university of groningen structure-based design of ligands ... · and residues in loops l1 and l3,...

University of Groningen

Structure-based design of ligands for vitamin transporters in bacteriaMonjas Gómez, Leticia

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Citation for published version (APA):Monjas Gómez, L. (2016). Structure-based design of ligands for vitamin transporters in bacteria.[Groningen]: University of Groningen.

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Chapter 2

Structure-based design of thiamine analogues as binders of ThiT:

modification of the thiazolium ring In this chapter, we describe the binding of thiamine derivatives to ThiT, a thiamine-

specific S-component of an energy-coupling factor (ECF) transporter. We designed the

new thiamine derivatives by modification of the thiazolium ring and the hydroxyethyl

side chain of thiamine. We synthesized and cocrystallized the compounds in complex with

ThiT. The structure of the substrate-binding site in ThiT remains almost unchanged

despite substantial differences in affinity. This work indicates that the structural

organization of the binding site is robust and suggests that substrate release, which is

required for transport, requires additional changes in conformation in ThiT that might

be imposed by the ECF module.

This chapter is adapted from the original publication:

Swier, L. J. Y. M.;* Monjas, L;* Guskov, A.; de Voogd, A. R.; Erkens, G. B.; Slotboom, D. J.; Hirsch, A. K. H. ChemBioChem 2015, 16, 819. (*Equal contribution)

Chapter 2

32

2.1 Introduction

The mechanism of binding and transport of energy-coupling factor (ECF) transporters is not fully elucidated. To investigate this mechanism, we focused on substrate binding to the S-component ThiT from Lactococcus lactis. ThiT is a 21 kDa protein that binds its natural substrate thiamine (vitamin B1) with subnanomolar affinity.1 Thiamine is a precursor of thiamine diphosphate (ThDP), which serves as a cofactor in many enzymes that perform decarboxylations and are involved in essential cellular processes.2 ThDP and its precursor thiamine monophosphate (ThMP) can bind to ThiT as well, but with lower affinities than thiamine.1 The crystal structure of ThiT in complex with thiamine (PDB ID: 3RLB)3 has been solved previously at a resolution of 2.0 Å. This crystal structure shows that the protein consists of six transmembrane helices (TMHs, Figure 1A). The substrate-binding pocket is located on the extracellular side of the membrane and is formed by residues in helices TMH4–6 and residues in loops L1 and L3, which are located between TMH1 and TMH2 and between TMH3 and TMH4, respectively. The flexible loop L1 shields the substrate-binding pocket from the surrounding solvent in a lid-like manner, with Trp34 playing a major role in substrate occlusion (Figure 1B).4 The substrate-binding site is connected to the exterior of the protein through an opening of a diameter of 7–9 Å. Thiamine interacts with several residues in the binding pocket (Figure 1C and 1D): the aminopyrimidine moiety acts as an anchor involved in hydrogen bonds with residues Glu84, His125, Tyr146 (via an ordered molecule of water) and Asn151, as well as a π–π-stacking interaction with Trp133. The thiazolium ring is forming π–π-stacking and cation–π interactions with Trp34 and His125, and the positively charged nitrogen atom is stabilized by an electrostatic interaction with Glu84. The hydroxyl group forms a hydrogen bond with Tyr85. In this chapter, we rationally designed derivatives of thiamine such that the various moieties of the original thiamine molecule were altered one by one, in order to explore the space within the substrate-binding pocket. The aim of this study was to trap the protein in previously undetected conformational states, in order to provide new insights into the mechanism of binding and transport. Furthermore, eukaryotic cells do not make use of ECF transporters, but instead use unrelated secondary transport systems.6 Given that the ECF transporter for thiamine is essential for the uptake of this vitamin in some pathogenic bacteria, this study is a first step on the way to new antibiotics capable of blocking thiamine transport and thereby interfering with essential cellular processes within these bacteria.

Thiamine analogues as binders of ThiT: modification of the thiazolium ring

33

Figure 1. Structure of thiamine-bound ThiT of Lactococcus lactis (chain A of structure PDB ID: 3RLB).3 A) Ribbon representation of ThiT, colored from the N terminus in blue to the C terminus in red. Transmembrane helices 1–6 are indicated by TMH1–6. Thiamine is shown in stick representation and colored using the following color code: C: green, O: red, N: blue, S: yellow. This color code for the oxygen, nitrogen and sulfur atoms is maintained throughout this thesis. B) Close-up of the substrate-binding pocket of ThiT with surface and ribbon representation. The surface of the loop L1 between TMH1 and TMH2 is colored blue, whereas the remainder of the surface is shown in gray. Residue Trp34 is shown in dark blue and stick representation, and thiamine is represented as in A. C) Residues involved in binding of thiamine shown in stick representation with their carbon atoms colored gray, whereas the other atoms and thiamine are colored as in A. The names of the residues are given using the three-letter codes for amino acids. The dashed lines indicate electrostatic interactions and hydrogen bonds, and the red sphere represents a molecule of water. D) Schematic representation of the binding mode of thiamine in the substrate-binding pocket of ThiT, generated with PoseView5 as implemented in the LeadIT suite.

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34

2.2 Results and discussion

2.2.1 Design of small-molecule binders

We designed potential binders of ThiT using the crystal structure of ThiT in complex with thiamine (1, PDB ID: 3RLB)3, in order to determine which functional groups are crucial for high-affinity binding (Figure 2). MOLOC software7 was used for molecular modeling, and the preferred orientation of the molecules in the binding pocket and the estimated free energies of binding were obtained with the FlexX docking module8 and with the scoring function HYDE9,10 in the LeadIT suite, respectively.

Figure 2. Small-molecule binders for ThiT. A) Structure of thiamine (1) with the names of the rings and the hydroxyethyl group indicated. B) Structures of pyrithiamine (2), thiamine monophosphate (3), thiamine diphosphate (4), and the designed and synthesized small-molecule binders (5–9). It has been previously reported that when the thiazolium ring of thiamine (1) is substituted by a pyridinium ring, as in 2, the affinity of the new molecule for ThiT is essentially the same (Table 1).1 When the hydroxyl group from the hydroxyethyl side chain is mono- or diphosphorylated (3 and 4), the binding affinity decreases by one order of magnitude (Table 1).1 First, we performed docking studies for compounds 1–4, for which the KD values had been determined previously, to benchmark how well the docking program LeadIT performs on our target protein and class of compounds. According to the estimated Gibbs free energies of binding (ΔGest), we could predict that compounds 1 and 2 should have higher binding affinity for ThiT than


35

compounds 3 and 4, and this is in agreement with the experimental data obtained from intrinsic-protein-fluorescence titration assays (Table 1). Table 1. Binding affinities of ThiT for compounds 1–4 as determined in the ligand-binding assay, with the errors indicated as standard deviations,1 together with the experimental (ΔGexp) and estimated Gibbs free energies of binding (ΔGest). The estimated values are based on the scoring function HYDE.

Compound KD ± S.D. (nM) ΔGexp (kJ mol–1) ΔGest (kJ mol–1) 1 0.122 ± 0.013 –57 –53 2 0.180 ± 0.070 –56 –54 3 1.01 ± 0.14 –51 –50 4 1.60 ± 0.00 –50 –52

In our design (compounds 5–9, Figure 2), we maintained the aminopyrimidine moiety of thiamine as an anchor in order to preserve the interactions between this part of the molecule and ThiT. We explored different modifications in the rest of the molecule: the thiazolium ring and the hydroxyethyl side chain. We substituted the thiazolium ring for a range of aromatic moieties that were chosen based on modeling, such as a thiophenyl (5) or a phenyl ring (6), to evaluate the effect on the π–π-stacking interaction of this moiety with residues Trp34 and His125. In order to analyze the importance of the hydroxyethyl side chain of thiamine (1), we designed derivatives of 5 and 6 featuring a hydroxymethyl side chain (7 and 8) or an aldehyde group (9) to evaluate the importance of the hydrogen bond with Tyr85. The predicted Gibbs free energies of binding indicate that the designed compounds should have a high binding affinity for ThiT (Table 2). Table 2. Estimated Gibbs free energies of binding (ΔGest) for the designed thiamine derivatives 5–9, based on the scoring function HYDE.

Compound ΔGest (kJ mol–1) 5 –52 6 –48 7 –46 8 –43 9 –40

The predicted binding poses of the new derivatives (Figure 3) are almost identical to that of thiamine (Figure 1), with differences mainly in the hydrogen bonds formed by the new groups introduced in place of the hydroxyethyl substituent of thiamine.

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36

Figure 3. Schematic representation of the top-ranked predicted binding modes of the small-molecule binders 2 and 5–9 in the substrate-binding pocket of ThiT, using the PDB ID: 3RLB and the program LeadIT. The figures were generated with PoseView5 as implemented in the LeadIT suite.


37

2.2.2 Synthesis of small-molecule binders

We synthesized deazathiamine (5) by optimizing a previously reported synthetic route (Scheme 1).11

Scheme 1. Synthesis of compounds 5, 7 and 9. Starting from 2,3,5-tribromo-4-methylthiophene (10), after treatment with sBuLi and quenching with methanol, we obtained intermediate 11 in 93% yield. In the second step, we introduced the hydroxyethyl side chain by treatment with sBuLi followed by reaction of the resulting anion with ethylene oxide and boron trifluoride diethyl etherate, affording the desired product 12 in 68% yield. In the third step, treatment with iPrMgCl (for the in situ protection of the hydroxyl group)12 followed by tBuLi, afforded an anion, which was treated with DMF to yield aldehyde 13 in 65% yield. Condensation of this aldehyde with 3-anilinopropionitrile under basic conditions afforded 14 in 71% yield. The aminopyrimidinyl ring was formed by cyclization of enamine 14 with acetamidine under basic conditions, affording the desired product 5 in 68% yield. We obtained compound 5 in 20% overall yield, an improvement on the 11% overall yield reported previously.11

Chapter 2

38

We synthesized compound 7 by the same route. In this case, in the second step, we treated 11 with sBuLi, and the resulting anion reacted with DMF, affording an aldehyde that was reduced to the corresponding alcohol 15 by using NaBH4 in 79% yield (over two steps). The following three steps were analogous to the ones for the synthesis of compound 5, affording compound 7 in 20% overall yield. We obtained compound 9 by oxidation of 7 with MnO2 in 70% yield. We synthesized compounds 6 and 8 by an analogous route (Scheme 2), and by modification of a previously reported synthesis of 6.13 Commercially available bromides 18 and 19 were converted into the corresponding aldehydes by treatment with iPrMgCl, tBuLi and DMF, in 70% and 74% yield for 20 and 21, respectively. These aldehydes were each condensed with 3-anilinopropionitrile under basic conditions to afford 22 and 23 in 24% and 45% yield, respectively. The final step, the cyclization of enamines 22 and 23 with acetamidine, afforded the desired products 6 and 8 in 61% and 66% yield, respectively. For the synthesis of these two compounds, the yields were not optimized.

Scheme 2. Synthesis of compounds 6 and 8.

2.2.3 Binding-affinity determination

We determined the KD values of ThiT for compounds 5–9 by monitoring the intrinsic protein fluorescence upon addition of ligand (Table 3). Replacement of the positively charged nitrogen atom in the thiazolium ring of thiamine (1) by a carbon atom in 5 resulted in an increase in KD to 4.23 nM. Replacement of the thiazolium ring of 1 by a phenyl ring in compound 6 led to a decrease in binding affinity, resulting in a KD of 266 nM. Variations of the hydroxyethyl side chain of 5 did not have a significant effect, as shown by the KD values of 5.21 and 7.44 nM for compounds 7 and 9, respectively. Shortening the hydroxyethyl side chain in


39

compound 6 by one carbon atom in compound 8 led to an increase in KD up to 528 nM. Table 3. Binding affinities of ThiT for compounds 5–9 as determined in the ligand-binding assay, with the errors indicated as standard deviations, together with the experimental (ΔGexp) and the estimated Gibbs free energies of binding (ΔGest). The estimated values are based on the scoring function HYDE, and were already reported on Table 2.

Compound KD ± S.D. (nM) ΔGexp (kJ mol–1) ΔGest (kJ mol–1) 5 4.23 ± 1.69c –48 –52 6 266 ± 131b –38 –48 7 5.21 ± 3.19a –47 –46 8 528 ± 135a –36 –43 9 7.44 ± 1.67a –46 –40

a–c The error represents the standard deviation obtained from a2, b3 or c4 experiments. Based on the KD values (Tables 1 and 3), we observe that the presence of the positively charged nitrogen atom in the central ring contributes to the picomolar binding affinity. Compounds 1 and 2 display comparable KD values of 122 pM and 180 pM, respectively. Measuring KD values in the low-nanomolar or picomolar range with high accuracy is difficult and with a ThiT concentration of 50 nM in the intrinsic-protein-fluorescence titration assay, these values cannot be considered significantly different. Replacement of the thiazolium ring by a thiophenyl ring (compounds 5, 7 and 9), and thereby removing the positive charge, lowered the binding affinity about 30-fold. Nonetheless, derivatives 5, 7 and 9 still bind with high affinity to ThiT (KD values between 4.2 and 7.4 nM) showing that these analogues are suitable thiamine mimics. Removal of the positively charged nitrogen atom and the methyl group in compounds 6 and 8, relative to 2, led to decreases in binding affinity, resulting in KD values in the upper nanomolar range. These results indicate that the presence of the charged nitrogen atom in the context of a six-membered ring provides a bigger contribution to interactions involved in binding than in the five-membered ring. On comparing compounds 5 and 7, each featuring a thiophenyl ring, with compounds 6 and 8, each with a phenyl ring, the binding affinity is about 60 to 100 times lower in the case of the phenyl ring. The difference in affinity could be explained by the sulfur atom in compounds 5 and 7, which can make S–π interactions with Trp34 and His125, and by the methyl group at C-4, which can also contribute through CH–π interactions with Tyr85. Removing the methyl group in compounds 6 and 8 leads to a loss of van der Waals interactions. Furthermore, different alignments of the dipole moments of the aromatic moieties could also account for the observed difference in affinity.

Chapter 2

40

Having investigated the importance of the central ring, we next examined the importance of the hydrogen-bonding interaction with Tyr85. To do so, we modified the hydroxyethyl group in compounds 7–9. In compounds 7 and 9, analogues to 5 but with a hydroxymethyl and an aldehyde group, respectively, the binding affinity does not change significantly relative to 5. In the case of compound 8, shortening the hydroxyethyl side chain by one carbon atom in comparison with 6, increases the KD value roughly 2-fold.

2.2.4 Cocrystallization of ThiT with small-molecule binders

To visualize how the thiamine derivatives bind to ThiT, we performed cocrystallization of substrate-free ThiT purified in the presence of compounds 2 and 5–9. All structures were solved to 2.0–2.5 Å resolution. A comparison of the overall crystal structures of ThiT complexed to the synthesized compounds with the structure of ThiT in complex with thiamine, revealed very subtle differences only in the spatial orientations of the α-helices. When zooming into the residues of ThiT involved in substrate binding, we noticed significant changes in the orientation of the side chains only for Trp34 in the complexes with compounds 6 and 8. Figure 4 shows the binding-site residues of ThiT cocrystallized with compounds 2 and 5–9.


41

Figure 4. Crystal structures of ThiT with small-molecule binders. The residues involved in binding are shown with use of the same format and color code as in Figure 1C. A–F) The carbon atoms of compounds 2, 5–9 are shown in magenta (PDB ID: 4MUU), cyan (4MHW), violet purple (4MES), lime (4POV), orange (4N4D) and deep salmon (4POP), respectively. This figure was created by using chain A of the corresponding PDB file. The aminopyrimidine moiety presents the same interactions in all the crystal structures: hydrogen bonds with residues Glu84, His125, Tyr146 (via a molecule of water) and Asn151, and a π–π-stacking interaction with Trp133. The π–π-stacking interactions of the thiazolium ring of thiamine (1), sandwiched between Trp34 and His125, are conserved in most of the crystal structures, as we predicted (Figure 3). In the cocrystallized structures with

Chapter 2

42

compounds 6 and 8, Trp34 has moved, and in the case of compound 6, it has moved in the direction of the pyrimidinyl ring, making a CH–π interaction with this ring (Figure 5). Despite the movement of the side chain of Trp34 and the broadening of the entrance to the binding pocket in this case, the backbone of the loop L1 maintains its position.

Figure 5. Superimposition of the crystal structures of ThiT in complex with thiamine (PDB ID: 3RLB), with compound 6 (4MES) and with compound 8 (4N4D). For ThiT-thiamine, same format as in Figure 1C (ThiT, C: gray; thiamine, C: green). For ThiT in complex with compound 6, both, residues and binder in purple. For ThiT in complex with compound 8, both, residues and binder in orange. The electrostatic interaction of Glu84 with the positively charged nitrogen atom in the thiazolium ring of thiamine (1) is only conserved in the complex with compound 2, given that it is the only derivative that maintains this nitrogen atom. In addition, the charged nitrogen atom also enables cation–π interactions with Trp34 and His125, and in its absence, only π–π-stacking interactions can be formed. As a result, the interactions of the ligand with Trp34 and His125 are weaker, which can explain the lower binding affinities of compounds 5–9. In addition, for compounds 6 and 8, the side chain of Trp34 has shifted compared to the structure of the complex with thiamine, making the π–π-stacking interactions with the phenyl ring in these compounds slightly different. Both modifications decreased the binding affinity, resulting in KD values of 266 and 528 nM for compounds 6 and 8, respectively. The hydroxyethyl group of compound 5 forms water-mediated hydrogen bonds with Asn29 and Tyr85. The same group of compound 6 forms hydrogen bonds through a single molecule of water with His72 and Tyr85. The hydroxymethyl group of compound 7 forms a hydrogen bond with Tyr85, and through a molecule of water with Glu84 and Lys121. In the case of compound 8, the phenyl ring has flipped over, relative to its position for compound 6, so as to allow the hydroxymethyl group to form a direct hydrogen bond with the backbone carbonyl group of Asn151. The aldehyde group of compound 9 forms water-mediated hydrogen bonds with Glu84, Tyr85 and Lys121. The hydrogen


43

bonds of the aldehyde group of compound 9 appears to contribute to the binding affinity almost as well as those of the hydroxyl group in compounds 5 and 7, therefore there is no real preference for a hydrogen-bond donor or acceptor on this side of the substrate, given that most interactions between the ligands and the residues of the binding pocket are water-mediated. Overall, the interaction between Tyr85 and thiamine (1) is not particularly important and does not make a major contribution to the high-affinity binding. The binding modes observed in the cocrystal structures (Figure 4) match the poses predicted by modeling and docking (Figure 3). An exception are the hydrogen bonds of the hydroxyl or aldehyde group, because we did not take into account the molecules of water in this part of the pocket during modeling, so in all the predicted binding poses the compounds are forming direct hydrogen bonds to the residues of ThiT. Besides the ordered molecules of water through which the ligands interact with the residues of the binding pocket, there are a few other molecules of water involved in interactions among residues lining the binding site. The backbone of Glu84 interacts with the side chain of Lys121 through an ordered molecule of water in all structures, which likely assures correct alignment of His125 with the ligand. In addition, the backbone of Trp34 makes interactions with the side chains of Tyr146 and Asn151 through ordered molecules of water, which could help the backbone of loop L1 to maintain its position. The only exception is the crystal structure with compound 8, in which the hydroxyl group is occupying virtually the position of the molecule of water that connects Trp34 and Asn151, and in this case, the hydroxyl group is forming a hydrogen bond with Asn151.

2.3 Conclusions

At the outset of this study, we hypothesized that the designed thiamine derivatives might trap ThiT in previously undetected conformational states, which might allow us to follow changes in conformation that take place for substrate binding and release. Remarkably, in spite of differences in affinity of over three orders of magnitude (compounds 1 and 8) the crystal structures of ThiT with the bound thiamine derivatives were almost identical. The only difference in the binding-site residues that we observed is a rotation of the side chain of Trp34 located in the loop L1 when compounds 6 and 8 are bound. The different orientation of Trp34 opens the entrance to the substrate-binding pocket slightly, but the backbone of loop L1 maintains its position, leaving the substrate-binding pocket intact. Although this observation is consistent with the studies on ThiT that showed that the main structural difference between apo ThiT and the thiamine-bound protein is the conformation of loop L1,4 it does not provide the anticipated mechanistic insight into the mechanism of binding. Apparently, the

Chapter 2

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structure of the binding site in the S-component is so robust that it stays intact, even if lower-affinity substrates bind. This robustness has direct consequences for the transport mechanism employed by ECF transporters. We hypothesize that the S-component topples in the membrane only if loop L1 is closed. After interaction with the ECF module, the binding site is exposed to the cytoplasmic side of the membrane, where the substrate can be released. Possibly ATP hydrolysis is needed for opening of the binding site and release of the substrate. Indeed, in the context of the entire complex (S-component and ECF module), the substrate-binding site of the S-component FolT (for folate) is disrupted and the loop L1 is displaced outward.14 We speculate that a substantial input of free energy is required to accomplish the changes in conformation in the binding site of the S-component, which are necessary for transport and release of the substrate. This energy is probably provided by the association with the ECF module and by binding and hydrolysis of ATP by the nucleotide-binding domains (NBDs). The observation that the thiamine derivatives bind to ThiT with high affinities is very promising for another reason. It suggests that the versatile scaffold of the derivatives that lack the thiazolium ring (compounds 5, 7 and 9) could be further modified, which is an important step in the design of new ligands. They can assist not only in the elucidation of the transport mechanism but also in the development of new antibiotics targeted against pathogenic bacteria that depend on this type of transporters.

2.4 Experimental section

2.4.1 Modeling and docking

The crystal structure of ThiT in complex with thiamine (PDB ID: 3RLB) was used.3 Thiamine derivatives were designed by using the program MOLOC,7 and the energy of the system was minimized by use of the MAB force field implemented in this software, while the protein coordinates and the crystallographically localized water molecule (HOH196) were kept fixed. Hydrogen bonds and hydrophobic interactions were measured in MOLOC. The designed thiamine derivatives were subsequently docked into the binding pocket of ThiT with the aid of the FlexX docking module in the LeadIT suite.8 During docking, the binding site in the protein was restricted to 8.0 Å around the cocrystallized thiamine, the 30 top-scored solutions were retained and subsequently post-scored with the scoring function HYDE.9,10 After careful visualization to exclude poses with significant inter- or intramolecular clash terms or unfavorable conformations, the resulting solutions were subsequently ranked according to their binding energies.


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2.4.2 Synthesis

General methods. All reagents were purchased from Sigma-Aldrich, Acros Organics, TCI Europe or Fluorochem, and were used without further purification unless noted otherwise. All solvents were reagent-grade, and if necessary, dried and distilled prior to use. All reactions were carried out under a nitrogen atmosphere (if not otherwise indicated), with use of dried glassware. Reactions were monitored either by GC-MS (GCMS-QP2010 Shimadzu) with a HP-5 column (Agilent Technologies) or by thin-layer chromatography (TLC) on silica-gel-coated aluminum foils (silica gel 60/Kieselguhr F254, Merck). Flash-column chromatography was performed on silica gel (SiliCycle 40–63 μm). Melting points were determined with a Büchi B-545 apparatus. NMR spectra were recorded on a Varian AMX400 spectrometer at 25 °C. Chemical shifts (δ) are reported in ppm relative to the residual solvent peak. Splitting patterns are indicated as (s) singlet, (d) doublet, (t) triplet, (q) quartet, (sept) septet, (m) multiplet and (br) broad. Coupling constants (J) are reported in Hertz (Hz). FT-IR spectra (neat) were recorded on a Perkin Elmer FT-IR spectrometer. High-resolution mass spectra (HRMS) were recorded on a Thermo Scientific LTQ Orbitrap-XL mass spectrometer. The purity of the compounds (≥95%) was determined on a Shimadzu HPLC-PDA (column: Agilent Eclipse XDB-C8, 5 μm, 4.6 x 150 mm; λ = 254 nm, method for compound 5: H2O/CH3CN 90:10 (0.1% TFA), 30 min, flow rate of 0.35 mL min–1; method for compounds 6–9: H2O/CH3CN 95:5 to 5:95 (0.1% TFA), 23 min, flow rate of 0.5 mL min–1). Pyrithiamine (compound 2) is commercially available. General procedure for the synthesis of aldehydes 13, 16, 20 and 21 (GP-A). To a solution of the corresponding bromide (1.0 eq) in anhydrous THF at 0 °C, iPrMgCl (2.0 M in THF, 1.1–1.2 eq) was added dropwise. The reaction mixture was stirred at 0 °C for 10 min, then cooled to –78 °C and tBuLi (1.7 M in pentane, 2.2 eq) was added dropwise. The reaction mixture was left to warm up from –78 to –40 °C for 1 h, then cooled down to –78 °C, anhydrous DMF (12–18 eq) was added and it was stirred from –78 °C to room temperature overnight (conversion by GC-MS with the following method: T0 = 100 °C, hold 5 min; ramp 6 °C min–1 to 250 °C, hold 20 min). The reaction was quenched with a saturated aqueous solution of NH4Cl, extracted 3 times with Et2O (for compounds 13 and 16) or EtOAc (for compounds 20 and 21), and the combined organic layers were washed once with water, once with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated under reduced pressure. The crude was purified by flash column chromatography.

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General procedure for the synthesis of enamines 14, 17, 22 and 23 (GP-B). To a solution of the corresponding aldehyde (1.0 eq) and 3-anilinopropionitrile (1.1–1.7 eq) in anhydrous DMSO, NaOEt in anhydrous EtOH (2.0 M, 1.2–1.8 eq) was added.a The reaction mixture was stirred at 40 °C for 16 h (for compounds 22 and 23) or in the microwave at 70 °C and 100 W for 30–90 min (for compounds 14 and 17).b The reaction mixture was diluted with water, extracted 3 times with EtOAc, and the combined organic layers were washed once with water, once with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated under reduced pressure. The crude was purified by flash column chromatography or by recrystallization. General procedure for the formation of the aminopyrimidinyl ring in

compounds 5–8 (GP-C). To a solution of the corresponding enamine (1.0 eq) in anhydrous EtOH, acetamidine·HCl (hygroscopic: before use co-evaporate with toluene 3 times, 2.0–3.0 eq) and NaOEt in anhydrous EtOH (2.0 M, 4.0–6.0 eq)a were added. The reaction mixture was stirred at reflux (90 °C, pre-heated oil bath) for 1–3 days. Then, the reaction mixture was cooled down, and the solvent evaporated under reduced pressure. The crude was purified by flash column chromatography. 2,4-Dibromo-3-methylthiophene (11):

To a solution of 2,3,5-tribromo-4-methylthiophene (10) (15.0 g, 44.8 mmol) in anhydrous Et2O (200 mL) at –78 °C, sBuLi (1.4 M in cyclohexane, 32.0 mL, 44.8 mmol) was added dropwise over 20 min,

resulting in a dark blue solution. The reaction mixture was stirred at –78 °C for 5 min, and then quenched with MeOH, resulting in a yellow solution. The mixture was allowed to warm to room temperature, and then the solvent was evaporated under reduced pressure. The crude was purified by distillation at 15 mbar and 120 °C with a Kugelrohr apparatus, to afford 11 as a colorless oil (10.9 g, 42.7 mmol, 93%) 1H-NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 2.20 (s, 3H). The spectroscopic data are consistent with those previously reported.11 2-(4-Bromo-3-methylthiophen-2-yl)ethanol (12):

To a solution of 11 (9.89 g, 38.6 mmol) in anhydrous THF (120 mL) at –78 °C, sBuLi (1.4 M in cyclohexane, 27.6 mL, 38.6 mmol) was added dropwise, resulting in a yellow

solution. The reaction mixture was stirred at –78 °C for 1 h, and then ethylene oxide (2.5–3.3 M in THF, 17.0 mL, ca. 42.5 mmol) was added. After 10 min, boron

a The reaction can also be done using NaOMe (2.0 M in MeOH). b The 4 compounds (enamines 14, 17, 22 and 23) can be synthesized either at 40 °C for ~16 h, or using a microwave (100 W) at higher temperature (70 °C) and lower reaction time (30–90 min).


47

trifluoride diethyl etherate (4.76 mL, 38.6 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 2 h (until full conversion by GC-MS was observed: T0 = 100 °C, hold 5 min; ramp 6 °C min–1 to 250 °C, hold 20 min. tR(prod, m/z = 222) = 15.0 min). The reaction was quenched with MeOH and a saturated aqueous solution of NaHCO3, and extracted 3 times with Et2O. The combined organic layers were washed once with water, once with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated under reduced pressure. The crude was purified by flash column chromatography (pentane/Et2O 7:3), to afford 12 as a pale yellow oil (5.82 g, 26.3 mmol, 68%). 1H-NMR (400 MHz, CDCl3) δ 7.11 (s, 1H), 3.83 (t, J = 6.4, 2H), 3.03 (t, J = 6.4, 2H), 2.17 (s, 3H). The spectroscopic data are consistent with those previously reported.11 5-(2-Hydroxyethyl)-4-methylthiophene-3-carbaldehyde (13):

This compound was synthesized according to GP-A, using 12 (2.91 g, 13.1 mmol), anhydrous THF (80 mL), iPrMgCl (2.0 M in THF, 7.58 mL, 15.2 mmol), tBuLi (1.7 M in pentane, 17.8 mL, 30.3 mmol) and anhydrous DMF (12.7 mL, 165 mmol). GC-

MS: tR(prod, m/z = 170) = 16.0 min. The crude was purified by flash column chromatography (pentane/Et2O 55:45), to afford 13 as a colorless oil (1.46 g, 8.55 mmol, 65%). 1H-NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 7.93 (s, 1H), 3.86 (t, J = 6.4, 2H), 3.03 (t, J = 6.4, 2H), 2.41 (s, 3H). The spectroscopic data are consistent with those previously reported.11

2-((5-(2-Hydroxyethyl)-4-methylthiophen-3-yl)methyl)-3-(phenylamino)-

acrylonitrile (14): This compound was synthesized according to GP-B, using 13 (1.46 g, 8.55 mmol), 3-anilinopropionitrile (1.50 g, 10.3 mmol), anhydrous DMSO (10 mL) and NaOEt (2.0 M in EtOH, 5.13 mL, 10.3 mmol). Reaction

in the microwave for 30 min. The crude was purified by flash column chromatography (CH2Cl2/MeOH 99:1), to afford 14 as a pale yellow solid (1.81 g, 6.06 mmol, 71%). 1H-NMR (400 MHz, CDCl3) δ 7.34 (d, J = 13, 1H), 7.29–7.25 (m, 2H), 7.00 (t, J = 7.4, 1H), 6.95 (s, 1H), 6.79 (d, J = 7.7, 2H), 6.51 (br d, J = 13, 1H), 3.81 (t, J = 6.5, 2H), 3.43 (s, 2H), 2.99 (t, J = 6.5, 2H), 2.13 (s, 3H). Only the signals corresponding to the major isomer are reported. The spectroscopic data are consistent with those previously reported.11

Chapter 2

48

3-Deazathiamine (5): This compound was synthesized according to GP-C, using 14 (1.81 g, 6.06 mmol), anhydrous EtOH (50 mL), acetamidine·HCl (1.72 g, 18.1 mmol) and NaOEt (2.0 M in EtOH, 15.1 mL, 30.3 mmol). Reaction

time: 72 h. The crude was purified by flash column chromatography (CH2Cl2/MeOH 94:6), to afford a pale yellow solid, that was washed with cold MeOH affording 5 as a white solid (1.09 g, 4.13 mmol, 68%). 1H-NMR (400 MHz, DMSO-d6) δ 7.58 (s, 1H), 6.76 (s, 1H), 6.57 (br s, 2H), 4.76 (t, J = 5.4, 1H), 3.56–3.51 (m, 4H), 2.83 (t, J = 7.1, 2H), 2.28 (s, 3H), 1.97 (s, 3H). The spectroscopic data are consistent with those previously reported.11

(4-Bromo-3-methylthiophen-2-yl)methanol (15):

To a solution of 11 (5.02 g, 19.5 mmol) in anhydrous Et2O (80 mL) at –78 °C, sBuLi (1.4 M in cyclohexane, 13.9 mL, 19.5 mmol) was added dropwise, and the reaction mixture was left to warm up

from –78 to –45 °C for 1 h. The mixture was cooled down to –78 °C, and anhydrous DMF (15.1 mL, 195 mmol) was added dropwise. The reaction mixture was stirred for 2 h (until full conversion by GC-MS was observed: T0 = 100 °C, hold 5 min; ramp 6 °C min–1 to 250 °C, hold 20 min. tR(prod, m/z = 205) = 11.5 min). A saturated aqueous solution of NH4Cl was added, and the aqueous phase was extracted 3 times with Et2O. The combined organic layers were washed once with water, once with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated under reduced pressure. The crude (4.15 g) was used without further purification: it was dissolved in MeOH (80 mL), and NaBH4 (0.370 g, 9.75 mmol) was added. The reaction mixture was stirred at room temperature for 1 h (no dry conditions needed), and then ice/water (~500 mL) was added. The mixture was extracted 3 times with CH2Cl2, washed once with water, once with a saturated aqueous solution of NaCl, dried over MgSO4, filtered and concentrated under reduced pressure. The crude was purified by flash column chromatography (pentane/Et2O 4:1), to afford 15 as a white solid (3.19 g, 15.4 mmol, 79% over 2 steps). M.p. 62–64 °C. 1H-NMR (400 MHz, CDCl3) δ 7.20 (s, 1H), 4.78 (s, 2H), 2.21 (s, 3H). 13C-NMR (101 MHz, CDCl3) δ 137.5 (C), 134.0 (C), 121.1 (CH), 113.7 (C), 58.8 (CH2), 13.7 (CH3). IR (cm–1) 3224, 3111, 2948, 2924, 2872, 1419, 1381, 1350, 1132, 1006, 988, 729, 683. HRMS (ESI+) calculated for C6H6BrS [M – OH]+ 188.9368, found 188.9367.


49

5-(Hydroxymethyl)-4-methylthiophene-3-carbaldehyde (16): This compound was synthesized according to GP-A, using 15 (2.50 g, 12.1 mmol), anhydrous THF (80 mL), iPrMgCl (2.0 M in THF, 6.64 mL, 13.3 mmol), tBuLi (1.7 M in pentane, 15.6 mL, 26.6 mmol) and anhydrous DMF (17 mL, 220 mmol). GC-MS:

tR(prod, m/z = 156) = 14.0 min. The crude was purified by flash column chromatography (pentane/Et2O 3:2) to afford 16 as a pale yellow oil (1.18 g, 7.53 mmol, 62%). 1H-NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 8.00 (s, 1H), 4.80 (s, 2H), 2.44 (s, 3H), 1.94 (br s, 1H). 13C-NMR (101 MHz, CDCl3) δ 186.5 (CHO), 141.3 (C), 140.5 (C), 138.3 (CH), 133.7 (C), 57.4 (CH2), 12.8 (CH3). IR (cm–1) 3384, 3093, 2928, 2873, 2805, 2729, 1673, 1409, 1099, 1001, 724. HRMS (ESI+) calculated for C7H9O2S [M + H]+ 157.0318, found 157.0317. 2-((5-(Hydroxymethyl)-4-methylthiophen-3-yl)methyl)-3-(phenylamino)-

acrylonitrile (17):

This compound was synthesized according to GP-B, using 16 (0.202 g, 1.28 mmol), 3-anilinopropionitrile (0.206 g, 1.40 mmol), anhydrous DMSO (3.0 mL) and NaOEt (2.0 M in EtOH, 1.28 mL, 2.40 mmol). Reaction in

the microwave for 1.5 h. The crude was purified by flash column chromatography (pentane/EtOAc 7:3) to afford 17 as a yellow, sticky solid (0.261 g, 0.918 mmol, 71%). 1H-NMR (400 MHz, CDCl3) δ 7.37 (d, J = 13, 1H), 7.30–7.26 (m, 2H), 7.07 (s, 1H), 7.01 (t, J = 7.4, 1H), 6.78 (d, J = 7.7, 2H), 6.27 (br d, J = 13, 1H), 4.78 (m, 2H), 3.46 (s, 2H), 2.21 (s, 3H). 13C-NMR (101 MHz, CDCl3) δ 141.0 (CH), 140.1 (C), 139.2 (C), 137.2 (C), 134.0 (C), 130.0 (2 CH), 123.1 (CH), 121.3 (CN), 120.7 (CH), 115.3 (2 CH), 81.3 (C), 58.4 (CH2), 28.2 (CH2), 12.4 (CH3). IR (cm–1) 3323, 3268, 3055, 2925, 2860, 2190, 1639, 1591, 1498, 1275, 997, 918, 749, 690. HRMS (ESI+) calculated for C16H15N2S [M – OH]+ 267.0951, found 267.0952. (4-((4-Amino-2-methylpyrimidin-5-yl)methyl)-3-methylthiophen-2-

yl)methanol (7): This compound was synthesized according to GP-C, using 17 (0.190 g, 0.670 mmol), anhydrous EtOH (1.5 mL), acetamidine·HCl (0.127 g, 1.34 mmol) and NaOEt (2.0 M in EtOH, 2.01 mL, 4.02 mmol). Reaction

time: 16 h. The crude was purified by flash column chromatography (CH2Cl2/MeOH 95:5) to afford 7 as a white solid (0.102 g, 0.408 mmol, 61%). M.p. 206–207 °C. 1H-NMR (400 MHz, CD3OD) δ 7.58 (s, 1H), 6.90 (s, 1H), 4.68 (s, 2H), 3.65 (s, 2H), 2.39 (s, 3H), 2.08 (s, 3H). 13C-NMR (101 MHz, CD3OD) δ 166.2 (C), 163.7 (C), 154.3 (CH), 140.0 (C), 138.7 (C), 134.6 (C), 121.6 (CH), 114.1 (C), 58.1 (CH2), 28.7 (CH2), 24.7 (CH3), 12.0 (CH3). IR (cm–1) 3341, 3086, 3034, 2951, 1667,

Chapter 2

50

1585, 1464, 1418, 1286, 999, 861, 683. HRMS (ESI+) calculated for C12H16N3OS [M + H]+ 250.1009, found 250.1009.

4-((4-Amino-2-methylpyrimidin-5-yl)methyl)-3-methylthiophene-2-

carbaldehyde (9): To a solution of 7 (40.0 mg, 0.160 mmol) in DMF (10 mL), MnO2 (0.209 g, 2.41 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h. MeOH was added, the solution was filtered over silica and

washed with cold MeOH. The crude was purified by flash column chromatography (CH2Cl2/MeOH 95:5) to afford 9 as a white solid (27.5 mg, 0.111 mmol, 70%). M.p. 224–226 °C. 1H-NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 7.68 (s, 1H), 7.50 (s, 1H), 6.64 (br s, 2H), 3.64 (s, 2H), 2.44 (s, 3H), 2.29 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ 183.8 (CHO), 164.9 (C), 161.7 (C), 154.5 (CH), 147.2 (C), 140.9 (C), 138.1 (C), 131.2 (CH), 110.8 (C), 26.5 (CH2), 25.1 (CH3), 12.4 (CH3). IR (cm–1) 3427, 3330, 3137, 3051, 3000, 2924, 2855, 1641, 1594, 1568, 1235, 975, 806, 772, 682. HRMS (ESI+) calculated for C12H14N3OS [M + H]+ 248.0852, found 248.0851. 3-(2-Hydroxyethyl)benzaldehyde (20):

This compound was synthesized according to GP-A, using 2-(3-bromophenyl)ethanol (18, 1.00 g, 4.97 mmol), anhydrous THF (50 mL), iPrMgCl (2.0 M in THF, 2.74 mL, 5.47 mmol), tBuLi (1.7 M in pentane, 6.40 mL, 10.9 mmol)

and anhydrous DMF (7.00 mL, 86.8 mmol). The crude was purified by flash column chromatography (pentane/EtOAc 1:1) to afford 20 as a colorless oil (0.519 g, 3.46 mmol, 70%). 1H-NMR (400 MHz, CDCl3) δ 10.02 (s, 1H), 7.78–7.74 (m, 2H), 7.54–7.47 (m, 2H), 3.92 (t, J = 6.5, 2H), 2.97 (t, J = 6.5, 2H). 13C-NMR (101 MHz, CDCl3) δ 192.6 (CHO), 140.0 (C), 136.8 (C), 135.4 (CH), 130.1 (CH), 129.3 (CH), 128.4 (CH), 63.4 (CH2), 38.9 (CH2). IR (cm–1) 3385, 3090, 2924, 2871, 2800, 2733, 1693, 1604, 1586, 1039, 899, 794, 692, 652. HRMS (ESI+) calculated for C9H9O [M – OH]+ 133.0648, found 133.0646. 2-(3-(2-Hydroxyethyl)benzyl)-3-(phenylamino)acrylonitrile (22):

This compound was synthesized according to GP-B, using 20 (0.400 g, 2.66 mmol), 3-anilinopropionitrile (0.467 g, 3.20 mmol), anhydrous DMSO (8.0 mL) and NaOEt (2.0 M in EtOH, 1.60 mL, 3.20 mmol). Reaction at

40 °C for 16 h. The crude was recrystallized from toluene to afford 22 as a yellow solid (0.180 g, 0.647 mmol, 24%). 1H-NMR (400 MHz, CDCl3) δ 7.39–7.26 (m, 4H), 7.21–7.11 (m, 3H), 7.01 (t, J = 7.5, 1H), 6.78 (d, J = 7.7, 2H), 6.17 (d, J = 13.4, 1H),


51

3.87 (t, J = 6.5, 2H), 3.56 (s, 2H), 2.88 (t, J = 6.5, 2H). The spectroscopic data are consistent with those previously reported.13 2-(3-((4-Amino-2-methylpyrimidin-5-yl)methyl)phenyl)ethanol (6):

This compound was synthesized according to GP-C, using 22 (0.150 g, 0.539 mmol), anhydrous EtOH (3.0 mL), acetamidine·HCl (0.102 g, 1.08 mmol) and NaOEt (2.0 M in EtOH, 1.08 mL, 2.16 mmol). Reaction

time: 48 h. The crude was purified by flash column chromatography (CH2Cl2/MeOH 9:1) to afford 6 as a white solid (0.795 g, 0.327 mmol, 61%). 1H-NMR (400 MHz, CD3OD) δ 7.74 (s, 1H), 7.24 (t, J = 7.5, 1H), 7.13–7.09 (m, 2H), 7.04 (d, J = 7.9, 1H), 3.76 (s, 2H), 3.73 (t, J = 7.0, 2H), 2.79 (t, J = 7.0, 2H), 2.39 (s, 3H). The spectroscopic data are consistent with those previously reported.13 3-(Hydroxymethyl)benzaldehyde (21):

This compound was synthesized according to GP-A, using 3-bromobenzylalcohol (19, 2.00 g, 10.7 mmol), anhydrous THF (100 mL), iPrMgCl (2.0 M in THF, 6.42 mL, 12.8 mmol), tBuLi (1.7 M in pentane, 13.8 mL, 23.5 mmol) and anhydrous DMF

(9.87 mL, 128 mmol). GC-MS: tR(prod, m/z = 136) = 11.5 min. The crude was purified by flash column chromatography (pentane/EtOAc 3:2), to afford 21 as a colorless oil (1.08 g, 79.5 mmol, 74%). 1H-NMR (400 MHz, CDCl3) δ 10.02 (s, 1H), 7.89 (s, 1H), 7.81 (d, J = 7.6, 1H), 7.65 (d, J = 7.6, 1H), 7.53 (t, J = 7.6, 1H), 4.80 (s, 2H). 13C-NMR (101 MHz, CDCl3) δ 192.5 (CHO), 142.1 (C), 136.7 (C), 133.0 (CH), 129.3 (CH), 129.1 (CH), 127.9 (CH), 64.6 (CH2). IR (cm–1) 3382, 3062, 2938, 2876, 2841, 2734, 1688, 1609, 1588, 1140, 1026, 903, 777, 687, 652. HRMS (ESI+) calculated for C13H16N3O [M – OH]+ 119.0491, found 119.0490. 2-(3-(Hydroxymethyl)benzyl)-3-(phenylamino)acrylonitrile (23):

This compound was synthesized according to GP-B, using 21 (0.300 g, 2.20 mmol), 3-anilinopropionitrile (0.387 g, 3.64 mmol), anhydrous DMSO (6.0 mL) and NaOEt (2.0 M in EtOH, 1.32 mL, 2.64 mmol). Reaction at 40 °C for 16 h.

The crude was purified by flash column chromatography (CH2Cl2/MeOH 99:1), to afford 23 as a colorless oil (0.259 g, 0.981 mmol, 45%). 1H-NMR (400 MHz, CD3OD) δ 7.55 (s, 1H), 7.33–7.20 (m, 6H), 7.12–7.08 (m, 2H), 7.00–6.95 (m, 1H), 4.60 (s, 2H), 3.64 (s, 2H). 13C-NMR (101 MHz, CD3OD) δ 142.6 (C), 139.8 (C), 130.6 (2 CH), 129.6 (CH), 128.4 (CH), 128.0 (CH), 126.4 (CH), 123.3 (CN), 116.5 (2 CH), 83.3 (C), 65.2 (CH2), 32.8 (CH2). One aromatic CH and two quaternary C are overlapping. IR (cm–1) 3372, 3320, 3062, 2929, 2872, 2184, 1643, 1594, 1502, 1443,

Chapter 2

52

1316, 1277, 1251, 1037, 901, 747, 687. HRMS (ESI+) calculated for C17H16N2ONa [M + Na]+ 287.1155, found 287.1157. (3-((4-Amino-2-methylpyrimidin-5-yl)methyl)phenyl)methanol (8):

This compound was synthesized according to GP-C, using 23 (0.240 g, 0.908 mmol), anhydrous EtOH (5.0 mL), acetamidine·HCl (0.172 g, 1.82 mmol) and NaOEt (2.0 M in EtOH, 1.81 mL, 3.63 mmol). Reaction

time: 16 h. The crude was purified by flash column chromatography (CH2Cl2/MeOH 92:8), followed by washing with CH2Cl2 and few drops of MeOH, to afford 8 as a white solid (0.138 g, 0.602 mmol, 66%). M.p. 168–169 °C. 1H-NMR (400 MHz, DMSO-d6) δ 7.78 (s, 1H), 7.25–7.21 (m, 1H), 7.18–7.08 (m, 3H), 6.54 (br s, 2H), 5.13, (t, J = 5.7, 1H), 4.44 (d, J = 5.7, 2H), 3.70 (s, 2H), 2.28 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ 156.8 (C), 154.2 (C), 145.2 (CH), 133.8 (C), 129.7 (C), 120.3 (CH), 119.2 (CH), 118.8 (CH), 116.9 (CH), 105.8 (C), 55.6 (CH2), 24.9 (CH2), 15.2 (CH3). IR (cm–1) 3379, 3344, 3144, 3038, 2934, 2907, 1665, 1598, 1560, 1472, 1420, 1021, 968, 900, 796, 778, 730, 693, 655, 583. HRMS (ESI+) calculated for C13H16N3O [M + H]+ 230.1288, found 230.1290.

2.4.3 Expression and purification of ThiT

The detergents n-dodecyl- -D-maltopyranoside (DDM), n-decyl- -D-maltopyranoside (DM) and n-nonyl- -D-glucopyranoside (NG) were purchased from Anatrace. Expression of native ThiT with an N-terminal His8-tag was performed as described previously1,3 with some modifications. Briefly, L. lactis NZ9000 cells15 with pNZnHisThiT plasmid were grown semi-anaerobically in chemically defined medium16 without thiamine and supplemented with glucose (2.0%, w/v) and chloramphenicol (5 μg mL–1) in a 10 L bioreactor at 30 °C and pH 6.5. At an OD600 of 1.5, expression of ThiT was induced by addition of culture supernatant (0.1%, v/v) from a Nisin-A-producing strain.15 The cells were induced for 3 h and harvested at a final OD600 of 7–8. After harvesting and washing by centrifugation at 6,000 rpm and 4 °C for 15 min, the cells were resuspended in buffer A (potassium phosphate buffer (KPi, pH 7.0, 50 mM)), frozen in liquid nitrogen and stored at –80 °C. The preparation of membrane vesicles was performed as described previously1 with some modifications. After the resuspended cells had been thawed, MgSO4 (5 mM) and DNase (375 μg mL–1) were added. The cells were lyzed by high-pressure disruption (Constant Cell Disruption Systems, Ltd, UK; twofold passage at 269 MPa and 4 °C), and the cell debris was separated from the membrane vesicles by low-speed centrifugation at 15,000 rpm (JA25.50 rotor) and 4 °C for 30 min. The membrane vesicles were harvested by high-speed


53

centrifugation for 2 h at 40,000 rpm (45Ti rotor) and 4 °C, resuspended in buffer A to a final concentration of 10 mg mL–1, frozen in liquid nitrogen and stored at –80 °C. For the purification of substrate-free ThiT, membrane vesicles were rapidly thawed and resuspended in buffer B (KPi (pH 7.0, 50 mM), KCl (200 mM), glycerol (10%, w/v)) to a final concentration of 6–10 mg mL–1. For the ThiT cocrystallization experiments, compounds 2 and 5–9 were added to all buffers used during the purification. The membrane vesicles were solubilized with DDM (1%, w/v) at 4 °C for 1 h with gentle rocking. The undissolved material was removed by centrifugation at 80,000 rpm (MLA-80 rotor) and 4 °C for 30 min. The supernatant was incubated with nickel-sepharose resin (column volume = 0.5 mL), equilibrated with buffer B, at 4 °C for 1 h with gentle rocking. Subsequently, the suspension was poured onto a 10 mL disposable column (Bio-Rad) and after collecting the flow-through, the column material was washed with 20 column volumes of buffer C (KPi (pH 7.0, 50 mM), KCl (200 mM), imidazole (50 mM), DM (0.15%, w/v)). In cases of purification of protein for crystallization, DM was replaced with NG (0.35%, w/v). The protein was eluted in three fractions of 350, 650 and 500 μL of buffer D (KPi (pH 7.0, 50 mM), KCl (200 mM,) imidazole (500 mM), DM (0.15%, w/v)). In cases of purification of protein for crystallization, DM was replaced with NG (0.35%, w/v). EDTA (1 mM) was added to the second elution fraction, which contained most of the purified protein, and this fraction was loaded on a Superdex 200 10/300 gel filtration column (GE Healthcare) equilibrated with buffer E (KPi (pH 7.0, 50 mM), KCl (150 mM), DM (0.15 %, w/v)). In cases of purification of protein for crystallization, buffer F (HEPES (pH 7.0, 20 mM), KCl (150 mM), NG (0.35%, w/v)) was used for gel-filtration chromatography. After gel-filtration chromatography, the fractions containing ThiT were used directly for further analysis or concentrated by use of a Vivaspin 2 concentrator device with a molecular weight cut-off of 50 kDa (Sartorius stedim) to a final concentration of 6–10 mg mL–1 and used for crystallization.

2.4.4 Binding-affinity determination

The ligand-binding measurements by intrinsic-protein-fluorescence titration were performed as described previously,1 by use of a Spec Fluorlog 322 fluorescence spectrophotometer (Jobin Yvon), with some modifications. Purified ThiT was diluted in buffer E to a concentration of about 50 nM in a final volume of 800 μL. The substrates were added in 1 μL steps using a Harvard apparatus syringe pump with a 500 μL gas-tight glass syringe (Hamilton). With use of an excitation wavelength of 280 nm, emission was measured at 350 nm and 25 °C. After each addition of substrate, 10 s were allowed for equilibration and mixing,

Chapter 2

54

and the signals were averaged over 15 s. The data analysis was performed as described.1

2.4.5 Cocrystallization of ThiT with small-molecule binders and

structure determination

The concentrated ThiT (purified in NG (0.35%, w/v)) in the presence of compounds 2 or 5–9 (Table 4) was used to set up 24-well hanging drop crystallization plates with use of a reservoir solution (NH4NO3 (0.15 M), PEG 3350 (20%, w/v)). Hanging drops of 2 μL were set up in a 1:1 ratio of concentrated-protein solution to reservoir solution. The plates were incubated at 5 °C, and after one to two weeks, crystals appeared. For cryoprotection, a cryoprotection solution (NH4NO3 (15 mM), PEG 3350 (40% or 50%, w/v)) was used when the crystals were fished. Diffraction data were collected at the DESY, Hamburg (ThiT cocrystallized with compounds 6, 7 or 9) or the ESRF, Grenoble (ThiT cocrystallized with compounds 2, 5 or 8). Data processing was carried out by use of XDS,17 and molecular replacement was performed with the aid of Phaser MR in the CCP4 suite,18 with use of the structure of thiamin-bound ThiT (PDB ID: 3RLB).3 Superimposition of the six structures with the corresponding chain of ThiT in complex with thiamine (PDB ID: 3RLB) gave RMSD values between 0.274 and 0.379 Å (Table 4). Refinements were performed by use of both Phenix refinement19 and Refmac.20 Manual model building and the placement of substrate, detergent and PEG molecules were done with Coot.21 Refinement statistics are shown in Table 5. The crystal structures were deposited in the PDB with the following PDB IDs: ThiT + compound 2: 4MUU, ThiT + compound 5: 4MHW, ThiT + compound 6: 4MES, ThiT + compound 7: 4POV, ThiT + compound 8: 4N4D, ThiT + compound 9: 4POP. Table 4. Conditions for cocrystallization of ThiT with small-molecule binders and diffraction results.

Compound Concentrationa

(μM) RMSD alignment (Å)

chain A chain B 2 100 0.282 0.276 5 10 0.341 0.322 6 40 0.310 0.379 7 10 0.305 0.274 8 10 0.332 0.361 9 10 0.287 0.317

a For purification and crystallization.


55

Table 5. Data collection and refinement statistics.

PDB ID 4MUU 4MHW 4MES 4POV 4N4D 4POP

Data collection (at 100 K)

Resolution (Å)

42.2-2.1 42.8-2.5 42.3-2.0 48.0-2.2 42.3-2.4 47.8-2.2

Space group C2 Cell

dimensions a, b, c (Å) α, , (°)

61.6, 84.6, 127.3

90.0, 95.6, 90.0

63.7, 84.1, 128.8

90.0, 94.6, 90.0

61.5, 84.2, 127.2

90.0, 93.5, 90.0

62.7, 84.4, 127.3

90.0, 96.1, 90.0

63.2, 84.3, 127.3

90.0, 94.2, 90.0

62.6, 84.1, 127.2

90.0, 95.1, 90.0

Robserved (%) 5.2

(41.2)* 13.5

(127.5) 8.6

(86.1) 7.2

(97.6) 11.7

(87.9) 8.4

(78.1)

I/Iσ 16.69 (3.12)

11.16 (1.57)

10.31 (1.59)

13.27 (1.36)

8.24 (1.36)

11.40 (1.47)

Complete-ness (%)

98.8 (95.8) 99.5 (99.9) 99.8 (99.9) 99.4 (98.5) 99.3 (98.9) 99.1 (98.6)

* Values in parentheses are for the highest resolution shell

Refinement Resolution

(Å) 2.1 2.5 2.0 2.2 2.4 2.2

No. reflections

37528 23444 43721 33341 25926 33165

Rwork/Rfree 0.194/ 0.236

0.188/ 0.250

0.174/ 0.208

0.185/ 0.237

0.204/ 0.257

0.199/ 0.248

Formula ligand

C14H19N4O- C13H17N3OS C14H17N3O C12H15N3OS C13H15N3O C12H13N3OS

Formula weight

ligand (Da) 259.3 263.4 243.3 249.3 229.3 247.3

No. atoms Protein 5719 5671 5682 5668 5660 5671 Ligand 38 36 70 34 64 34 Water 110 48 111 57 23 66

B-factors Protein 39.39 57.36 38.40 52.75 46.80 41.00 Ligand 25.09 50.47 34.32 37.05 37.07 30.10 Water 41.28 53.09 44.03 57.31 42.66 48.49 R.m.s.

deviations

Bond lengths (Å)

0.012 0.013 0.012 0.013 0.014 0.013

Bond angles (°)

1.409 1.492 1.314 1.345 1.480 1.390

Chapter 2

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2.5 Contributions from co-authors

The biochemical and cocrystallization experiments were performed by L. J. Y. M. Swier, Dr. A. Guskov and Dr. G. B. Erkens from the group of Prof. D. J. Slotboom. Part of the synthesis was done by A. R. de Voogd during his Master’s project.

2.6 References

(1) Erkens, G. B.; Slotboom, D. J. Biochemistry 2010, 49, 3203.

(2) Kluger, R.; Tittmann, K. Chem. Rev. 2008, 108, 1797.

(3) Erkens, G. B.; Berntsson, R. P.-A.; Fulyani, F.; Majsnerowska, M.; Vujičić-Žagar, A.; ter Beek, J.; Poolman, B.; Slotboom, D. J. Nat. Struct. Mol. Biol. 2011, 18, 755.

(4) Majsnerowska, M.; Hänelt, I.; Wunnicke, D.; Schäfer, L. V.; Steinhoff, H. J.; Slotboom, D. J. Structure 2013, 21, 861.

(5) Stierand, K.; Rarey, M. ACS Med. Chem. Lett. 2010, 1, 540.

(6) Manzetti, S.; Zhang, J.; van der Spoel, D. Biochemistry 2014, 53, 821.

(7) Gerber, P. R.; Müller, K. J. Comput.-Aided Mol. Des. 1995, 9, 251.

(8) LeadIT (version 2.1.8), BioSolveIT GmbH, An Der Ziegelei 79, 53757 Sankt Augustin, Germany, 2014.

(9) Reulecke, I.; Lange, G.; Albrecht, J.; Klein, R.; Rarey, M. ChemMedChem 2008, 3, 885.

(10) Schneider, N.; Hindle, S.; Lange, G.; Klein, R.; Albrecht, J.; Briem, H.; Beyer, K.; Claußen, H.; Gastreich, M.; Lemmen, C.; Rarey, M. J. Comput.-Aided Mol. Des. 2012, 26, 701.

(11) Zhao, H.; De Carvalho, L. P. S.; Nathan, C.; Ouerfelli, O. Bioorg. Med. Chem. Lett. 2010, 20, 6472.

(12) Ko, J.; Ham, J.; Yang, I.; Chin, J.; Nam, S. J.; Kang, H. Tetrahedron Lett. 2006, 47, 7101.

(13) Mann, S.; Perez Melero, C.; Hawksley, D.; Leeper, F. J. Org. Biomol. Chem. 2004, 2, 1732.

(14) Xu, K.; Zhang, M.; Zhao, Q.; Yu, F.; Guo, H.; Wang, C.; He, F.; Ding, J.; Zhang, P. Nature 2013, 497, 268.

(15) Kuipers, O. P.; de Ruyter, P. G. G. A.; Kleerebezem, M.; de Vos, W. M. J.

Biotechnol. 1998, 64, 15.

(16) Berntsson, R. P.-A.; Alia Oktaviani, N.; Fusetti, F.; Thunnissen, A.-M. W. H.; Poolman, B.; Slotboom, D. J. Protein Sci. 2009, 18, 1121.

(17) Kabsch, W. Acta Crystallogr. D. Biol. Crystallogr. 2010, 66, 125.

(18) Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S. Acta Crystallogr. D. Biol. Crystallogr. 2011, 67, 235.


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(19) Adams, P. D.; Afonine, P. V; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L.-W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr. D. Biol. Crystallogr. 2010, 66, 213.

(20) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr. D. Biol. Crystallogr. 1997, 53, 240.

(21) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr. D. Biol.

Crystallogr. 2010, 66, 486.

university of groningen structure-based design of ligands ... · and residues in loops l1 and l3,...

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