news & views In this orientation, the distance between the sp-hybridized carbon atoms in adjacent molecules is 3.42 Å, which is below the minimal distance (approximately 4 Å) for a topochemical reaction to occur. After only 40 minutes of UV irradiation at room temperature, up to 80% of the sp-hybridized carbon atoms were converted to sp2-hybridized carbons, yielding graphenelike nanosheets that are functionalized on both sides. This process opens up the possibility of modulating the properties of the semiconducting carbon layer, and offers a significant advantage over traditional graphene functionalization, which often involves harsh conditions that partially destroy the sp2 carbon network5. These carbon nanosheets are robust, uniform and are semiconducting with an optical bandgap of around 2.2 eV, all of which brings new opportunities for applications such as membrane materials, sensors and coatings. A few other methods for preparing twodimensional graphene-like nanosheets with unique properties, involving carbon-rich precursors prearranged within either xerogels6 or monolayers7, have also been reported recently. However, none provide good control over the sheet thickness under mild conditions — the xerogel approach offers no control over the nanosheet thickness, whereas the monolayer approach requires
very high temperatures. The combination of both control over the thickness and mild reaction conditions is what makes the work of Frauenrath and co-workers a significant advance in carbon nanomaterials. So, what comes next? How can this method be implemented or adapted to produce large quantities of well-defined materials? Can varying the length of the molecular rods modulate the thickness of the nanosheets? Some of these questions are not easy to answer at this point. Obviously, this method will not be suitable for the production of large quantities of graphene-like materials, mostly because the relatively complex experimental set-up would be difficult to replicate at industrial scales. Moreover, this approach has not yet proved to be suitable for the preparation of graphene-like materials with tunable electronic, optical and structural properties; on the other hand, the possibility of functionalizing both sides of the nanosheets opens up interesting avenues of exploration in this direction. It could be argued that this synthetic method could be beneficial for niche applications in which small quantities of structurally well-defined graphene-like materials are desired. Unquestionably, this method shows great promise for the preparation of semiconducting layers on substrates of
interest because the whole process is accomplished at room temperature without the need for any additional chemical reagents. For this method to transcend the realms of pure academic interest, however, an efficient and reliable method for transferring either the monolayer or the final nanosheets onto different substrates would have to be developed. More studies will also be needed to assess the real potential (and generality) of this new synthetic method, and a thorough investigation of the properties of the resulting material is yet to be performed. Nonetheless, the strategy offers new opportunities for the preparation of one of the most studied classes of materials in the history of materials science. ❐ Jean-François Morin is in the Department of Chemistry, Université Laval, 1045 Ave de la Médecine, Pavillon Alexandre-Vachon, local 1250B, Québec G1V 0A6, Canada. e-mail: [email protected] References 1. Chernick, E. T. & Tykwinski, R. R. J. Phys. Org. Chem. 26, 742–749 (2013). 2. Schrettl, S. et al. Nature Chem. 6, 468–476 (2014). 3. Wegner, G. Natureforsch. B: Chem. Sci. 24, 824–832 (1969). 4. Hoheisel, T. N., Schettl, S., Szilluweit, R. & Frauenrath, H. Angew. Chem. Int. Ed. 49, 6496–6515 (2012). 5. Chua, C. K. & Pumera, M. Chem. Soc. Rev. 42, 3222–3233 (2013). 6. Levesque, I. et al. Chem. Sci. 5, 831–836 (2014). 7. Angelova, P. et al. ACS Nano 7, 6489–6497 (2013).
NATURAL PRODUCTS
DNA double whammy
The lomaiviticins are exceedingly potent antibiotic agents, but the mechanism responsible for this activity has so far been unclear. Now, efficient generation of double-strand breaks in DNA by lomaiviticin A has been linked to the remarkable cytotoxicity of these diazobenzofluorene-containg natural products.
Kent S. Gates
D
NA-damaging natural products are an important category of anticancer drugs1,2. The ability of mitomycin C, bleomycin and calicheamicin (as an antibody conjugate) to induce DNA crosslinking or double-strand cleavage — thereby killing rapidly dividing cancer cells — underpins the clinical use of these natural products as chemotherapeutics1. In addition to their therapeutic importance, studies of highly active natural products also have the potential to reveal new fundamental insights regarding the mechanisms by which small molecules modulate biological processes3. One structurally interesting class of natural products — the lomaiviticins — display very potent antibiotic properties 464
(IC50 values in the range of 7 pM–70 nM against human cancer cell lines)4. The original communication4 describing the structure and antibiotic activity of lomaiviticin A and B noted that the compounds showed potent DNA-cleaving activity in the biochemical induction assay, and mentioned an “ongoing study” that showed the lomaiviticin A cleaved duplex DNA under reducing conditions. However, no data was shown and almost fourteen years have passed without any description of the DNA-damaging properties of the lomaiviticins emerging in the literature. Writing in this issue of Nature Chemistry, Herzon and co-workers report 5 breakthrough data showing that low
concentrations of lomaiviticin A (Fig. 1a) generate double-strand breaks in DNA under physiological conditions. The lomaiviticins are part of a larger structural family of diazobenzofluorenecontaining natural products. The first compounds in this class to be identified were the kinamycins. Initially, kinamycins were assigned as N-cyanocarbazoles6; however, efforts towards the total synthesis of these structures ultimately led to reassignment of the kinamycins as the structurally novel diazobenzofluorenes7,8 (Fig. 1b,c). The first suggestion of DNA-damaging activity for the kinamycins came from Moore in 1977. This was, however, based on the N-cyanocarboazole structure of the natural
NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
news & views product that is now known to be incorrect, and rested on similarities between this structure and reductively activated agents like mitomycin C (ref. 9). Nonetheless, interest persisted in the possible DNAdamaging properties of the kinamycins, even after structures of these natural products were revised. This interest was driven, at least in part, by the knowledge that other diazoand diazonium-containing compounds display DNA-damaging properties1,10. Results from several different groups in the context of the kinamycin analogues led to a wide array of proposals for the mechanism by which DNA-damaging reactive intermediates might arise from the diazobenzofluorene unit. In most of these proposals, generation of reactive intermediates is preceded by one- or twoelectron reduction of the quinone unit in the natural product. The requirement for reductive activation by intracellular enzymes or thiols is a rather common feature among DNA-damaging natural products, and studies of the activation mechanisms often reveal interesting strategies for the selective generation of reactive intermediates inside cells1,11. The work reported in this issue by Herzon and co-workers was enabled by a semisynthetic route to lomaiviticin A previously reported by the same group12. They have now found that low micromolar concentrations of lomaiviticin A in the presence of thiols directly generate strand breaks in double-stranded DNA. Mechanistic tests suggested that strand cleavage was initiated by a natural-product-derived radical intermediate. Importantly, the team observed efficient generation of double-strand breaks by lomaiviticin A, rather than just a cut in one of the strands of duplex DNA. Doublestrand DNA breaks are highly cytotoxic, and provide a reasonable explanation for the potent bioactivity of lomaiviticin A. The data further showed that the double-strand breaks probably arise from a single binding event rather than by a simple accumulation of single-strand cleavage events that ultimately yield two closely spaced breaks. The results suggest that each of the diazobenzofluorene units in lomaiviticin A initiate strand cleavage. In contrast, kinamycin C — an analogue containing only a single diazobenzofluorene moiety — displayed no ability to generate double-strand breaks in DNA. Herzon and co-workers also detected biochemical markers of double-strand DNA breaks within K562 cells after treatment with lomaiviticin A, which showed that such breaks also occurred in live cells. The ability of the natural product to generate doublestrand breaks in cells was further supported
a N HO
HO
O
N2
O
O
O
N2
OH
O
O
OAc CN
O
Original kinamycin structure
O O
c
O
HO
OH
N
O OH
O
O
OAc
AcO
O
O
O
HO O
b
OH
HO
O
OAc
AcO
OH
O
HO
(–)-Lomaiviticin A
OAc
N
OH
O
N
N Correct kinamycin structure
d OH
O
OH
OH –RS –N2
RSH
OH
O
N N
OH
O
N
Diazosulfide
N SR
OH
OH
OH
O
Vinyl radical
Figure 1 | Structures and reactions of diazobenzofluorene-containing natural products. a, Structure of (–)-lomaiviticin A. b, Original structure proposed for kinamycin C. c, Revised structure of kinamycin C. d, Proposed mechanism for thiol-mediated generation of a hydrogen-abstracting radical from the diazofluorene unit of lomaiviticin A.
by the observation that a cell line deficient in the protein BRCA2 — which plays a role in the cellular repair of double-strand breaks — was hypersensitive to lomaiviticin A. This facile generation of double-strand DNA breaks within cells provides an explanation for the higher potency of lomaiviticins compared with structurally related natural products such as kinamycins. Herzon and co-workers also provide considerable insight regarding the chemical reaction mechanisms by which lomaiviticin A causes DNA damage. Isotopic labelling experiments were consistent with the generation of the vinyl radical intermediate (Fig. 1d) of the type first proposed by Feldman and co-workers in 2005. This radical is envisaged to initiate strand cleavage by abstraction of hydrogen atoms from the DNA backbone13. Radical formation is suggested to be activated by nucleophilic attack of a thiol on the diazo unit, followed by fragmentation of the intermediate diazosulfide (a Stadler– Ziegler-type reaction)14. In the absence of thiol, Herzon and co-workers propose that DNA itself may serve as a nucleophile to activate one of the diazofluorene units (but apparently not both, as doublestrand cleavage is not seen in the absence of thiol). Nucleophilic activation of this sort has previously been discussed by the Dmietrienko group in 200215.
NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
The report by Herzon and co-workers sheds fresh light on the mechanisms of DNA cleavage by lomaiviticin A and possibly foreshadows additional significant advances in the understanding of the covalent and non-covalent interactions of the diazobenzofluorene natural products with DNA. ❐ Kent S. Gates is in the Departments of Chemistry and Biochemistry at the University of Missouri, Columbia, Missouri 65211, USA. e-mail: [email protected]
References
1. Gates, K. S. in Comprehensive Natural Products Chemistry Vol. 7 (ed. Kool, E. T.) 491–552 (Pergamon, 1999). 2. Newman, D. J. & Cragg, G. M. J. Nat. Prod. 75, 311–335 (2012). 3. Clardy, J. & Walsh, C. T. Nature 432, 829–837 (2004). 4. He, H. et al. J. Am. Chem. Soc. 123, 5362–5363 (2001). 5. Colis, L. C. et al. Nature Chem. 6, 504-510 (2014). 6. Omura, S. et al. Chem. Pharm. Bull. 21, 931–940 (1973). 7. Gould, S. J., Tamayo, N., Melville, C. R. & Cone, M. C. J. Am. Chem. Soc. 116, 2207–2208 (1994). 8. Mithani, S., Weeratunga, G., Taylor, N. J. & Dmitrienko, G. I. J. Am. Chem. Soc. 116, 2209–2210 (1994). 9. Moore, H. W. Science 197, 527–532 (1977). 10. Nawrat, C. C. & Moody, C. J. Nat. Prod. Res. 28, 1426–1444 (2011). 11. Fekry, M. et al. J. Am. Chem. Soc. 132, 17641–17651 (2011). 12. Woo, C. M., Beizer, N. E., Janso, J. E. & Herzon, S. B. J. Am. Chem. Soc. 134, 15285–15288 (2012). 13. Gates, K. S. Chem. Res. Toxicol. 22, 1747–1760 (2009). 14. Abeywickrema, A. N. & Beckwith, A. L. J. J. Am. Chem. Soc. 108, 8227–8229 (1986). 15. Laufer, R. S. & Dmitrienko, G. I. J. Am. Chem. Soc. 124, 1854–1855 (2002).
465
very high temperatures. The combination of both control over the thickness and mild reaction conditions is what makes the work of Frauenrath and co-workers a significant advance in carbon nanomaterials. So, what comes next? How can this method be implemented or adapted to produce large quantities of well-defined materials? Can varying the length of the molecular rods modulate the thickness of the nanosheets? Some of these questions are not easy to answer at this point. Obviously, this method will not be suitable for the production of large quantities of graphene-like materials, mostly because the relatively complex experimental set-up would be difficult to replicate at industrial scales. Moreover, this approach has not yet proved to be suitable for the preparation of graphene-like materials with tunable electronic, optical and structural properties; on the other hand, the possibility of functionalizing both sides of the nanosheets opens up interesting avenues of exploration in this direction. It could be argued that this synthetic method could be beneficial for niche applications in which small quantities of structurally well-defined graphene-like materials are desired. Unquestionably, this method shows great promise for the preparation of semiconducting layers on substrates of
interest because the whole process is accomplished at room temperature without the need for any additional chemical reagents. For this method to transcend the realms of pure academic interest, however, an efficient and reliable method for transferring either the monolayer or the final nanosheets onto different substrates would have to be developed. More studies will also be needed to assess the real potential (and generality) of this new synthetic method, and a thorough investigation of the properties of the resulting material is yet to be performed. Nonetheless, the strategy offers new opportunities for the preparation of one of the most studied classes of materials in the history of materials science. ❐ Jean-François Morin is in the Department of Chemistry, Université Laval, 1045 Ave de la Médecine, Pavillon Alexandre-Vachon, local 1250B, Québec G1V 0A6, Canada. e-mail: [email protected] References 1. Chernick, E. T. & Tykwinski, R. R. J. Phys. Org. Chem. 26, 742–749 (2013). 2. Schrettl, S. et al. Nature Chem. 6, 468–476 (2014). 3. Wegner, G. Natureforsch. B: Chem. Sci. 24, 824–832 (1969). 4. Hoheisel, T. N., Schettl, S., Szilluweit, R. & Frauenrath, H. Angew. Chem. Int. Ed. 49, 6496–6515 (2012). 5. Chua, C. K. & Pumera, M. Chem. Soc. Rev. 42, 3222–3233 (2013). 6. Levesque, I. et al. Chem. Sci. 5, 831–836 (2014). 7. Angelova, P. et al. ACS Nano 7, 6489–6497 (2013).
NATURAL PRODUCTS
DNA double whammy
The lomaiviticins are exceedingly potent antibiotic agents, but the mechanism responsible for this activity has so far been unclear. Now, efficient generation of double-strand breaks in DNA by lomaiviticin A has been linked to the remarkable cytotoxicity of these diazobenzofluorene-containg natural products.
Kent S. Gates
D
NA-damaging natural products are an important category of anticancer drugs1,2. The ability of mitomycin C, bleomycin and calicheamicin (as an antibody conjugate) to induce DNA crosslinking or double-strand cleavage — thereby killing rapidly dividing cancer cells — underpins the clinical use of these natural products as chemotherapeutics1. In addition to their therapeutic importance, studies of highly active natural products also have the potential to reveal new fundamental insights regarding the mechanisms by which small molecules modulate biological processes3. One structurally interesting class of natural products — the lomaiviticins — display very potent antibiotic properties 464
(IC50 values in the range of 7 pM–70 nM against human cancer cell lines)4. The original communication4 describing the structure and antibiotic activity of lomaiviticin A and B noted that the compounds showed potent DNA-cleaving activity in the biochemical induction assay, and mentioned an “ongoing study” that showed the lomaiviticin A cleaved duplex DNA under reducing conditions. However, no data was shown and almost fourteen years have passed without any description of the DNA-damaging properties of the lomaiviticins emerging in the literature. Writing in this issue of Nature Chemistry, Herzon and co-workers report 5 breakthrough data showing that low
concentrations of lomaiviticin A (Fig. 1a) generate double-strand breaks in DNA under physiological conditions. The lomaiviticins are part of a larger structural family of diazobenzofluorenecontaining natural products. The first compounds in this class to be identified were the kinamycins. Initially, kinamycins were assigned as N-cyanocarbazoles6; however, efforts towards the total synthesis of these structures ultimately led to reassignment of the kinamycins as the structurally novel diazobenzofluorenes7,8 (Fig. 1b,c). The first suggestion of DNA-damaging activity for the kinamycins came from Moore in 1977. This was, however, based on the N-cyanocarboazole structure of the natural
NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
news & views product that is now known to be incorrect, and rested on similarities between this structure and reductively activated agents like mitomycin C (ref. 9). Nonetheless, interest persisted in the possible DNAdamaging properties of the kinamycins, even after structures of these natural products were revised. This interest was driven, at least in part, by the knowledge that other diazoand diazonium-containing compounds display DNA-damaging properties1,10. Results from several different groups in the context of the kinamycin analogues led to a wide array of proposals for the mechanism by which DNA-damaging reactive intermediates might arise from the diazobenzofluorene unit. In most of these proposals, generation of reactive intermediates is preceded by one- or twoelectron reduction of the quinone unit in the natural product. The requirement for reductive activation by intracellular enzymes or thiols is a rather common feature among DNA-damaging natural products, and studies of the activation mechanisms often reveal interesting strategies for the selective generation of reactive intermediates inside cells1,11. The work reported in this issue by Herzon and co-workers was enabled by a semisynthetic route to lomaiviticin A previously reported by the same group12. They have now found that low micromolar concentrations of lomaiviticin A in the presence of thiols directly generate strand breaks in double-stranded DNA. Mechanistic tests suggested that strand cleavage was initiated by a natural-product-derived radical intermediate. Importantly, the team observed efficient generation of double-strand breaks by lomaiviticin A, rather than just a cut in one of the strands of duplex DNA. Doublestrand DNA breaks are highly cytotoxic, and provide a reasonable explanation for the potent bioactivity of lomaiviticin A. The data further showed that the double-strand breaks probably arise from a single binding event rather than by a simple accumulation of single-strand cleavage events that ultimately yield two closely spaced breaks. The results suggest that each of the diazobenzofluorene units in lomaiviticin A initiate strand cleavage. In contrast, kinamycin C — an analogue containing only a single diazobenzofluorene moiety — displayed no ability to generate double-strand breaks in DNA. Herzon and co-workers also detected biochemical markers of double-strand DNA breaks within K562 cells after treatment with lomaiviticin A, which showed that such breaks also occurred in live cells. The ability of the natural product to generate doublestrand breaks in cells was further supported
a N HO
HO
O
N2
O
O
O
N2
OH
O
O
OAc CN
O
Original kinamycin structure
O O
c
O
HO
OH
N
O OH
O
O
OAc
AcO
O
O
O
HO O
b
OH
HO
O
OAc
AcO
OH
O
HO
(–)-Lomaiviticin A
OAc
N
OH
O
N
N Correct kinamycin structure
d OH
O
OH
OH –RS –N2
RSH
OH
O
N N
OH
O
N
Diazosulfide
N SR
OH
OH
OH
O
Vinyl radical
Figure 1 | Structures and reactions of diazobenzofluorene-containing natural products. a, Structure of (–)-lomaiviticin A. b, Original structure proposed for kinamycin C. c, Revised structure of kinamycin C. d, Proposed mechanism for thiol-mediated generation of a hydrogen-abstracting radical from the diazofluorene unit of lomaiviticin A.
by the observation that a cell line deficient in the protein BRCA2 — which plays a role in the cellular repair of double-strand breaks — was hypersensitive to lomaiviticin A. This facile generation of double-strand DNA breaks within cells provides an explanation for the higher potency of lomaiviticins compared with structurally related natural products such as kinamycins. Herzon and co-workers also provide considerable insight regarding the chemical reaction mechanisms by which lomaiviticin A causes DNA damage. Isotopic labelling experiments were consistent with the generation of the vinyl radical intermediate (Fig. 1d) of the type first proposed by Feldman and co-workers in 2005. This radical is envisaged to initiate strand cleavage by abstraction of hydrogen atoms from the DNA backbone13. Radical formation is suggested to be activated by nucleophilic attack of a thiol on the diazo unit, followed by fragmentation of the intermediate diazosulfide (a Stadler– Ziegler-type reaction)14. In the absence of thiol, Herzon and co-workers propose that DNA itself may serve as a nucleophile to activate one of the diazofluorene units (but apparently not both, as doublestrand cleavage is not seen in the absence of thiol). Nucleophilic activation of this sort has previously been discussed by the Dmietrienko group in 200215.
NATURE CHEMISTRY | VOL 6 | JUNE 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
The report by Herzon and co-workers sheds fresh light on the mechanisms of DNA cleavage by lomaiviticin A and possibly foreshadows additional significant advances in the understanding of the covalent and non-covalent interactions of the diazobenzofluorene natural products with DNA. ❐ Kent S. Gates is in the Departments of Chemistry and Biochemistry at the University of Missouri, Columbia, Missouri 65211, USA. e-mail: [email protected]
References
1. Gates, K. S. in Comprehensive Natural Products Chemistry Vol. 7 (ed. Kool, E. T.) 491–552 (Pergamon, 1999). 2. Newman, D. J. & Cragg, G. M. J. Nat. Prod. 75, 311–335 (2012). 3. Clardy, J. & Walsh, C. T. Nature 432, 829–837 (2004). 4. He, H. et al. J. Am. Chem. Soc. 123, 5362–5363 (2001). 5. Colis, L. C. et al. Nature Chem. 6, 504-510 (2014). 6. Omura, S. et al. Chem. Pharm. Bull. 21, 931–940 (1973). 7. Gould, S. J., Tamayo, N., Melville, C. R. & Cone, M. C. J. Am. Chem. Soc. 116, 2207–2208 (1994). 8. Mithani, S., Weeratunga, G., Taylor, N. J. & Dmitrienko, G. I. J. Am. Chem. Soc. 116, 2209–2210 (1994). 9. Moore, H. W. Science 197, 527–532 (1977). 10. Nawrat, C. C. & Moody, C. J. Nat. Prod. Res. 28, 1426–1444 (2011). 11. Fekry, M. et al. J. Am. Chem. Soc. 132, 17641–17651 (2011). 12. Woo, C. M., Beizer, N. E., Janso, J. E. & Herzon, S. B. J. Am. Chem. Soc. 134, 15285–15288 (2012). 13. Gates, K. S. Chem. Res. Toxicol. 22, 1747–1760 (2009). 14. Abeywickrema, A. N. & Beckwith, A. L. J. J. Am. Chem. Soc. 108, 8227–8229 (1986). 15. Laufer, R. S. & Dmitrienko, G. I. J. Am. Chem. Soc. 124, 1854–1855 (2002).
465
