, and while their lipophilic character increases cell permeability, accumulation and anti-target inhibition occur once they are ionized and trapped inside the cell due to the high homology of the CA proteases, leading to poor therapeutic efficacy, dose escalation, drug resistance, and intensified side effects Radiation therapy uses high doses of radiation to kill cancer cells but causes side effects such as hair loss, nausea, and motion sickness.
To minimize risks and costs in future therapies, optimizing drug exposure is essential As researchers explore current methodologies, new technologies are continually emerging, including photo-activated chemotherapy (PACT) In this approach, a drug is caged to a chaperone, enabling spatial and temporal control over drug delivery at the infected site The localized abnormal proteolysis by CST-K makes it a prime target for PACT.
This study describes the investigation of a PACT RuII complex that enables spatiotemporal control of drug release and exposure for protein inhibition, advancing a strategy with potential to impact cancer pharmacology The approach has been under development by this research team for about five years, with pharmacological interest in cancer treatment remaining central Previously, inhibitors were bound to the chaperone via a ruthenium–nitrile (RuII–N≡CR) dative bond, a binding mode that the current work seeks to supersede with more precise activation and delivery.
‡ Attached by a dative or covalent bond § A Ru II complex, organic protecting group, nanoparticle, or vesicle that can be separated from a drug by photolysis
However, as a result of the instability of these complexes in growth media, a new binding arrangement was sought that will produce similar or better dissociative quantum yields, dark/light
IC50 ratios (DLIR), binding affinities, and growth-media stability were evaluated for derivatives of known cysteine protease inhibitors The derivatives were synthesized and assessed for inhibition of recombinant human cathepsin K, with the most potent derivative subsequently complexed to a Ru(II) chaperone via a pyridyl substituent and analyzed under both dark and light conditions Light-induced photo-activation of the Ru(II)-inhibitor complex releases the cathepsin K inhibitor, enabling controlled delivery and activity.
** DLIR = IC 50 (pre-irradiation) ÷ IC 50 (post-irradiation)
†† [ ] = complex; exempli gratia: [Ru II ] = Ru II complex
Overview of the Investigation
The project aimed to assess the impact of a new Ru(II)–inhibitor binding arrangement on CST-K inhibition by comparing Ru(II)–N≡CR binding with Ru(II)–pyR coordination To enable the dative (L-type) binding required for photodissociation, an N-heteroatom was introduced into a known CST-K inhibitor by substituting the parent inhibitor’s phenyl groups with a pyridyl substituent, yielding two derivatives, 2 and 3 These derivatives were synthesized to explore how increasing ligand basicity influences CST-K inhibition.
Figure [1.2.0].1 The inhibitors being investigated
To do this, I synthesized a previously established compound (1) and two new derivatives
Enzyme inhibition assays identified the most potent derivative, which was caged onto a RuII chaperone to form complex 4 (Fig 1.2.0.2) After thorough characterization and photolysis evaluation, CST-K inhibition was measured in the dark (pre-irradiation) and in the light (post-irradiation) The resulting inhibition profiles were then compared to those reported in previous studies.
Figure [1.2.0].2 The Ru II complex being investigated.
Review of the Literature
Enzymes
Proteases are lysosomal proteolytic enzymes that regulate and enable a wide range of physiological processes, from immune responses and growth to cell signaling They fall into five main families defined by their active site residues: aspartic proteases (e.g., CST-D), serine proteases (e.g., trypsin), threonine proteases (e.g., ornithine acetyltransferase), cysteine proteases (e.g., CST-K), and metalloproteases (e.g., gluzincins) These enzymes are tightly regulated and responsible for cleaving substrates necessary for cellular and structural maintenance When proteolysis is unregulated or mislocalized, it can drive disease processes such as inflammation, emphysema, and cancer.
Proteases are organized into four main clans based on structure, fold, and function: papain- and calpain-like proteases (CA); caspases and related proteases (CD); picornaviral and related proteases (PA); and carboxy- and related proteases as organized by Rawlings and Barrett.
These enzymes typically possess an active-site nucleophile, such as a thiol or hydroxyl, and a basic residue that can act as an acid during catalysis Cysteine proteases remain stable under the acidic conditions found in abnormal cells and at sites of bone resorption They are usually recognized and denoted by their S2, S1, and S1' binding sites according to the Schechter and Berger method, as shown in Fig 2.1.1.
2.1.2CAPAPAIN-LIKE (CA)CYSTEINE PROTEASES
Cysteine proteases are enzymes found in animals, plants, and microorganisms, sharing similar sequences and folds This family includes plant proteases such as papain, parasite proteases like cruzipain, and mammalian lysosomal cathepsins There are 13 described lysosomal cathepsins, of which 11 are sequentially known and are expressed in various cells within lysosomes or extracellularly The named members are CST-B, CST-C, CST-F, CST-H, CST-K, CST-L, CST-O, CST-S, CST-V, CST-W and CST-X, while the two that remained sequentially unknown have been identified as CST-N and CST-T.
Of the sequentially known cathepsins, CST-B and CST-L have the highest lysosomal concentation 27 CST- V and S are tissue-specific CST-V is found in the testis/thymus 27 and CST-
CST-F, CST-L, and CST-X are widely distributed in lymphatic tissues, including lymph nodes and the spleen Among CA proteases, CST-L shows the highest proteolytic activity among lysosomal proteases The majority of these enzymes are endopeptidases, with CST-C representing an exception.
Cysteine proteases are an oligomeric family of enzymes that function outside the lysosome, with CST-B, CST-H, CST-L, CST-S, CST-X and CST-C showing distinct activities CST-B and CST-X can act as carboxypeptidases, CST-H as an aminopeptidase, and these proteases process key proteins beyond canonical lysosomal roles, such as thyroglobulin, a thyroid hormone source In addition to proteolysis, cysteine proteases participate in apoptosis (CST-B and CST-C) and in MHC class II–mediated antigen presentation (CST-S and CST-L) Mislocalization of these enzymes is linked to pathologies including Alzheimer’s disease, multiple sclerosis, muscular dystrophy, cancers and bone-related diseases Notably, CST-B, CST-H and CST-L promote cancer progression by directly degrading the extracellular matrix or by activating other proteases, thereby facilitating invasion and metastasis Their substrate interactions follow Schechter–Berger subsites, where substrate residues bind to the corresponding active sites, defining the binding modes that drive their proteolytic activity.
Enzyme–inhibitor binding interactions show that the S2 and S1' binding sites contribute most to the selectivity and diversity of these enzymes Interaction of the P2, P1, and P1' inhibitor residues with their respective enzyme subsites involves both main- and side-chain atoms, whereas interaction with P3 involves only side-chain atoms Structure characterization has revealed that these binding patterns underlie binding affinity and specificity, offering actionable insights for inhibitor design and optimization.
In cysteine proteases, binding sites beyond S4 and S3' are not present, although P4 and P5 residues can interact with the enzyme Consequently, the substrate binding site is typically defined by the S2-S2' region.
CST-K is a significant lysosomal cysteine protease, whose recombinant form was characterized in 1996 after CST-B, CST-H, and CST-L The human CST-K enzyme comprises 329 amino acids, including a 215-amino-acid catalytic domain, a 99-amino-acid propeptide, and a 15-amino-acid N-terminal sequence, with its X-ray crystal structure shown in Fig 2.1.3 Inhibitors for this enzyme are rarely selective because the amino acid sequence is about 60% identical to CST-L, CST-S, and CST-V, and about 35% identical to CST-B, CST-F, CST-H, CST-O, and CST-W All cathepsins share conserved features across the family.
The Major Histocompatibility Complex (MHC) common fold features two domains—the N-terminal and the C-terminal—and forms a V-shaped active-site cleft between the S1 and S1' subsites, as depicted in Figure 2.1.1; enzymes bind substrates in this pocket, the active site, which is lined with specific amino acid residues and subsites also shown in Figure 2.1.1 In CST-K, the catalytic site comprises His159, Cys25, and Asn175, and enzymatic cleavage occurs at this catalytic region within the V-shaped cleft The surrounding subsites are likewise occupied by amino acids, shaping the substrate-binding environment For example, the S2 subsite is defined by Tyr67, Met68, Ala133, Leu157, Ala160, and Leu205 according to papain residue numbering.
Cathepsin K is predominantly expressed in macrophages and osteoclasts, with macrophages showing roughly a 100-fold higher concentration than Cathepsins L and S After diffusing across the cell membrane, Cathepsin K is ionized and sequestered by lysosomes, yet it is transported between endosomes, lysosomes, and phagosomes It can also be secreted as inactive glycosylated preproenzyme precursors from osteoclasts, macrophages, fibroblasts, and malignant cells, and can be sequestered into the extracellular resorption lacuna.
Cathepsin K (CST-K) plays a major role in skeletal development and bone remodeling by driving collagen degradation during bone resorption Deficiency of CST-K can lead to pycnodysostosis, a condition of abnormally dense bones caused by reduced bone resorption Inhibition of CST-K causes accumulation of undigested collagen fibrils in lysosomes, highlighting its central role in collagenolysis While CST-K has regulated functions in normal cells, its primary responsibilities are collagenolytic activity and bone maintenance, making it a critical bone-resorbing protease that degrades type I and II collagen as well as bone matrix proteins like osteopontin and osteonectin After acid-induced demineralization of bone, CST-K is secreted from osteoblasts, promoting proteolytic degradation of the organic matrix, notably type I collagen fibers When CST-K activity is misregulated in abnormal cells, its imbalance and secretion are associated with pathological conditions and diseases including rheumatoid arthritis, osteoporosis, atherosclerosis, inflammation, and cancer Increased CST-K secretion and corresponding collagen matrix degradation drive osteoporotic processes, and CST-K expression is upregulated by RANKL during osteoclastogenesis and down-regulated by estrogen.
Figure [2.1.3].1 Crystal structure of Cathepsin K (A) by amino acid residue (JSmol-Javascript), (B) by secondary structure (PV-WebGL) Image from the RCSB PDB (www.rcsb.org) of PDB ID 5TUN (S Law, P.M Andrault, A Aguda, N Nguyen, N Kruglyak, G Brayer, D Bromme (2017) Identification of mouse cathepsin
K structural elements that regulate the potency of odanacatib Biochem J 474: 851-864)
Inhibitors
The ideal cysteine protease inhibitor has a low molecular weight, minimal peptide character, high selectivity, high specificity, lipophilic character (to allow cell permeability) and reversible target-binding ability (to avoid antigenic and immunologic complications) 11 Additionally, inhibitors are most useful if they have good pharmacokinetic properties such as good oral bioavailability, high membrane permeability, low toxicity, slow elimination rate and align with Lipinski’s rule of 5 Most importantly, inhibitors should be unreactive under physiological conditions, but selective and reactive enough to interact with the target Selectivity of the cysteine protease inhibitors is one of the most important and difficult properties to design due to the high homology of the CA clan Successful and selective inhibitors of CST-K are used to treat osteoporosis, osteoarthritis, as well as, metastatic bone diseases associated with breast, lung, thyroid and prostate cancers
The peptide aldehydes, leupeptin and antipain (5 & 6; Fig [2.2.0].1), were the first synthetic cysteine protease inhibitors designed 11 They are capable of inhibiting both serine and cysteine proteases E64 and peptidyl diazomethanes (7 & 8; Fig [2.2.0].1) were synthesized shortly after, for the selective inhibition of CST- B, H and L These were the only known cathepsins of the time
Protease inhibitors can suppress enzyme activity through diverse mechanisms—alkylation, acylation, phosphonylation, sulfonylation, metalloid binding, hemiacetal formation, pinner-type reactions, and other interactions (see Table 2.2.0) Given CST-K’s extracellular localization in cancers and metastases, non-lysosomotropic inhibitors may offer greater selectivity by limiting inhibition of intracellular CSTs.
Peptide Phosphonates Phosphonyl Fluorides Sulfonyl Fluorides Metalloid binding Hemiacetal formation
Table [2.2.0].1 Major CA cysteine protease inhibitors, categorized by their mechanism of attack
Irreversible covalent binders, including epoxysuccinyl derivatives, ketones, and vinyl sulfones, are often impractical for many pharmacological applications, yet they have proven particularly useful in combating drug-resistant cell lines and diseases At the same time, compounds bearing reactive functional groups may trigger acute or delayed toxic responses by binding glutathione and/or DNA, underscoring the need for thorough study The inhibitors described above are commonly referred to as covalent modifiers because they inhibit cysteine residues in their targets.
Figure 2.2.0.1 illustrates four of the first protease inhibitors synthesized, which inhibit proteases by forming either irreversible or reversible covalent bonds By contrast, some inhibitors lack an electrophilic warhead and are classified as non-covalent modifiers Among these, arylaminoethylamides (compound 9 in Figure 2.2.0.2) are the most common and were previously investigated by Novartis Pharma.
Pinner-type inhibitors have drawn significant interest because their nitrile warhead covalently binds the substrate, conferring potent and selective inhibition The authentic Pinner reaction is an acid-catalyzed reaction between a nitrile and an alcohol that yields an amidine or an ester; in contrast, enzyme–inhibitor Pinner-type reactions are reversible, forming a thioimidate adduct that dissociates to regenerate the nitrile and substrate Three major, structurally related classes exist: cyanamides, aryl-nitriles, and aminoacetonitriles Cyanamides (R2N-C≡N) feature a nitrile linked to an N-heteroatom, which increases electrophilicity through inductive effects and enhances CST-K activity, though selectivity remains moderate In aryl-nitriles (R5Ph-C≡N), the warhead is appended to an aromatic ring, and electrophilicity increases with increasingly electron-withdrawing substituents on the ring.
Figure [2.2.0].2 Pinner-type inhibitors, the first aminoacetonitrile inhibitors and an example of an azapeptide inhibitor. withdrawing substituents and heterocycles In aminoacetonitriles (R2N-R2C-C≡N; exempli gratia
Nitrile warheads terminating at an amino group act as potent, selective inhibitors of cysteine proteases, though electrophilicity varies from intermediate to low among compounds Clinically developed odanacatib (17) exemplifies this inhibitor class The first nitrile-based cysteine protease inhibitors were acetamidoacetonitrile (12) and benzamidoacetonitrile (13) Currently, dipeptide aminonitriles and azapeptides (14) are being competitively developed for CST-K inhibition.
In azapeptides, the CH group of a peptide is replaced by an N-heteroatom When this is done at
Compared with the carboanalogue, a more potent and stable inhibitor was observed, enhancing potential therapeutic efficacy against target enzymes Increasing the nitrile's electrophilicity and reactivity can promote irreversible covalent binding to cysteine proteases, a mechanism linked to sustained inhibition Naturally occurring CST-K inhibitors, including proteins from the cystatin, stefin, kininogen, thyropin, serpin families, and alpha-2-macroglobulin, illustrate the diversity of endogenous protease inhibitors that regulate cysteine proteases.
To date, three of the most promising CST-K inhibiting drugs were Balicatib (15), Relacatib
(16) and Odanacatib (17), see Fig [2.2.1].3 11 Balicatib (AAE-581) is a highly potent, basic peptidic nitrile having an IC50 of 1.4 nM with, however, low selectivity in cell based assays 2, 11 In
Novartis halted phase II testing of a drug in 2006 after the appearance of rare dermatological adverse events, including lesions, pruritus, and skin thickening Relcatib (SB-462795) is a monobasic, highly potent azepanone analogue developed by GlaxoSmithKline, with a Ki(app) of 41 pM and about 89% oral bioavailability among other favorable pharmacokinetic properties; its development toward approval was discontinued in 2007, possibly due to low selectivity, lysosomal accumulation, and off-target effects Odanacatib (MK-0822) showed promise and remained on a path toward approval up to 2016, as a non-basic, non-lysosomotropic, nitrile-based inhibitor with high potency and selectivity for cathepsin K.
An IC50 of 0.2 nM demonstrates high potency, with selectivity attributed to the 4-fluoroleucine side chain at the P2 position The compound also displayed favorable pharmacokinetic properties, including a long half-life and slow elimination and metabolism After 12 years of clinical development, Merck & Co discontinued further development due to an increased risk of cardiovascular events observed in clinical patients.
This thesis is based on the use of nitriles inhibitors, because they are both selective for the
Within the CA cysteine protease clan, inhibitor structure dictates how binding occurs as the inhibitor locks into the active-site cleft and is stabilized by several amino acid residues, where it is engaged by the catalytic thiol Nitrile inhibitors show higher inhibitory activity against cysteine proteases than serine proteases, a pattern explained by several theories: the Hard-Soft Acid-Base (HSAB) principle predicts that the soft cyano (C≡N) electrophile binds more tightly to the soft sulfur nucleophile than to the hard oxygen nucleophile; peptide nitriles reversibly bind the cathepsin thiol through a covalent thioimidate linkage, formed when the thiol attacks the nitrile carbon to yield a trigonal planar thioimidate that resists hydrolysis and resembles an acyl-enzyme intermediate rather than the hydrolysis-prone tetrahedral intermediate of carbonyls; a third factor involves transition-state stabilization of the nitrile pathway, contributing to the overall potency of nitrile inhibitors against cysteine proteases.
Figure 2.2.0.3 shows three clinically developed CST-K inhibitors The interaction in nitrile inhibition is less constrained than the serine-nitrile transition state because the sulfur-containing heteroatom has a large atomic radius, making the adduct more likely to revert than to hydrolysis This contrasts with serine proteases, where the small size of the oxygen atom can steer the reaction toward irreversible hydrolysis of the substrate Additionally, the nitrile-inhibition intermediate does not require hydrogen-bond stabilization since no oxyanion hole is formed as with carbonyl inhibition.
Figure 2.2.0.4 depicts the X-ray co-crystal structure in which inhibitor 10 sits in the active-site cleft of cathepsin K (CST-K) and is bound by Cys139 through a reversible Cys139-S-C=N-R bond Panel A shows the surface representation, Panel B provides a magnified frontal view of the active site, and Panel C highlights the Cys139-S-C=N-R bond from a magnified side perspective This image is sourced from the RCSB Protein Data Bank (www.rcsb.org) under PDB ID 1YK8, based on the 2005 study by Barretta et al describing acyclic cyanamide-based inhibitors of cathepsin K.
Figure [2.2.0.].6 Depiction of the intermediate oxyanion hole that results from enzyme-carbonyl interactions
Structural implications of the dipeptide inhibitor are substantial: Lüser et al show that the P2 residue dictates different IC50 values for cathepsins K, L, and S depending on the substituent’s nature (aliphatic/aromatic, bulky/compact, short/long, etc.), with P2 exerting greater influence than P1 or P3 For cathepsin K, the leucinyl group at P2 yields the highest affinity for subsite S2, and the phenylalanyl substituent also demonstrates favorable binding and inhibitory activity Table 2.2.0.1 indicates a specific length and bulk requirement for strong S2 binding, while the IC50 increases with aromatic bulk on the P3 N-terminal group, and inhibitory activity is enhanced when a carboxybenzyl (Cbz) substituent is used at P3 rather than a tert-butyloxycarbonyl (Boc) substituent Hydrogen-bond donation from the N-H amide backbone significantly contributes to adduct stabilization.
Figure 2.2.0.5 outlines a proposed mechanism for cysteine versus serine inhibition Methylation of the amide N-heteroatom (N-Me) markedly reduces the inhibitor’s affinity for cells expressing CST-K, by about three orders of magnitude The data also show that strong cooperativity between covalent and non-covalent inhibitor–protease interactions plays a significant role, even though the observed inhibition is time-independent CST-K displays substrate specificity with leucine favored over phenylalanine at the P2 position Inhibition of cathepsin K with a basic P3 substituent increases enzyme selectivity and lysosomal concentration, a rise that can boost anti-target potency but may reduce selectivity over time (reference 30).
Table [2.2.0].2 Comparison of the inhibition constants of dipeptide inhibitors by the length and bulk of P 2 binding site.
Photo-caging: Metal-Based Alternatives
Research on organic-based chromophores and fluorophores far exceeds studies of metal-based systems, and these organic options are continually developed as alternatives to metals in renewable energy and medicine Like metal-based chromophores, organic chromophores enable controlled, light-assisted protein inactivation and other cellular functions Among the most promising and longstanding approaches are CALI and FALI (chromophore-assisted light inactivation and fluorescence-assisted light inactivation).
Chromophore/ Fluorophore-assisted light inactivation 42-46 This methods has primarily been used to elucidate biological structure and function; it is useful in high-throughput applications because of its characteristic efficiency
Protein inhibition can be achieved by using an antibody labeled with malachite green dye that binds the target protein; irradiation with laser light of suitable energy excites the dye, which then generates singlet oxygen and irreversibly damages the bound protein along with nearby proteins, though inactivation is confined to the antibody-bound target This photodynamic strategy has been applied to a range of proteins, including membrane receptors, components of signal transduction pathways, and transcription factors.
Fluorescein is commonly used for protein labeling due to its water solubility and stability at pH 7 and above, and it is commercially available as an isothiocyanate derivative for convenient conjugation Its UV–visible absorbance increases linearly with dye concentration When fluorescein is bound to an antibody in an air-saturated solution, the triplet lifetime increases slightly, in contrast to the decrease observed under anaerobic conditions, indicating that oxygen collisions can quench the chromophore’s excited states Binding the antibody provides spatial shielding that helps preserve photo-efficiency by limiting oxygen quenching, but the method remains susceptible to light-induced protein inactivation depending on irradiation energy, enzyme context, fluorophore concentration, and oxygen quenching, among other factors Photo-bleaching is an undesired side reaction independent of protein inactivation.
Organic fluorophores have also been used as drug chaperones or photo-responsive drug delivery systems (PDDSs), affording spatio-temporal control over biological activity or inhibition
A wide range of organic groups are useful for application as PDDSs 47-48 , these include but are not limited to o-nitrobenzyl 15 , acridin-9-ylmethyl 49 , acetylpyrene 50 , hydroxyquinoline 51 , hydroxylnapthyl 52 and coumarinyl 19, 53 derivatives
BODIPY-derived photo-caging groups have emerged as an attractive alternative to traditional o-nitrobenzyl cages due to their superior optical properties, ease of synthesis, and biocompatibility (Figure 2.3.1) Goswami et al reported successful photo-release of a carboxylic acid from an organic chaperone using visible light, with a concomitant increase in fluorescence under green light excitation—likely because the carboxylate quenches fluorescence when attached, an effect that is relieved upon release.
Fig [2.3.1].1 Organic photo-activatable molecules described in this section
Hybrid strategies now leverage dual PDDS platforms that enable the simultaneous photo-triggered release of two therapeutics Venkatesh and colleagues explored the acetylcarbazole framework in combination with carboxylic acids and amino acids, highlighting how carbazole units can support dual delivery Carbazoles are attractive PDDS components due to their wide band gaps, high luminescence efficiency, and flexible functionalization that facilitates fluorescence-guided delivery Acetylcarbazole, depicted as a representative agent, functions both as a fluorescent imaging probe and as a prospective PDDS element In this system, caffeic acid, with anti-tumor and anti-metastatic activity, and chloroambucil, a methylating agent used in leukemia, serve as the photo-releasable cargo, as illustrated in the referenced figure.
Synergistic effects are reported in the system After 60 minutes of UV irradiation at 365 nm, 91% and 94% of the drugs were released, with quantum yields of 0.046 and 0.051, respectively A drawback noted is the photo-decomposition of the free carbazole following the same irradiation The study also reports biocompatibility and cellular uptake, demonstrated by confocal microscopy in U87MG glial cancer cells, with cell viability remaining high before irradiation but decreasing after exposure.
Organic-CALI enables the generation of chimera molecules that can selectively bind and inhibit target proteins, but polypyridyl-[Ru II]-CALI outperforms it in several respects Both organic- and Ru II-based CALI systems are highly efficient for time-resolved studies of cellular dynamics, yet organic chromophores suffer from self-bleaching, poor cell permeability, and generally lower affinity and selectivity for the proteins of interest In contrast, polypyridyl-[Ru II]-CALI chromophores resist photo- and self-bleaching (stable to singlet oxygen), are cell-permeable, and can quantitatively inactivate selected proteins They also feature a longer triplet-state lifetime, higher turnover of singlet oxygen, and stronger affinity to synthetic binding proteins due to advantageous photophysical and photochemical properties.
Lanthanide-doped up-converting nanoparticles (LD-UCNPs) are increasingly studied for spatio-temporal controlled therapy due to their anti-Stokes up-conversion, a photophysical process in which a photon is up-converted to a higher energy level by the lanthanide ions This up-conversion, rooted in the distinctive lanthanide photophysics, has long been exploited in renewable energy and data storage and is now being probed for biological applications LD-UCNPs are renowned for high photostability, deep tissue penetration, and activation with low-energy near-infrared (NIR) light, features that make them especially attractive for bio-imaging and for accommodating a diverse range of functional groups.
LD-UCNPs offer a versatile platform for studying biological processes, regenerative medicine, and cell isolation therapies, enabling non-invasive, spatio-temporally controlled modulation of cell–material interactions through photo-controlled cell adhesion In this approach, cells are immobilized to TmYb-doped, silica-coated NaYF4 nanoparticles via a photo-cleavable linker, a polyethylene glycol spacer, and a bio-adhesive ligand before irradiation The up-conversion property is essential because ultraviolet light is required to trigger the controlled photo-release of adhered cells As a result, near-infrared (NIR) light can be used to activate UV emission through up-conversion, enabling precise, light-triggered manipulation of cell adhesion.
980 nm irradiation activates LD-UCNP to emit UV light locally, triggering cleavage of photo-labile linkers and changing the surface cell-binding state to dissociate adhesive cells Direct UV light suffers from limited tissue penetration and can induce cellular and DNA damage; by contrast, this upconversion approach offers deep tissue penetration with minimal cell damage The rate and total number of cells released can be tuned by adjusting the exposure duration and the light power used.
LD-UCNPs are being developed for uncaging photo-labile compounds and for bioluminescence imaging studies Yang et al report using the same upconversion nanoparticles (UCNPs) described earlier to enhance bio-imaging protocols, demonstrated with a D-luciferin–conjugated LD-UCNP that integrates the luciferin substrate with the LD-UCNP platform to boost luminescent signals and imaging performance.
Figure 2.3.2.1 shows a system activated by near-infrared (NIR) irradiation, which converts the energy into UV emission to trigger the release of D-luciferin The released D-luciferin enhances fluorescence and bioluminescence signals in vitro and in vivo, with tissue penetration reaching up to 10 mm This non-invasive approach enables real-time monitoring of dynamic biological systems.
Other methods to improve the bio-imaging and photolysis protocols include multiphoton photolysis 20, 58-60 , uncaging of biological stimuli from nanocapsules 61 and photo-induced electron transfer 62
Two-photon photolysis is the most common form of multiphoton photolysis, and it occurs when a photoactivated system simultaneously absorbs two photons from pulsed, low-energy light in the visible, near-infrared (NIR), or far-infrared (FIR) range, enabled by sufficiently intense laser illumination.
PEG, a polyethylene glycol linker, is used with the D-luciferin–conjugated LD-UCNP described above The excitation of this construct yields results equivalent to single-photon excitation, yet the processes are quantum-mechanically different: single-photon excitation is linear, while multiphoton (two-photon) excitation is quadratic, with fluorescence emission increasing as the square of the excitation intensity Contrary to common belief, single- and two-photon excitation can produce equivalent results when the combined energy of the two photons exceeds the energy gap between the ground and excited states.
Two-photon excitation offers depth discrimination and enhanced 3D resolution that improve tissue penetration and imaging quality The latest methods broaden the range of usable fluorophores while limiting cellular damage, provided the probes have long-wavelength absorption properties Although two-photon excitation minimizes photobleaching and photodamage, cells and biological systems still experience these effects; two-photon-related damage can arise from reactive oxygen species production, thermal damage due to high-power infrared irradiation, and dielectric breakdown of proteins caused by the intense electromagnetic field of the femtosecond laser Additionally, two-photon excitation often exhibits lower conversion efficiency due to a narrow absorption cross-section, and it requires more elaborate instrumentation.
Photo-caging: Metal-Based
2.4.1ABOUT METAL-BASED PACT&PDT
Ru II complexes are being developed extensively for use in renewable energy production and medicine The use of Ru II -complexes ([Ru II ]) in renewable energy includes the design of photo-sensitizers and catalysts for high power output 66 and for carbon dioxide reduction 67 In medicine, they are used as chemotherapeutics, 68 and are being developed as photodynamic therapy (PDT) agents 69 and photo-activated chemotherapeutics (PACT) 70-72 The interest in metal-based therapeutics increased shortly after 1978, 73-74 when cisplatin became the first metal-based anticancer therapy approved by the USFDA 75 Interest in [Ru II ] in particular, began with investigations into transition metal
Photodynamic therapy (PDT) is an evolving alternative to photo-activatable chemotherapy for cancer, dermatology, and infections Like PACT, PDT begins with photoexcitation of a metal complex or organic photosensitizer to a singlet excited state, from which it can relax back to ground state by fluorescence or non-radiative decay; efficient photosensitizers undergo intersystem crossing to a long-lived triplet state that transfers energy to ground-state molecular oxygen, generating reactive singlet oxygen that damages cellular components and triggers cell death However, PDT's reliance on molecular oxygen limits its effectiveness in hypoxic microenvironments PDT agents also suffer from photobleaching, poor solubility, and limited tissue retention Additional PDT approaches include photo-initiated DNA binding, where metal complexes release a ligand upon light exposure to bind DNA, a strategy that can function under hypoxic conditions.
Figure [2.4.1].1 The essential electronic mechanism for the excitation of the 1 O 2 species to 3 O 2 in PDT
Understanding the electronic dynamics of Ru(II) complexes in photoactivated cancer therapy (PACT) requires recognizing their strong UV–visible absorption, long-lived excited states, and chemical stability in solution, while remaining relatively inert in the ground state Upon photon absorption, an electron is promoted from the ground state to a higher-energy metal‑to‑ligand charge transfer (1 MLCT) state, after which intersystem crossing produces the triplet MLCT (3 MLCT) state The 3 MLCT can decay back to the ground state via radiative or non‑radiative pathways or be thermally populated into accessible triplet ligand‑field (3LF) states Ligand dissociation is controlled by the population of these 3LF states, so the 3LF manifold is a key determinant of the photoinduced reactivity of Ru(II) in PACT.
This thesis presents an investigation grounded in a proven, refined photochemical approach developed in collaboration with the Turro group at Ohio State University The Turro group has tuned the 3 LF state of Ru(II) so that bound ligands such as cyanide and pyridyl groups readily dissociate from Ru(II) upon irradiation In the most recently optimized Ru(II) complex, a tridentate terpyridine (terpy) ligand is paired with a bidentate bipyridine (bpy) ligand, and the terpy ligand promotes distortion of the octahedral bond angles, facilitating selective, light-driven ligand dissociation.
Excited-state pathways in ruthenium(II) complexes involve electronic mechanisms that populate the 3LF excited state, driving depopulation and promoting ligand dissociation The octahedral distortion observed is modest and largely attributed to the steric bulk of the bidentate bipyridine ligand rather than large angular deviations from 90 to 180 degrees Increasing the steric bulk on the bpy ligand raises the propensity for 3LF population, thereby increasing the lability of the axial, monodentate ligand under low-energy light Experiments show that substituting bpy with 6,6′-dimethyl-2,2′-bipyridine (dmbpy) increases the quantum yield for pyridine dissociation of the [Ru(II)] complexes These complexes remain stable in the dark.
Raising the steric bulk of the bidentate ligand further distorts the pseudo-octahedral geometry of the complex and tilts the active ligand A 15-degree decrease in the dihedral angle of the dmbpy ligand, relative to the analogous bpy complex, is observed As a result, the Ru(II)-pyridine overlap diminishes, weakening the Ru(II)-py bond and accelerating pyridine dissociation Accordingly, the energy of the 3LF state is lowered.
Figure [2.4.1].3 Structural geometry of 32 & 33 via X-ray crystal structures, 32X & 33X Image 32X from the Cambridge Structural Database CSD-JIMJUK C R Hecker, P E Fanwick, D R McMillin, Inorganic
Chemistry, 1991, 30, 659 DOI: 10.1021/ic00004a013 Image 33X from the Cambridge Structural Database CSD-ABAHUJ J D Knoll, B A Albani, C B Durr, C Turro, Journal of Physical Chemistry A, 2014, 118,
According to the study (DOI: 10.1021/jp5057732), the excited state is more readily populated; the sigma and pi donation from pyridine is diminished, and bond dissociation is activated by photons The energy of the lowest MLCT state remains largely unchanged because that state is unreactive toward substitution.
2.4.2PREVIOUS WORK DONE BY THE KODANKO GROUP
An ideal chaperone is cleanly removable but has a high binding affinity to its substrate 62
The material remains stable before irradiation but can be selectively removed by exposing it to light at wavelengths that aren’t absorbed by nearby components It also exhibits a high quantum yield for dissociation, enabling efficient cleavage when illuminated.
Achieving all the key features with organic chaperones remains challenging Theoretically, when an inhibitor and a chaperone are co-optimized for potent inhibitory activity and favorable dissociation, their coupling can provide spatiotemporal control over protein inhibition Photo-activated compounds are continually developed because they offer localized, specific, selective, time-gated therapeutics This strategy is increasingly valuable for both in vitro and in vivo research and is advancing toward clinical use.
[Ru II (bpy)2] 2+ is one of the most commonly used chaperones today Prior to the developments to be discussed in this thesis, senior authors of the Kodanko lab presented
[Ru II (bpy)2] 2+ as a chaperone for cysteine protease inhibitors 5, 79, 81 When [Ru II (bpy)2] 2+ is used, two molecules of the monodentate inhibitors are bound to the metal center The chaperone therefore has dual-release capacity 5
In 2014, CST-K activity in osteoclast cells was targeted in an effort to spatially control apoptosis, inflammation and cell signaling 79 The developed complex (34 & 35) were bound to the
Ru II metal center via a dative bond to the nitrile (Ru II N≡CR) and had lower quantum yields than the parent acetonitrile complex, 36, much like the CST-B complex described below 34 & 35 showed rapid and efficient dissociation of the first molecule, but inefficient dissociation of the second identical ligand The decreased efficiency for the release of the second inhibitor is a result of (a) steric bulk around the complex preventing aquation, (b) the polarity and (c) larger size of the monodentate inhibitor compound In spite of this, the authors observed spatial and temporal control over inhibition; the uncaged inhibitor and the irradiated complex inhibited the enzyme to the same degree and the compound showed neither toxicity or growth inhibitory effects of BMM or PC-3 cells by MTT assays
In a related study, a similar complex (37) was synthesized for the inhibition of CST-B CST-B is one of the most abundant proteases, overexpressed in human breast cancer Upon irradiation with 395 nm visible light, the protease inhibitors were released in vitro
Figure [2.4.2].1 The first generation nitrile-bound inhibitors synthesized and investigated by the Kodanko group
IC50 values were determined using the CST‑B enzyme assay, and the potency of the free inhibitor was similar to that of the caged inhibitor, indicating that inhibition is blocked when the inhibitor is tethered to the chaperone fragment The DLIR of the [RuII–inhibitor] complex was 12 in CST‑B assays, and the inhibitory activity increased upon irradiation, providing further evidence that the dissociation of the inhibitor molecules is light‑controlled Because two inhibitor molecules are attached to the chaperone, the IC50 would be expected to be at least half that of the free inhibitor; however, dual release was limited by the low dissociation efficiency of the second caged inhibitor The inhibitor also demonstrated selectivity for cathepsin B in vitro assays In the triple negative breast cancer cell line MDA-MB‑231, the DLIR increased to 79.6, whereas in Hs578T cells it did not change, likely due to differences in protein complexation across cell lines Live cell proteolysis assays showed that the irradiated complex is more potent than the free inhibitor and can be effective at lower concentrations; in a 3D MAME culture, 1 μM of the photo‑activated compound significantly reduced DQ‑collagen IV degradation (green fluorescence), though at higher concentrations significant inhibition was observed.
Figure [2.4.2].2 The second generation nitrile bound [Ru II -inhibitor] synthesized and investigated by the
Kodanko group dark This may be due to the release of inhibitor over the 4-day timescale of the experiment The complexes were not found to be toxic
While advancing photo-activatable inhibitors for CST-K82, we observed that the stability of their Ru(II) complexes in growth media declines when a Ru(II)–N≡CR bond is present compared with a Ru(II)–pyridyl bond, based on unpublished results from Li and Kodanko This finding prompted us to focus on pyridyl-containing CST inhibitors and their derivatives to determine whether pyridyl coordination enhances complex stability and informs inhibitor design.
Our first publication presents a pyridyl-bound Ru(II)–inhibitor complex aimed at Cathepsin L (CST-L) and papain inhibition through spatiotemporally controlled irreversible binding Cathepsin L is a target due to its influence in inflammation, cancer and other pathologies Although the warhead was not directly bound to Ru(II) as in previously described complexes, the bulk of the metal scaffold was expected to inhibit binding until photo-release Previously established epoxysuccinyl CLIK inhibitors were synthesized (38 and 41) and modified for caging (39 and 42) The second-order rate constants for CST-L inhibition (Ki) by 38, 39, 40 (pre-irradiation) were experimentally equivalent within error Complexes 41 and 42 had similar inhibitory effects, but pre-irradiation 43 showed much lower inhibition than 42, with 43 (λmax = 471 nm) being effective only post-irradiation.
Methodology
Materials
All declared materials were used as received from commercial suppliers without further purification Cbz-Phe-OH, Boc-4PA-OH, Boc-Phe-OH, HBTU and 4-nitrophenyl carbonate were obtained from Chem-Impex International, Inc Trifluoroacetic acid, 12 N hydrochloric acid, sodium bicarbonate, sodium sulfate, sodium hydroxide, N-methylmorpholine, terpyridine, acetone, ethanol, methanol and dry diethyl ether were obtained from Sigma-Aldrich Co LLC Triethylamine, sodium chloride, dichloromethane, ethyl acetate and hexanes were obtained from Fisher Chemical-Thermo Fisher Scientific, Inc Celite and dry dimethylformamide were obtained from Merck & Co., Inc Formic acid, lithium chloride and toluene were obtained from EMD Millipore Corporation Diisopropylethylamine, benzyl chloroformate and sodium carbonate were obtained from Acros Organics-Thermo Fisher Scientific, Inc Aminoacetonitrile hydrochloride was obtained from Alfa Aesar-Thermo Fisher Scientific, Inc Pyridine-4-methanol was obtained from Matrix Scientific Ruthenium (III) chloride trihydrate was obtained from Strem Chemicals, Inc Dmbpy was obtained from Ark Pharma, Inc Deuterated NMR solvents were obtained from Cambridge Isotope Laboratories, Inc Recombinant human procathepsin K (0.023 mg/mL; purity unspecified).
Reagents, including Cbz-Phe-Arg-AMC (10 mg, purity > 99%), were commercially obtained from Enzo Life Sciences, Inc., and the appropriate aliquots were prepared and frozen Air-sensitive reactions were conducted under an inert atmosphere of nitrogen or argon supplied by Airgas, Inc Column chromatography was performed using silica gel from Sigma-Aldrich Co LLC and activated neutral alumina gel (Brockmann Grade I) from EMD Millipore Corporation.
Synthesis of compounds 1, 2, 3 and 4 were accomplished using advanced intermediates All compounds were prepared using methods as stated in the literature, or using slightly modified versions.
Instrumentation
Thin-layer chromatography (TLC) analyses were performed on silica gel glass plates with a UV-254 fluorescent indicator and on alumina gel glass plates with a UV-254 fluorescent indicator, both sourced from Merck & Co.; 1H NMR spectra were acquired on a Varian FT-NMR Mercury 400, processed with Varian Vnmrj 2.2D, or on a Varian FT-NMR/Agilent 400 system, processed with Varian Vnmrj 3.2A; mass spectra were obtained with a Waters HPLC ZQ2000 single-quadrupole MS equipped with electrospray ionization (ESI); UV–visible spectra were recorded on a Varian Cary 50 spectrophotometer; enzymatic assay plates were read on a Tecan Infinite M200 microplate reader with Tecan i-control 1.10.4.0 software; enzyme assays were irradiated using an Osram Xenophot HLX 24 V/250 W tungsten halogen lamp, and a 10 cm water cell was used to absorb unwanted infrared light; inhibition data were fit using Igor Pro 7.00 (32-bit).
Procedures
Inhibitor 1 was prepared following a previously established method 81 A 10 mL round bottom flask containing 48, Cbz-Phe-OH (250 mg; 0.84 mmol), was purged with N2 gas for 5 minutes; the flask was maintained under N2 gas for the remainder of the experiment Dry DMF (6 mL) was then added to the flask and the mixture was stirred until dissolution of 48 HBTU (380 mg; 1.0 mmol), aminoacetonitrile hydrochloride (92.7 mg; 1.00 mmol) and triethylamine (0.28 mL; 2.0 mmol) were then added The reaction mixture was stirred at room temperature (23 °C) for
After 16 h, complete conversion of the reactant to the product was verified by TLC analysis The organic layer was extracted with DCM (8 mL) and washed with 0.1 M HCl (10 mL; twice), followed by saturated sodium bicarbonate (10 mL; twice), then brine (10 mL) and dried using sodium sulfate The organic phase was filtered, concentrated in vacuo, and the product was recrystallized from ethyl acetate/hexanes, yielding 204 mg (72%) The product was characterized by 1H NMR and MS, as described in Chapter 4, and the characterization data agreed with the previously reported data.
Figure [3.3.1].1 Reaction scheme for synthesis of Inhibitor 1
Reagent 50 was prepared following a previously established method 90 A 50 mL round bottom flask containing pyridine-4-methanol (1.00 g; 0.00916 mmol) and 49, 4- nitrophenylcarbonate (3.07 g; 0.0100 mmol), was purged with N2 gas for 5 minutes and maintained under an N2 atmosphere DCM (17 mL) was added, followed by N-methylmorpholine (1.00 mL; 0.00916 mmol) The mixture was stirred at room temperature (23 °C) for 76.5 h The reaction mixture changed from a cloudy off-white colloid to a dark red-orange solution Upon completion, as determined by TLC analysis, the solvent was evaporated in vacuo The residue was then dissolved in ethyl acetate (40 mL) and filtered through sterile cotton The compound was extracted using 1 M HCl (50 mL; 4 times) The aqueous layers were combined, the pH increased to 9 using saturated sodium carbonate The pH was monitored with universal indicator paper The product was then extracted with DCM (30 mL; 4 times) then washed with brine (50 mL), dried over anhydrous sodium sulfate and concentrated in vacuo The product was recrystallized from ethyl acetate/hexanes Yield: 1.92g; 77% The product was characterized by 1 H NMR and MS, see chapter 4 The characterization data agrees with previously reported data
Figure [3.3.1].2 Reaction scheme for synthesis of Reagent 42
Compound 52 was prepared following a method modified from the literature 81 A 100 mL round bottom flask containing 51, Boc-Phe-OH (3.00 g; 0.0113 mol) was purged with N2 gas for
5 minutes; the flask was maintained under N2 gas for the remainder of the experiment Dry DMF
Approximately 60 mL was added and the mixture stirred until dissolution of 51 HBTU (5.15 g; 0.0136 mol), aminoacetonitrile hydrochloride (1.15 g; 0.0124 mol) and DIPEA (7.10 mL; 0.0396 mol) were then added The reaction mixture was stirred at room temperature (24 °C) for 19 h Complete conversion was verified by TLC analysis The organic layer was extracted using ethyl acetate (75 mL) and washed with saturated sodium bicarbonate (100 mL; 2 times), then brine (100 mL) and dried over anhydrous sodium sulfate The organic layer was then filtered, concentrated under reduced pressure and recrystallized from ethyl acetate to yield white crystals Yield: 2.93 g; 85% The product was characterized by 1H NMR, see chapter 4 The characterization data agrees with previously reported data.
Compound 53 was prepared following previously established methods 25, 91 Compound 52
(550 mg; 1.8 mmol) was added to a 20 mL round bottom flask Formic acid (8 mL) was added and
Figure 3.3.1 depicts the reaction scheme for the synthesis of Inhibitor 2 The reaction mixture was allowed to stir at room temperature (25 °C) for 16.5 hours, after which dichloromethane (DCM, 5 mL) was added and the mixture was evaporated in vacuo Water (10 mL) was added and the solution basified to pH 9 with saturated sodium bicarbonate, with pH monitored using universal indicator paper Compound 53 was extracted with DCM (10 mL) The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo Yield: 224 mg (61%) The product was characterized by 1H NMR (Chapter 4), and the characterization data agree with the previously reported data.
Inhibitor 2 was prepared following a method modified from the literature 90 A 10 mL round bottom flask containing 50 (148.5 mg; 0.5416 mmol) and 53 (135 mg; 0.492 mmol) was purged with N2 gas for 5 minutes; the flask was maintained under N2 gas for the remainder of the experiment Dry DMF (5 mL) was added and the mixture stirred until dissolution of 50 and 53
Triethylamine (0.08 mL, 0.5 mmol) was added to the reaction and the mixture was stirred at room temperature (24 °C) for 12 h, with complete conversion verified by TLC The organic phase was extracted with ethyl acetate (10 mL), washed with saturated sodium bicarbonate (10 mL; two times), then brine (10 mL) and dried over anhydrous sodium sulfate After filtration, the organic layer was concentrated in vacuo and recrystallized from ethyl acetate/hexanes to yield white crystals (120 mg, 72%) The product was characterized by 1H NMR and MS (see chapter 4); the characterization data agrees with previously reported data.
Compound 55 was prepared following a previously established method 81 A 25 mL round bottom flask containing 54, Boc-4PA-OH (500 mL; 1.88 mmol), was purged with N2 gas for 5 minutes; the flask was maintained under N2 gas for the remainder of the experiment Dry DMF (10 mL) was added and the mixture stirred until dissolution of 54 HBTU (854 mg; 2.25 mmol), aminoacetonitrile hydrochloride (191 mg; 2.07 mmol) and DIPEA (1.10 mL; 2.25 mmol) were then added The reaction mixture was stirred at room temperature (23 °C) for 18 h Complete conversion was verified by TLC analysis The organic layer was extracted using ethyl acetate (15 mL) and washed with saturated sodium bicarbonate (20 mL; 2 times), brine (20 mL) and dried over anhydrous sodium sulfate The organic layer was then filtered, concentrated in vacuo and recrystallized from ethyl acetate/hexanes to yield white crystals Yield: 563 mg; 99% The product was characterized by 1 H NMR, see chapter 4 The characterization data agrees with previously reported data
Inhibitor 3 was prepared following previously established methods 25, 91 Compound 55
(20.0 mg; 0.657 mmol) was added to a 5 mL round bottom flask Excess formic acid (1 mL) was
Figure 3.3.1.4 illustrates the reaction scheme for the synthesis of Inhibitor 3 After the reagents were added, the mixture was stirred at room temperature (23 °C) for 18.5 hours Dichloromethane (DCM, 5 mL) was then added to the round-bottom flask, and the mixture was evaporated under reduced pressure.
Concentrated product was basified to pH 9 with saturated sodium bicarbonate and cooled to 5 °C, with pH monitored by universal indicator paper while the flask was kept on ice Benzyl chloroformate was then added dropwise to the mixture; upon completion, as monitored by TLC, the product was extracted with dichloromethane (DCM, 5 mL), dried over anhydrous sodium sulfate, evaporated in vacuo, and recrystallized from ethyl acetate/hexanes to yield white crystals (8 mg, 36%) The product was characterized by 1H NMR and MS (see Chapter 4); the characterization data agree with previously reported data.
Synthesis of Complex 4 [Ru II (terpy)(bpy)(2)](PF 6 ) 2
Complex 57 was prepared following a method modified from the literature 92 A 150 mL sealed flask containing 56, Ru III Cl3ã3H20 (500 mg; 1.91 mmol) and terpy (446 mg; 1.91 mmol) was purged with argon gas for 10 minutes in the dark Ethanol (134 mL) was then added and the flask was sealed The reaction was allowed to proceed under reflux (82 °C) for 4 h The mixture was then allowed to cool to room temperature and the deep purple crystals were collected after centrifuging and washing with ethanol The crystals were allowed to dry in vacuo for 16 h Yield:
796 mg; 94% The product was characterized by 1 H NMR The characterization data agrees with previously reported data
Complex 58 was prepared following a method modified from the literature 93 A 75 mL sealed flask containing compound 57 (262 mg; 0.589 mmol), dmbpy (163 mg; 184.24 mmol), lithium chloride (250 mg; 5.89 mmol) was purged with argon gas for 15 minutes Ethanol (32 mL)
Reaction scheme for the synthesis of Complex 4 proceeds by adding water (16 mL) under continual purging, followed by TEA The reaction is heated to reflux (85 °C) for 21 h, then filtered through Celite while hot and the filtrate evaporated in vacuo Purification is carried out over alumina in the dark (solid phase: alumina; mobile phase: 0–5% methanol/DCM) with recrystallization from 1% methanol/DCM/hexanes Yield: 148.8 mg (43%) The product was characterized by 1H NMR, and the data agree with previously reported data.
Complex 4 was prepared following a method modified from the literature 78 A 15 mL sealed flask containing compound 58 (10 mg; 0.017 mmol) and 2 (17 mg; 0.050 mmol) were purged with argon gas for 15 minutes Ethanol (2.5 mL) and distilled water (2.5 mL) were then added The reaction was allowed to proceed under reflux (97 °C) for 16.5 h The solvent was evaporated in vacuo and the product was washed with toluene Product was purified via column chromatography (solid phase: alumina; mobile phase: 0–10% methanol/DCM), and solvent layering (ethanol/diethyl ether, 3 times) and vapor diffusion (ethanol/diethyl ether; 2 times) Yield:
7 mg; 49% The product was characterized by 1 H NMR and MS, see chapter 4 The characterization data agrees with previously reported data
3.3.2CHARACTERIZATION:SAMPLE PREPARATION AND DATA COLLECTION
Approximately 20 mg or twenty drops of the compound was dissolved in 0.6–0.8 mL of the deuterated NMR grade solvent in an appropriately sized vial The solution was then filtered through cotton into another via then transferred to a 7 inch, borosilicate glass NMR tube (with an inner diameter of 0.5 mm and an outer diameter of 10 mm) 1 H NMR solvents were either methanol-D4 or chloroform-D1 and the resonances were referenced to the residual methanol and chloroform signals on the Varian FT-NMR Mercury 400 or the Varian FT-NMR Agilent 400 for all applicable samples Mass spectroscopy was performed in methanol for all applicable samples See chapter 4 for details specific to each compound and section 3.2 for details specific to the instrument