Photochemical activation of enediyne warheads: A potential tool for

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Photochemical activation of enediyne warheads: A potential tool for targeted antitumor therapy Prabuddha Bhattacharya, Amit Basak, Adam Campbell, and Igor V. Alabugin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00911 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 6, 2018

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Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

Photochemical

activation

of

enediyne

warheads: A potential tool for targeted antitumor therapy Prabuddha Bhattacharya, Department of Chemistry, Adamas University, Kolkata 700126, India Amit Basak, Department of Chemistry, Indian Institute of Technology Kharagpur 721302, India Adam Campbell, Igor V. Alabugin, Department of Chemistry & Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, FL, 32306, USA Contents: 1. Introduction 2. Enediyne warheads 3. Photodynamic Therapy (PDT) 3.1. In vivo light delivery 3.2. NIR activation 4. Photo-triggered diradical generation 4.1. Enediynes Activated toward Photo-Bergman Cyclization 4.2. Photoinduced Electron Transfer (PET) mediated C1-C5 cyclization of enediynes 4.3. The photochemical C1-C5 cycloaromatization reaction of enynes 4.4. Enediyne metal complexes as photoinduced diradical generators 4.5. Photoactivation of locked or acyclic enediyne 4.6. Photoactivation via reduction of ring size 4.7. Photoactivation via conversion of diazoketone to ketene 4.8. Development of enediyne/enyne - amino acid hybrids: the unique properties of lysinconjugates. 4.8.1. N-lysine conjugates versus C-lysine conjugates 4.8.2. Application of Photoinduced Electron Transfer (PET) based lysine conjugates as DNA cleaving agents 5. Multiple mechanisms of DNA damage 6. Miscellaneous reports of photoinduced DNA damage

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7. New Strategy to Generate Double Strand Breaks: photochemical ssds break conversion. Towards photochemical RNA interference 8. Cellular uptake and intracellular DNA-damage 9. Cytotoxicity toward cancer cell lines 10. Conclusion 1. Introduction Natural enediyne antibiotics have been hailed as the most potent antineoplastic agents ever discovered. Their cytotoxicity is attributed to the ability of the (Z)-3-hexene-1,5-diyne (enediyne) motif to cycloaromatize via Bergman cyclization (BC)1, producing cytotoxic benzenoid diradicals (Scheme 1). However, the lack of anti-tumor selectivity of this class of natural products results in high general toxicity and hampers their clinical applications.2

Scheme 1. Bergman cyclization leading to the formation of 1,4-diradical

On the other hand, photo-chemotherapy and photodynamic therapy offer a unique approach to localized activation of the drug directly at the targeted tumor using tissue-penetrating photons.3 Merging the two approaches led to the design of photo switchable enediynes, which afforded temporal control over the triggering process of the enediyne warhead. A large body of chemical literature documents DNA as the therapeutic cellular targets for photoactivated drugs.4 Photo-activated “chemical nucleases,” known also as “photocleavers,” interact with DNA and cause its cleavage when excited with light. Since a chemical reaction occurs only when the mixture of the photo-cleaver and DNA is irradiated, there is no need for external chemical initiator. The excited photo-cleaver sensitizes the reactions which via various mechanistic pathways may lead to either the readily repairable single-strand (ss) DNA damage, and/or double-strand (ds) DNA cleavage. The latter type of damage is more difficult to repair and is more likely to cause efficient apoptosis (self-programmed cell death), making this approach a promising tool for cancer therapy. This detailed review outlines the reports on applications of DNA cleavage mediated by photoactivated enediynes and illustrates that photochemical reactivity can be 2 ACS Paragon Plus Environment

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complemented by additional factors such as the release of internal strain, chelation, pH changes, intramolecular H-bonds and substituent effects.5 We will discuss the design and reactivity of photoactivated enediynes/enynes along with the analysis of the challenges and successes in application in the use of these molecular designs in biological systems. 2. Enediyne warheads Enediynes have their origin6 in various marine and terrestrial plant sources, and comprise a substantial class of potent antitumor and antimicrobial agents with high cytotoxic activities. The initial reports7 of the enediyne anticancer antibiotics in the late 1980s led to an upsurge in the research in this area with a broad focus on any facets of enediyne chemistry and biology. The antitumor activity of these compounds is due to the presence of a highly reactive hex-3-ene-1,5-diyne subunit that undergoes Bergman cyclization (BC) and generates a benzene-1,4-diradical. This diradical can abstract two H-atoms from the DNA backbone and lead to the subsequent cell death (Figure 1). Despite the notable cytotoxity of natural enediynes, their ds:ss ratios for the DNA cleavage favour the repairable ss-cleavage. This reactivity trend implies that improvements over nature are possible! Apart from their DNA damaging activity, the enediyne-derived diradicals have been shown to rupture the protein structure via oxidative cleavage of the peptide bond.8 This proteolytic activity also explains the resistance mechanism employed in pathogenic bacteria against such toxic secondary metabolites, e.g., calicheamicin γ1. While the acyclic enediynes undergo thermal cycloaromatization only at elevated temperatures (200 °C), the natural enediynes are mostly embedded in a cyclic skeleton and cyclize at the physiological temperatures.9 A better understanding of their biochemical mechanism of action became possible when the structures of calicheamicin – γ1 (Figure 1) and esperamicin A1, the first representatives of the enediyne antibiotics, were elucidated. The very heart of these molecules is contained in the same enediyne unit; and therein lies the means by which these unprecedented molecules exhibit their remarkable biological properties. Dynemicin A and kedarcidin chromophore, representing structurally distinct classes of enediyne antibiotics, were subsequently reported (Figure 2). Neocarzinostatin chromophore is often grouped with the enediyne antibiotics due to the similarity of its structure and mode of action.10



enediyne

Tri

Trigger

OMe

O

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gge

r

H

NH H O

O MeO

O

H

O O

OH

NH HO HN

DNA-binder

SSSCH3

OH

O OH MeO

O

Calicheamicin

O

OMe O

S

DNA-binder

OMe OH

Unrepaired Double Strand Cleavage

I O

Apoptosis Figure 1. Structure of Calicheamicin, highlighting the interplay of structural elements in the naturally occurring enediynes and mechanism for inducing double-strand (ds) DNA cleavage. The enediyne warhead is shown in red, the trigger is shown in blue.

Incorporation of locking11 devices into these systems ensures the safe delivery of the molecule to the target before the enediyne functionality is activated toward diradical generation. Only after the triggering mechanism is activated, hydrogen-atom abstraction from the sugar-phosphate backbone of DNA becomes possible. The chemistry of the triggering or activation process for the natural enediynes can involve a change of hybridization at a carbon center in the proximity of the enediyne moiety or an opening of the epoxide ring fused onto the enediyne-containing cycle.12 This structural change is believed to lower the activation barrier for the cycloaromatization process either by bringing the terminal acetylenic carbon atoms

closer

or

by

easing

the

restrictions.


overall

conformational

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Figure 2. Examples of some naturally occurring enediyne antibiotics

The prediction of the kinetics of BC considering the c and d distance factor as proposed by Nicolaou et al.13 mainly relies on the ground-state configuration of the enediynes, and the rule (critical c and d distance for spontaneous BC is 3.31-3.20 Å) applies well for simple monocyclic enediynes without much strain. A deeper analysis is based on the effect of strain on the energy difference between the transition state (TS) and ground state for the cycloaromatization process. This analysis paved the way for the subsequent introduction of the electronic factors in the design of reactive synthetic analogues of the natural enediynes. Unfortunately, the increased reactivity of cyclic enediynes has a negative consequence as well. It introduces a challenge because, in the absence of an additional control element, only a narrow time window would exist for separating the production of these compounds from their transformation into the highly reactive diradical. In calicheamicin, the additional control is achieved via reductive S-S bond cleavage of the allylic trisulfide trigger. Formation 5 ACS Paragon Plus Environment


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of the nucleophilic cyclic group initiates intramolecular Michael addition that indirectly facilitates Bergman cyclization. The remaining functionalities have multiple roles in addition to contributing to the solubility and DNA binding (Figure 3). O

O

O NHCO2Me

NHCO2Me HO

HO

NHCO2Me HO

O

O

O

sugar

sugar

..

S

B

A Bergman Cyclization

O NHCO2Me

O

DNA

NHCO2Me

HO

.

HO O

S

sugar

HS

S calicheamicin S S Me Nu

sugar

D

DNA diradical O2

.

O S

sugar

C

DNA double strand cleavage

Figure 3. Mechanism by which calicheamicin cleaves DNA

Calicheamicin γ1 contains two distinct structural regions, each playing a specific role in its biological activity. The larger region consists of a sugar residue comprising four monosaccharide units and a hexasubstituted benzene ring which are joined together through a highly unusual sequence of glycosidic, thioester, and hydroxylamine linkages. The second structural region, the aglycon (termed calicheamicinone), contains a highly functionalized bicyclic core housing a strained enediyne unit within a bridging 10-membered ring. First, calicheamicin γ1 reacts with glutathione to form a free thiol A and then undergoes a Michael addition to form a more strained intermediate B (Figure 3). The latter rearranges to the diradical C, which then abstracts hydrogen atoms from the ribose backbone of DNA.11 Other mechanisms for the control of reactivity are likely to exist (Scheme 2). For example, the sugar residues of esperamicins were suggested to play a role in the autoresistance to natural enediyne antibiotics by the enediyne-producing microorganisms by making the Bergman cyclization step irreversible via conformation-gated intramolecular Hatom transfer.14


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trapping OMe

O

a)

O

NH O R'' O

O O

HO

HO HN R

Esperamicin A1

b)

O

N

S X

R'' .

OH R''

O

O .

H

OH

R Fragmentation

O

"aromatic core" + "carbohydrate"

R'' conformationally p-benzyne gated H-atom transfer

SSSCH3

Mechanistic study: H

O

O H

"aromatic core"

O

O

O

O

H

H O

c)

+

Fragmentation "carbohydrate"

trapping

Pr ot ein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RG = Reactive Group RG

RG

X

X

RG*

RG

-Protein

X BC

Conformational change

.

Intramolecular . reaction

Y

Z

. . fragmentation / irreversible p-benzyne interception

Scheme 2. a) The suggested Esperamicin A1 fragmentation, b) Esperamicin mimics undergo intramolecular Habstraction/fragmentation cascade, c) Conformation-gated hypothesis for autoprotection in enediyne-producing microorganisms from natural enediynes.

Neocarzinostatin (NCS) is a member of a family of chromoprotein antitumor antibiotics which consists of two noncovalently bound components, a labile chromophore component (NCS-chrom) with biological activities such as DNA cleavage and a protein component that stabilizes NCS-chrom. The mechanism for the activation of NCS-chrom involves the nucleophilic attack of the thiol leading to the highly reactive enyne cumulene moiety (Scheme 3). The latter undergoes Myers Saito15 cycloaromatization to form a biradical that cleaves DNA by abstracting hydrogen atoms from the deoxyribose sugar.16



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O

O

O

CH3

O

O H3CO

O

OH O

O O

OH

SR

O

O H

O O

O

C

C

Myers Saito cyclization

Hydrogen abstraction DNA (H)

SR

Ar

O

Sugar O

O

Sugar O

Neocarzinostatin NHCH3

HO

OH

SR

Ar

RSH

O

H3C

Ar

O

O H

.

O

O

O

.

Sugar O

Scheme 3. Thiol mediated NCS-Chrom activation and subsequent DNA cleavage

Myers Saito cyclization15 is an intramolecular cyclization of eneyne-allene via a σ,πbiradical intermediate generated by the bonding between C2 and C7 (Scheme 4). Compared to the Bergman Cyclization, this reaction takes place smoothly even below room temperature, benefiting from a cyclic electron delocalization. Similarly, the partially conjugated σ,πbiradical is less reactive than the σ,σ-biradical in the Bergman Cyclization. This reaction has been reported to be exothermic reaction and to have a relatively low activation energy of ∼ 23 kcal/mol. Besides the thermal initiation, this reaction can also be triggered by light, acid, or base; however, the reaction rate is sensitive to the substituents on alkyne and allene moieties. R1 R2 C

Myers Saito Cyclization R3

H

.

R1

H-source 2

.R

C2-C7 cyclization

R2

R1 R2 H R2

Scheme 4. Myers Saito Cyclization

Based on Myers Saito pathway for the diradical generation, an alternative mechanism for the photoactivation of NCS was reported17 by Gomibuchi and Hiramal. They reported that photoactivated NCS undergoes Myers Saito cycloaromatization via naphthoate participation without a thiol trigger affording an indacene derivative. Product F supported the Norrish Type II fragmentation of E. Furthermore, the new product K strongly indicated the existence of an alternative photo-decomposition pathway via the intramolecular rearrangement of the activated naphthoate as shown in Scheme 5.


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O O O hν

(T1)*

. O . O

OH

(S1)*

NEOCARZINOSTATIN

O

O H3C

OCH3

Sugar

E

Norrish Type II fragmentation

naphthoate migration

1,5-H shift O

O

O

OH O

O

O OH O H3C

H

O

O

F

O H3C

H

H3C

naphthoate migration

O

OCH3

OCH3

Sugar

H3C

H

O

O

C H

Sugar Myers-Saito Cycloaromatization O

OH

O

Hydrogen abstraction DNA (H)

O

O

O

H3C

O

.

OH

O

H3CO O slow hydrolysis

C

H3CO

O

O

OH

O O

G

O O

O

O

.. ..

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


.

H3CO

H

O

Sugar

Sugar

J

H3C

I

O OH H3CO

O

O

H

O

O

OH

HO O

H Sugar

K

Scheme 5. Photoactivation of NCS-Chrom

The naphthoate migration to C-12 with the concomitant epoxide opening leading to the formation of the enyne cumulene H, is likely to occur from the photo-excited triplet state E which may have a radical character. Formation of the enyne cumulene could be also invoked via the photo-enol intermediate F. The formed ketene acetal with eneyne-cumulene structure H undergoes the cycloaromatization giving the biradical intermediate I, which leads to 9 ACS Paragon Plus Environment


indacene J through hydrogen abstraction from the media. Slow hydrolysis of the ketene acetal group in J provides K and F. In spite of varying the triggering mechanism of the enediyne core, the basic therapeutic mode of action involves DNA cleavage. In general, efficient ds DNA cleavage is hard to achieve as illustrated by the fact that even calicheamicin, the best ds DNA cleaver among anticancer drugs, induces only about 25% of ds cleavage (a ∼3:1 ss:ds ratio).18 Remarkably, not only does such a seemingly low ds:ss ratio far surpass other popular DNAcleavers, such as bleomycins (1:6–1:20)19, but is still sufficient to account for their recordbreaking biological activity because of the connection between ds-DNA cleavage and selfprogrammed cell death (apoptosis). These data provide a compelling rationale for the development of DNA-damaging species as a strategy towards new cancer therapies but also illustrate that improvement of the ds DNA damage efficiency is possible. With so many arguments in favour of the enediyne usage as possible DNA cleavers, one of the major deterrents in their clinical application is their inability to distinguish between the healthy and cancerous cells. This indiscriminate cytotoxicity negates the advantages. Thus, this pressing problem of target selectivity evokes the need for some triggering mechanism present in the drug which would specifically respond to the physiochemical environment of the cancerous cell and initiate the diradical forming process. Finding the right balance between reactivity and selectivity of the enediyne warheads has been the focus of concentrated research efforts. The fine-tuning of reactivity can be achieved through strain, chelation, and/or electronic effects whereas the selectivity of enediynes was increased via a variety of approaches including conjugation to monoclonal antibodies, use of the reductive environment of hypoxic tumors, designing pH-sensitive warheads for acidified solid tumors, and photochemical triggering.20 3. Photodynamic Therapy (PDT)21 The quest for spatial and temporal control over prodrug activation led to the inception of the therapeutic technique that involved the application of tissue penetrating low energy


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(long wavelength) light directly to the tumor with a high concentration of the prodrug. Tissue Oxygen Photosensitizer (excited state) Free radicals, singlet oxygen

Light

Photosensitizer (ground state)

Cellular Toxicity

Figure 4. Mechanism of action of Photodynamic therapy (PDT)

PDT involves three individually non-toxic components that are combined to induce cellular and tissue effects in an oxygen-dependent manner. The first component of PDT is a photosensitizer — a photosensitive molecule that localizes to a target cell and/or tissue, (porphyrin happens to be the most extensively studied photosensitizer). The second component involves the administration of light of a specific wavelength that activates the sensitizer. The third component is oxygen. The photosensitizer transfers the photochemical energy to molecular oxygen (present in tissues) to generate reactive oxygen species (ROS) (Figure 4). These reactions occur in the immediate locale of the light-absorbing photosensitizer. Therefore, the biological responses to the photosensitizer are activated only within the diffusion lengths of ROS from the particular areas of tissue that have been exposed to light. Other photochemical reactions that do not rely on oxygen, such as photoaddition to DNA, have also been developed. These reactions are called ‘photo-chemotherapy’.



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Photosensitizer

Light

Type I reaction

Activated photosensitizer

Radicals, Radical ions

O2

Type II reaction

Substrate

1

O2

O2

Substrate

Products of oxidation

Products of oxidation

Figure 5. Type I and type II reaction in Photodynamic therapy (PDT)

Following the absorption of light, the sensitizer is transformed from the ground state into an excited state. The activated sensitizer can mediate a subsequent cascade of chemical transformations via two distinct scenarios.22 In the first of them, the excited state is transformed into reactive species (e.g., radicals) that can interact with oxygen to produce oxygenated products (type I reaction). Alternatively, the activated sensitizer can transfer its energy directly to oxygen, to form singlet oxygen (1O2). These highly reactive oxygen species can oxidize various substrates (type II reaction) (Figure 5). PDT has a number of advantages23 over conventional cancer treatments. It avoids systemic treatment and minimizes collateral damage of the nearby healthy non-cancerous tissues. This is repeatable, low-cost, minimally invasive localized treatment that can be helpful when the malignant tissue cannot be removed surgically. However, there are a few limitations24 associated with this method as well. Since the photosensitising drug used for PDT stays in the human body for up to several weeks, its presence leads to continuous generation of singlet oxygen when the patient is exposed to sunlight. As a result, damage to healthy tissues may continue even after the target cancer cells have already been destroyed. Additionally, since the conventional PDT approach requires oxygen, it is less effective in hypoxic tumors. 3.1. In vivo light delivery Biological cells span the range of sizes from submicron to over 20 mm. In such cases, interaction with light can lead to both scattering and absorption. Since Rayleigh scattering is inversely proportional to the fourth power of wavelength, the light of longer wavelength is absorbed more (scattered less), and penetrates deeper into a biological specimen. 12 ACS Paragon Plus Environment

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A very active area of current research is in vivo optical excitation for spectroscopic analysis, bioimaging, laser surgery, and light-activated therapy. The research involves the study of various methods of employing light for inducing photoexcitation in a specific section of a tissue or an internal organ of a live biological specimen or a human subject. The four principal methods used for the light delivery are shown in Figure 6.25

Figure 6. Various modes of in vivo light delivery

i) Free Space Propagation: Here the light is delivered directly to the photoexcitation site by propagating it through free space. 26 This method is used for UV–visible light photoexcitation of external tissues/organs such as skin, the eyes, and so on. With the availability of solid-state diode lasers, which can now cover from blue to near IR and are very small in size, one can even use them as hand-held units for in vivo photoexcitation. ii) Optical Fiber Delivery System: The optical fibers are used for light delivery in the wavelength range of 200 nm (UV) to 1600 nm (IR) and are made of glass or fused silica. 27 They trap light entering at one end by total internal reflection from the interface between the fiber edge and an outer coating material, called cladding (generally a plastic). iii) Articulated Arm Delivery: This optomechanical solution for the beam delivery is used for mid-infrared range, such as 10.6 µm radiation from a CO2 laser.28 In the case of using an articulated arm to propagate a CO2 laser beam that is in the infrared region, a co-propagating visible beam (usually a red beam from a He–Ne laser or a diode laser) is used as an aiming beam. iv) Hollow Tube Waveguides: This method involves a hollow tube made of a metal, ceramic or plastic. The inside wall of the tube is coated with a high reflector. The laser light is propagated down the tube by reflection from the inner wall.29 For use in high-power laser delivery applications the guides have been shown to be capable of transmitting up to 3 kW of CO2 laser power. They are also finding uses in both temperature and chemical fiber sensor applications. 3.2. NIR activation 13 ACS Paragon Plus Environment


Although the focus of this review on photodamage, interaction of light and matter has been of interest in a variety of other fields. For example, bioimaging with optical methods is an actively developing field of biophotonics.30 We will discuss the key lessons from this field in this section because such lessons are of the general importance for any application of the tissue penetrating photons in biology. Optical bioimaging is applicable to a wide range of biological specimens, from cells to ex vivo tissue samples, to in vivo imaging of live objects. Optical bioimaging also covers a broad range of length scale, from submicron size viruses and bacteria, to macroscopic-sized live biological species. In vivo optical imaging makes use of molecular probes to visualize the underlying mechanisms of biological processes at the cellular and molecular levels. The near infrared (NIR) spectral zone from 650 – 950 nm (defined as tissue optical window, also known as the therapeutic window)31 falls in the region of the spectrum with the deepest tissue penetration. The NIR optical window has been conventionally used for imaging studies. To operate effectively, the imaging agents must absorb and emit in this longwavelength window. Imaging agents (Figure 7) comprise both aqueous soluble and insoluble species, both organic and inorganic, and unimolecular and supramolecular constructs. Nearinfrared fluorescent probes provide optical detection of cells targeted by real-time nanoparticle-distribution studies within the organ compartments of live, anesthetized animals. By combining different imaging modalities, one can carry out deep-body imaging by magnetic resonance imaging or computed tomography, and by using optical imaging, get down to the resolution required for real-time fluorescence-guided surgery. Complex nanomedical systems have been designed and constructed to contain drugs for gene therapy, and simultaneously provide high-resolution three-dimensional (3-D) multimodal imaging of diseased cells in the body for very early detection of diseases.32 Three main factors contribute to the ever growing interest in long-wavelength, near-IR (NIR), and IR dyes: (i) These NIR dyes require excitation near IR which produces practically no autofluorescence from any endogenous cellular components. Hence, the sensitivity of detection, often limited by the autofluorescence background, is significantly improved; (ii) the longer excitation wavelength and the corresponding near-IR emission also produce reduced scattering in the tissue, and thus increase both the penetration depth and the efficiency of collection of emission; and (iii) commercially available, low-cost, and highly compact red, NIR, and IR diode lasers (e.g., 650 nm, 800 nm, 970 nm, etc.) can be used as convenient excitation sources for these dyes.


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Owing to its excellent sensitivity and temporal resolution, optical imaging in the NIR window has enormous potential for detecting diseases and non-invasive monitoring of the therapeutic process. The assessment of treatment efficacy should allow to adjust and customize treatment in vivo. One of the most interesting applications of NIR dyes is the photodynamic therapy (PDT) of malignant tumours.33,34 This method is based on photodynamic generation of singlet oxygen (1O2) due to the interaction of a photoexcited molecule of photosensitizer with common triplet oxygen. The selectively localizing in-tumour dye is administered by intravenous injection of its solution, preferably of aqueous one, 1-48 hours prior to the light treatment. The 1O2 formed under laser excitation is a powerful oxidant leading to necrosis of tumour tissue. O

cyanine dye HN

NH

O n = 3, 4 R = COOH, H N n(H2C) O3S

O

porphyrin based dye

O

N

N

N R squarine dye

HN O

Zn NH

N

N

N

N N

N N

O CF3 O

N

N

O

B F

F OMe

MeO BODIPY (borondipyrromethane) dye

Figure 7. Structures of a few NIR photosensitizers

However, there are certain disadvantages associated with this NIR method. Aqueous insolubility and ease of aggregate formation are problems often encountered with phthalocyanine and squaraine dyes in biological systems. Squaraine dyes are also highly chemically reactive. Cyanine dyes are excellent NIR dyes that have high molar absorptivity, strong fluorescence, and good photostability. However, their intrinsically small Stokes shifts may produce excitation and scattered light interferences. Another important technique for bio-imaging is based on the principle of fluorescence resonance energy transfer (FRET).35 It has emerged as a powerful technique for biomedical



research. Its applications cover a broad range such as study of protein–protein interactions, calcium metabolism, protease activity, and high-throughput screening assays. One of the significant advantages of probes with FRET modulation is that these can enable ratiometric measurement in living cells, which reduces the artefacts from microscopic imaging systems. Apart from this, FRET affords real-time imaging of enzyme activities in vivo which in turn gives valuable information in understanding living systems and in developing medicine for various types of diseases. One of the most recent successful clinical applications36 has involved the FRET based probe for 19F MRI. The fundamental principle involves the use of Förster excitation energy transfer from an excited molecule of higher energy (donor) to another molecule of lower excitation energy (acceptor). This energy transfer occurs nonradiatively through dipole–dipole interaction, showing a distance dependence of R-6. It is maximized when there is a significant overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. Apart from these methods, two-photon (TP) excitation constitutes another highly used imaging technique.37 It is known that conventional one-photon excited fluorescence microscopy techniques for fluorescence excitation make use of a UV or visible photon that is able to match the energy gap between the ground and excited state. TP excitation of fluorescent molecules is a nonlinear process related to the simultaneous absorption of two, typically IR, photons whose total energy is sufficient to produce a molecular transition to an excited electronic state. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantized event. Since the energy of a photon is inversely proportional to its wavelength, the two photons should be about twice the wavelength required for single-photon excitation. Since this process depends on the simultaneous absorption of two infrared photons, the probability of two-photon absorption by a fluorescent molecule is a quadratic function of the excitation radiance. Two-photon excitation provides optical sectioning for three-dimensional imaging, but in contrast to confocal microscopy there is no absorption and fluorescence (and thus no photobleaching and phototoxicity) above and below the plane of focus. Consequently, it can be less perturbing to live samples due to the reduced phototoxicity incurred throughout the sample. In addition, the ability to image at depth in the sample is degraded less by sample scattering of excitation and emission photons. Thus, two-photon excitation microscopy is used, preferably over confocal microscopy, for experiments that require large image depths in live tissue or in small animals. However, because the photophysics involved with two-photon excitation is different from conventional fluorescence excitation, deleterious effects may 16 ACS Paragon Plus Environment

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occasionally be observed with two-photon excitation for certain fluorophores, and this in turn can limit the applicability of this method for optical sectioning in thin samples. Two-photon excitation microscopy works well for dynamic imaging of living cells, often for experiments where conventional imaging is not possible. Since there are no major technical limitations to the implementation of two-photon excitation microscopy, its use should continue to increase as cheaper and more reliable ultrafast mode-locked lasers are developed. Two-photon microscopy has had a major impact in areas such as physiology, neurobiology, embryology and tissue engineering, for which imaging of highly scattering tissue is required. Highly opaque tissues such as human skin have been visualized with cellular detail. Clinically, two-photon microscopy finds application in non-invasive optical biopsy, for which high speed imaging is required. In cell biology, the most promising applications are those that rely on two-photon excitation to produce localized chemical reactions, such as in 3D resolved uncaging and photobleaching recovery studies. 4. Photo-triggered diradical generation The relative clinical success of PDT paves the way for the introduction of other phototherapies, such as photochemically activated enediynes, in cancer treatment.38Again, these therapies can take advantage of time- and space-resolved drug activation with the drug being harmless until activated with light. Some of these strategies involve direct photochemical induction of BC. Others use irradiation to induce a structural change in the prodrug, that promotes the thermal BC. A combination of several methods can be used for a greater therapeutic effect. These strategies can be classified into the categories shown in Figure 8.



Category 1

Page 18 of 64

h

Enediynes with suitable appendages

Photo-BC

Diradical

. .

hv

Category 2 h

Enediyne

PET

Diradical

C1-C5 cyclization

Photo excitation of enediyne

hv

.

C1-C5 cyclization Category 3 h

Enediyne-Metal complex

Photo excited enediyne

Metal to Ligand or Ligand to Metal Charge Transfer

X X

. X = donor atom, M

n+

M

n+

Diradical

. .

.

hv

X X Mn+ X X

Thermal-BC

X X

= chelated metal ion

Category 4 h

Locked enediyne or Acyclic enediyne

Unlocked enediyne or Reactive enediyne

. .

hv O

deprotection

..

..

..

X PG

locked

XH

..

XH

XH

r.t.

HO

HO

O

Nu

unlocked

Diradical

Thermal-BC

HO

.. Nu Nu XH PG = Protecting Group X = O, NH

Category 5 Prodrug

h

Diradical

Photo BC/Thermal Myer Saito Cyclization

Reactive enediyne or enyne-ketone

.

hv

. prodrug Category 6 Enediyne with azo functionalization

h Activation

Photo-isomerization cis-trans isomerization N hv N trans N

N

N

cis

N

activated enediyne

Thermal BC

Diradical

.

.

Figure 8. Selected strategies for photoactivated diradical generation from enediynes and related compounds

4.1. Use of Enediynes Activated toward Photo-Bergman cyclization


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Scheme 6. Photo-Bergman cyclization (Turro and Nicolaou)

Although the photo-Bergman cyclization has been known since the 1968 report of Campbell and Eglington (as long as the thermal ring closure!),39 more targeted efforts were undertaken only after the importance of the enediyne warhead had been discovered. Acylic enediynes undergo cis-trans isomerisation of the central double bond40 but benzannelated enediynes were found to undergo photo-Bergman Cyclization (photo-BC) by Turro, Evanzahav, and Nicolaou.41 Upon photo irradiation (λ ≥ 313 nm) of isopropanol solutions of n-propyl- or n-phenyl-substituted enediyne 1, the naphthalene derivatives 2, (in addition to enynes 3 and 4) similar to those obtained in a thermal BC (Scheme 6), was reported. This finding illustrated that embedding of enediyne moiety into a cycle enables the photocyclization.

hν (λ>300nm) a 6

5

a) 1,4-CHD, Solvent: acetonitrile, diethyl ether, acetone

8a: dialkynyl naphthalene (38% conversion) 8b: dialkynyl phenanthrene (82% yield)

7a: dialkynyl benzene (15% conversion) 7b: dialkynyl naphthalene (no reaction)

Scheme 7. Photo-Bergman cyclization of ortho-dialkynylarenes (Funk)



Page 20 of 64

Funk et al. reported the photo-BC of dialkynylarenes 5, 7, 8 (Scheme 7).42 Except the naphthalene derivative 7b, all other dialkynylarenes underwent a photochemical cycloaromatization upon irradiation (Hanovia 450 W, Pyrex, Et2O, 18 h) in the presence of 1,4-cyclohexadiene (30 equiv). The cyclization of the dialkynylpyrene 5 to the benzpyrene 6 was the most efficient of the transformations, which could be also affected in sunlight (CH3CN, 3 h, Pyrex). In order to study the DNA photocleaving properties of these compounds, attachment of water-solubilizing side chains at the homopropargylic positions of 8 and 5, e.g., the (S,S) enantiomers 9a-b (Scheme 8), respectively, were synthesized. DNA photocleavage by dialkynylarenes 9a-b were investigated using supercoiled plasmid pUC19 DNA (38 µM in base pairs) in Pyrex reaction vessels with a Hanovia 450 W light source. From agarose gel electrophoresis, it was found that both dialkynylarenes 9a-b affected predominantly single-strand cleavage (conversion to form II DNA) upon irradiation but were ineffective in the dark. However, the dialkynyl pyrene 9b was more effective in this regard since DNA cleavage could be accomplished at a lower concentration (2 µM) and rapidly consumed the DNA at 20 µM concentration producing both form II and form III cleavage products which reflected either the superior binding affinity (better intercalator) and/or the more efficient photochemistry of dialkynyl pyrene 9b. Moreover, the negligible photocleavage of DNA with a water soluble dialkyl phenanthrene (12) demonstrated that alkynyl substituted arenes are required for efficient photocleavage. Finally, attenuation of DNA photocleavage (as evident from the gel electrophoresis) by dialkynyl phenanthrene 9a in the presence of a water soluble dialkyl phenanthrene 12 was observed which suggested that cleavage arises from bound (intercalated) 9b. O

Me N Me Me

O

O O

hν (λ>300nm) O 9a-b

O

9a: dialkynyl phenanthrene 9b: dialkynyl pyrene

10a-b

O H O

DNA

O Cleaved DNA

O

Me Me N Me

H 11a-b

O

O


Me Me N Me

Me Me N Me Me N Me Me

Me N Me Me Me Me N Me

Me N Me Me

12

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Scheme 8. Photo-Bergman cyclization of ortho-dialkynyl arenes tethered to water soluble motifs at homopropargylic position (Funk)

In 1999, Hirama and co-workers reported the photo-BC of several non-benzenoid enediynes 13 (Scheme 9) and suggested that photo-BC of these enediynes could play an important role in 1,4-didehydrobenzene chemistry because it could be carried out at the ambient temperatures.

Scheme 9. Photo-Bergman cyclization of diethynyl cyclopentene derivatives in hexane (Hirama)

The product obtained from the photo-BC of 1,2-diethynylcyclopentene derivatives 13 afforded the cyclized products 14 in very low yield (∼3%), except for the dipropynyl derivative (R = Me) in which case the cyclized product was isolated in yields up to 71%. Interestingly, no photoreduction products were observed unlike the photochemistry of the benzenoid analogue.43 After irradiation of the strained, ten-membered cyclic enediyne 15 with a low-pressure mercury lamp (4 x 20 W) at room temperature for 3 h (Scheme 10), tetrahydronaphthalene (16) was obtained. An appreciable amount of 1,2-diethynylcyclohexene (17) (a retro-BC product) was also formed in all solvents except iPrOH. The overall transformation is an example a strain-driven photo-Cope rearrangement.44 The retro-BC product was not isolated under the thermal conditions, suggesting a different kinetic competition for the ring opening and H-atom abstraction at the excited surface.

Scheme 10. Photolysis of cyclodeca-1,5-diyn-3-ene (Hirama)

In another example, Branda and coworkers reported an elegant design of a cyclic photochromic enediyne 18. This compound undergoes hexatriene electrocyclization when activated with 365 nm light (Scheme 11).45 The cyclised product 19 can be reopened with the 21 ACS Paragon Plus Environment


Page 22 of 64

higher energy > 525 nm light. The absence of photochemical Bergman cyclization suggests that this process is slower than the electrocyclic ring closure.

hv X

365 nm > 525 nm Ph

S

S

Ph

S

S Ph

18

Ph Ph

19

Ph S 20 Bergman Product S

Scheme 11. Photo electrocyclization is preferred over Bergman cyclization in photochromic cyclic enediynes (Branda).

In 2000, Russel and co-workers reported the synthesis of the cyclic pyrimidine enediynes (21 and 23) capable of undergoing photo-BC (Scheme 12).46 Both the ketone 23 and the alcohol 21 were able to affect DNA cleavage at reasonable concentrations at physiological temperatures. As expected from the cyclization studies, 21 gave better DNA cleavage under photochemical conditions while 23 was superior under thermal conditions alone. Flow cytometric studies showed that while both compounds caused an increase in the number of cells with less G0/G1 DNA content (possibly apoptotic cells), their effects on cell cycle traverse were different. While alcohol 21 caused accumulation of cells in G0/G1-early S-phase of the cell cycle, ketone 23 caused accumulation of cells in the G2/M part of the cell cycle. Thus, it is evident that the two molecules intervene at two different stages of the cell division. OH OMe

OMe hν (313 nm) isopropanol

N N

MeO

OH

N N

MeO

21

22 O OMe

OMe N MeO

N

hν (313 nm) isopropanol

O

N MeO

N 24

23 ∆

Scheme 12. Photo-Bergman cyclization of pyrimidine based enediyne (Russel)

The enediynes are also known to display protein-cleavage ability which can be used as a self-defense mechanism. In this mechanism, microorganisms producing enediynes protect themselves through the sacrifice of a protein that is secreted by the same organism. Jones and co-workers reported the likely mechanism of enediyne mediated protein cleavage and then went on to design and synthesize photoactivated enediynes and study their protein-cleaving ability.47 The mechanism of protein cleavage involved the formation of radical 26 at the 22 ACS Paragon Plus Environment

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captodatively stabilized α-carbon. This radical then reacted with molecular oxygen to form the peroxo radical 29. This radical 26 could undergo strand scission, adduction or crosslinking (Scheme 13). Jones’s group provided considerable support for the proposed mechanism. For example, the cycloaromatization in the presence of labelled amino acids, such as dideuterated glycine (Scheme 14), indicated that the deuterium got abstracted by the diradical to form compound 35. The isolation of the dimerization product 37 and the amide 40 together with the deuterated aldehyde 39 could be explained on the basis of the formation of glycyl radical 36.

Scheme 13. Potential pathways for peptide degradation (Jones)

Scheme 14. Atom transfer using d2-Gly mimic (Jones)

Jones and coworkers also studied47 the effect of ring size on photo-BC for a series of enediyne 41with terminal Ph groups (Scheme 15).



Page 24 of 64

Ph Ph

hν (254 nm) n(H2C)

41

isopropanol

n(H2C)

Ph 42

Ph

n 2 3 4 5 6

yield 0% 13% 22% 16% 14%

Scheme 15. Effect of ring size on photo-Bergman cyclization (Jones)

Finally, the same group synthesized photochemically activated enediynes (43, 45, 46, 47) and used these compounds for specific protein targeting (Scheme 16).47 On the basis of the target-based molecular design, three independent classes of enediynes with defined protein targets were identified and elegantly synthesized (albumin, histone and estrogen receptor). Upon irradiation, these molecules could cause degradation of bovine serum albumin (targeted by molecule 43), histone (targeted by molecule 45), and an estrogen receptor (targeted by molecules 46, 47), thus opening a new application of enediynes as chemical proteases.

Scheme 16. Photo-activated enediynes targeted against bovine serum albumin, histone H1 and estrogens (Jones)

Peterson et al. reported the synthesis and reactivity of imidazole-fused cyclic enediynes toward photo-BC.48 The more conformationally rigid analogues gave higher yields of cycloaromatized products upon irradiation at ambient temperature. For example, the bicyclic analogue 48 underwent photo-BC to produce the cycloaromatized product 49


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(Scheme 17) and consequently induced single-strand breaks in supercoiled DNA at micromolar concentrations.

Scheme 17. Photoactivated imidazole-fused enediynes (Peterson)

One of the earliest PET (Photoinduced Electron Transfer) triggered photoactivations of enediyne was reported by Schmittel and co-workers. This approach provides an interesting photochemical route for activating enediynes for DNA cleavage at a relatively long wavelength (Scheme 18).49 Upon photoexcitation of the acridinium unit 51, an intramolecular ET is expected to afford an acridine radical and a guaiacol radical cation (Scheme 18). As silyl enol and silyl phenol radical cations (52) are prone to rapid O– Si bond cleavage with concomitant loss of a ‘silyl cation’, it should photochemically afford 52. Alternatively, 53 should also be formed via desilylation of 51 in presence of a “silophile” (fluoride or hydroxide). OMe

OMe OTIPS

OTIPS hν (419 nm) PET

N

N

51

52

BF4

F or OH

-TIPS OMe

OMe

O

O

C C

C

C

N

N

53

54



Page 26 of 64

Scheme 18. PET triggered photo-activation of acridine tethered enediyne (Schmittel)

The DNA photocleavage activity of enediyne 51 was examined at 300, 350 and 419 nm. Irradiation at 419 nm was more efficient than at shorter wavelengths leading to almost complete destruction of the supercoiled pBR322 DNA. Importantly, apart from single strand cleavage, double strand cleavage is also observed. It was proposed that 51 was activated for DNA cleavage via the reactive bis-cumulene 54. Treatment of 51 with NaOH removed the TIPS group (Scheme 18). As expected, reaction of 51 with NaOH led to extremely efficient cleavage of pBR322 DNA. An interesting variation of BC was reported by the Matzger group in the photochemistry of diethynyl sulfide. Photo-irradiation of bis(phenylethynyl) sulfide 55 in hexane in the presence of 1,4-cyclohexadiene (1,4-CHD, standard DNA surrogate) produced 3,4-diphenyl thiophene 57 through the presumed intermediacy of the 2,5didehydrothiophene diradical 56 (Scheme 19).50 This report constituted the first example of a 5-membered ring cycloaromatization that took advantage of the aromaticity of a heterocyclic ring. S R

hν (300 nm)

.

S

.

1,4-CHD

S

H

H

R 55

R

R 56

R

R 57

Scheme 19. Photo-activated bis(phenylethynyl) sulfide (Matzger)

4.2. Photoinduced Electron Transfer (PET) mediated C1-C5 cyclization of enediynes Alabugin and Kovalenko reported that the PET induced cyclization of enediynes 58 tethered to highly electron-withdrawing tetrafluoropyridyl (TFP) substituents at the alkyne termini can yield substituted indenes 59 and 60 via fulvene intermediate (Scheme 20).51 In spite of having the combination of structural elements suitable for BC, a unique C1–C5 cyclization (a radical-anionic version of the elusive Schreiner–Pascal cyclization)52 was observed which led to the formal abstraction of four H-atoms (compared to only two Habstractions in the BC).


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F F

F

N

N F

F

Y

F F

X

hν (320 nm) 1,4-CHD, CH3CN

F F +

F 58

N

F F

F

F F

F

Y

F N

Y

F

F

F

N

N

X

X 59

X = H, Me, Cl Y = H, Me, Cl

F

F

F

60

F

Scheme 20. Novel C1-C5 photochemical cyclization of 1,2-bis(tetrafluoropyridine) enediynes

This transformation is sensitive to the substrate structure. For example, the pyrazine variant 61 of the substrate underwent a cycloaddition (producing products 62 and 63) with the DNA surrogate (1,4-CHD) instead of expected C1-C5 cyclization (Scheme 21). This transformation provided a key to the understanding of a surprising finding of unexpectedly efficient DNA damage by TFP-monoacetylenes incapable of either the BC or C1–C5 cyclisation (vide infra) as it illustrated the potential of electrophilic alkyne triplet states to alkylate electron-rich π-systems. Subsequently, this observation laid the foundation for development of mono TFP-substituted alkynes as pharmacophores for DNA alkylation. TFP

TFP N

hν (313 nm) 1,4-CHD, CH3CN

N 61

TFP

TFP hν (313 nm)

N

CH3CN

N 62

TFP

F

N

N

TFP =

N 63

TFP

F

F

F

Scheme 21. Cycloaddition products obtained for pyrazine derivative

These reactivity features can be explained by the fact that the presence of strongly electron-withdrawing TFP substituents renders photoinduced electron transfer (PET) from 1,4-cyclohexadiene to the singlet excited states of the enediynes 58 highly exergonic (>25 kcal/mol). At this exergonicity, PET should be nearly diffusion-controlled. Indeed, efficient quenching of fluorescence of enediyne by cyclohexadiene was observed. PET induced C1-C5 cycloaddition can be rationalized through the mechanism outlined in Scheme 19. Unlike the cyclization of neutral enediynes, the C1-C5 cyclization of the enediyne radical anions led to an intermediate 69 stabilized through resonance involving cyclopentadienyl anion, thus rendering this cyclization mode possible. The mechanistic studies revealed that the four Hatoms abstracted (shown in blue in Scheme 22) by the enediyne warhead from the environment are delivered through a combination of H-atom transfers, electron transfers and proton transfers. 27 ACS Paragon Plus Environment


F F

F

Y

F F

X

F

X

65 F

H

Y

i)

H

R ii)

X

X

67

68

R

R X

Y R X

F

R

+

60 H

H

H

X H H

X

X

R

R=

70

H

F N

F

59 H H

R

Y

69 H

R H R

Y

Y

+

H

H H

R

hν

H H

R

Y

R

H H

H

R

Y

+

66

N

F

F R

+ F

N

F

F

71

F F F

X

F 64

N

Y

F F

N

F

F F

N

Y

hν F

58

*

F F

N

Page 28 of 64

F

Scheme 22. Possible mechanism for the C1-C5 cyclization

4.3 The photochemical C1-C5 cycloaromatization reaction of enynes Subsequently, the utility of photochemical activation for the discovery of new cycloaromatization reactions was expanded to aromatic enynes. The four ways for enediynes and enynes to transform into 1,4-diradicals are shown in Scheme 23. In each of these unusual processes, one chemical bond is created at the expense of two chemical bonds that are sacrificed. Bergman C1-C6 6-endo-dig / 6-endo-dig

Hopf C1-C6 6-endo-dig / 6-endo-trig

R R

R

R

R R

R Schreiner Pascal C1-C5 5-exo-dig / 5-endo-dig

R

R

R

R

C1-C5 5-exo-dig / 5-endo-trig

R

Scheme 23. The possible cycloaromatization modes for enediynes (left) and enynes (right).

The last member of the cycloaromatization reaction family, a C1-C5 cyclization of enynes, had remained elusive until, in 2015, Mohamed et al. reported53 a new "selfterminating" strategy to trapping the diradical product of this process (Scheme 24).


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Ar

Ar Ar

hν (300 nm) OR 72

+

DCM (1mM) 73 (major)

R = H, Me

74 (minor)

Quantum Yield: 0.01 to 0.04

Quantum yields for the reactions were determined by comparing the conversion rate of 50 µM solution of enyne and the benzophenonebenzohydrol actinometer system (reduction of a 0.1 M solution of benzophenone in a 1 M solution of benzhydrol, Φ = 0.57)

Scheme 24. Photochemical cyclization of enynes resulting mainly in the formation of benzofulvenes.

In this light-promoted reaction cascade, C-C fragmentation was coupled with an intramolecular H-atom abstraction made possible by the introduction of a CH2OH moiety in the enyne 72 which converted the diradical directly into a closed-shell species via a single concerted step. It was observed that the formation of benzofulvenes 73 is the preferred path for

the

photocyclization

for

most

of

the

studied

enynes.

Generally,

the

photocyclization/fragmentation was completed in 4-6 hours to cleanly provide benzofulvenes, as the only isolable products, in 83-95% yields. Only for certain enynes where the alkynyl substituent

Ar

=

Ph,

tolyl,

pyridine

and

p-fluorophenyl

was

the

photocyclization/fragmentation inefficient and produced a mixture of naphthalene derivatives 74 along with the benzofulvene products 73. The addition of benzophenone (a triplet sensitizer) to the reaction mixture of these enynes successfully suppressed naphthalene formation and promoted exclusively the formation of fulvene products. These results strongly suggested that the fulvene formation proceed from the triplet manifold.

Scheme 25. Possible mechanism for the C1-C5 cyclization of enynes

Photochemically-induced alkene twisting directly accounts for the efficiency and low barrier for five-membered ring formation in the present system. The cyclization reaction benefits from a highly favorable combination of two factors: a) the twisted alkene π-bond projects a radical orbital toward the alkyne and b) the alkyne has two orthogonal π-systems, so it can accept the incoming radical without breaking its conjugation with the benzene πsystem (Scheme 25). This combination of factors led to a surprisingly facile 5-endo-trig closure that bypasses the usual stereoelectronic limitations for this process which, in the ground state, requires significant distortion to reach the favorable Burgi-Dunitz trajectory. It 29 ACS Paragon Plus Environment


was found that twisting of the olefin double bond resulted in relief of T1 state antiaromaticity and gain of some closed-shell Hückel aromaticity of the phenyl group, as evidenced by a series of aromaticity indices, changes in bond length alternation, and spin densities. An interesting feature of the cyclization/fragmentation cascades described above is that they provide an approach to photochemical release (“uncaging”) of formaldehyde. It would be interesting to expand this work to the photo release of other aldehydes and ketones.

4.4. Enediyne metal complexes as photoinduced diradical generators In the quest for therapeutic strategies involving application of long-wavelength (λ > 600 nm) with higher tissue penetration, two approaches could be considered for initiating the BC. First, increasing conjugation of the substrates could decrease excitation energy but would likely lead to solubility problems. Alternatively, one can explore the photophysics associated with the presence of the metal ligands with variable oxidation states and donor/acceptor redox potentials. Zaleski and co-workers developed a number of creative approaches for phototriggering BC of enediyne/metal complexes.54 For example, they took advantage of a strongly absorbing oxygen-to-V(V) (75) LMCT transition, which when pumped with near-IR photons (λ = 785 nm), led to the formation of polymeric material 77 with spectral characteristics consistent with the formation of metallo-poly(p-phenylene) products derived from BC (Scheme 26).

Scheme 26. Photo-excitation via Ligand-to-Metal Charge Transfer (Zaleski)

In another interesting approach55, this group used an MLCT-facilitated photo-activated BC of of Cu(I) and Cu(II) metalloenediynes (78 and 81 respectively) and showed that the intermediates perform double-stranded DNA cleavage (Scheme 27).


Page 30 of 64

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+ + O

O N N

N

a

N

O

N

N

λ ≥ 395 nm

N Cu

O

O Cu

N O

O

O

79

OCOMe

78 O N

b

O

N O

80

O N N

N Zn

N

O

O 82

S

2+

O

OCOMe

O N N

N Cu

O

O λ ≥ 395 nm

N

N

b

a O

N O 80

S

a) 1,4-CHD or 2-propanol, CH3CN; b) EDTA, DMF, H2, CH2Cl2

81 Quantum Yield for photocyclization of 78 (Φ366 ) = 4.2 ( ±0.6) x 10-5

S = Solvent

Scheme 27. Photo-excitation via Metal-to-Ligand Charge Transfer (MLCT) (Zaleski)

In contrast, the uncomplexed ligand and Zn(II) compound 82 were photochemically inert under the same conditions. It is also important to note that the intermediates produced upon photolysis of 78 and 81 degrade both pUC19 plasmid DNA as well as a 25 base pair double stranded oligonucleotide via C-4’ hydrogen-atom abstraction. Both single- and double-strand cleavages were observed in micromolar concentrations. 4.5. Photoactivation of locked or acyclic enediyne This approach involves the administration of a prodrug with photocleavable protecting group(s) that mask the nucleophilic character of an amine or a phenolic hydroxyl group and thus inactivate the enediyne warhead. Photolytic deprotection changes the molecular structure and enables the flow of electrons that results in the release of strain by the opening of an epoxide ring and triggers the diradical generation. As a consequence, the enediynes get activated toward BC under ambient conditions.



O O

Page 32 of 64

O

O N

hν (365 nm)

O

O

N

O

Nu

O

a

NO2 OH

O

OH

HO

85 H

O

Nu O

room temperature

N HO

O

N HO O

OH HO

86

Nu

H

O

BC Nu

HO

OH

O

84

83

N HO

OH

87

Me

a) THF/H2O (10:1), argon, 0 °C, 40 min; b) EtOH/THF/phosphate buffer (pH 8.0) (1:1:1), air, 25 °C, 1.5 h

Scheme 28. Activation of the Dynemicin model by photodeprotection (Nicolaou)

The underlying concept was exemplified through an elegant design of model compound 83 by Nicolaou and coworkers56 that resulted in chemistry depicted in Scheme 28. As anticipated, after the photolytic deprotection of the o-nitrobenzyl group, the phenolic intermediate 84 showed propensity toward epoxide opening due to flow of electron from phenolic –OH group. The isomerization yielded to p-quinone methides 85 which could be intercepted with a variety of nucleophiles. The derived cis products 86 underwent spontaneous BC at ambient temperatures to yield 87. Wender et al. reported57 a similar synthetic route leading to BC triggered by photolytic removal of N-Voc (photolabile functional group). The photochemical activation (λ > 300 nm) of N-Voc dynemicin analogue 88 (Scheme 29) induced both ss and ds DNA cleavage. The deprotected analogue underwent epoxide ring opening (to produce 89) followed by subsequent interception by suitable nucleophile (MeOH or chloride). Subsequent BC occurred under ambient conditions. The enediyne was also activated toward thermal BC by an acid-catalyzed epoxide opening. Thus, the molecule is equipped with a dual triggering mechanism (pH as well as light).


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MeO

NO2

MeO

a O

88

H N O

hν (365 nm)

H

O H

N O

HO H

89

MeOH

88

hν (365 nm) b

H

MeO HO H 91

BC

H N

90

HO H H N

HO H H N HO

H

N HO

H H

HO Cl HO H H

H

HO MeO HO H 92

.

H N DNA

. cleaved DNA

94

H H

HO MeO HO H H 93

a) THF/MeOH; b) AcCl, THF, MeOH

Scheme 29. Activation of the Dynemicin model by Photodeprotection (Wender)

An unorthodox approach based on a photo-removable protecting group was also reported by Basak et al.58 One of the earlier problems59 involving inherent reactivity of the substrate leading to intramolecular nucleophilic attack was solved by using a photolabile protecting group to reduce the N-nucleophilicity (Scheme 30).

Scheme 30. Photo-activation of enediyne through removal of photo-labile protecting group (Basak)

The 5-nitro veratryl carbamate (N-Voc) was chosen because of its electron withdrawing ability and the ease of its removal under photolytic conditions (irradiation at λmax= 350 nm). As expected, compound 95 was able to induce single-strand cleavage of plasmid DNA upon irradiation at 365 nm. The ketone 95 also caused partial DNA damage in the dark. However, the efficiency of cleavage was ∼2.5 times lower. This observation ruled out the cleavage via the Maxam Gilbert mechanism60 as the major pathway.



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4.6. Photoactivation via reduction of ring size In 2005, Popik and co-workers reported61 synthesis and photochemistry of a cyclopropenone containing enediyne precursor, 2,3-benzobicyclo[8.1.0]undec1(10)-en-4-yn11-one (99). The method takes advantage of photochemical activation of a highly thermally stable enediyne precursor. This compound has high thermal stability and shows no signs of decomposition after heating at 84 °C for 7 days (Scheme 31). However, it readily undergoes an efficient in situ activation via the photochemical generation of one of the triple bonds (in 100). UV irradiation of the substrate 99 resulted in an efficient (Φ300nm = 0.45) and quantitative decarbonylation producing benzannulated enediyne 100. This enediyne was found to undergo BC at 84 °C. O H hν (300 nm)

a

(- CO) 101 H

100

99 Quantum Yield (Φ300) = 0.45

a) 1,4-CHD, 80 °C

Scheme 31. Photo-activation of benzene based enediyne via decarbonylation (Popik)

In the following year, the same group expanded62 this triggering strategy via photolysis of p-quinonoid cyclopropenone-containing enediyne prodrug102 (Scheme 32). It was observed that the cyclopropenone derivative 102 was stable up to 90 °C but readily produced reactive enediyne 103 upon single-photon (Φ300nm = 0.46) or two-photon (σ800nm = 0.5 GM) photolysis. O O

hν (300 nm) or (hν)2 (800 nm)

O

O

H

O

H 104

a

(- CO) O

O

102 a) 1,4-CHD, 40 °C

103 Quantum Yield = 0.46

Scheme 32. Photo-activation of p-quinone based enediyne via decarbonylation (Popik)

The photo product 103 underwent BC at 40 °C with the lifetime of 88 h to afford napththoquinone 104. The two-photon excitation provides a conceptually interesting approach for using light in the “photo-therapeutic window” (a region of relative tissue transparency between 650 and 950 nm). In this particular case, the two-photon induced 34 ACS Paragon Plus Environment

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photochemical reaction of 102 is much cleaner because it is not accompanied by the secondary photochemistry. O N2

O C

O hν (350 nm)

O

C O

O +

a

105

107

106 a) ROH, 1,4-CHD

CO2R

O

O

CO2R

110

108 H

OH

H CO2R

Quantum Yield (Φ350) = 0.36

109

H

CO2R OH

111 H

Scheme 33. Photo-activation of enediyne via ring contraction (Popik)

More recently, Popik and coworkers reported another interesting recipe63 for generating enediyne through the photolysis of a prodrug containing α-diazo-β-diketone moiety. This work reported the first example of triggering of the thermal BC by the photochemical ring contraction. Upon irradiation or thermolysis, compound 105 underwent Wolff rearrangement to produce reactive 10-membered enediynes 106 and 107 (Scheme 33). Both of the enediynes underwent spontaneous BC. Solutions containing various concentrations of 105 and ȹX174 supercoiled circular DNA (30 µg/µL) were irradiated at 351 nm using an argon ion laser at ca. -5 °C. Diazodiketone 105 induced substantial singlestrand cleavage (RF II) of ȹ X174 DNA upon irradiation, although the linearized form (RF III) was observed only at higher concentrations of the cleaving agent (>500 µM) and prolonged irradiation. König et al. reported64 a remarkable increase in the activity towards BC for bipyridyl containing enediyne due to decrease in the distance between the acetylenic carbons undergoing covalent connection (c, d-distance) upon complexation with mercury (II). Inspired by this report, Basak and co-workers came up with a strategy65 based on photoswitchable enediynes with variable reactivity. Cyclic enediynes 112 and 114 containing stable E-azo moiety (azoenediynes) (Scheme 34) isomerized to their corresponding Z-isomers (113 35 ACS Paragon Plus Environment


Page 36 of 64

and 115) upon irradiation with 350nm light. The structural change affected by the photoisomerization is expected to decrease the c, d-distance between the alkynes. Reactivity studies toward BC using Differential Scanning Calorimetry (DSC) predictably indicated higher reactivity for the Z-isomers as shown in Scheme 34.

N N N N

N N N N

λ (350 nm)

O

O

O

λ (350 nm)

O

O

heat

O

O

114

113

112

181 °C

O

heat

151 °C

115

93.8 °C

69.6 °C

N N

N N

N N

N N O

O

O

O

O

O

O

O

117

116

119

118

Scheme 34. Triggering of BC via photo-isomerization azo-enediyne (Basak)

4.7. Photoactivation via conversion of diazoketone to ketene The research group of Nakatani and Saito designed and synthesized the diazoketone 120. which upon photo-irradiation (high-pressure Hg lamp), rearranged to produce the ketene 121

in

situ

via

Wolff

rearrangement.

This

intermediate

underwent

Moore

cycloaromatization66 to produce the phenoxy diradical. Upon H-atom transfer, this diradical produced the final product 121 (Scheme 35).67 This process was shown to induce cleavage of ds plasmid DNA (pBR322). H

Ph

Ph

120

OH

C O

O R

Ph 1,4-CHD

hν (λ>300nm)

R

R

N2 121

R = H, Me


122

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Scheme 35. Photochemical conversion of diazoketone to ketene leading to Moore Cyclization (Nakatani and Saito)

4.8. Development of enediyne/enyne - amino acid hybrids: the unique properties of lysine conjugates. After the potential of TFP substituted alkynes was disclosed by Alabugin and coworkers,51 continuous research efforts directed towards increasing water-solubility and the biocompatibility of various enediyne/enyne based pharmacophores were undertaken. The great potential of the amino acid conjugates of the enediyne/enynes, acted as the driving force behind this scientific journey. The amino acids with their diverse chemical architecture can be expected to provide broad variations in charge, hydrogen bonding ability, lipophilicity, steric bulk, etc.68 Tumor cells actively convert glucose and other substrates to lactic acid with the concomitant decrease in the extracellular pH as most of the additional H+ ions are transported outside of the cell.69 However, it is possible to equilibrate the extracellular H+ ions with the cell contents, thus lowering intracellular pH of cancer cells, using certain drugs such as amiloride, nigericin and hydralyzine.70 Under such conditions, one may take advantage of the acidic environment of cancer cells for the design of pH gated drugs which would selectively respond to the lower pH of cancerous cells. Further, the pH controlled target selectivity of the drug is expected to increase in presence of hyperglycemia and/or hypoxia, as cell pH under such conditions can get as low as 5.5. Considering the acidic micro-environment of a cancerous cells and tissues, tethering lysine to the ‘warhead’ had a number of advantageous properties. The ammonium salt formed via the protonation of the side-chain amino group would facilitate71 pH selective electrostatic-binding to the negatively charged phosphate backbone of the DNA. This interaction would mimic72 the well-known interaction between cellular DNA and lysine-rich histone proteins. Interestingly, these conjugates are able to selectively73 bind and cleave at Gsites flanking A-T tracks. Furthermore, lysine can “up-convert” the oxidative DNA damage74 via cross-linking with guanine radical cation, 8-oxo-guanine, or other oxidised 8-oxoguaninederivatives (Scheme 36).



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Lysine-mediated "processing" of base damage: O N

oxidation

NH

N N R G 123

O

O

H N

NH

O N R

[Ox]

NH2

-

Lys NH2

-e

N R

N

G+.

NH2 125

OGox

124

Lys NH2

O

N

hv, [Ox]

N or

O

NH2

N OG

Base damage via lysine oxidation: Lys NH2

N

or

cross-linking, spiro-adducts, etc

NH

N N R G

NH2

Scheme 36. Oxidative DNA damage caused by lysine

Alternatively, formation of lysine-DNA adducts can also proceed through initial oxidation of lysine followed by reaction of an N-centered radical with DNA. This cross-linking can block cell replication and promote cell death. In an interesting scenario, lysine can mimic75 8oxoguanine-DNA glycosylases (OGG1) by creating abasic sites and converting them into strand cleavage (Scheme 37). Lysine-mediated "processing" of backbone damage = creation of abasic sites and accelerated strand scission: DNA O

O

O

O

H N

O

DNA O NH

N

DNA

N

NH2

129

DNA O

OH

O

128 DNA O

OH N

Lys

OH

N

Lys NH2 DNA 127 Abasic site

126 DNA O

Strand scission

O

Lys NH2

Lys

Lys NH2

130

O

DNA

OH O

Lysine/TFP-enediyne conjugates [Ox] Lys-NH2

?

DNA

hv Hybrid molecules combining photoxidant with lysine moiety

Scheme 37. Creation of abasic site by lysine through mimicking 8-oxoguanine-DNA glycosylases (OGG1)

4.8.1. N-lysine conjugates versus C-lysine conjugates There are two possible ways of attaching lysine to the enediyne/enyne chromophore via peptide linkage: N-conjugate (linking the α-NH2 group of the amino acid to the chromophore), C-conjugate (linking the α-COOH group of the amino acid the chromophore) (Figure 9)76. All examples in this section involve C-conjugates. A practical example of Nconjugate was reported77 by Zhou et al. while studying visible-light-induced cleavage of 4-αamino acid substituted naphthalimides and its application in DNA photocleavage (vide infra, 38 ACS Paragon Plus Environment

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section 6). Importantly, both amino groups of the lysine residue of C-conjugates are free for facilitation of DNA-damage. It was experimentally found (via NMR and fluorescence quenching) that the α-amine has the considerably lower pKa amongst the two. We will show below that this feature bestows upon it the ability to distinguish between the cancerous and healthy tissues.78 Thus, the presence of the free α-amine is particularly important for the design of pH-controlled DNA-cleavers. Furthermore, the di-cationic conjugate acid of the Cconjugate is expected to have greater DNA binding capability. Based on these aspects, one can expect C-conjugates to have unique advantages over the more common N-conjugates.

O

NH2

NH2

O

Chromophore N H

HN

Chromophore

HO

Only one amino NH2 group is available for protonation

-NH2 group -NH2 group (More basic amine) (Less basic amine) pKa = 9.5 - 10.8 pK = 6.9 - 7.6 a

C-lysine conjugate

N-lysine conjugate

Figure 9. Comparative study between C-lysine and N-lysine

Furthermore, the ability of the C-lysine conjugates to recognize a single phosphate monoester in the presence of multiple phosphate diesters of the DNA backbone makes them a privileged choice in the domain of amino acid conjugates (vide infra).73b,79 Potentially, these compounds can be used for the development of possible RNA interference involving a chemical analogy to siRNA with a potential for photochemical triggering.79 Based on this background, one can easily comprehend the potential of pH gated (to ensure spatial control) photo-triggered (to ensure temporal control) enediyne/enyne based chemotherapeutics. This strategy is expected to afford prodrugs which can be photochemically activated and are selective towards low-pH cancerous cells. 4.8.2. Application of Photoinduced Electron Transfer (PET) based lysine conjugates as DNA cleaving agents The evolution of PET-induced pH-gated amino acid-enediyne/enyne hybrids led to the development of conjugates with high DNA damaging efficiency.80 The molecular design of such amino acid hybrids was based on two factors. Firstly, the basicities of the two amino groups (α-NH2 and ε-NH2) differ considerably. While the ε-ammonium group has a pKa of ∼10–11, remaining protonated at all physiologically relevant pH, the α-ammonium group has 39 ACS Paragon Plus Environment


a pKa of ∼6.5–7. This lower basicity of the α-amino group allows for a change in protonation at a pH threshold separating healthy and cancer cells. Protonation of the less basic α-amino group enhances the DNA-cleaving activity via a combination of synergistic effects. First, it increases the binding interaction of the drug with DNA by the virtue of enhanced electrostatic interaction between the phosphate backbone and the conjugate acid of the drug. Interestingly, experimental observations suggested that the change in pH also modified the basic mode of drug-DNA binding (intercalation/groove binding). By having the α-amino acid protonated and bound to DNA, the warhead is positioned closer to the intended damage site, increasing cleavage activity. Second, the difference in protonation results in marked photophysical changes. If an amino group remains non-protonated, then its lone pair of electrons can quench the warhead’s excited state via electron transfer. Intramolecular back electron transfer of the initially formed charge-separated state should quickly restore the ground state of the enediyne. This deactivation path is effectively a way to “waste a photon” by converting photochemical excitation into thermal energy. Its presence dramatically reduces the DNA-damaging efficiency of the warhead (Figure 10).80 Photophysical experimental determined that the lone pair of electrons over α-NH2 quenches the photoexcited state of the chromophore most efficiently due to its spatial proximity. Since this amino group gets protonated at lower pH than the remote ε-NH2 group, it plays the key role in the pH-selectivity of the observed DNA cleavage. One has to keep in mind though that protonation of both amino groups is essential for increasing the overall efficiency of DNA damage because cationic nature of the protonated amines ensures stronger electrostatic binding to the negatively charged phosphate backbone. Based on the above principles, various synthetic variations of this family of molecules were tested by Alabugin and coworkers in an effort to connect the effect of the chromophore (TFP substituted enediyne/enyne) and the DNA binding part (lysine and other amino acid bridges with varying chain length) structure with the overall ability to cause DNA-cleavage and apoptosis.


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DNA damage

DNA damage DNA

DNA

Excited state Chromophore (DNA cleaver) PET

XH3N

no internal quenching

NH O

H3N ACTIVE FORM

X

Theshold cancerous cell pH

-H +

H

(no DNA damage)

Excited state Chromophore (DNA cleaver) PET

..

H2N

internal quenching of the excited state via intamolecular PET H3N

NH O

INACTIVE FORM

hν ν

After internal quenching

.-

Quenched Chromophore (DNA cleaver)

.+

H3N

hν ν

NH O

Chromophore (DNA cleaver)

Chromophore (DNA cleaver) H3N

H3N

NH

H2N

O

H3N

NH O

Back electron transfer

H3N

Figure 10. Activation/Deactivation of lysine conjugates viaPhotoinduced Electron Transfer (PET)

Synthesis and reactivity of this class of molecules were first reported in 2005 (Figure 11).81 The ability of synthesized C-amino acid-enediyne hybrids to cleave DNA under irradiation (λ >305 nm) was investigated using conversion of supercoiled plasmid DNA (plasmid pB322) into the respective relaxed circular and linear forms (Forms II and III). The relative amounts of the three DNA forms were determined by densitometric analysis of the gel electrophoresis bands at different irradiation times. Subsequent statistical analysis of DNA-photocleavage by Povirk test82 for compound 132 (for R=TFP) revealed that the obtained range of n1/n2 values (14–53) is significantly smaller than that expected from a completely random process (where n1 and n2signifies the ss and ds-breaks respectively per DNA molecule). The obtained results established that more double-strand breaks are produced than can be accounted for by coincident single-strand breaks and that the DNA-photocleaver interaction played an important role in the DNA cleavage. Further, n1/n2 values were found to be smaller for compounds 131, than for compounds 132. This finding established that the longer length of 41 ACS Paragon Plus Environment


the spacer between the chromophore and the ammonium group may play a role in ensuring a better alignment of the enediyne for interaction with opposing DNA strands.

Figure 11. Lysine–enediyne conjugates as photochemically triggered DNA double-strand cleavage agents

The 2011 report disclosed a more potent dipeptide variant of C-lysine-enediyne (or enyne) conjugate (133-136).83 In the second generation of hybrid molecules, the warheads were tethered to two lysine groups (Figure 12). The conjugates were equipped with either two α- and one ε-amino groups (133-134), or with one α- and two ε-amino groups (135-136).

Figure 12. Structures of bis-lysine conjugates

At physiologically relevant pH thresholds, these conjugates switch either from monocation to tri-cation (for two α- and one ε-amino group) (133-134) or from di-cation to trication (for one α- and two ε-amino groups) (135-136). As expected, the greater electrostatic binding of the bis-lysine conjugate with the DNA phosphate backbone than the mono-lysine conjugate manifested as much better DNA cleaving efficiency of the former. The observed ds/ss ratios of up to 2:1 by far exceed those of the naturally occurring calicheamicin and 42 ACS Paragon Plus Environment

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bleomycin, indicating that not only is the cleavage activity increased at low pH, but also the recognition of initial ss break sites by these conjugates, creating subsequent damage that leads to an overall ds break, have also strongly enhanced. These conjugates were also capable of the efficient penetration through cellular membranes and induction of intracellular DNA damage. The high phototoxicity displayed toward melanoma cells with CC50 in the 10−7 M range suggested that this class of hybrid molecules has potential value for the development of future light-activated anticancer agents. Subsequently, this strategy was successfully expanded by Hatial et al.84 who described the design, synthesis and DNA cleaving study of two 10-membered benzo-fused Nsubstituted cyclic enediynes (Figure 13), one an amino methyl (regio-isomeric mixture of 137 and 138) and the other, a C-lysine conjugated derivative (regio-isomeric mixture of 139 and 140). NH2 NH2 NH3 2CF3COO

NH3

137

2CF3COO

138

NH2 NH2 R

R 139 3CF3COO

140 3CF COO 3

NH R=

H3N

O NH3

Figure 13. Structures of benzo-fused N-substituted cyclic enediynes

The BC onset temperature for these derivatives was reduced to around 65 °C as confirmed Differential Scanning Calorimetry (DSC). For compound 137/138, the ss:ds ratio was 2.26:1 at pH 5.5. Compound 139/140 showed even better cleavage efficiency at lower pH, ss:ds ratio was1.35:1 at pH 6.5 to 0.80:1 at pH 5.5. Although this reaction was not phototriggered, the excellent results obtained from the DNA cleavage study of the C-lysineenediyne hybrid provided another indication of potential value of such amino acid conjugates for controlled DNA binding.



Page 44 of 64

Mono acetylene-Lysine conjugates TFP

A TFP

TFP

NH2

H2N

N H pH 6 34% ds DNA

141

N H

143 NH2

pH 6 15% ds DNA

R = Ph, TFP

TFP H2N

N H pH 6 0% ds DNA

R

C meta

TFP

O 144

N H

151

N H

145 hν

1,4-CHD

O

O

O

hν

H2N

Acetamides

TFP

B

NH2

142

For 144-146, Samples were placed on ice at a distance of 20 cm from 200 W Hg-Xe lamp, with long pass filter with 324 nm cut-on wavelength

N H

O

O

O H2N

1,4-CHD

146

hν

1,4-CHD

NH

O

NH2

N H

meta

R

O O O

TFP N H

147

HN

N +

TFP

TFP

148 149

O 150

TFP

Scheme 38. (A) DNA cleaving activity of mono-acetylene conjugates of lysine (Yang); (B) Reactivity of monoacetylene acetamide derivatives under photochemical condition (Kaya); (C) Structure of C-lysine-m,m-enediyne hybrid (Kaya)

Inspired by the interesting results85 obtained by Yang et al. regarding the photochemistry and photophysics responsible for the highly efficient DNA photocleavage activity of the mono acetylene-lysine conjugates (141-143) (Scheme 38A), Kaya et al. tested86 the relative photoreactivity of the acetylenic o-, m- and p- acetamidyls (146, 145 and 144 respectively) towards 1,4-cyclohexadiene (1,4-CHD) as a DNA surrogate (Scheme 38B). The conjugates of amino acids and aromatic alkynes were expected to damage the DNA through a combination of photoinduced electron transfer and base alkylation. This study found that both p- and m- alkynes (144 and 145) alkylate 1,4-CHD to form the formal photocycloaddition products (147 and 148) whereas o-isomer (146) underwent intramolecular cyclization with 1,4-CHD to give product 150. It was also found that only the p- and malkyne isomers were capable of causing the ds DNA cleavage. To gain further insight into the biochemical process of DNA damage, associated with such hybrids, the authors designed and synthesized the C-lysine-m,m-enediyne hybrids (151) which were structurally impaired to undergo BC or the C1-C5 cyclization (Scheme 38C). The alkyne substitution was varied from 44 ACS Paragon Plus Environment

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H to TFP-group, in order to study the effect of electronic distribution on the overall process. Standard methods to monitor the DNA photocleavage were used while studying the photoinduced interaction with supercoiled plasmid DNA. The conversion to relaxed circular and linear DNA forms was determined by gel electrophoresis followed by densitometric analysis. The hybrids showed much better DNA cleaving activity at the lower pH (pH = 6, conjugate 2 ss:ds 1.0:1.2). Scavenger experiments for reactive oxygen species (ROS) showed that generation of singlet oxygen is an important contributor to the observed DNA dsdamage. DNA binding assays using UV/Vis-absorption titration and fluorescence based ethidium bromide displacement assay revealed that for phenyl substituted derivative 151 (for R = Ph), intercalative binding of the chromophore to the DNA is observed. For the corresponding TFP derivative of 151 (For R = TFP), it behaved as an intercalator at the lower pH, but became a groove binder at higher pH. Regarding the modification of the DNA binding part, a third generation of lysine hybrids comprising of a further variation in the binding part was reported78 in 2016 where two α-amino groups were grafted into the hybrid enyne (Figure 14).

Dipeptide acetylene conjugates with two α-amine: dual contribution to pH switching R

O

DNA cleaver

N H O NH2 (neutral molecule in normal tissues)

+

R

H

H2N

O

H3N

-

H

Cancer threshold pH

O

NH3

DNA cleaver N H

(dicationic species in cancer cells)

Figure 14. Dual protonation of the two α-NH2 groups at lower pH (Kaya)

The rationale behind the design was that at the cancer pH threshold, the neutral hybrids will be converted into their conjugate dications. This protonation-gated behaviour was expected to increase the selectivity of the conjugates. In order to study the effect of the spacer length between the two amino groups ornithine and α-alanine were also incorporated as the covalent linkers with length different of that provided by lysine (Figure 15). The mode of binding for the chromophore with the DNA was studied using UV/Vis-absorption titration and fluorescence-based ethidium bromide displacement assay. At pH=6, intercalation mode of DNA binding was preferred but at higher pH (pH=8), mixed mode of binding was observed. Almost all compounds exhibited ds DNA cleavage, with compound 152 (with phenyl alanine; i.e, R = CH2Ph) giving the best results in terms of pH-gated selectivity (change from neutral to pH 6). Statistical analysis shows that the ds cleavage was caused by a 45 ACS Paragon Plus Environment


Page 46 of 64

coordinated ds cleavage event instead of two random ss cleavage events. Scavenger experiments revealed that there is no significant contribution of reactive oxygen species (ROS) on DNA damage. The molecules were also found to be able to form the abasic sites in DNA. Ornithine Bridge

Lysine Bridge

TFP

TFP R

H N

H2N O

R = Me, CH2Ph,

O

O

NH2

R

N H

152

NH2

O N H

NH2

N H

153

R = H, Me, CH2Ph

CH2 N H

α-Amino Alanine Bridge Bridge TFP O R NH2

O N H

NH2

N H

R = Me, CH2Ph 154

Figure 15. Structures of dipeptide acetylene conjugates with two α-NH2 groups with varying bridge lengths (Kaya)

5. Multiple mechanisms of DNA damage With a few references made to application of scavenger experiments in this review, it becomes pertinent to highlight the significance of this experimental tool in understanding molecular mechanisms of the observed DNA damage. Apart from the diradical formation by the photo-triggered BC, or four formal H-atom abstractions by the C1-C5 cyclization, there are other pharmaceutically relevant chemical reactions that can also damage DNA. This list is broad and includes processes like cross-link formation between dipeptides and DNA, alkylation of DNA nucleobases, nucleobase oxidative damage via electron transfer, hydrogen abstraction from the sugar moieties, and other reactions with ROS. Such complicated biochemical processes are often followed by the generation of reactive free radicals. It is through scavenger experiments87 that one gets the necessary insights into the extent of the involvement of various reactive species in the process. Figure 16 provides a schematic representation of the various photoinduced free radical-generating mechanisms of potential biological significance. All of them start with photochemical excitation that transforms the aryl acetylene conjugates into the excited singlet 46 ACS Paragon Plus Environment

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states. Subsequently, triplet excited state can be generated via the inter system crossing (ISC) of the initially formed singlet excited states. Both singlet and triplet states of alkynes are sufficiently electrophilic for alkylating reactive π-systems such as DNA bases. In addition, the triplet state is capable of sensitizing the formation of singlet oxygen (1O2). The latter highly reactive form of oxygen can oxidize DNA bases, especially guanine. Another path to cleave DNA is via electron transfer (ET) from DNA that generates alkyne radical anion, capable of transferring an electron to molecular oxygen with the formation of superoxide.87 The latter species can undergo further reactions with the formation of reactive oxygen species (ROS) including the hydroxyl radical. Reactive Oxygen Species (ROS)

3

O1

Sp

..

Hydrogen abstraction/base modification

OH

3

O2

R S1

ISC

NH3

O

.

O2

Guanine modification

R'

Sp

..

R

NH3

O R'

. .

O2

R

DNA Sp

ET

NH3

O

+ DNA

.

Base modification

R'

T1

hv intermolecular proton-transfer

intramolecular proton-transfer

.

R Sp NH3

O R'

Base alkylation/ hydrogen abstraction

H

Sp

NH2

O R

.

R

'

R

H

Sp

NH3

O R

'

S0

Figure 16. Possible mechanistic pathways for radical mediated DNA photocleavage.

For example, scavenger experiments were carried out to investigate the origin of DNA damage by molecule 151 (Scheme 38C).86 There was no significant effect on the efficiency of this process by hydroxyl radical scavengers (DMSO and glycerol) for both compounds (R = Ph, TFP). On the other hand, the ds DNA photocleavage activity of the conjugate R = TFP was noticeably inhibited (70%) by singlet oxygen scavenger (NaN3). This inhibition was even 47 ACS Paragon Plus Environment


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more pronounced for conjugate 151 with R = Ph, where singlet oxygen is responsible for a significant part of DNA photocleavage.

6. Miscellaneous reports of photoinduced DNA damage In 2013, Mandal et al. reported88 the synthesis and DNA cleaving study of a series of enediynes differing by the length of the alkyl chain (Figure 17). These enediynes (155-157) were grafted onto Au-NPs and their photo-assisted cyclization was investigated. The photoactivated reactivity of the enediynes coordinated to the gold surface seemed to be enhanced by the presence of the aromatic ring. Interestingly, the fastest cyclization process was observed with the shortest spacer where it led to polymerization of the coating agent (which was observed using TEM). On the contrary, elongation of the alkyl chain reduced the cyclization rate and inhibited polymerization. When tested as DNA cleavage agents at the optimized wavelength of 350 nm, Au-NPs were unable to inflict any DNA cleavage, which was attributed to the inability of the diradicals to approach the DNA sugar backbone. Interestingly, mixing the free ligand with Au-NPs enabled DNA cleavage, both thermally and photochemically induced. It was suggested that the electrostatic interactions between DNA and Au-NPs in solution may induce a conformational change of DNA, which promoted the DNA damage. SH

SH

155

SH

SH

156

SH

157

SH

Figure 17. Structures of enediynes which were grafted onto Au-NPs (Mandal)

In 2015, Zhou et al. described77 the photocleaving properties of 4-α-amino acid substituted naphthalimides 158 upon blue light irradiation. We include these molecules to illustrate a new potentially interesting role of lysine (in its N-conjugate form) in photoreactivity. The photoactivation of these molecules occurred at the C–N bond between 4amino group and the amino acid residue. Release of a fluorescent 4-aminonaphthalimide product helped in monitoring the reaction progress. Scavenger experiments suggested that the DNA photocleavage in the presence of DNLys (158) may involve the reactive oxygen species, especially the singlet oxygen. The photoactivation of DNLys showed the photocleavage ability towards DNA, suggesting its potential application in phototherapy after


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further modifying the 4-α-amino acid substituted naphthalimides to enhance the DNA binding ability (Figure 18).

Figure 18. Structures of DNLys and DNNH (Zhou)

7. New Strategy to Generate Double Strand Breaks: photochemical ss ds break conversion. Towards photochemical RNA interference In this section, we will discuss a unique and potentially very useful property of CLysine conjugates of photochemically-activate enediynes. It was found that these molecules can bind to partially broken DNA (i.e., an ss-damage site) and “upconvert” this partial damage into a full double-strand cleavage.89 A variety of damage sites was tested (both nicked and gapped DNA with phosphates at 3′ and/or 5′ locations) at different locations of the target within the DNA strand. The results suggested that such binding is quite general and can provide a basis for the photochemical transformation of an easily repairable ss damage to a therapeutically more important ds damage that lead efficiently to cell apoptosis (Figure 19).



Figure 19. Photochemical conversion of ss DNA damage to ds DNA damage based on recognition of DNA ss cleavage by a lysine conjugate. (Left) Comparison of photocleavage efficiency and selectivity in an intact DNA 54-mer and DNA with a single break introduced across a minor damage site (G26). The data are shown for a gapped DNA with two phosphates flanking the gap. Similar increase in selectivity is also observed for nicked and gapped DNA with a varying number of phosphate monoester moieties at the damage site. (Right) Possible mechanism for the lysine-mediated transformation of ss damage into ds-damage.

Because a terminal phosphate group could be used to direct the lysine-conjugates to specific locations within DNA, these findings suggest that recognition of DNA damage sites does not need to be the exclusive domain of large (repair) enzymes and some rather elaborate natural products. This study, illustrates that selective recognition of partial DNA cleavage is possible with small molecules (MV ≤ 500). It is also an intriguing possible explanation of the high ds/ss cleavage ratio observed in the reactions of lysine conjugates with DNA and a harbinger for a new strategy in the development of chemotherapy agents. Furthermore, these transformations provide a chemical analogy to siRNA with a potential for photochemical triggering (Figure 20).


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Figure 20. Potential design for photo RNA interference experiments. By annealing a single strand nucleotide with complementary oligonucleotides bearing terminal phosphate groups, a target site for lysine recognition is created on the targeted nucleotide backbone. Lysine conjugates are then photochemically excited to perform photocleavage at that site, akin to the DNA experiments shown earlier.

8. Cellular uptake and intracellular DNA-damage Determining the DNA-cleaving ability of compounds against isolated DNA is the first step in quantifying cleavage activity. It is also useful for comparing a variety of lead compounds and for optimising the chemistry responsible for DNA cleavage. However, in order to determine whether a compound could become a viable pharmaceutical tool, tests against more realistic and more complex targets are essential.

Figure 21. Left: the low auto-fluorescence of A375 cells. Center: increased fluorescence due to the penetration of acetylene-bis-lysine conjugate into A375 cells. Right: SCGE (Comet) assays for A375 cells + UV (365 nm) + compound 135. The characteristic “comet” shape confirms DNA fragmentation due to the presence of the conjugate. All photochemical irradiations were carried out for 10 minutes.

Targeting intracellular DNA is more challenging than cleaving isolated DNA. Not only is DNA protected by the cell and nucleus membranes, the intracellular DNA is also compactly organized by histone proteins. Single cell gel electrophoresis (SGGE, or “Comet”) assays (Figure 21) were used to confirm that intracellular DNA damage occurs in the presence of lysine conjugates and light.73(a) Indeed, compound 141 and 135 were able to penetrate into the cell nucleus and damage highly compacted DNA upon photoactivation. Exposure to light does not produce efficient DNA damage in the absence of the photoactivated conjugates. In order to further elucidate the cellular effects of these compounds and their ability to cause apoptosis, we studied time dependence of DNA damage in A375 human melanoma cells (Figure 22). Although the DNA degradation was observed at all times, it progressively increased even in the dark, after irradiation had been stopped. This is an interesting observation because the continuous increase in DNA degradation is considered as one of the 51 ACS Paragon Plus Environment


signs of apoptosis. It is propagated by the release of endogenous endonucleases.90 The appearance of fragmented DNA on the gel was similar to that reported earlier for apoptotic cell lysates where histone proteins were cross-linked to the nucleus.91

Figure 22. UV activated bis-lysine conjugate 135 causes DNA damage in A375 cells. 30 µM of 135 was added to cells and incubated for 3 hours. Treated and untreated cells were exposed to UV radiation (365 nm) for 20 minutes and harvested at 6, 24 and 48 hours post UV radiation Smaller – and + symbols indicates absence or presence of conjugate 135. Time points are indicated by numbers in hours; M stands for DNA marker. Bigger – and + indicates whether cells were UV irradiated.

9. Cytotoxicity toward cancer cell lines The cytotoxicity of lysine-conjugates of enediynes and acetylenes against several cancer lines was tested in cell proliferation assays both in the dark and under UV irradiation (Figure 23). While all compounds showed strong photocytotoxicity, some compounds were toxic in the dark as well. The mechanism of the undesired dark cytotoxicity remains unknown. Because the enediynes are thermally stable, the toxicity is unlikely to be associated with a ground state cycloaromatization process (Bergman or C1-C5 cyclization). Several results are encouraging. For example, the lysine-TFP-acetylene conjugate 141 displayed high activity in presence of light, while not inhibiting cell growth in the dark. More than 90% of LNCaP cells were destroyed after a single 10 min photochemical treatment whereas no cell toxicity was observed in absence of light. A strong activity against LNCaP human prostate adenocarcinoma cells was observed at concentration as low as 10 nM. In a similar way, all three isomeric lysine alkyne conjugates inhibited human melanoma cell growth under photoactivation but the p-conjugate has the lowest CC50 (50% cell cytotoxicity) value of 1.49 × 10-7 M.


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Figure 23. Left: Cell proliferation assay using LNCaP cells and acetylene-lysine conjugate 141after 10 min. of UVC (254 nm) irradiation. Right: Cell proliferation assay using A375 cells (human melanoma) and p- (green square), m- (red up-pointing triangle) and o- (blue down-pointing triangle) alkyne lysine conjugates 141, 142 and 143 after 10 min. of photoactivation (365 nm) at concentration where toxicity in the dark is very low.

These levels of activity are comparable to the activity displayed in cell assays by Photofrin.92 This is noteworthy because Photofrin, a commonly used photodynamic therapy agent, acts as a catalyst for the production of singlet oxygen, whereas the lysine/alkyne conjugates act stoichiometrically– they are inactivated after a single DNA-cleavage reaction. The comparison with other chemotherapy agents is also favourable. For example, the DNA crosslinker cisplatin requires ~100-fold higher concentrations to achieve LD90.93 Even though Taxol is effective at approximately equal concentrations as the lysine-acetylene conjugate, it requires treatment times that are longer by 2-3 orders of magnitude.94 10. Conclusion The rational design and studies of the enediyne/enyne DNA photocleavers has empowered chemists and biologists with a promising strategy for targeted antitumor drug delivery. The various mechanisms of phototriggered diradical generation invokes the need for design and synthesis of newer molecular scaffolds capable of potential biomedical application, with greater biocompatibility and enhanced antineoplastic activity. Enediyne-amino acid hybrids offer temporal and spatial control over the photoactivation of the enediyne warhead. These hybrids show profound anti-cancer activities against various cancer cell lines. The DNA-cleaving ability of these compounds is enhanced by the ability of lysine residues to detect the sites of initial (ss) DNA damage sites and convert them to the therapeutically important ds breaks. The cytotoxicity is further reinforced by the slightly acidic pH of the hypoxic cancer tissues. One of the major challenges in modern day PDT is the inaccessibility of visible light to deep-seated tumors. This drawback is caused by irradiating currently approved photosensitizers below 700 nm, where light penetration is only a few millimeters from the 53 ACS Paragon Plus Environment


tissue surface. Phenomenona like scattering and auto fluorescence further hinder the process. Another limitation is the difficulty in delivering the drug in physiological mediums because of their high lipophilicity. In addition, the poor selectivity of conventional drugs for the targeted pathologic sites minimizes PDT efficacy in vivo and increases the potential phototoxicity to normal tissues. Since scattering decreases, and tissue penetration is maximized in the “phototherapeutic window” which ranges between 650 to 900 nm, it becomes imperative to tune the in vivo imaging parameters away from potential interference from endogenous fluorescence factors and into the near-infrared (NIR) spectral region (650-900 nm). Hence designing enediynes capable of undergoing photoexcitation in the therapeutic window and further increasing the selectivity towards cancerous cells and tissues, are perhaps the next stepping stones in this domain of research. This mode of triggering the enediyne warhead is expected to provide greater spatial control and minimize autofluorescence from any endogenous cellular components. Application of enediyne based NIR photosensitizers and two-photon photo excitations of enediyne scaffolds95 are thus becoming one of the most important biomedical techniques in PDT. In conclusion, it may be inferred that photoactivated DNA-cleavers have the potential to provide a minimally invasive “on-demand” chemical tool for cancer chemotherapy. With the advent of NIR photoactivable warheads equipped with suitable diradical generating biocompatible molecular architectures, photoactivated enediynes have the potential to hold a much greater prominence in the field of photochemical anti-cancer therapeutics. Acknowledgements Research at FSU was supported by the National Science Foundation (CHE-1465142). AB thanks DST, Government of India for JC Bose Fellowship. References and Notes (1) a) Jones, R. R.; Bergman, R. G. p-Benzyne. Generation as an intermediate in a thermal isomerization reaction and trapping evidence for the 1,4-benzenediyl structure. J. Am. Chem. Soc. 1972, 94, 660–661; b) Bergman, R. G. Reactive 1,4-dehydroaromatics. Acc. Chem. Res. 1973, 6, 25–31. (2) Galm, U.; Hager, M. H.; Lanen, S. G. V.; Ju, J.; Thorson, J. S.; Shen, B. Antitumor Antibiotics: Bleomycin, Enediynes, and Mitomycin. Chem. Rev. 2005, 105, 739–758. (3) Dalla, V. L.; Marciani, M. S. Photochemotherapy in the treatment of cancer. Curr. Med. Chem. 2001, 8, 1405–1418. (4) Dougherty, T. J.;Gomer, C. J.; Henderson, B. W.;Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. Photodynamic Therapy. J. Natl. Cancer Inst.1998, 90, 889–905.


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