View Article Online View Journal
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. Hasserodt and M. Prost, Chem. Commun., 2014, DOI: 10.1039/C4CC07147F.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 5
ChemComm View Article Online
DOI: 10.1039/C4CC07147F
Journal Name
RSCPublishing
COMMUNICATION
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x
Maxime Prost,[a] and Jens Hasserodt*[a]
www.rsc.org/
A new archetype of activatable imaging probe is described that responds in an off-on mode with a fluorescent signal only after being consecutively converted by two different enzymes. Pre-pro-proteins are a well-established concept employed by Nature, but “pre-pro-fluorescent” molecular probes have been unknown until now, even though they promise to furnish precious extra information and added specificity over habitual probe technologies. Our prototype discriminates cells that express both active β-D-galactosidase and leucine amino-peptidase from those which lack one of these enzymes thanks to the release of a precipitating fluorescent tag. We wish to disclose a new mode of operation for imaging probes responding to enzyme activity that we term “double gating”. Our prototype example is a silent probe that requires chemical conversion both by a glycosidase and by an exopeptidase in order to become activated. An ever increasing number of single-activation small-molecule imaging probes (and prodrugs) are being reported in the recent literature.1–8 Upon conversion by the targeted analyte they release their signalophore (or the drug) that thus adopts its active form. This strategy has been widely applied to the design of targeted diagnostic or therapeutic compounds that are selectively activated by enzymes known to be up-regulated in diseased tissue.9,10 However, protein overexpression ratios for diseased over healthy cells frequently do not exceed a factor of 2 to 3.11,12 Also, overproduction of a particular enzyme may be characteristic for more than just one pathology.13–15 The level of
This journal is © The Royal Society of Chemistry 2012
tissue selectivity achieved by single-activation probes may be unsatisfactory, as a false positive signal (or detrimental side effects for prodrugs) would be perceived also in healthy tissue. To overcome this problem, recent efforts have focused on adding a second criterion to the selection process, mostly one that takes advantage of molecular recognition by cell surface receptors rather than catalytic enzyme turnover. The recognition unit on the probe can be as diverse as an antibody (leading to well-established antibody-drug conjugates),16,17 a nucleic-acid aptamer,18 a peptide,8 a small drug-like molecule,19 or even polymers.20 When these units bind to their partner on the cell surface, the intact probe is processed by the target enzyme either directly outside the cell, or inside after internalization. Even though these probe constructs often achieved better selectivity, their fairly complex structure requires particular efforts in synthesis and purification. The incorporation of antibodies or polypeptides also renders ADME properties sub-optimal compared to small-molecule constructs.21 To our knowledge, probes that need to fulfil two successive selection criteria in the form of two catalytically active enzymes, thus forming an “and” type logic gate, are not yet described in the literature, even if elegant technologies approaching this idea were reported in the past few years. For example, one concept consists in producing luciferin in situ from two separate probes that have to be activated by H2O2 (an analyte that needs to be present in stoichiometric amounts) and
J. Name., 2012, 00, 1-3 | 1
ChemComm Accepted Manuscript
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
Cite this: DOI: 10.1039/x0xx00000x
„Double gating“ – a concept for enzymeresponsive imaging probes aiming at high tissue specificity
ChemComm
Caspase-8 (an enzyme), respectively.22 The principal drawback of this concept is the need for the administering of two independent probe molecules and the unlikelihood that they accumulate in areas of interest at concentrations conducive to optimal detection sensitivity. One case of single-probe fluorescence activation by two enzymes has also been reported, but the two enzyme-susceptible trigger moieties on this probe23 can be processed independently, and in this way the underlying concept has to be regarded as an “or” logic gate rather than an “and” logic gate as we wish to report herein. Other probes somewhat approaching our concept comprise a probe whose initial enzyme conversion is followed by a spontaneous hydrolysis of an anilide bond;24 or a probe that reacts nonspecifically with any esterase before becoming susceptible to light activation.25 A few peptide-trigger comprising prodrugs require multiple enzyme conversion for activation, but these constructs are neither based on rational design nor are the target enzymes clearly defined.26,27 By contrast, Nature has pursued the strategy of double enzyme activation for a long time, and this in the form of so-called “pre-pro-proteins”.28–30 Indeed, certain zymogens require conversion by two different peptidases thus allowing the organism to precisely control in which location it develops its enzymatic activity. In view of the above, we set out to design and validate the first example of an off-on probe that has to be converted by two different enzymes (has to pass two regulating gates) in order to generate a fluorescent signal (figure 1).
Journal Name DOI: 10.1039/C4CC07147F piperidine cyclizing spacer.41 This spacer showed very good immolation kinetics (t1/2 = 7 sec) in a recently reported probe of ours (2) to which it also confers exemplary stability.42 The phenolic fluorophore (9)43,44 we employed there and in the present probe 1 is unique in that it is silent when carbamylated via its hydroxyl group, but is highly insoluble and precipitates in the form of intensely fluorescent microcrystals once released. This unusual property allows for effective tagging of the cells capable of converting this probe.42 Probe 1 is prepared in a convergent synthetic pathway in only 5 steps starting with Koenigs-Knorr coupling of α-acetobromogalactose 3 and parahydroxybenzaldehyde 4 to give glycoside 5 in its β configuration (Scheme 1). The latter is reduced to alcohol 6 before being coupled to para-nitrophenol chloroformate to yield carbonate 7 in very good yields. The key step consists in the establishment of a carbamate link between amine 2 (in itself a LAP-responsive probe) and 6. Surprisingly, the reaction took ten days at room temperature to be complete, but then furnished probe precursor 8 in very good yields. Final deprotection resulted in a sample of probe 1, ready for testing.
Figure 1. Principle of double activation of a prepro-fluorescent probe 1.
The design of such a probe requires the masking of one trigger component susceptible to enzymatic cleavage by another. The habitual chemistry that allows for the probe to (a) become susceptible to the targeted enzyme reactivity and to (b) transduce the cleavage event to the signalophore relies principally on two classes of self-immolative spacers, either operating by elimination31–35 or by cyclization.36–40 We chose to make use of both in order to target β-galactosidase (β-Gal), a glycosidase, and leucine aminopeptidase (LAP), an exopeptidase. A β-galactose unit (“substrate 1”) is linked via an eliminating para-hydroxybenzylcarbamyl spacer to the leucine unit (“substrate 2”), which in return is linked to the silenced fluorophore via our recently introduced aminomethyl-
2 | J. Name., 2012, 00, 1-3
Scheme 1. Synthesis of the pre-pro-fluorescent probe 1 (A) and its activation by successive cleavage with β-D-Galactosidase and Leucine AminoPeptidase (B).
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
COMMUNICATION
Page 2 of 5 View Article Online
Page 3 of 5
ChemComm View Article Online
COMMUNICATION DOI: 10.1039/C4CC07147F
B)
F / RFU
A) 4E+5
8 +LAP + b-Gal 8 + LAP 8 + b-Gal 8 - no enzyme
2E+5
entry with the acetyl groups being removed inside the cytosol by esterases. This strategy can however not be applied to our present probe since the corresponding molecule 8 is not soluble in water, a property that can be fairly easily deduced from its structure. When this same cell line was preincubated with L-leucinethiol, a LAP inhibitor (β-Gal + / LAP -), prior to reaction with probe 1, then not a trace of fluorescence was observed (Figure 3.C). HeLa cells by contrast, that do not produce β-galactosidase, but of course contain LAP activity (β-Gal - / LAP +), did not show any response to the presence of probe 1 either (Figure 3.D). These results perfectly corroborate our concept of a probe that requires double activation in order to generate a detectable signal in a live cell.
4E+5
8 +LAP 8 +b-Gal 8 - no enzyme
2E+5
0E+0
0E+0
0
20
40
t / min
5
t/h
10
15
10µM 5µM 2.5µM 1µM no enzyme
C) 6E+4
F / RFU
0
60
4E+4 2E+4 0E+0
0
30
60
t / min
90
Figure 2. A) Response by 1 (100µM) to either LAP (0.007U) + β-gal (1U), LAP (0.007U) alone, β-gal (1U) alone, or without enzyme; B) stability of 1 (100µM) toward spontaneous hydrolysis or non-specific enzyme activation; C) response by decreasing concentrations of 1 with LAP (0.007U) and β-gal (1U).
These encouraging in vitro results prompted us to test probe 1 with live cells. Incubation of C17.2 cells that are stably transfected with LacZ gene and are thus competent in the production of both target enzymes (β-Gal + / LAP +; LAP being ubiquitous in all mammalian cell lines) led to the formation of characteristic, intensely fluorescent spots in the cytoplasm as we had observed in previous work (figure 3.A and B).42 It is important to note that we had to increase probe concentration and incubation time compared to the case of previously reported probe 2 in order to observe intracellular fluorescence appearing. This is probably due to the increased polarity conferred onto the probe by the galactosyl moiety, thus reducing the probe’s capacity to partition with the cytosol. This phenomenon is well-known, and it has been reported45 that fully acetylated galactosyl units allow for more efficient cell
This journal is © The Royal Society of Chemistry 2012
Figure 3. C17.2 cells (β-gal+ / LAP+ ) were incubated with probe 8 (50µM) for 4h (A: merged bright field and fluorescence channels, B: fluorescent channel alone). This experiment was repeated with C17.2 cells pre-treated with L-Leucinethiol (5µM ;β-gal+ / LAP- , C: fluorescence channel) or with HeLa cells (β-gal- / LAP+ , D: fluorescence channel). Scale bar: 20µm.
Conclusions In conclusion, we report a first proof-of-concept of a new kind of activatable imaging probe that holds great promise for increased precision in the identification of specific cell lines. The mode of operation of this molecular device may be regarded as an “AND-type logic gate”. Its simplicity bodes well for widespread investigation and may see extension to triplegating probes. While applied here to a diagnostic end, we are convinced that this concept will turn out to be useful also for the design of prodrugs for greatly improved targeted therapy. Efforts are currently in progress in our laboratory to target more disease-relevant enzymes as well as NIR fluorophores for future in vivo imaging.
Acknowledgements
J. Name., 2012, 00, 1-3 | 3
ChemComm Accepted Manuscript
Initially, probe 1 was incubated with purified enzymes in an in vitro setting. Four different scenarios were tested under physiological conditions (PBS at 37°C): (i) presence of both LAP and β-Gal, (ii) LAP alone, (iii) β-Gal alone, and (iv) without enzyme (Figure 3.A). To our delight, a fluorescent signal (precipitate) is only observed when both enzyme activities are present. Interestingly, the fact that four successive chemical reactions have to occur before the fluorophore is released does not compromise rapid signal built-up. Probe 1 also proved to be completely robust when tested in an overnight experiment at 37°C with only one or the other enzyme present (Figure 2.B). A fluorescent response can be observed all the way down to 1µM probe concentration, but with an increasingly delayed onset (Figure 2.C). This is not surprising when taking into account that free phenol 9 only precipitates once having reached its solubility limit, and only then does it become fluorescent.42
F / RFU
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
Journal Name
ChemComm
We gratefully acknowledge J. Samarut and L. Canaple for technical assistance.
Keywords molecular probe • enzyme activity • molecular imaging • cell microscopy • activatable probe
Notes and references Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
[a]
M. Prost, Prof. Dr. J. Hasserodt Laboratoire de Chimie, UMR CNRS 5182 University of Lyon – ENS 46 allée d’Italie - 69364 Lyon Cedex 07 - France E-mail: [email protected]
Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/c000000x/ 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17.
18. 19.
20. 21. 22. 23. 24.
J. B. Grimm, L. M. Heckman, and L. D. Lavis, Prog. Mol. Biol. Transl. Sci., 2013, 113, 1–34. D. Ding, K. Li, B. Liu, and B. Z. Tang, Acc. Chem. Res., 2013, 46, 2441–53. Z. Guo, S. Park, J. Yoon, and I. Shin, Chem. Soc. Rev., 2014, 43, 16–29. A. Razgulin, N. Ma, and J. Rao, Chem. Soc. Rev., 2011, 40, 4186–216. X. Li, X. Gao, W. Shi, and H. Ma, Chem. Rev., 2014, 114, 590–659. I. Tranoy-Opalinski, T. Legigan, R. Barat, J. Clarhaut, M. Thomas, B. Renoux, and S. Papot, Eur. J. Med. Chem., 2014, 74C, 302–313. S.-D. Clas, R. I. Sanchez, and R. Nofsinger, Drug Discov. Today, 2014, 19, 79–87. X. Tian, K.-H. Baek, and I. Shin, Chem. Sci., 2013, 4, 947–956. M. M. Mohamed and B. F. Sloane, Nat. Rev. Cancer, 2006, 6, 764–75. E. Hadler-Olsen, J.-O. Winberg, and L. Uhlin-Hansen, Tumour Biol., 2013, 34, 2041–51. G. Kristiansen, F. R. Fritzsche, K. Wassermann, C. Jäger, a Tölls, M. Lein, C. Stephan, K. Jung, C. Pilarsky, M. Dietel, and H. Moch, Br. J. Cancer, 2008, 99, 939–48. C. Morales-Gutiérrez, a Abad-Barahona, E. Moreno-González, R. Enríquez de Salamanca, and I. Vegh, Eur. J. Surg. Oncol., 2011, 37, 526–31. L. M. Coussens, B. Fingleton, and L. M. Matrisian, Science, 2002, 295, 2387–92. A. Dufour and C. M. Overall, Trends Pharmacol. Sci., 2013, 34, 233– 42. B. Korkmaz, M. S. Horwitz, D. E. Jenne, and F. Gauthier, Pharmacol. Rev., 2010, 62, 726–59. J. a Flygare, T. H. Pillow, and P. Aristoff, Chem. Biol. Drug Des., 2013, 81, 113–21. P. J. Burke, P. D. Senter, D. W. Meyer, J. B. Miyamoto, M. Anderson, B. E. Toki, G. Manikumar, M. C. Wani, D. J. Kroll, and S. C. Jeffrey, Bioconjug. Chem., 2009, 20, 1242–50. T. C. Chu, J. W. Marks, L. A. Lavery, S. Faulkner, M. G. Rosenblum, A. D. Ellington, and M. Levy, Cancer Res., 2006, 66, 5989–92. T. Legigan, J. Clarhaut, I. Tranoy-Opalinski, A. Monvoisin, B. Renoux, M. Thomas, A. Le Pape, S. Lerondel, and S. Papot, Angew. Chem. Int. Ed. Engl., 2012, 51, 11606–10. A. Gopin, N. Pessah, M. Shamis, C. Rader, and D. Shabat, Angew. Chem. Int. Ed. Engl., 2003, 42, 327–32. N. Krall, J. Scheuermann, and D. Neri, Angew. Chem. Int. Ed. Engl., 2013, 52, 1384–402. G. C. Van de Bittner, C. R. Bertozzi, and C. J. Chang, J. Am. Chem. Soc., 2013, 135, 1783–95. Y. Li, H. Wang, J. Li, J. Zheng, X. Xu, and R. Yang, Anal. Chem., 2011, 83, 1268–74. N.-H. Ho, R. Weissleder, and C.-H. Tung, Chembiochem, 2007, 8, 560–6.
4 | J. Name., 2012, 00, 1-3
Journal Name DOI: 10.1039/C4CC07147F 25. A. M. L. Hossion, M. Bio, G. Nkepang, S. G. Awuah, and Y. You, ACS Med. Chem. Lett., 2013, 4, 124–127. 26. A. Trouet, A. Passioukov, K. Van derpoorten, A. M. Fernandez, J. Abarca-Quinones, R. Baurain, T. J. Lobl, C. Oliyai, D. Shochat, and V. Dubois, Cancer Res., 2001, 61, 2843–6. 27. A. M. Fernandez, K. Van Derpoorten, L. Dasnois, K. Lebtahi, V. Dubois, T. J. Lobl, S. Gangwar, C. Oliyai, E. R. Lewis, D. Shochat, and A. Trouet, J. Med. Chem., 2001, 44, 3750–3. 28. O. Zschenker, D. Oezden, and D. Ameis, J. Biochem., 2004, 136, 65–72. 29. T. Burster, H. Macmillan, T. Hou, B. O. Boehm, and E. D. Mellins, Mol. Immunol., 2010, 47, 658–65. 30. B. Korkmaz, T. Moreau, and F. Gauthier, Biochimie, 2008, 90, 227–42. 31. J.-A. Richard, Y. Meyer, V. Jolivel, M. Massonneau, R. Dumeunier, D. Vaudry, H. Vaudry, P.-Y. Renard, and A. Romieu, Bioconjug. Chem., 2008, 19, 1707–18. 32. E. W. P. Damen, T. J. Nevalainen, T. J. M. van den Bergh, F. M. H. de Groot, and H. W. Scheeren, Bioorg. Med. Chem., 2002, 10, 71–7. 33. R. Erez and D. Shabat, Org. Biomol. Chem., 2008, 6, 2669–72. 34. N. Karton-Lifshin, E. Segal, L. Omer, M. Portnoy, R. Satchi-Fainaro, and D. Shabat, J. Am. Chem. Soc., 2011, 133, 10960–5. 35. M.-R. Lee, K.-H. Baek, H. J. Jin, Y.-G. Jung, and I. Shin, Angew. Chem. Int. Ed. Engl., 2004, 43, 1675–8. 36. S. S. Chandran, K. A. Dickson, and R. T. Raines, J. Am. Chem. Soc., 2005, 127, 1652–3. 37. M. A. DeWit and E. R. Gillies, Org. Biomol. Chem., 2011, 9, 1846–54. 38. X.-B. Zhang, M. Waibel, and J. Hasserodt, Chem. - a Eur. J., 2010, 16, 792–5. 39. M. Waibel, X.-B. Zhang, and J. Hasserodt, Synthesis (Stuttg)., 2008, 2009, 318–324. 40. Y. Meyer, J.-A. Richard, B. Delest, P. Noack, P.-Y. Renard, and A. Romieu, Org. Biomol. Chem., 2010, 8, 1777–80. 41. O. Thorn-Seshold, M. Vargas-Sanchez, S. McKeon, and J. Hasserodt, Chem. Commun., 2012, 48, 6253–6255. 42. M. Prost, L. Canaple, J. Samarut, and J. Hasserodt, Chembiochem, 2014, 15, 1413–7. 43. Z. Huang, E. Terpetschnig, W. You, and R. P. Haugland, Anal. Biochem., 1992, 207, 32–9. 44. V. B. Paragas, J. A. Kramer, C. Fox, R. P. Haugland, and V. L. Singer, J. Microsc., 2002, 206, 106–19. 45. L. Lavis, ACS Chem. Biol., 2008, 3, 203–206.
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
COMMUNICATION
Page 4 of 5 View Article Online
Page 5 of 5
ChemComm View Article Online
DOI: 10.1039/C4CC07147F
ChemComm Accepted Manuscript
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
A pro-fluorescent probe requires the presence of two target biomolecules to act on it in order to generate a signal.
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. Hasserodt and M. Prost, Chem. Commun., 2014, DOI: 10.1039/C4CC07147F.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 5
ChemComm View Article Online
DOI: 10.1039/C4CC07147F
Journal Name
RSCPublishing
COMMUNICATION
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x
Maxime Prost,[a] and Jens Hasserodt*[a]
www.rsc.org/
A new archetype of activatable imaging probe is described that responds in an off-on mode with a fluorescent signal only after being consecutively converted by two different enzymes. Pre-pro-proteins are a well-established concept employed by Nature, but “pre-pro-fluorescent” molecular probes have been unknown until now, even though they promise to furnish precious extra information and added specificity over habitual probe technologies. Our prototype discriminates cells that express both active β-D-galactosidase and leucine amino-peptidase from those which lack one of these enzymes thanks to the release of a precipitating fluorescent tag. We wish to disclose a new mode of operation for imaging probes responding to enzyme activity that we term “double gating”. Our prototype example is a silent probe that requires chemical conversion both by a glycosidase and by an exopeptidase in order to become activated. An ever increasing number of single-activation small-molecule imaging probes (and prodrugs) are being reported in the recent literature.1–8 Upon conversion by the targeted analyte they release their signalophore (or the drug) that thus adopts its active form. This strategy has been widely applied to the design of targeted diagnostic or therapeutic compounds that are selectively activated by enzymes known to be up-regulated in diseased tissue.9,10 However, protein overexpression ratios for diseased over healthy cells frequently do not exceed a factor of 2 to 3.11,12 Also, overproduction of a particular enzyme may be characteristic for more than just one pathology.13–15 The level of
This journal is © The Royal Society of Chemistry 2012
tissue selectivity achieved by single-activation probes may be unsatisfactory, as a false positive signal (or detrimental side effects for prodrugs) would be perceived also in healthy tissue. To overcome this problem, recent efforts have focused on adding a second criterion to the selection process, mostly one that takes advantage of molecular recognition by cell surface receptors rather than catalytic enzyme turnover. The recognition unit on the probe can be as diverse as an antibody (leading to well-established antibody-drug conjugates),16,17 a nucleic-acid aptamer,18 a peptide,8 a small drug-like molecule,19 or even polymers.20 When these units bind to their partner on the cell surface, the intact probe is processed by the target enzyme either directly outside the cell, or inside after internalization. Even though these probe constructs often achieved better selectivity, their fairly complex structure requires particular efforts in synthesis and purification. The incorporation of antibodies or polypeptides also renders ADME properties sub-optimal compared to small-molecule constructs.21 To our knowledge, probes that need to fulfil two successive selection criteria in the form of two catalytically active enzymes, thus forming an “and” type logic gate, are not yet described in the literature, even if elegant technologies approaching this idea were reported in the past few years. For example, one concept consists in producing luciferin in situ from two separate probes that have to be activated by H2O2 (an analyte that needs to be present in stoichiometric amounts) and
J. Name., 2012, 00, 1-3 | 1
ChemComm Accepted Manuscript
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
Cite this: DOI: 10.1039/x0xx00000x
„Double gating“ – a concept for enzymeresponsive imaging probes aiming at high tissue specificity
ChemComm
Caspase-8 (an enzyme), respectively.22 The principal drawback of this concept is the need for the administering of two independent probe molecules and the unlikelihood that they accumulate in areas of interest at concentrations conducive to optimal detection sensitivity. One case of single-probe fluorescence activation by two enzymes has also been reported, but the two enzyme-susceptible trigger moieties on this probe23 can be processed independently, and in this way the underlying concept has to be regarded as an “or” logic gate rather than an “and” logic gate as we wish to report herein. Other probes somewhat approaching our concept comprise a probe whose initial enzyme conversion is followed by a spontaneous hydrolysis of an anilide bond;24 or a probe that reacts nonspecifically with any esterase before becoming susceptible to light activation.25 A few peptide-trigger comprising prodrugs require multiple enzyme conversion for activation, but these constructs are neither based on rational design nor are the target enzymes clearly defined.26,27 By contrast, Nature has pursued the strategy of double enzyme activation for a long time, and this in the form of so-called “pre-pro-proteins”.28–30 Indeed, certain zymogens require conversion by two different peptidases thus allowing the organism to precisely control in which location it develops its enzymatic activity. In view of the above, we set out to design and validate the first example of an off-on probe that has to be converted by two different enzymes (has to pass two regulating gates) in order to generate a fluorescent signal (figure 1).
Journal Name DOI: 10.1039/C4CC07147F piperidine cyclizing spacer.41 This spacer showed very good immolation kinetics (t1/2 = 7 sec) in a recently reported probe of ours (2) to which it also confers exemplary stability.42 The phenolic fluorophore (9)43,44 we employed there and in the present probe 1 is unique in that it is silent when carbamylated via its hydroxyl group, but is highly insoluble and precipitates in the form of intensely fluorescent microcrystals once released. This unusual property allows for effective tagging of the cells capable of converting this probe.42 Probe 1 is prepared in a convergent synthetic pathway in only 5 steps starting with Koenigs-Knorr coupling of α-acetobromogalactose 3 and parahydroxybenzaldehyde 4 to give glycoside 5 in its β configuration (Scheme 1). The latter is reduced to alcohol 6 before being coupled to para-nitrophenol chloroformate to yield carbonate 7 in very good yields. The key step consists in the establishment of a carbamate link between amine 2 (in itself a LAP-responsive probe) and 6. Surprisingly, the reaction took ten days at room temperature to be complete, but then furnished probe precursor 8 in very good yields. Final deprotection resulted in a sample of probe 1, ready for testing.
Figure 1. Principle of double activation of a prepro-fluorescent probe 1.
The design of such a probe requires the masking of one trigger component susceptible to enzymatic cleavage by another. The habitual chemistry that allows for the probe to (a) become susceptible to the targeted enzyme reactivity and to (b) transduce the cleavage event to the signalophore relies principally on two classes of self-immolative spacers, either operating by elimination31–35 or by cyclization.36–40 We chose to make use of both in order to target β-galactosidase (β-Gal), a glycosidase, and leucine aminopeptidase (LAP), an exopeptidase. A β-galactose unit (“substrate 1”) is linked via an eliminating para-hydroxybenzylcarbamyl spacer to the leucine unit (“substrate 2”), which in return is linked to the silenced fluorophore via our recently introduced aminomethyl-
2 | J. Name., 2012, 00, 1-3
Scheme 1. Synthesis of the pre-pro-fluorescent probe 1 (A) and its activation by successive cleavage with β-D-Galactosidase and Leucine AminoPeptidase (B).
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
COMMUNICATION
Page 2 of 5 View Article Online
Page 3 of 5
ChemComm View Article Online
COMMUNICATION DOI: 10.1039/C4CC07147F
B)
F / RFU
A) 4E+5
8 +LAP + b-Gal 8 + LAP 8 + b-Gal 8 - no enzyme
2E+5
entry with the acetyl groups being removed inside the cytosol by esterases. This strategy can however not be applied to our present probe since the corresponding molecule 8 is not soluble in water, a property that can be fairly easily deduced from its structure. When this same cell line was preincubated with L-leucinethiol, a LAP inhibitor (β-Gal + / LAP -), prior to reaction with probe 1, then not a trace of fluorescence was observed (Figure 3.C). HeLa cells by contrast, that do not produce β-galactosidase, but of course contain LAP activity (β-Gal - / LAP +), did not show any response to the presence of probe 1 either (Figure 3.D). These results perfectly corroborate our concept of a probe that requires double activation in order to generate a detectable signal in a live cell.
4E+5
8 +LAP 8 +b-Gal 8 - no enzyme
2E+5
0E+0
0E+0
0
20
40
t / min
5
t/h
10
15
10µM 5µM 2.5µM 1µM no enzyme
C) 6E+4
F / RFU
0
60
4E+4 2E+4 0E+0
0
30
60
t / min
90
Figure 2. A) Response by 1 (100µM) to either LAP (0.007U) + β-gal (1U), LAP (0.007U) alone, β-gal (1U) alone, or without enzyme; B) stability of 1 (100µM) toward spontaneous hydrolysis or non-specific enzyme activation; C) response by decreasing concentrations of 1 with LAP (0.007U) and β-gal (1U).
These encouraging in vitro results prompted us to test probe 1 with live cells. Incubation of C17.2 cells that are stably transfected with LacZ gene and are thus competent in the production of both target enzymes (β-Gal + / LAP +; LAP being ubiquitous in all mammalian cell lines) led to the formation of characteristic, intensely fluorescent spots in the cytoplasm as we had observed in previous work (figure 3.A and B).42 It is important to note that we had to increase probe concentration and incubation time compared to the case of previously reported probe 2 in order to observe intracellular fluorescence appearing. This is probably due to the increased polarity conferred onto the probe by the galactosyl moiety, thus reducing the probe’s capacity to partition with the cytosol. This phenomenon is well-known, and it has been reported45 that fully acetylated galactosyl units allow for more efficient cell
This journal is © The Royal Society of Chemistry 2012
Figure 3. C17.2 cells (β-gal+ / LAP+ ) were incubated with probe 8 (50µM) for 4h (A: merged bright field and fluorescence channels, B: fluorescent channel alone). This experiment was repeated with C17.2 cells pre-treated with L-Leucinethiol (5µM ;β-gal+ / LAP- , C: fluorescence channel) or with HeLa cells (β-gal- / LAP+ , D: fluorescence channel). Scale bar: 20µm.
Conclusions In conclusion, we report a first proof-of-concept of a new kind of activatable imaging probe that holds great promise for increased precision in the identification of specific cell lines. The mode of operation of this molecular device may be regarded as an “AND-type logic gate”. Its simplicity bodes well for widespread investigation and may see extension to triplegating probes. While applied here to a diagnostic end, we are convinced that this concept will turn out to be useful also for the design of prodrugs for greatly improved targeted therapy. Efforts are currently in progress in our laboratory to target more disease-relevant enzymes as well as NIR fluorophores for future in vivo imaging.
Acknowledgements
J. Name., 2012, 00, 1-3 | 3
ChemComm Accepted Manuscript
Initially, probe 1 was incubated with purified enzymes in an in vitro setting. Four different scenarios were tested under physiological conditions (PBS at 37°C): (i) presence of both LAP and β-Gal, (ii) LAP alone, (iii) β-Gal alone, and (iv) without enzyme (Figure 3.A). To our delight, a fluorescent signal (precipitate) is only observed when both enzyme activities are present. Interestingly, the fact that four successive chemical reactions have to occur before the fluorophore is released does not compromise rapid signal built-up. Probe 1 also proved to be completely robust when tested in an overnight experiment at 37°C with only one or the other enzyme present (Figure 2.B). A fluorescent response can be observed all the way down to 1µM probe concentration, but with an increasingly delayed onset (Figure 2.C). This is not surprising when taking into account that free phenol 9 only precipitates once having reached its solubility limit, and only then does it become fluorescent.42
F / RFU
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
Journal Name
ChemComm
We gratefully acknowledge J. Samarut and L. Canaple for technical assistance.
Keywords molecular probe • enzyme activity • molecular imaging • cell microscopy • activatable probe
Notes and references Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
[a]
M. Prost, Prof. Dr. J. Hasserodt Laboratoire de Chimie, UMR CNRS 5182 University of Lyon – ENS 46 allée d’Italie - 69364 Lyon Cedex 07 - France E-mail: [email protected]
Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/c000000x/ 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17.
18. 19.
20. 21. 22. 23. 24.
J. B. Grimm, L. M. Heckman, and L. D. Lavis, Prog. Mol. Biol. Transl. Sci., 2013, 113, 1–34. D. Ding, K. Li, B. Liu, and B. Z. Tang, Acc. Chem. Res., 2013, 46, 2441–53. Z. Guo, S. Park, J. Yoon, and I. Shin, Chem. Soc. Rev., 2014, 43, 16–29. A. Razgulin, N. Ma, and J. Rao, Chem. Soc. Rev., 2011, 40, 4186–216. X. Li, X. Gao, W. Shi, and H. Ma, Chem. Rev., 2014, 114, 590–659. I. Tranoy-Opalinski, T. Legigan, R. Barat, J. Clarhaut, M. Thomas, B. Renoux, and S. Papot, Eur. J. Med. Chem., 2014, 74C, 302–313. S.-D. Clas, R. I. Sanchez, and R. Nofsinger, Drug Discov. Today, 2014, 19, 79–87. X. Tian, K.-H. Baek, and I. Shin, Chem. Sci., 2013, 4, 947–956. M. M. Mohamed and B. F. Sloane, Nat. Rev. Cancer, 2006, 6, 764–75. E. Hadler-Olsen, J.-O. Winberg, and L. Uhlin-Hansen, Tumour Biol., 2013, 34, 2041–51. G. Kristiansen, F. R. Fritzsche, K. Wassermann, C. Jäger, a Tölls, M. Lein, C. Stephan, K. Jung, C. Pilarsky, M. Dietel, and H. Moch, Br. J. Cancer, 2008, 99, 939–48. C. Morales-Gutiérrez, a Abad-Barahona, E. Moreno-González, R. Enríquez de Salamanca, and I. Vegh, Eur. J. Surg. Oncol., 2011, 37, 526–31. L. M. Coussens, B. Fingleton, and L. M. Matrisian, Science, 2002, 295, 2387–92. A. Dufour and C. M. Overall, Trends Pharmacol. Sci., 2013, 34, 233– 42. B. Korkmaz, M. S. Horwitz, D. E. Jenne, and F. Gauthier, Pharmacol. Rev., 2010, 62, 726–59. J. a Flygare, T. H. Pillow, and P. Aristoff, Chem. Biol. Drug Des., 2013, 81, 113–21. P. J. Burke, P. D. Senter, D. W. Meyer, J. B. Miyamoto, M. Anderson, B. E. Toki, G. Manikumar, M. C. Wani, D. J. Kroll, and S. C. Jeffrey, Bioconjug. Chem., 2009, 20, 1242–50. T. C. Chu, J. W. Marks, L. A. Lavery, S. Faulkner, M. G. Rosenblum, A. D. Ellington, and M. Levy, Cancer Res., 2006, 66, 5989–92. T. Legigan, J. Clarhaut, I. Tranoy-Opalinski, A. Monvoisin, B. Renoux, M. Thomas, A. Le Pape, S. Lerondel, and S. Papot, Angew. Chem. Int. Ed. Engl., 2012, 51, 11606–10. A. Gopin, N. Pessah, M. Shamis, C. Rader, and D. Shabat, Angew. Chem. Int. Ed. Engl., 2003, 42, 327–32. N. Krall, J. Scheuermann, and D. Neri, Angew. Chem. Int. Ed. Engl., 2013, 52, 1384–402. G. C. Van de Bittner, C. R. Bertozzi, and C. J. Chang, J. Am. Chem. Soc., 2013, 135, 1783–95. Y. Li, H. Wang, J. Li, J. Zheng, X. Xu, and R. Yang, Anal. Chem., 2011, 83, 1268–74. N.-H. Ho, R. Weissleder, and C.-H. Tung, Chembiochem, 2007, 8, 560–6.
4 | J. Name., 2012, 00, 1-3
Journal Name DOI: 10.1039/C4CC07147F 25. A. M. L. Hossion, M. Bio, G. Nkepang, S. G. Awuah, and Y. You, ACS Med. Chem. Lett., 2013, 4, 124–127. 26. A. Trouet, A. Passioukov, K. Van derpoorten, A. M. Fernandez, J. Abarca-Quinones, R. Baurain, T. J. Lobl, C. Oliyai, D. Shochat, and V. Dubois, Cancer Res., 2001, 61, 2843–6. 27. A. M. Fernandez, K. Van Derpoorten, L. Dasnois, K. Lebtahi, V. Dubois, T. J. Lobl, S. Gangwar, C. Oliyai, E. R. Lewis, D. Shochat, and A. Trouet, J. Med. Chem., 2001, 44, 3750–3. 28. O. Zschenker, D. Oezden, and D. Ameis, J. Biochem., 2004, 136, 65–72. 29. T. Burster, H. Macmillan, T. Hou, B. O. Boehm, and E. D. Mellins, Mol. Immunol., 2010, 47, 658–65. 30. B. Korkmaz, T. Moreau, and F. Gauthier, Biochimie, 2008, 90, 227–42. 31. J.-A. Richard, Y. Meyer, V. Jolivel, M. Massonneau, R. Dumeunier, D. Vaudry, H. Vaudry, P.-Y. Renard, and A. Romieu, Bioconjug. Chem., 2008, 19, 1707–18. 32. E. W. P. Damen, T. J. Nevalainen, T. J. M. van den Bergh, F. M. H. de Groot, and H. W. Scheeren, Bioorg. Med. Chem., 2002, 10, 71–7. 33. R. Erez and D. Shabat, Org. Biomol. Chem., 2008, 6, 2669–72. 34. N. Karton-Lifshin, E. Segal, L. Omer, M. Portnoy, R. Satchi-Fainaro, and D. Shabat, J. Am. Chem. Soc., 2011, 133, 10960–5. 35. M.-R. Lee, K.-H. Baek, H. J. Jin, Y.-G. Jung, and I. Shin, Angew. Chem. Int. Ed. Engl., 2004, 43, 1675–8. 36. S. S. Chandran, K. A. Dickson, and R. T. Raines, J. Am. Chem. Soc., 2005, 127, 1652–3. 37. M. A. DeWit and E. R. Gillies, Org. Biomol. Chem., 2011, 9, 1846–54. 38. X.-B. Zhang, M. Waibel, and J. Hasserodt, Chem. - a Eur. J., 2010, 16, 792–5. 39. M. Waibel, X.-B. Zhang, and J. Hasserodt, Synthesis (Stuttg)., 2008, 2009, 318–324. 40. Y. Meyer, J.-A. Richard, B. Delest, P. Noack, P.-Y. Renard, and A. Romieu, Org. Biomol. Chem., 2010, 8, 1777–80. 41. O. Thorn-Seshold, M. Vargas-Sanchez, S. McKeon, and J. Hasserodt, Chem. Commun., 2012, 48, 6253–6255. 42. M. Prost, L. Canaple, J. Samarut, and J. Hasserodt, Chembiochem, 2014, 15, 1413–7. 43. Z. Huang, E. Terpetschnig, W. You, and R. P. Haugland, Anal. Biochem., 1992, 207, 32–9. 44. V. B. Paragas, J. A. Kramer, C. Fox, R. P. Haugland, and V. L. Singer, J. Microsc., 2002, 206, 106–19. 45. L. Lavis, ACS Chem. Biol., 2008, 3, 203–206.
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
COMMUNICATION
Page 4 of 5 View Article Online
Page 5 of 5
ChemComm View Article Online
DOI: 10.1039/C4CC07147F
ChemComm Accepted Manuscript
Published on 08 October 2014. Downloaded by Tufts University on 08/10/2014 15:58:25.
A pro-fluorescent probe requires the presence of two target biomolecules to act on it in order to generate a signal.
