Cover Page

Cover Page

The handle http://hdl.handle.net/1887/123227 holds various files of this Leiden University dissertation.

Author: Maurits, E.

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Figure 1. General overview of combining proteasome inhibition with chemotherapeutic drugs to accelerate cell death. Cleavage

of the linker is explored throughout this Thesis.

Chapter 1 provides a literature survey on mammalian proteasomes, their structure, function and

physiological roles, and provides a background on proteasome-targeting drugs, the haematological cancers that are treated by these, and the problem of emerging drug resistance that necessitates the development of conceptually new therapeutic strategies.

Chapter 2 focuses on the development of selective fluorogenic substrates for each of the catalytic

activities inherent to human constitutive proteasomes and immunoproteasomes. It was shown that by taking the oligopeptide recognition element of previously developed subunit selective proteasome inhibitors, fluorogenic substrates reporting on the proteasome catalytic activity, also targeted by the parent inhibitor, could be made. Though not generally applicable (β5c and β2i substrates remaining elusive), effective fluorogenic substrates reporting on the individual activity of β1c, β1i, β2c and β5i in Raji (human B cell) lysates and purified 20S proteasomes were identified in this manner. This work thus adds to the expanding proteasome research toolbox through the identification of new and/or more effective subunit-selective fluorogenic substrates.

Chapter 3 describes a new strategy aimed to selectively deliver a toxic cargo to cells expressing

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This showed the need for a self-immolative linker between the toxic moiety and the proteasome inhibitor scaffold, in order for the conjugate to induce apoptosis. However, cell death was also induced in β5i pre-inhibited cells, indicating that substrate hydrolysis was prompted by other proteasome subunits or other proteases altogether. In a related study, sulfonate ester/amide based covalent proteasome inhibitors were equipped with a self-immolative linker connected to doxorubicin. These compounds however proved not to inhibit proteasomes and thus this design was abandoned. Finally, a proteasome inhibitor having an epoxyketone warhead was equipped with a potential self-immolative linker and functionalized with a FRET-pair to determine the effectiveness of cleavage. Initial comparison of its potency against intermediate and control compounds revealed a loss of selectivity and potency concomitant with observed complete inhibition of all proteasome subunits at 10 µM. To summarize the results, the conjugates proved neither potent nor selective proteasome inhibitors, and new conjugate designs are needed. One way to approach this would be to take the advantageous properties of the individual constructs and combine these in a new design. The substrates described in Chapter 3 proved to effectively induce apoptosis, however their selectivity was compromised, possibly because of the lack of an inhibitor moiety. The constructs based on proteasome inhibitors on the other hand, proved inactive, arguably because of the ligation site chosen. Shifting the covalent electrophilic trap from targeting the catalytic site to a different position might leave room for a toxic substrate-based release by catalytic proteasome activity. 4-CA (1, Figure 2) is a proteasome inhibitor that covalently binds to Cys48 in the β5i proteasome subunit pocket and thereby selectivity inhibits it without utilizing the catalytic site.5 A combination of 4-CA with the here described toxic substrates might therefor lead to a covalent proteasome inhibitor (2, 4-CA-Doxo), that can still utilize the catalytic site for cleavage of the toxic moiety.

Figure 2. Proposed new proteasome inhibitor based on 4-CA that utilizes the catalytic threonine to release doxorubicin.

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Figure 3. Proposed mechanism of self-immolation upon inhibition of the proteasome using an aziridine ketone warhead. A)

inhibition via the classical 6-membered morpholine ring intermediate, B) inhibition via the recently proposed 7-membered oxazepane ring formation.

To begin investigating the validity of this hypothesis, the synthesis of an alpha-keto-aziridine was developed (Scheme 1). The synthesis started with similar steps as described for making the epoxyketone from Boc-Leu-OH 3, leading to compound 6.8 Alcohol 6 was then reacted with trichloroacetonitrile to form imidate 7. Boc-protected trichloro-oxazole 8 was then constructed by creating an iodonium ion on the nearby olefin which is immediately opened upon intramolecular closing of the five-membered ring by the nucleophilic imine. Theoretically Boc-protected trichloro-oxazole 8 can be deprotected, in the absence of H2O, using TFA and anhydrous DCM and subsequently protected with a Cbz group using

benzylchloroformate via nucleophilic catalysis of pyridine. In practice, partial hydrolysis is observed resulting in the formation of two products, the Cbz-protected trichloro-oxazole 9a and trichloro-ester 9b. Isolating the two products proved to be difficult without also isolating impurities. Therefore, a crude of both products was exposed to 6M HCl in a ratio of 4:3 H2O:MeOH to fully hydrolyse both to the NH3Cl salt 10. This compound was then treated with IRA67 base resin to deprotonate the amine and immediately

prompts intramolecular cyclisation, affording aziridine 11. LC-MS analysis confirmed the formation of the aziridine 11, but purification by neutralized silica gel column chromatography or ion exchange chromatography proved to be troublesome. When performing TEMPO-PIDA oxidation using crude aziridine 11, no oxidation was observed.

Scheme 1. Attempted alpha-keto-aziridine synthesis. Reagents and conditions: a) N,O,-dimethylhydroxylamine, HCTU, DIPEA,

DCM, 75%; b) vinylmagnesium bromide, THF, 0oC, 90%; c) NaBH4, CeCl3•7 H2O, MeOH, 85%; d) i. NaH, 0oC; ii.

trichloroacetonitril, DCM, RT, 85%; e) NIS, MeCN, 30%; f) CbzCl, pyridine, DCM, 30oC; g) 6M HCl in 4:3 H2O:MeOH, 40oC; h) IRA67

basic resin, MeOH, 30oC; i) TEMPO, PIDA, MeCN, 0oC.

Chapter 4 describes an immunoproteasome inhibitor-doxorubicin conjugate that targets multiple

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approach was described that combines two drug classes into a single molecule: anthracyclines and proteasome inhibitors. Doxorubicin was conjugated to an immunoproteasome-selective inhibitor via light-cleavable linkers, yielding peptide epoxyketone-doxorubicin prodrugs that remained selective and active towards immunoproteasomes in both lysates and living AMO-1 cells. Upon cellular uptake and immunoproteasome inhibition, doxorubicin was released from the immunoproteasome inhibitor through photo-irradiation. Multiple myeloma derived cells in this way take a double hit: immunoproteasome inhibition and doxorubicin-induced toxicity.

Chapter 5 describes how a trans-cyclooctene (TCO) is utilized for the release of a toxic payload via a click

to release reaction in conjugation with selective proteasome targeting. TCOs were used in order to facilitate toxin activation via an IEDDA reaction with tetrazine cycloaddition. Five different bifunctionalized TCOs were constructed with either a β5i, β1i or pan-subunit selective proteasome inhibitor attached to the non-eliminating end of the TCO. On the eliminating end, a toxin was attached, consisting of doxorubicin or monomethyl auristatin F. For the auristatin, also the synthesis was described. Validation of the TCO conjugates in biological assays showed a lack of inhibitory activity for β1i targeting conjugate while the other constructs showed selectivity and inhibition towards their targeted proteasome subunits. Labelling of the inhibited proteasome subunits proved possible by clicking them to a BODIPY-FL tetrazine. Then, the cell permeability and inhibition profiles in living AMO-1 cells were determined for some of the conjugates, indicating a similar inhibition profile compared to the lysates. Finally, cell survival showed enhanced cytotoxicity for the doxorubicin eliminating conjugates when the cells were treated with tetrazine. However, the monomethyl auristatin F conjugate showed to be already cytotoxic regardless of tetrazine cycloaddition.

Maximizing toxin release with the click to release reaction is highly depended on tetrazine specifications. In order to successfully apply click to release in biology, the identification of optimized tetrazines is crucial. Current studies lack the use of eliminating alkylamines (as used in the here presented constructs), are very concentration depended or are not representing physiological conditions.9–12 Therefore, a FRET-based method to rapidly determine the overall alkylamine release rate caused by any tetrazine without stringent concentration requirements would be of great importance. TCO-FRET reporter 13 was designed and synthesized based on the bifunctional TCO introduced by Robillard and co-workers (Figure 4A, see experimental for details on the synthesis and characterisation of 13).13 _{By linking}

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Figure 4. Design of a TCO-based FRET pair. A) Structure of DABCYL-TCO-EDANS 13. B) The rationale behind the loss of FRET

caused by tetrazine cycloaddition.

Besides attaching the here described toxic moieties to the proteasome inhibitor-TCO constructs, biotin-linked conjugates could also be envisioned to enhance affinity purification procedures (Figure 5). An oft-occurring problem in pull-downs is the high background signal caused by the formation of the biotin-streptavidin bond.14 _{Harsh denaturing conditions have to be used to break the formed bond, causing the}

eluted sample to contain large amounts of streptavidin and endogenously biotinylated biomolecules, resulting in an undesirably large background signal. To obtain a cleaner result cleavable linkers in ABPP have been proposed to ensure that only the ABP-reacted enzymes bound to the streptavidin are released.15,16 Here, the click to release approach could be used as complimentary method for the cleavage of targeted protein-streptavidin bond.

Conjugates bearing a biotin on one end and a small molecule inhibitor on the other end of the TCO were constructed (see experimental for details on the synthesis and characterisation of all conjugates). Four mechanism-based, covalent and irreversible inhibitors were selected for these studies: a broad-spectrum proteasome inhibitor (as in 14), a β1i selective inhibitor (as in 15), a β-glucocerebrosidase inhibitor (as in

16) and a cathepsin B (as in 17, GB111) inhibitor. Affinity purifications conducted with the conjugates

showed the incompatibility with high thiol (such as dithiothreitol) concentrations causing the TCO to isomerize quickly towards the CCO (confirmed by LC-MS, data not shown). Further affinity purifications concluded to be inconclusive at this point and additional experiments have to be conducted.

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Inhibitors with dissimilar P3 and fixed P4 sites, and vice versa, were made in an attempt to improve the pan-immuno subunit selectivity based on BocPip or HCH P4 and Ser or Ser(OBn) P3. Cationic side chains at P3 revealed potency and selectivity for β2/β5 and the extension of P3 hydrophobic residues in the S3 pocket showed a redundant increase of β5c potency. Future research may focus on the β5 S5 pocket, and P4 Chg and P4 free amino acids could be considered the most promising approach. Additionally, evidence of the ‘’locked mode’’ provides a systematic way to design β5 potent compounds which might be an alternative starting point to discover new pan-immunoproteasome subunit selective inhibitors based on β1i-β2i potent scaffolds.

Figure 6. Schematic representation of discovered best pan-immuno proteasome subunit selective inhibitor, with the affinity of

each side chain to specificity pocket (S1-S5) indicated. The R groups indicate possible sites at which pan-immuno selectivity could be further improved. P4 = BocPip and P3 = Ser. Lead compound 18 consists of R1 = CH2 and R2 = H.

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Figure 7. Inhibition profiles of epoxomicin derivatives with increasing sized P1 amino acid side chains, determined by

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Experimental

Synthetic procedures

All reagents were of commercial grade and used as received unless stated otherwise. Solvents used in synthesis were dried and stored over 4Å molecular sieves, except MeOH and ACN which were stored over 3Å molecular sieves. Triethylamine (Et3N) and Di-isopropylethylamine (DiPEA) were stored over

KOH pellets.

Column chromatography was performed on silica gel 60 Å (40-63 µm, Macherey-Nagel). TLC analysis was

performed on Macherey-Nagel aluminium sheets (silica gel 60 F254).

TLC was used to monitor reactions with visualization by UV at wavelength 254 nm and by one of

following treatments: spraying with either cerium molybdate spray (25 g/L (NH4)6Mo7O24, 10 g/L

(NH4)4Ce(SO4)4·H2O in 10% H2SO4 water solution) or KMnO4 spray (20 g/L KMnO4 and 10 g/L K2CO3 in

water) followed by charring at c.a. 250°C. TLC-MS analysis was performed on a Advion ExpressionL _EMS

in combination with a Plate Express interface (eluted with MeOH/H2O 90%/10% v/v + 0.1% formic acid,

flow rate 0.20 mL/min).

LC-MS analysis was performed on a Finnigan Surveyor HPLC system with a Nucleodur C18 Gravity 3um 50

x 4.60 mm column (detection at 200-600 nm) coupled to a Finnigan LCQ Advantage Max mass spectrometer with ESI or coupled to a Thermo LCQ Fleet Ion mass spectrometer with ESI. The Method used was 10→90% 13.5 min (0→0.5 min: 10% MeCN; 0.5→8.5 min: 10% to 90% MeCN; 8.5→ 11 min: 90% MeCN; 11→13.5 min: 10% MeCN).

High-resolution mass spectrometry (HRMS) was performed on a Thermo Scientific Q Exactive HF

Orbitrap mass spectrometer equipped with an electrospray ion source in positive-ion mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 275°C) with resolution R = 240.000 at m/z 400 (mass range of 150-6000) correlated to an external calibration, or on a Waters Synapt G2-Si (TOF) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV) and LeuEnk (m/z = 556.2771) as internal lock mass.

1

H and 13C NMR spectra were recorded on a Bruker AV-400 NMR, a Bruker DMX-400 NMR instrument

(400 and 101 MHz respectively), a Bruker AV-500 NMR instrument (500 and 126 MHz respectively), and Bruker AV-600 NMR instrument (600 and 151 MHz respectively). Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard or the residual signal of the deuterated solvent. Coupling constants (J) are given in Hz. All given 13_{C-APT spectra are proton decoupled. Assignment of}

NMR spectra was based on 1_{H-COSY and} 1_H-13_C-HSQC.

HPLC purification was performed on a Gilson HPLC system coupled to a Magerey-Nagel Nucleodur C18

Gravity 5 μm 250 ×10 mm column, or on an Agilent 1200 HPLC/6130 MS system coupled to a Magerey-Nagel Nucleodur C18 Gravity 5μm 250×10 mm column or on a Waters autopurifier HPCL/MS system coupled to a Phenomenex Gemini 5μm 150×21.2 mm column.

General Procedure 1 (GP1) for the functionalization of TCOs: A 2 mL Eppendorf tube was charged with

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twice and subsequently washed with H2O twice and brine, dried over MgSO4 and concentrated in vacuo.

Column purification (1:10 → 2:1 EtOAc:PE, v:v) yielded 4 as a transparent oil (12.3 mmol, 3.79 g, 82%). TLC Rf 0.3 (1:2, EtOAc:PE, v:v). LC-MS (linear gradient 10->90 MeCN, 0.1% TFA, 12.5 min): Rt(min) 7.48

ESI-MS (m/z): 308.93 (M+H+_).

Vinyl ketone 5. Compound 4 (12.3 mmol, 3.79 g) was co-evaporated with toluene twice

and then dissolved in anhydrous THF (30 mL). The solution was then cooled to 0o_{C and}

purged with N2. Next vinylmagnesium bromide (50 mL, 1.0 M in THF) was added drop

wise to the solution and the reaction mixture was left stirring for 2 h under N2

atmosphere at 0o_{C. Next the reaction mixture is added to a cooled HCl (aq. 2.0 M)}

solution. Simultaneously EtOAc is used to dilute the mixture. The aqueous layer was back extracted with EtOAc twice. The EtOAc is then washed with H2O and brine, dried over MgSO4 and concentrated in vacuo.

Column purification (1:20 → 1:8 EtOAc:PE, v:v) yielded 11 in a colourless oil, (10.1 mmol, 2.78 g, 82%). TLC Rf 0.5 (1:7, EtOAc:PE, v:v). 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.30 (m, 5H), 6.52 – 6.34 (m, 2H), 5.87

(dd, J = 10.0, 1.8 Hz, 1H), 5.53 (d, J = 8.5 Hz, 1H), 5.09 (s, 2H), 4.73 (ddd, J = 9.8, 8.5, 3.9 Hz, 1H), 1.80 – 1.67 (m, 1H), 1.56 (ddd, J = 13.4, 9.2, 3.9 Hz, 1H), 1.39 (ddd, J = 14.2, 9.8, 4.6 Hz, 1H), 1.00 (d, J = 6.5 Hz, 3H), 0.91 (d, J = 6.7 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 198.8, 156.2, 136.4, 133.2, 128.5, 128.1, 66.9,

56.3, 41.5, 24.9, 23.4, 21.8. LC-MS (linear gradient 10->90 MeCN, 0.1% TFA, 12.5 min): Rt(min) 8.00

ESI-MS (m/z): 275.80 (M+H+_).

Vinyl alcohol 6. Compound 5 (4.9 mmol, 1.36 g) was dissolved in MeOH (30 mL) and

CeCl3 (8.40 mmol, 3.13 g, 1.7 eq) was added and stirred until dissolved. Then the

solution was cooled to 0oC and NaBH4 (5.93 mmol, 0.22 g, 1.2 eq) was added in portions

and the solution was stirred for 1.5 h. Afterward the solvent was evaporated and subsequently acidified with HCl (aq 1.0 M). The product was back extracted twice using EtOAc and then washed with H2O and Brine. The resulting organic layer was then dried over MgSO4 and

concentrated in vacuo. Column purification (1:200 → 1:50 MeOH:DCM, v:v) yielded 6 as a transparent oil (4.9 mmol, 1.35 g, quant.). TLC Rf 0.25 (1:1, EtOAc:PE, v:v). 1H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 4.6 Hz,

4H), 5.84 (dddd, J = 22.5, 16.3, 10.5, 5.5 Hz, 1H), 5.36 – 5.11 (m, 2H), 5.07 (d, J = 4.6 Hz, 2H), 5.00 (d, J = 9.0 Hz, 1H), 4.25 – 4.02 (m, 1H), 3.92 – 3.65 (m, 1H), 1.63 (tq, J = 13.3, 6.6 Hz, 1H), 1.48 – 1.18 (m, 2H), 0.96 – 0.85 (m, 6H). 13_{C NMR (101 MHz, CDCl}

3) δ 157.1, 138.2, 136.8, 136.4, 128.6, 128.1, 116.7, 116.3,

75.5, 74.8, 66.9, 66.7, 53.9, 53.4, 40.9, 38.7, 24.7, 23.6, 21.7. LC-MS (linear gradient 10→90 MeCN, 0.1% TFA, 12.5 min): Rt(min) 7.39 ESI-MS (m/z): 277.93, (M+H+).

Imidate 7. Compound 6 (11.5 mmol, 2.31 g) was co-evaporated with toluene

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mmol, 1.73 mL, 1.5 eq) was added drop wise. The reaction mixture was left stirring overnight under N2

atmosphere at RT. The reaction mixture was then diluted with DCM and acidified with HCl (aq 1.0 M), washed with H2O and brine, dried over MgSO4 and concentrated in vacuo. Column purification (0:1 →

1:100 DCM:MeOH, v:v) yielded 7 as an orange oil (9.75 mmol, 3.78 g, 85%). TLC Rf 0.7 (1:10, EtOAc:PE,

v:v). 1_{H NMR (400 MHz, CDCl}

3) δ 8.41 (d, J = 12.3 Hz, 1H), 5.93 – 5.78 (m, 1H), 5.51 – 5.24 (m, 3H), 4.64 (d,

J = 9.8 Hz, 1H), 4.03 (ddt, J = 10.1, 6.5, 4.0 Hz, 1H), 1.69 (dq, J = 13.8, 6.9 Hz, 1H), 1.53 – 1.22 (m, 11H), 0.98 – 0.87 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 174.6, 168.0, 161.6, 155.4, 132.5, 118.3, 81.0, 80.1,

79.2, 77.5, 51.1, 40.8, 24.6, 23.6, 23.1, 22.1, 21.6.

Boc trichloro-oxazole 8. Compound 7 (1.27 mmol, 0.44 g) was dissolved in

anhydrous MeCN (3.5 mL) and purged with N2. The flask is protected from UV

light using aluminium foil and NIS then (3.18 mmol, 0.715 g, 2.5 eq) is added and the reaction mixture is left stirring overnight under N2 atmosphere at RT. Next

the solution is diluted with EtOAc and washed with saturated Na2S2O3, H2O and

brine. The resulting organic layer was dried over MgSO4 and concentrated in

vacuo. Column purification (1:10 → 1:1 EtoAc:PE, v:v) yielded 8 as an orangey powder (2.8 mmol, 1.36 g, 30%). TLC Rf 0.85 (1:10, EtOAc:PE, v:v). 1H NMR (400 MHz, CDCl3) δ 4.56 (t, J = 5.5 Hz, 1H), 4.51 (d, J = 9.4

Hz, 1H), 4.21 (q, J = 5.1 Hz, 1H), 3.92 – 3.81 (m, 1H), 3.40 (d, J = 5.3 Hz, 2H), 1.72 (dt, J = 13.5, 6.5 Hz, 1H), 1.45 (s, 10H), 1.38 (dd, J = 8.5, 5.8 Hz, 2H), 0.94 (dd, J = 9.7, 6.6 Hz, 7H). 13C NMR (101 MHz, CDCl3) δ

163.3, 155.5, 91.2, 80.2, 68.6, 51.3, 39.0, 28.5, 24.6, 23.7, 21.6, 9.9. LC-MS (linear gradient 10→90 MeCN, 0.1% TFA, 12.5 min): Rt(min) 9.05 ESI-MS (m/z): 512.93 (M+H+).

Cbz trichloro-oxazole 9. Compound 8 (0.5 mmol, 0,186 g) was co-evaporated

with toluene three times and then dissolved in anhydrous DCM (1 mL). TFA (1 mL) was added and the solution was left stirring for 1.5 h at RT. The solution concentrated in vacuo and co-evaporated with toluene three times. The free amine was then dissolved in anhydrous DCM (1 mL) and onto this solution anhydrous pyridine (1.5 mmol, 0.125 mL 3 eq) was added. The solution was cooled to 0o_{C and Cbz-Cl (0.75 mmol, 0.11 mL, 1.5 eq) was added drop wise. The reaction mixture was}

left stirring overnight under N2 atmosphere at RT. Next the solution was diluted with DCM and washed

with HCl (1.0 M aq.), H2O and brine. The resulting organic layer was dried over MgSO4 and concentrated

in vacuo. Column purification (1:20 → 1:1 EtOAc:PE, v:v) yielded 9 as a yellow oil and was immediately used in the next reaction as a crude. TLC Rf 0.3 (1:7, EtOAc:PE, v:v).

Aziridine alcohol 10. Compound 9 (0.16 mmol, 0.09 g) was dissolved in MeOH (3

mL) and cooled to 0o_{C. HCl (4 mL, 6.0 M aq.) was added and the reaction mixture}

was left stirring under N2 atmosphere at 50oC until LC-MS indicated full

conversion. The reaction mixture is then diluted with H2O, concentrated in vacuo

and co-evaporated with toluene three times. The crude product is then dissolved in 10% NH3 in MeOH and left stirring for 1 h. The mixture is then concentrated in vacuo thereby obtaining 11. LC-MS (linear gradient 10→90 MeCN, 0.1% TFA, 12,5 min): Rt(min) 9.05 ESI-MS (m/z): 293.13 (M+H+).

NHS-TCO-EDANS (23). Starting from EDANS-NH2 (15.3 mg, 57

µmol) and DiNHS-TCO (20.0 mg, 47 µmol), the reaction was carried out following GP1 to afford the desired mono-functionalized TCO (21.2 mg, 0.037 mmol, 79%). 1_{H NMR (500}

BocHN OH

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(m/z): 574.20 (M+H)). HRMS: calculated for C27H32N3O8S 558.19101 [M+H]; found 558.19014.

DABCYL-TCO-EDANS (13). Starting from DABCYL-NH2

(3.36 mg, 10.8 µmol, see Chapter 3 for the synthesis) and NHS-TCO-EDANS (4.87 mg, 10.3 µmol), the reaction was carried out following GP1 and purified by prep-HPLC (60-70% MeCN-H2O) yielding

DABCYL-TCO-EDANS (4.76 mg, 6.2 µmol, 60%). 1_{H NMR (500 MHz, MeOD) δ 8.21 – 8.08 (m, 3H), 7.98 – 7.89 (m, 3H),}

7.88 – 7.80 (m, 6H), 7.37 (ddd, J = 8.5, 7.4, 6.0 Hz, 2H), 6.79 (d, J = 9.1 Hz, 2H), 6.66 (d, J = 7.7 Hz, 1H), 5.89 (ddd, J = 15.1, 9.8, 4.4 Hz, 1H), 5.65 (d, J = 17.3 Hz, 1H), 5.09 (s, 1H), 3.60 – 3.36 (m, 7H), 2.19 (d, J = 7.4 Hz, 2H), 2.13 – 2.02 (m, 1H), 1.97 – 1.82 (m, 4H), 1.71 (d, J = 13.3 Hz, 1H), 1.61 (d, J = 14.1 Hz, 2H), 1.47 (dd, J = 15.7, 6.3 Hz, 1H), 1.29 (s, 7H), 1.11 (s, 3H), 0.89 (q, J = 6.6 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 174.4, 167.8, 157.1, 155.2, 152.9, 143.7, 141.6, 134.1, 132.1, 131.7, 131.3, 128.6, 128.1, 125.7, 125.5, 122.3, 119.7, 111.6, 110.1, 72.5, 67.2, 54.2, 46.3, 44.5, 44.4, 42.5, 41.6, 40.8, 40.4, 36.6, 35.9, 31.6, 30.6, 30.5, 29.8, 25.7, 18.7, 18.0, 17.6, 12.2, 8.8. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5

min): Rt (min): 5.45 (ESI-MS (m/z): 770.07(M+H+)). HRMS: calculated for C40H48N7O7S 770.33304 [M+H]+;

found 770.33203.

NHS-TCO-DABCYL (24). Starting from DABCYL-NH2 (9.0 mg, 29

µmol) and DiNHS-TCO (12.5 mg, 29 µmol), the reaction was carried out following GP1 to afford DABCYL-TCO-NHS (20.9 mg, 0.028 mmol, 96%). 1_{H NMR (500 MHz, CDCl} 3) δ 10.34 (s, 1H), 8.01 (s, 1H), 7.95 – 7.86 (m, 2H), 7.85 (d, J = 8.6 Hz, 1H), 7.73 (dd, J = 8.9, 0.7 Hz, 2H), 7.48 (dd, J = 1.9, 0.8 Hz, 2H), 7.22 (dd, J = 8.9, 1.8 Hz, 2H), 6.76 (d, J = 9.1 Hz, 1H), 5.87 (dd, J = 17.0, 9.5 Hz, 1H), 5.65 – 5.54 (m, 1H), 5.18 (s, 1H), 3.75 – 3.60 (m, 3H), 3.63 (s, 1H), 3.54 – 3.46 (m, 1H), 3.21 – 3.08 (m, 2H), 3.11 (s, 4H), 3.04 (s, 1H), 2.96 (s, 2H), 2.88 (d, J = 0.7 Hz, 2H), 2.81 (d, J = 3.0 Hz, 2H), 2.27 (dd, J = 17.6, 10.4 Hz, 2H), 2.12 – 1.98 (m, 2H), 1.96 – 1.82 (m, 1H), 1.37 (dd, J = 8.4, 7.1 Hz, 12H), 1.33 – 1.22 (m, 4H). 13_{C NMR (126 MHz, CDCl3) δ 174.4, 167.8, 157.1, 155.2, 152.9, 143.7,} 141.6, 134.1, 132.1, 131.7, 131.3, 128.6, 128.1, 125.7, 125.5, 122.3, 119.7, 111.6, 110.1, 72.5, 67.2, 54.2, 46.3, 44.5, 44.4, 42.5, 41.6, 40.8, 40.4, 36.6, 35.9, 31.6, 30.6, 30.5, 29.8, 25.7, 18.7, 18.0, 17.6, 12.2, 8.8. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 7.08 (ESI-MS (m/z): 564.22

(M+H+_{)). HRMS: calculated for C}

30H42N7O4 564.32928 [M+H]+; found 564.32888.

EDANS (25). Starting from

DABCYL-TCO-NHS (3.24 mg, 5.2 µmol) and EDANS-NH2 (1.58 mg, 5.5

µmol), the reaction was carried out following GP1 to afford DABCYL-TCO-EDANS (3.55 mg, 4.6 µmol, 88%).

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C40H48N7O7S 770.33304 [M+H]+; found 770.33212.

Biotin-TCO-NHS (26). Starting from DiNHS-TCO (63.5 mg, 148 µmol) and

biotin-amine (42.3 mg, 148 µmol), the reaction was carried out following GP1 to afford Biotin-TCO-NHS (31.6 mg, 53 µmol, 36%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.01 (ESI-MS (m/z): 593.93 (M+H+)).

HRMS: calculated for C27H39N5O8S 593.25193 [M+H]+; found 593.25214.

Biotin-TCO-LU-Epoxomicin (14). Starting from Biotin-TCO-NHS

(3.50 mg, 5.4 µmol) and epoxomicin (2.41 mg, 3.6 µmol, synthesized in Chapter 5), the reaction was carried out following

GP1 and purified by prep-HPLC to afford Biotin-TCO-epoxomicin

(1.27 mg, 1.1 µmol, 30%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.23 (ESI-MS (m/z):

1157.40 (M+H+)). HRMS: calculated for C59H53N12O12S 1157.67556 [M+H]+; found 1157.67490.

Biotin-TCO-LU-001i (15). Starting from Biotin-TCO-NHS (2.20 mg, 3.7

µmol) and LU-001i-amine (2.36 mg, 3.3 µmol, synthesized in Chapter 5), the reaction was carried out following GP1 and purified by prep-HPLC to afford Biotin-TCO-LU001i (1.38 mg, 1.2 µmol, 35%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min):

6.99 (ESI-MS (m/z): 1187.73 (M+H+)). HRMS: calculated for C57H89F2N12O11S 1187.64625 [M+H]+; found 1187.64578.

TCO-LU-cyclophellitol-aziridine (16). Starting from

Biotin-TCO-NHS (3.24 mg, 5.2 µmol) and cyclophellitol-aziridine-amine (3.16 mg, 5.3 µmol), the reaction was carried out following GP1 and purified by prep-HPLC to afford Biotin-TCO-cyclophellitol-aziridine (0.92 mg, 1.0 µmol, 19%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 4.67 (ESI-MS (m/z): 918.40

(M+H+_{)). HRMS: calculated for C}

44H72N9O10S 918.51229 [M+H]+; found 918.51321.

Biotin-TCO-GB111 (17). Starting from Biotin-TCO-NHS (3.89 mg, 6.6

µmol) and GB111-amine (4.00 mg, 6.0 µmol), the reaction was carried out following GP1 and purified by prep-HPLC to afford Biotin-TCO-GB111 (2.43 mg, 2.3 µmol, 38%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 7.67 (ESI-MS (m/z):

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After 4 h of stirring at -30°C, H2N-Phe-EK (0.014 g, 0.03 mmol, 1.1 eq.) dissolved in anhydrous DMF (0.5

mL) was added to the reaction mixture, followed by DiPEA (0.1 mL, 0.6 mmol, 10 eq.). The reaction mixture was stirred for 16 h and slowly warmed to RT. TLC analysis was used to monitor completion of the reaction, before DMF was removed from the reaction mixture in vacuo. The residue was dissolved in EtOAc and washed with H2O (1x). The water layer was extracted with EtOAc (3x) and the combined

organic layers were washed with brine (1x), dried over NaSO4, filtered and concentrated. After

purification by silica gel column chromatography (DCM → 2% MeOH in DCM), the tert-butyl protected product intermediate was obtained, which was deprotected by adding TFA:DCM (1:1, 1 mL). After stirring for 2 h, the reaction mixture was co-evaporated with toluene (5x) and CHCl3 (1x) and

concentrated in vacuo. Purification by prep-HPLC (C18, 35-75% MeCN, 0.2% TFA, 10 min gradient) followed by evaporation on speed vacuum afforded N3-Ac-Ile2-Thr-Phe-EK (20) as a white solid (0.016 g,

0.026 mmol, 43%). 1_{H NMR (600 MHz, CDCl}

3) δ 7.69 (s, 1H), 7.26 (s, 4H), 7.25 – 7.20 (m, 1H), 7.18 – 7.13

(m, 2H), 4.81 (q, J = 7.7, 7.0 Hz, 1H), 4.64 (s, 2H), 4.46 (t, J = 8.5 Hz, 1H), 4.15 (s, 1H), 3.95 (d, J = 16.2 Hz, 1H), 3.33 (d, J = 5.0 Hz, 1H), 3.12 (dd, J = 14.0, 4.6 Hz, 1H), 2.93 (d, J = 4.9 Hz, 1H), 2.79 (dd, J = 13.9, 8.1 Hz, 1H), 2.01 (s, 6H), 1.51 (s, 3H), 1.25 (s, 3H), 1.09 (d, J = 6.4 Hz, 3H), 0.85 (td, J = 7.3, 5.1 Hz, 8H), 0.78 (d, J = 6.7 Hz, 3H). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.90 (ESI-MS

(m/z): 616.20 (M+H+)).

N3-Ac-Ile2-Thr-Cha-EK (21). N3-Ac-Ile2-Thr(tBu)-OMe (0.291 g, 0.58

mmol, synthesized in Chapter 5) was dissolved in 10 mL MeOH followed by addition of NH2NH2×H2O (1 mL, 32 mmol, 55 eq.). The

reaction mixture was stirred for 16 h at RT. After coevaporation with toluene (5x) and concentration in vacuo, N3-Ac-Ile2-Thr(tBu)-NHNH2

was obtained. In the next reaction, N3-Ac-Ile2-Thr(tBu)-NHNH2 (0.030 g, 0.06 mmol) was dissolved in

anhydrous DMF (0.5 mL) and cooled to -30°C, followed by the addition of tBuONO (16 µL, 0.13 mmol, 2.2 eq.) and 5 M HCl in EtOAc (67 µL, 0.34 mmol, 5.6 eq.). After 4 h of stirring at -30°C, H2N-Cha-EK (0.013 g,

0.03 mmol, 1.1 eq.) dissolved in anhydrous DMF (0.5 mL) was added to the reaction mixture, followed by DiPEA (0.1 mL, 0.6 mmol, 10 eq.). The reaction mixture was stirred for 16 h and slowly warmed to RT. TLC analysis was used to monitor completion of the reaction, before DMF was removed from the reaction mixture in vacuo. The residue was dissolved in EtOAc and washed with H2O (1x). The water layer was

extracted with EtOAc (3x) and the combined organic layers were washed with brine (1x), dried over NaSO4, filtered and concentrated. After purification by silica gel column chromatography (DCM → 2%

MeOH in DCM), the tert-butyl protected product intermediate was obtained, which was deprotected by adding TFA:DCM (1:1, 1 mL). After stirring for 2 h, the reaction mixture was co-evaporated with toluene (5x) and CHCl3 (1x) and concentrated in vacuo. Purification by prep-HPLC (C18, 35-75% MeCN, 0.2% TFA,

10 min gradient), followed by evaporation on speed vacuum afforded N3-Ac-Ile2-Thr-Cha-EK (21) as a

white solid (0.013 g, 0.020 mmol, 33%). 1_{H NMR (600 MHz, CDCl}

3) δ 7.97 (s, 1H), 7.51 (d, J = 25.7 Hz,

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3.28 (d, J = 5.0 Hz, 1H), 2.90 (d, J = 5.0 Hz, 1H), 1.77 (d, J = 12.9 Hz, 3H), 1.68 (d, J = 14.8 Hz, 1H), 1.62 (dt, J = 13.0, 4.3 Hz, 3H), 1.53 (s, 3H), 1.30 (dd, J = 10.1, 2.8 Hz, 1H), 1.25 (s, 1H), 1.18 – 1.12 (m, 1H), 1.13 – 1.08 (m, 3H), 0.85 (ddt, J = 11.0, 6.3, 4.0 Hz, 12H). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1%

TFA, 12.5 min): Rt (min): 7.58 (ESI-MS (m/z): 622.33 (M+H+)).

N3-Ac-Ile2-Thr-HomoPhe-EK (22). N3-Ac-Ile2-Thr(tBu)-OMe (0.291 g,

0.58 mmol, synthesized in Chapter 5) was dissolved in 10 mL MeOH followed by addition of NH2NH2×H2O (1 mL, 32 mmol, 55 eq.). The

reaction mixture was stirred for 16 h at RT. After coevaporation with toluene (5x) and concentration in vacuo, N3-Ac-Ile2-Thr(tBu)-NHNH2

was obtained. In the next reaction, N3-Ac-Ile2-Thr(tBu)-NHNH2 (0.030

g, 0.06 mmol) was dissolved in anhydrous DMF (0.5 mL) and cooled to -30°C, followed by the addition of tBuONO (16 µL, 0.13 mmol, 2.2 eq.) and 5 M HCl in EtOAc (67 µL, 0.34 mmol, 5.6 eq.). After 4 h of stirring at -30°C, H2N-HomoPhe-EK (18c, 0.011 g, 0.03 mmol, 1.1 eq.) dissolved in anhydrous DMF (0.5 mL) was

added to the reaction mixture, followed by DiPEA (0.1 mL, 0.6 mmol, 10 eq.). The reaction mixture was stirred for 16 h and slowly warmed to RT. TLC analysis was used to monitor completion of the reaction, before DMF was removed from the reaction mixture in vacuo. The residue was dissolved in EtOAc and washed with H2O (1x). The water layer was extracted with EtOAc (3x) and the combined organic layers

were washed with brine (1x), dried over NaSO4, filtered and concentrated. After purification by silica gel

column chromatography (DCM → 2% MeOH in DCM), the tert-butyl protected product intermediate was obtained, which was deprotected by adding TFA:DCM (1:1, 1 mL). After stirring for 2 h, the reaction mixture was co-evaporated with toluene (5x) and CHCl3 (1x) and concentrated in vacuo. Purification by

prep-HPLC (C18, 46-52% MeCN, 0.2% TFA, 12 min gradient) followed by evaporation on speed vacuum afforded N3-Ac-Ile2-Thr-HomoPhe-EK (20d) as a light yellow solid (0.009 g, 0.014 mmol, 24%). 1H NMR (600 MHz, CDCl3) δ 7.28 (s, 3H), 7.21 (d, J = 7.1 Hz, 1H), 7.15 (d, J = 7.2 Hz, 2H), 4.54 (s, 1H), 4.25 (s, 3H),

3.71 (s, 3H), 3.21 (s, 3H), 2.89 (s, 1H), 2.69 (s, 1H), 2.61 (s, 1H), 1.68 (s, 1H), 1.49 (s, 4H), 1.44 (s, 1H), 1.39 (s, 4H), 1.25 (s, 18H), 1.12 (s, 4H), 0.91 – 0.79 (m, 10H). LC-MS (linear gradient 10 → 90% MeCN/H2O,

0.1% TFA, 12.5 min): Rt (min): 7.15 (ESI-MS (m/z): 639.27 (M+H+)).

N3-Ac-Ile2-Thr-HomoCha-EK (22). N3-Ac-Ile2-Thr(tBu)-OMe (0.291

g, 0.58 mmol, synthesized in Chapter 5) was dissolved in 10 mL MeOH followed by addition of NH2NH2×H2O (1 mL, 32 mmol, 55

eq.). The reaction mixture was stirred for 16 h at RT. After coevaporation with toluene (5x) and concentration in vacuo, N3

-Ac-Ile2-Thr(tBu)-NHNH2 was obtained. In the next reaction, N3

-Ac-Ile2-Thr(tBu)-NHNH2 (0.030 g, 0.06 mmol) was dissolved in anhydrous DMF (0.5 mL) and cooled to

-30°C, followed by the addition of t-BuONO (16 µL, 0.13 mmol, 2.2 eq.) and 5 M HCl in EtOAc (67 µL, 0.34 mmol, 5.6 eq.). After 4 h of stirring at -30°C, H2N-HomoCha-EK (0.011 g, 0.03 mmol, 1.1 eq.) dissolved in

anhydrous DMF (0.5 mL) was added to the reaction mixture, followed by DiPEA (0.1 mL, 0.6 mmol, 10 eq.). The reaction mixture was stirred for 16 h and slowly warmed to RT. TLC analysis was used to monitor completion of the reaction, before DMF was removed from the reaction mixture in vacuo. The residue was dissolved in EtOAc and washed with H2O (1x). The water layer was extracted with EtOAc (3x)

and the combined organic layers were washed with brine (1x), dried over NaSO4, filtered and

concentrated. After purification by silica gel column chromatography (DCM → 2% MeOH in DCM), the tert-butyl protected product intermediate was obtained, which was deprotected by adding TFA:DCM (1:1, 1 mL). After stirring for 2 h, the reaction mixture was co-evaporated with toluene (5x) and CHCl3

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Raji cell lysate was diluted to 1.4 mg/mL total protein concentration in a buffer containing 50 mM Tris pH 7.5, 1 mM DTT, 5 mM MgCl2, 10% (v/v) glycerol and 2 mM ATP. An inhibitor dilution series (0.3 µM, 1

µM, 3 µM, 10 µM, 30 µM, 100 µM, 300 µM, 1000 µM) was prepared in DMSO. 10 µl of 1.4 mg/mL cell lysate solution was exposed to 1 µl inhibitor DMSO solution in the dilution series and incubated at 37°C for 1 h. Afterwards, 1 µl 10x ABPs cocktail (Cy2: BODIPY(FL)-LU-112, Cy3: BODIPY(TMR)-NC-005VS, Cy5: Cy5-NC-001) was added to each incubated vial, and the vials were incubated for an additional 1 h at 37°C, followed by 5 min boiling with 3.34 µL 4x gel-loading buffer. Electrophoresis was performed on 12.5% SDS-PAGE gel for 15 min at 80 V and then 2 h at 130 V. On each gel, 2.5 µl of page ruler was used. Right after the electrophoresis (before the protein diffuse), multiplex fluorescent detection of residual ABPs was performed on a ChemiDoc™ MP System with Cy5, Cy3 and Cy2 channels.

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