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Synlett 2017; 28(16): 2169-2173
DOI: 10.1055/s-0036-1588875
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© Georg Thieme Verlag Stuttgart · New York

Scalable and Purification-Free Synthesis of a Myristoylated Fluoro­genic Sirtuin Substrate

Iacopo Galleano
a   Center for Biopharmaceuticals, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark   Email: cao@sund.ku.dk
b   Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
,
John Nielsen
b   Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
,
Andreas S. Madsen 
a   Center for Biopharmaceuticals, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark   Email: cao@sund.ku.dk
b   Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
,
Christian A. Olsen  *
a   Center for Biopharmaceuticals, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark   Email: cao@sund.ku.dk
b   Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
› Author AffiliationsThis work was supported by The University of Copenhagen, The Lundbeck Foundation (Young Group Leader Fellowship, C.A.O.), The Danish Independent Research Council-Production and Technical Sciences (Sapere Aude grant no. 12-132328, A.S.M.), the Carlsberg Foundation (2011_01_0169 and 2013_01_0333), and the Novo Nordisk Foundation (NNF15OC0017334).
Further Information

Publication History

Received: 28 March 2017

Accepted after revision: 17 May 2017

Publication Date:
29 June 2017 (online)

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Abstract

Sirtuins are NAD+-dependent deacylases involved in the regulation of fundamental cellular processes in both normal and diseased cells. Therefore, the development of selective inhibitors for this class of enzymes is of great interest. Herein, we report an optimized synthesis of Ac-ETDK(myristoyl)-AMC, a sirtuin 2 substrate that can be used to profile inhibitors in fluorescence-based assays. The presented synthesis is scalable and does not involve chromatographic purification, making this substrate readily available in few steps.

Key words

sirtuins - enzyme substrates - purification-free synthesis - posttranslational modifications - deacylases - SIRT2

Supporting Information

    Supporting information for this article is available online at https://doi.org/10.1055/s-0036-1588875.
  • Supporting Information
 

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  • 20 Ac-Glu( t Bu)-Thr( t Bu)-Asp( t Bu)-Lys-AMC (11) Ac-Glu( t Bu)-Thr( t Bu)-Asp( t Bu)-Lys(Fmoc)-AMC (10, 277 mg, 0.26 mmol, 1.0 equiv) was dissolved in DMF (4.8 mL). 1-Octane­thiol (187 mg, 1.3 mmol, 5.0 equiv) was added, followed by a mixture of piperidine (22 mg, 0.26 mmol, 1 equiv) and DBU (1.2 mg, 0.008 mmol, 0.03 equiv) in DMF (50 μL). The reaction mixture was stirred at r.t. for 3 h, where LC–MS indicated full conversion of starting material. The solvent was removed under reduced pressure, and the resulting residue was split in two 50 mL Falcon tubes. Cold Et2O (30 mL for each Falcon tube) was added, resulting in precipitation of a white solid, which was isolated by centrifugation. Trituration with cold Et2O (4 × 20 mL) afforded Ac-Glu( t Bu)-Thr( t Bu)-Asp( t Bu)-Lys-AMC as a white solid (11, 167 mg, 76%). 1H NMR (600 MHz, DMSO-d 6): δ = 8.12 (d, J = 7.8 Hz, 1 H), 8.05 (d, J = 7.3 Hz, 1 H), 7.99 (d, J = 8.1 Hz, 1 H), 7.76 (d, J = 1.8 Hz, 1 H), 7.72 (d, J = 8.7 Hz, 1 H), 7.59 (d, J = 7.7 Hz, 1 H), 7.50 (dd, J = 8.6, 1.8 Hz, 1 H), 6.27 (s, 1 H), 4.71–4.63 (m, 1 H), 4.41–4.29 (m, 2 H), 4.25 (dd, J = 7.3, 3.7 Hz, 1 H), 3.94–3.85 (m, 1 H), 2.70 (dd, J = 15.9, 5.0 Hz, 1 H), 2.57–2.53 (m, 1 H, peak overlaps with solvent), 2.50 (m, 2 H, peak overlaps with solvent), 2.40 (s, 3 H), 2.26–2.19 (m, 2 H), 1.93–1.83 (m, 4 H), 1.76–1.58 (m, 3 H), 1.41–1.22 (m, 22 H), 1.15 (s, 9 H), 1.01 (d, J = 6.3 Hz, 3 H). 13C NMR (151 MHz, DMSO): δ = 171.7, 171.14, 171.06, 170.0, 169.5, 169.3, 169.0, 160.0, 153.6, 153.0, 142.1, 125.9, 115.2, 115.1, 112.3, 105.7, 80.3, 79.6, 74.0, 66.8, 57.1, 53.8, 51.8, 49.4, 41.4, 37.5, 32.9, 31.7, 31.4, 27.9, 27.7, 27.6, 27.2, 22.7, 22.4, 18.6, 17.9. The data are in agreement with literature.11d Using compound 10 (82 mg, 0.08 mmol) from the Rink-acid resin sequence as starting material afforded compound 11 in 63% yield (41 mg).
  • 21 Ac-Glu-Thr-Asp-Lys(myristoyl)-AMC (7) Ac-Glu( t Bu)-Thr( t Bu)-Asp( t Bu)-Lys-AMC (11, 271 mg, 0.32 mmol, 1.0 equiv) was dissolved in anhydrous CH2Cl2 (10.0 mL), then i-Pr2NEt (122 mg, 0.95 mmol, 3.0 equiv) was added, and the solution was cooled on an ice-bath. Myristoyl chloride (94 mg, 0.38 mmol, 1.2 equiv) was added, and the reaction mixture was allowed to reach r.t. Conversion of starting material was monitored by TLC (CH2Cl2–MeOH (9:1, v/v); Rf (starting material) = 0.0; Rf (myristoylated compd) = 0.7, achieving full conversion in approximately 1 h. The reaction mixture was then diluted with CH2Cl2 (40 mL) and washed with aq HCl (0.5 M, 2 × 40 mL), sat NaHCO3 (2 × 40 mL), and brine (50 mL). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to afford an off-white solid. The Ac-Glu( t Bu)-Thr( t Bu)-Asp( t Bu)-Lys(myristoyl)-AMC, containing excess myristic acid, was used in the next step without further purification (MALDI-TOF: m/z calcd for C57H92N6NaO13 + [M + Na]+: 1091.66; found: 1091.40). The solid residue was dissolved in TFA–CH2Cl2–H2O (12 mL, 50:48:2) for 90 min to remove protecting groups. The reaction mixture was then concentrated under reduced pressure, a solid was precipitated from cold Et2O (40 mL), and triturated with cold Et2O (2 × 25 mL). The resulting residue was then dissolved in aq NaOH (0.1 M, 30 mL) and stirred at r.t. for 90 min. The solution was then cooled on an ice-bath, and a solid was precipitated by aq HCl (0.2 M) to pH = 2. The solid was triturated with cold water (2 × 5 mL). The residue was lyophilized, affording Ac-Glu-Thr-Asp-Lys(myristoyl)-AMC (7) as a white solid (230 mg, 81%). 1H NMR (600 MHz, DMSO-d 6): δ = 12.28 (br s, 2 H), 10.36 (s, 1 H), 8.28 (d, J = 7.7 Hz, 1 H), 8.18 (d, J = 7.7 Hz, 1 H), 7.99 (d, J = 7.5 Hz, 1 H), 7.81 (d, J = 1.9 Hz, 1 H), 7.79–7.73 (m, 2 H), 7.71 (d, J = 8.7 Hz, 1 H), 7.56 (dd, J = 8.7, 1.9 Hz, 1 H), 6.26 (d, J = 1.0 Hz, 1 H), 5.10 (s, 1 H), 4.63–4.57 (m, 1 H), 4.36–4.28 (m, 2 H), 4.25 (dd, J = 7.9, 4.4 Hz, 1 H), 4.06–3.99 (m, 1 H), 3.06–2.96 (m, 2 H), 2.74 (dd, J = 16.6, 5.6 Hz, 1 H), 2.59 (dd, J = 16.6, 7.4 Hz, 1 H), 2.40 (d, J = 1.0 Hz, 3 H), 2.31–2.21 (m, 2 H), 2.00 (t, J = 7.5 Hz, 2 H), 1.96–1.84 (m, 4 H), 1.80–1.71 (m, 2 H), 1.68–1.60 (m, 1 H), 1.45–1.14 (m, 26 H), 1.05 (d, J = 6.3 Hz, 3 H), 0.84 (t, J = 7.0 Hz, 3 H).13C NMR (151 MHz, DMSO): δ = 173.9, 172.0, 171.9, 171.5, 171.2, 170.6, 169.9, 169.6, 160.0, 153.6, 153.0, 142.2, 125.8, 115.4, 115.1, 112.3, 105.7, 66.7, 58.0, 53.9, 52.2, 49.6, 38.2, 35.9, 35.4, 31.3, 31.2, 30.3, 29.04, 29.02, 29.00, 28.99, 28.93, 28.75 (2C), 28.69, 28.67, 27.1, 25.3, 22.8, 22.4, 22.1, 19.2, 18.0, 13.9. The data are in agreement with literature.11d Using compound 11 (37 mg, 0.04 mmol) from the Rink-acid resin sequence as starting material afforded 7 in 72% yield (28 mg).
  • 22 Zoom of the α-proton region of the compound 7 1H NMR spectrum (Figure 2).
  • 23 For comparison, commercial SIRT2 substrate 3 required for performance of a 96-well microtiter assay (0.5 μmol) is priced at €150/$190 ( http://www.enzolifesciences.com).