View Article Online View Journal
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: E. Oueis, F. Nachon, C. Sabot and P. Renard, Chem. Commun., 2013, DOI: 10.1039/C3CC48871C.
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 3
ChemComm
Journal Name
View Article Online
DOI: 10.1039/C3CC48871C
RSCPublishing
Cite this: DOI: 10.1039/x0xx00000x
First Enzymatic Hydrolysis/Thio-Michael Addition Cascade Route to AChE Inhibitors Emilia Oueis,a Florian Nachon,b Cyrille Sabot*a and Pierre-Yves Renard*a
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
The irreversible Michael addition of thiols to acrylamides is reported as a new tool for the kinetic target-guided synthesis. In an unprecedented enzymatic hydrolysis/thio-Michael addition procedure, potent and selective acetylcholinesterase inhibitors are assembled by the enzyme using both its esterasic and templating ability. Target-guided synthesis (TGS) is an unconventional fragment-based lead discovery methodology that relies on the multi-binding biological target (BT) itself to build its own inhibitors.1 Indeed, the reaction that occurs between ligands bearing complementary reactive functions is dramatically accelerated in the presence of the BT, leading to the formation of highly selective inhibitors. Different TGS approaches have already been developed and proved efficient for the discovery of potent inhibitors of various enzymes. Worth noting among them, are the dynamic combinatorial chemistry (DCC)2 which involves reversible bond formations, and the kinetically controlled target-guided synthesis (KTGS) which is based on irreversible reactions.1, 3 As an eminent example of KTGS, the in situ click chemistry tool developed by Sharpless et al. uses the 1,3dipolar cycloaddition, between the bioorthogonal azides and alkynes (Scheme 1, A) as the irreversible templated reaction to create new CN bonds and obtain triazole-based heterodimeric inhibitors.4 Aside from the Huisgen reaction, which was used on dozen occasions for the discovery of enzymatic triazole-based inhibitors, only a handful of other irreversible reactions have been engaged in KTGS approaches.3, 5 Notably, the synthesis of amide bonds via the sulfoclick reaction between thio acids and sulfonyl azides was achieved by Manetsch et coll. for the discovery of inhibitors of a Bcl-2 protein family member (Scheme 1, B).5b-d Huc et al. explored the utility of the nucleophilic substitution of alkyl chlorides by mercaptoarylsulfonamide that generates a new C-S bond (Scheme 1, C) to identify inhibitors of the zinc-containing metalloenzyme bovine carbonic anhydrase (CAII).5a However, due to the high reactivity of alkyl chlorides generating a background activity, only
This journal is © The Royal Society of Chemistry 2012
binary competition assays were conducted, in which the enzyme favoured the assembly of the best inhibitors.
Scheme 1. KTGS reactions: A Huisgen reaction between azides and alkynes. B Amidation reaction between sulfonyl azides and thio acids. C SN2 reaction between mercaptoarylsulfonamides and alkyl chlorides. D Thio-Michael addition of thiols to acrylamides (this study).
Even though the efficiency of the KTGS approach in drug discovery has been proven, its common use is still hampered by the low availability of adapted reactions. In this context, we considered a new amenable reaction for KTGS: the Michael addition of thiols to acrylamide derivatives. Maleimides are widely used for thio-mediated conjugation of biomolecules due to their high reactivity, specificity and strong C-S bond formation. We questioned whether less reactive acrylamide derivatives would limit the formation of 1,4-adducts in the absence of the target enzyme, and remain suitable Michael acceptors for KTGS. Interestingly, this reaction proved to be irreversible (see also ESI),6 in sharp contrast to the reaction carried out with enones which utility has already been established for the dynamic inhibitors discovery.7 However, in the latter strategy cautious reaction conditions are required:
J. Name., 2012, 00, 1-3 | 1
Chemical Communications Accepted Manuscript
Published on 20 December 2013. Downloaded by St. Petersburg State University on 31/12/2013 08:27:24.
COMMUNICATION
ChemComm
sequential additions, and freezing of the final equilibrium before analysis. In this communication, we reported a new KTGS based on the irreversible thio-Michael addition for the discovery of acetylcholinesterase (AChE) inhibitors. Furthermore, in order to avoid the preparation of highly reactive thiols and lessen their amount in the biological medium, an unprecedented cascade deacetylation/thio-Michael reaction is also explored, taking advantage of the hydrolase activity of this enzyme. AChE that plays a key role in the treatment of Alzheimer’s disease (AD), has a buried catalytic site separated by a narrow hydrophobic gorge from a peripheral site located at the surface of the enzyme.8 In our previous work, we reported that both huprine (HUP) and 6,7-dimethoxy-1-phenyl-1,2,3,4tetrahydroisoquinoline (PIQ) derivatives were respective catalytic site and peripheral site ligands suitable for the TGS approach.4g In this present work, molecular docking studies (see ESI), showed that the thio-Michael reaction was more likely when HUP and PIQ derivatives were bearing the acrylamide function and the thiol moiety, respectively.9 We investigated the effect of the chain length spacer on the reaction specificity within the enzyme. The corresponding HUP building blocks ()-Hn and PIQ building blocks ()-Pm, both with two to four carbon-atom chain (Table 1), were incubated as binary mixtures in the presence of m-AChE in citrate buffer pH=7.4 at 37 °C, and monitored by LC-MS. Control experiments consisted of the same mixtures in the absence of m-AChE, and non-specific reaction assays with BSA. Table 1. m-AChE templated assembly of ()-Hn-Pm heterodimers via the irreversible Michael addition of thiols to acrylamide derivatives.
Entry Hn[a] Reaction Product IC50 (nM) Pm 1 NO 11.4±0.7 2 2 (±)-H2-P2 2 NO 10.6±0.1 2 3 (±)-H2-P3 3 NO 14.6±1.1 2 4 (±)-H2-P4 4 YES[b] 0.9±0.1 3 2 (±)-H3-P2 5 NO 3.2±0.1 3 3 (±)-H3-P3 6 YES[b] 4.0±0.2 3 4 (±)-H3-P4 7 NO 1.6±0.1 4 2 (±)-H4-P2 8 NO 2.1±0.4 4 3 (±)-H4-P3 9 NO 3.2±0.7 4 4 (±)-H4-P4 [a] IC50-H2 = 64.9±0.1 nM; IC50-H3 = 56.9±0.9 nM; IC50-H4 = 5.4±0.1 nM. [b] Product detected after 6 h of incubation.
First, ()-H2 and H4 failed to give any heterodimeric product with either one of the three thiol-based PIQ derivatives (±)-P2, (±)-P3, or (±)-P4 (entries 1-3 and 7-9) even after 15
2 | J. Name., 2012, 00, 1-3
Page 2 of 3
Journal Name days of incubation with m-AChE. No reaction was observed in the control experiments either. A moderate 4 to 6 fold gain of inhibitory activity is observed between the ()-H2-Pm heterodimeric products and monomeric ()-H2 (64.9 nM). The synergic gain in activity of (±)-H4-Pm is, at best, only 3-fold better (1.6 nM) compared to the starting acrylamide (±)-H4 (5.4 nM). Finally, in situ assembly of ()-H3 was observed with thiols (±)-P2 and (±)-P4 after only 6 h of incubation (entries 46), but not with (±)-P3 (entry 5). Interestingly, the corresponding heterodimers ()-H3-Pm are 14 to 57-fold more active towards m-AChE with IC50 values in the low nanomolar range compared to the starting huprine ()-H3 (56.9 nM). The control experiments in the absence of the enzyme or in the presence of BSA resulted in no reaction at all or a low background activity, which in any case was insignificant compared to the reaction in the presence of m-AChE (see ESI). The contribution of the active centre of m-AChE in the formation of the heterodimer (±)-H3-P4 was clearly established, since the incubation of (±)-H3 and (±)-P4 in the presence of (±)-huprine X10 did not furnish ()-H3-P4. The irreversible Michael addition of thiols to acrylamides proved thus to be efficient for the linear synthesis of templated inhibitors. However, the high reactivity of thiols is also considered as their major drawback especially for their preparation, as they readily dimerize to form disulfide bonds, particularly under oxidative and basic conditions. This was also observed in all blank and click experiments. Hence, the extraordinary enzymatic efficiency of AChE as an esterase was taken into consideration for the engineering of a cascade double catalysis assay: an enzymatic deacetylation followed by the templated in situ thio-Michael addition. The in situ experiments were conducted using thioacetate ()-P4-Ac, and acrylamidebased huprine ()-H3 in the presence of m-AChE, affording the desired products ()-H3-P4. Kinetic studies of the m-AChE catalytic activity were conducted. As depicted in Figure 1A, the templated reaction of ()-H3-P4 took place solely in the presence of m-AChE, indicating that the double catalysis strategy was successful. In order to examine whether the thioester hydrolysis step into the corresponding thiol intermediate (±)-P4 was promoted by AChE, a study of the hydrolysis kinetics was undertaken with and without the enzyme (Figure 1, B). A significant hydrolysis of the thioacetate was observed in the presence of AChE after only one day of incubation. More than 99% of the thioacetate was hydrolysed on the third day of incubation. Non-specific hydrolysis of thioacetate (±)-P4-Ac was significantly slower in aqueous buffered solution (Figure 1, B). Likewise, in situ assembly of ()-H3-P2 from (±)-P2-Ac was successful despite a low background reactivity (see ESI) due to the non-specific hydrolysis of (±)-P2-Ac. Accordingly, these results demonstrated that the enzyme catalysed both the hydrolysis of thioesters (±)-Pm-Ac and the formation of the heterodimeric inhibitors ()-H3-Pm.
This journal is © The Royal Society of Chemistry 2012
Chemical Communications Accepted Manuscript
Published on 20 December 2013. Downloaded by St. Petersburg State University on 31/12/2013 08:27:24.
COMMUNICATION
View Article Online
DOI: 10.1039/C3CC48871C
Page 3 of 3
View Article Online
ChemComm
DOI: 10.1039/C3CC48871C
COMMUNICATION
Journal Name
B
In situ double catalysis kinetics comparison in the presence and the absence of m-AChE for the assembly of ()-H3-P4
35000000
500000
30000000
AChE
400000 300000
Blank
200000 100000
25000000
AChE (enzymatic hydrolysis) Blank (non-specific hydrolysis)
20000000 15000000 10000000 5000000
0
0 0
1
2
3
4
5
6
7
0
2
4
6
8
10
Time (days)
Time (days)
Figure 1. A: Kinetics comparison over 6 days for the assembly of heterodimer ()-H3-P4 in the double catalysis process. B: In situ thioacetate ()-P4-Ac hydrolysis kinetics comparison between the catalytic process in the presence of AChE and the non-specific catalysis in the buffered solution.
In summary, we have reported the first use of the irreversible Michael addition of thiols to acrylamide derivatives for the kinetic templated synthesis of AChE inhibitors. More importantly, we have developed an unprecedented double catalysis reaction involving: 1) the hydrolase activity of AChE, converting the thioester into the corresponding thiol intermediate; 2) the hosting faculty of the enzyme bringing together active site and peripheral site ligands. Finally, this study adds a useful linear reaction that could be convenient in the case of a limited place within a BT for the click reaction, and validates the proof-of-concept for the double enzymatic and templated catalysis of inhibitors.
Notes and references
5.
a
Normandie Univ, COBRA, UMR 6014 & FR 3038; Univ Rouen; INSA Rouen; CNRS, 1 rue Tesnière 76821 Mont-Saint-Aignan, Cedex, France. Fax: (+33) 2 35 52 29 71. E-mail: [email protected]. b Institut de Recherche Biomédicale des Armées, BP73, F-91993 Brétigny-sur-Orge, France. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/c000000x/ 1. K. B. Sharpless and R. Manetsch, Expert Opin. Drug Discovery, 2006, 1, 525-538. 2. P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor, J. K. M. Sanders and S. Otto, Chem. Rev., 2006, 106, 3652-3711. 3. X. Hu and R. Manetsch, Chem. Soc. Rev., 2010, 39, 1316-1324, and references cited therein. 4. a) S. K. Mamidyala and M. G. Finn, Chem. Soc. Rev., 2010, 39, 1252-1261, and references cited therein; b) T. Suzuki, Y. Ota, Y. Kasuya, M. Mutsuga, Y. Kawamura, H. Tsumoto, H. Nakagawa, M. G. Finn and N. Miyata, Angew. Chem., Int. Ed., 2010, 49, 6817-6820; c) N. P. Grimster, B. Stump, J. R. Fotsing, T. Weide, T. T. Talley, J. G. Yamauchi, Á. Nemecz, C. Kim, K.-Y. Ho, K. B. Sharpless, P. Taylor and V. V. Fokin, J. Am. Chem. Soc., 2012, 134, 6732-6740; d)
This journal is © The Royal Society of Chemistry 2013
6.
7. 8. 9. 10.
J. G. Yamauchi, K. Gomez, N. Grimster, M. Dufouil, A. Nemecz, J. R. Fotsing, K.-Y. Ho, T. T. Talley, K. B. Sharpless, V. V. Fokin and P. Taylor, Mol. Pharmacol., 2012, 10.1124/mol.112.080291; e) C. Peruzzotti, S. Borrelli, M. Ventura, R. Pantano, G. Fumagalli, M. S. Christodoulou, D. Monticelli, M. Luzzani, A. L. Fallacara, C. Tintori, M. Botta and D. Passarella, ACS Med. Chem. Lett., 2013, 4, 274-277; f) W. Tieu, T. P. Soares da Costa, M. Y. Yap, K. L. Keeling, M. C. J. Wilce, J. C. Wallace, G. W. Booker, S. W. Polyak and A. D. Abell, Chem. Sci., 2013, 10.1039/c3sc51127h; g) E. Oueis, G. Santoni, C. Ronco, O. Syzgantseva, V. Tognetti, L. Joubert, A. Romieu, M. Weik, L. Jean, C. Sabot, F. Nachon and P.-Y. Renard, Org. Biomol. Chem., 2014, DOI: 10.1039/C3OB42109K. a) R. Nguyen and I. Huc, Angew. Chem., Int. Ed., 2001, 40, 17741776; b) X. Hu, J. Sun, H.-G. Wang and R. Manetsch, J. Am. Chem. Soc., 2008, 130, 13820-13821; c) S. S. Kulkarni, X. Hu, K. Doi, H.G. Wang and R. Manetsch, ACS Chem. Biol., 2011, 6, 724-732; d) N. K. Namelikonda and R. Manetsch, Chem. Commun., 2012, 48, 15261528; e) M. Gelin, G. Poncet-Montange, L. Assairi, L. Morellato, V. Huteau, L. Dugué, O. Dussurget, S. Pochet and G. Labesse, Structure, 2012, 20, 1107-1117. a) R. H. Nonoo, A. Armstrong and D. J. Mann, ChemMedChem, 2012, 7, 2082-2086; b) J. Xiao, P. Broz, A. W. Puri, E. Deu, M. Morell, D. M. Monack and M. Bogyo, J. Am. Chem. Soc., 2013, 135, 9130-9138. B. Shi, R. Stevenson, D. J. Campopiano and M. F. Greaney, J. Am. Chem. Soc., 2006, 128, 8459-8467. I. Silman and J. L. Sussman, Chem-Biol inter., 2008, 175, 3-10. Huprine and PIQ derivatives were synthesized as racemic mixtures. P. Camps, R. E. Achab, J. Morral, D. Muñoz-Torrero, A. Badia, J. E. Baños, N. M. Vivas, X. Barril, M. Orozco and F. J. Luque, J. Med. Chem., 2000, 43, 4657-4666.
J. Name., 2013, 00, 1-3 | 3
Chemical Communications Accepted Manuscript
Hydrolysis kinetics comparison of P4-Ac in the presence and the absence of m-AChE
600000
Intensity (AU)
Intensity (AU)
Published on 20 December 2013. Downloaded by St. Petersburg State University on 31/12/2013 08:27:24.
A
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: E. Oueis, F. Nachon, C. Sabot and P. Renard, Chem. Commun., 2013, DOI: 10.1039/C3CC48871C.
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 3
ChemComm
Journal Name
View Article Online
DOI: 10.1039/C3CC48871C
RSCPublishing
Cite this: DOI: 10.1039/x0xx00000x
First Enzymatic Hydrolysis/Thio-Michael Addition Cascade Route to AChE Inhibitors Emilia Oueis,a Florian Nachon,b Cyrille Sabot*a and Pierre-Yves Renard*a
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
The irreversible Michael addition of thiols to acrylamides is reported as a new tool for the kinetic target-guided synthesis. In an unprecedented enzymatic hydrolysis/thio-Michael addition procedure, potent and selective acetylcholinesterase inhibitors are assembled by the enzyme using both its esterasic and templating ability. Target-guided synthesis (TGS) is an unconventional fragment-based lead discovery methodology that relies on the multi-binding biological target (BT) itself to build its own inhibitors.1 Indeed, the reaction that occurs between ligands bearing complementary reactive functions is dramatically accelerated in the presence of the BT, leading to the formation of highly selective inhibitors. Different TGS approaches have already been developed and proved efficient for the discovery of potent inhibitors of various enzymes. Worth noting among them, are the dynamic combinatorial chemistry (DCC)2 which involves reversible bond formations, and the kinetically controlled target-guided synthesis (KTGS) which is based on irreversible reactions.1, 3 As an eminent example of KTGS, the in situ click chemistry tool developed by Sharpless et al. uses the 1,3dipolar cycloaddition, between the bioorthogonal azides and alkynes (Scheme 1, A) as the irreversible templated reaction to create new CN bonds and obtain triazole-based heterodimeric inhibitors.4 Aside from the Huisgen reaction, which was used on dozen occasions for the discovery of enzymatic triazole-based inhibitors, only a handful of other irreversible reactions have been engaged in KTGS approaches.3, 5 Notably, the synthesis of amide bonds via the sulfoclick reaction between thio acids and sulfonyl azides was achieved by Manetsch et coll. for the discovery of inhibitors of a Bcl-2 protein family member (Scheme 1, B).5b-d Huc et al. explored the utility of the nucleophilic substitution of alkyl chlorides by mercaptoarylsulfonamide that generates a new C-S bond (Scheme 1, C) to identify inhibitors of the zinc-containing metalloenzyme bovine carbonic anhydrase (CAII).5a However, due to the high reactivity of alkyl chlorides generating a background activity, only
This journal is © The Royal Society of Chemistry 2012
binary competition assays were conducted, in which the enzyme favoured the assembly of the best inhibitors.
Scheme 1. KTGS reactions: A Huisgen reaction between azides and alkynes. B Amidation reaction between sulfonyl azides and thio acids. C SN2 reaction between mercaptoarylsulfonamides and alkyl chlorides. D Thio-Michael addition of thiols to acrylamides (this study).
Even though the efficiency of the KTGS approach in drug discovery has been proven, its common use is still hampered by the low availability of adapted reactions. In this context, we considered a new amenable reaction for KTGS: the Michael addition of thiols to acrylamide derivatives. Maleimides are widely used for thio-mediated conjugation of biomolecules due to their high reactivity, specificity and strong C-S bond formation. We questioned whether less reactive acrylamide derivatives would limit the formation of 1,4-adducts in the absence of the target enzyme, and remain suitable Michael acceptors for KTGS. Interestingly, this reaction proved to be irreversible (see also ESI),6 in sharp contrast to the reaction carried out with enones which utility has already been established for the dynamic inhibitors discovery.7 However, in the latter strategy cautious reaction conditions are required:
J. Name., 2012, 00, 1-3 | 1
Chemical Communications Accepted Manuscript
Published on 20 December 2013. Downloaded by St. Petersburg State University on 31/12/2013 08:27:24.
COMMUNICATION
ChemComm
sequential additions, and freezing of the final equilibrium before analysis. In this communication, we reported a new KTGS based on the irreversible thio-Michael addition for the discovery of acetylcholinesterase (AChE) inhibitors. Furthermore, in order to avoid the preparation of highly reactive thiols and lessen their amount in the biological medium, an unprecedented cascade deacetylation/thio-Michael reaction is also explored, taking advantage of the hydrolase activity of this enzyme. AChE that plays a key role in the treatment of Alzheimer’s disease (AD), has a buried catalytic site separated by a narrow hydrophobic gorge from a peripheral site located at the surface of the enzyme.8 In our previous work, we reported that both huprine (HUP) and 6,7-dimethoxy-1-phenyl-1,2,3,4tetrahydroisoquinoline (PIQ) derivatives were respective catalytic site and peripheral site ligands suitable for the TGS approach.4g In this present work, molecular docking studies (see ESI), showed that the thio-Michael reaction was more likely when HUP and PIQ derivatives were bearing the acrylamide function and the thiol moiety, respectively.9 We investigated the effect of the chain length spacer on the reaction specificity within the enzyme. The corresponding HUP building blocks ()-Hn and PIQ building blocks ()-Pm, both with two to four carbon-atom chain (Table 1), were incubated as binary mixtures in the presence of m-AChE in citrate buffer pH=7.4 at 37 °C, and monitored by LC-MS. Control experiments consisted of the same mixtures in the absence of m-AChE, and non-specific reaction assays with BSA. Table 1. m-AChE templated assembly of ()-Hn-Pm heterodimers via the irreversible Michael addition of thiols to acrylamide derivatives.
Entry Hn[a] Reaction Product IC50 (nM) Pm 1 NO 11.4±0.7 2 2 (±)-H2-P2 2 NO 10.6±0.1 2 3 (±)-H2-P3 3 NO 14.6±1.1 2 4 (±)-H2-P4 4 YES[b] 0.9±0.1 3 2 (±)-H3-P2 5 NO 3.2±0.1 3 3 (±)-H3-P3 6 YES[b] 4.0±0.2 3 4 (±)-H3-P4 7 NO 1.6±0.1 4 2 (±)-H4-P2 8 NO 2.1±0.4 4 3 (±)-H4-P3 9 NO 3.2±0.7 4 4 (±)-H4-P4 [a] IC50-H2 = 64.9±0.1 nM; IC50-H3 = 56.9±0.9 nM; IC50-H4 = 5.4±0.1 nM. [b] Product detected after 6 h of incubation.
First, ()-H2 and H4 failed to give any heterodimeric product with either one of the three thiol-based PIQ derivatives (±)-P2, (±)-P3, or (±)-P4 (entries 1-3 and 7-9) even after 15
2 | J. Name., 2012, 00, 1-3
Page 2 of 3
Journal Name days of incubation with m-AChE. No reaction was observed in the control experiments either. A moderate 4 to 6 fold gain of inhibitory activity is observed between the ()-H2-Pm heterodimeric products and monomeric ()-H2 (64.9 nM). The synergic gain in activity of (±)-H4-Pm is, at best, only 3-fold better (1.6 nM) compared to the starting acrylamide (±)-H4 (5.4 nM). Finally, in situ assembly of ()-H3 was observed with thiols (±)-P2 and (±)-P4 after only 6 h of incubation (entries 46), but not with (±)-P3 (entry 5). Interestingly, the corresponding heterodimers ()-H3-Pm are 14 to 57-fold more active towards m-AChE with IC50 values in the low nanomolar range compared to the starting huprine ()-H3 (56.9 nM). The control experiments in the absence of the enzyme or in the presence of BSA resulted in no reaction at all or a low background activity, which in any case was insignificant compared to the reaction in the presence of m-AChE (see ESI). The contribution of the active centre of m-AChE in the formation of the heterodimer (±)-H3-P4 was clearly established, since the incubation of (±)-H3 and (±)-P4 in the presence of (±)-huprine X10 did not furnish ()-H3-P4. The irreversible Michael addition of thiols to acrylamides proved thus to be efficient for the linear synthesis of templated inhibitors. However, the high reactivity of thiols is also considered as their major drawback especially for their preparation, as they readily dimerize to form disulfide bonds, particularly under oxidative and basic conditions. This was also observed in all blank and click experiments. Hence, the extraordinary enzymatic efficiency of AChE as an esterase was taken into consideration for the engineering of a cascade double catalysis assay: an enzymatic deacetylation followed by the templated in situ thio-Michael addition. The in situ experiments were conducted using thioacetate ()-P4-Ac, and acrylamidebased huprine ()-H3 in the presence of m-AChE, affording the desired products ()-H3-P4. Kinetic studies of the m-AChE catalytic activity were conducted. As depicted in Figure 1A, the templated reaction of ()-H3-P4 took place solely in the presence of m-AChE, indicating that the double catalysis strategy was successful. In order to examine whether the thioester hydrolysis step into the corresponding thiol intermediate (±)-P4 was promoted by AChE, a study of the hydrolysis kinetics was undertaken with and without the enzyme (Figure 1, B). A significant hydrolysis of the thioacetate was observed in the presence of AChE after only one day of incubation. More than 99% of the thioacetate was hydrolysed on the third day of incubation. Non-specific hydrolysis of thioacetate (±)-P4-Ac was significantly slower in aqueous buffered solution (Figure 1, B). Likewise, in situ assembly of ()-H3-P2 from (±)-P2-Ac was successful despite a low background reactivity (see ESI) due to the non-specific hydrolysis of (±)-P2-Ac. Accordingly, these results demonstrated that the enzyme catalysed both the hydrolysis of thioesters (±)-Pm-Ac and the formation of the heterodimeric inhibitors ()-H3-Pm.
This journal is © The Royal Society of Chemistry 2012
Chemical Communications Accepted Manuscript
Published on 20 December 2013. Downloaded by St. Petersburg State University on 31/12/2013 08:27:24.
COMMUNICATION
View Article Online
DOI: 10.1039/C3CC48871C
Page 3 of 3
View Article Online
ChemComm
DOI: 10.1039/C3CC48871C
COMMUNICATION
Journal Name
B
In situ double catalysis kinetics comparison in the presence and the absence of m-AChE for the assembly of ()-H3-P4
35000000
500000
30000000
AChE
400000 300000
Blank
200000 100000
25000000
AChE (enzymatic hydrolysis) Blank (non-specific hydrolysis)
20000000 15000000 10000000 5000000
0
0 0
1
2
3
4
5
6
7
0
2
4
6
8
10
Time (days)
Time (days)
Figure 1. A: Kinetics comparison over 6 days for the assembly of heterodimer ()-H3-P4 in the double catalysis process. B: In situ thioacetate ()-P4-Ac hydrolysis kinetics comparison between the catalytic process in the presence of AChE and the non-specific catalysis in the buffered solution.
In summary, we have reported the first use of the irreversible Michael addition of thiols to acrylamide derivatives for the kinetic templated synthesis of AChE inhibitors. More importantly, we have developed an unprecedented double catalysis reaction involving: 1) the hydrolase activity of AChE, converting the thioester into the corresponding thiol intermediate; 2) the hosting faculty of the enzyme bringing together active site and peripheral site ligands. Finally, this study adds a useful linear reaction that could be convenient in the case of a limited place within a BT for the click reaction, and validates the proof-of-concept for the double enzymatic and templated catalysis of inhibitors.
Notes and references
5.
a
Normandie Univ, COBRA, UMR 6014 & FR 3038; Univ Rouen; INSA Rouen; CNRS, 1 rue Tesnière 76821 Mont-Saint-Aignan, Cedex, France. Fax: (+33) 2 35 52 29 71. E-mail: [email protected]. b Institut de Recherche Biomédicale des Armées, BP73, F-91993 Brétigny-sur-Orge, France. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/c000000x/ 1. K. B. Sharpless and R. Manetsch, Expert Opin. Drug Discovery, 2006, 1, 525-538. 2. P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor, J. K. M. Sanders and S. Otto, Chem. Rev., 2006, 106, 3652-3711. 3. X. Hu and R. Manetsch, Chem. Soc. Rev., 2010, 39, 1316-1324, and references cited therein. 4. a) S. K. Mamidyala and M. G. Finn, Chem. Soc. Rev., 2010, 39, 1252-1261, and references cited therein; b) T. Suzuki, Y. Ota, Y. Kasuya, M. Mutsuga, Y. Kawamura, H. Tsumoto, H. Nakagawa, M. G. Finn and N. Miyata, Angew. Chem., Int. Ed., 2010, 49, 6817-6820; c) N. P. Grimster, B. Stump, J. R. Fotsing, T. Weide, T. T. Talley, J. G. Yamauchi, Á. Nemecz, C. Kim, K.-Y. Ho, K. B. Sharpless, P. Taylor and V. V. Fokin, J. Am. Chem. Soc., 2012, 134, 6732-6740; d)
This journal is © The Royal Society of Chemistry 2013
6.
7. 8. 9. 10.
J. G. Yamauchi, K. Gomez, N. Grimster, M. Dufouil, A. Nemecz, J. R. Fotsing, K.-Y. Ho, T. T. Talley, K. B. Sharpless, V. V. Fokin and P. Taylor, Mol. Pharmacol., 2012, 10.1124/mol.112.080291; e) C. Peruzzotti, S. Borrelli, M. Ventura, R. Pantano, G. Fumagalli, M. S. Christodoulou, D. Monticelli, M. Luzzani, A. L. Fallacara, C. Tintori, M. Botta and D. Passarella, ACS Med. Chem. Lett., 2013, 4, 274-277; f) W. Tieu, T. P. Soares da Costa, M. Y. Yap, K. L. Keeling, M. C. J. Wilce, J. C. Wallace, G. W. Booker, S. W. Polyak and A. D. Abell, Chem. Sci., 2013, 10.1039/c3sc51127h; g) E. Oueis, G. Santoni, C. Ronco, O. Syzgantseva, V. Tognetti, L. Joubert, A. Romieu, M. Weik, L. Jean, C. Sabot, F. Nachon and P.-Y. Renard, Org. Biomol. Chem., 2014, DOI: 10.1039/C3OB42109K. a) R. Nguyen and I. Huc, Angew. Chem., Int. Ed., 2001, 40, 17741776; b) X. Hu, J. Sun, H.-G. Wang and R. Manetsch, J. Am. Chem. Soc., 2008, 130, 13820-13821; c) S. S. Kulkarni, X. Hu, K. Doi, H.G. Wang and R. Manetsch, ACS Chem. Biol., 2011, 6, 724-732; d) N. K. Namelikonda and R. Manetsch, Chem. Commun., 2012, 48, 15261528; e) M. Gelin, G. Poncet-Montange, L. Assairi, L. Morellato, V. Huteau, L. Dugué, O. Dussurget, S. Pochet and G. Labesse, Structure, 2012, 20, 1107-1117. a) R. H. Nonoo, A. Armstrong and D. J. Mann, ChemMedChem, 2012, 7, 2082-2086; b) J. Xiao, P. Broz, A. W. Puri, E. Deu, M. Morell, D. M. Monack and M. Bogyo, J. Am. Chem. Soc., 2013, 135, 9130-9138. B. Shi, R. Stevenson, D. J. Campopiano and M. F. Greaney, J. Am. Chem. Soc., 2006, 128, 8459-8467. I. Silman and J. L. Sussman, Chem-Biol inter., 2008, 175, 3-10. Huprine and PIQ derivatives were synthesized as racemic mixtures. P. Camps, R. E. Achab, J. Morral, D. Muñoz-Torrero, A. Badia, J. E. Baños, N. M. Vivas, X. Barril, M. Orozco and F. J. Luque, J. Med. Chem., 2000, 43, 4657-4666.
J. Name., 2013, 00, 1-3 | 3
Chemical Communications Accepted Manuscript
Hydrolysis kinetics comparison of P4-Ac in the presence and the absence of m-AChE
600000
Intensity (AU)
Intensity (AU)
Published on 20 December 2013. Downloaded by St. Petersburg State University on 31/12/2013 08:27:24.
A
