Accepted Manuscript Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa Mette R. Hansen, Tim H. Jakobsen, Claus G. Bang, Anders Emil Cohrt, Casper L. Hansen, Janie W. Clausen, Sebastian T. Le Quement, Tim Tolker-Nielsen, Michael Givskov, Thomas E. Nielsen PII: DOI: Reference:

S0968-0896(15)00062-0 http://dx.doi.org/10.1016/j.bmc.2015.01.038 BMC 12041

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

12 December 2014 21 January 2015 22 January 2015

Please cite this article as: Hansen, M.R., Jakobsen, T.H., Bang, C.G., Cohrt, A.E., Hansen, C.L., Clausen, J.W., Le Quement, S.T., Tolker-Nielsen, T., Givskov, M., Nielsen, T.E., Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.01.038

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Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa

Mette R. Hansen a, Tim H. Jakobsen b, Claus G. Bang a, Anders Emil Cohrt a, Casper L. Hansen a, Janie W. Clausen a, Sebastian T. Le Quement a, Tim Tolker-Nielsen b, Michael Givskov b,c, and Thomas E. Nielsen a,c,* a Department of Chemistry, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark b Costerton Biofilm Center, Department of International Health, Immunology and Microbiology, University of Copenhagen, DK-2200 Copenhagen, Denmark c Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore 637551, Singapore O

AA1 AA2 NH 2 H N

N

O O

N N

O

O O

N O

O

N N

H N

O

O R

H N

N N N R

N N N R

H N

O

O O

O O

H N

O O

N N N

R

N N N

lasB-gfp (E. coli) agonist EC 50 = 2 µM

Library of triazole-containing HSL analogues and mimics

1

Bioorganic & Medicinal Chemistry j o ur n al h o m e p a g e : w w w . e l s e v i e r . c o m

Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa Mette R. Hansen a,Tim H. Jakobsen b,Claus G. Bang a, Anders Emil Cohrt a, Casper L. Hansen a, Janie W. Clausen a, Sebastian T. Le Quement a, Tim Tolker-Nielsen b, Michael Givskov b,c, and Thomas E. Nielsen a,c,* a

Department of Chemistry, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark Costerton Biofilm Center, Department of International Health, Immunology and Microbiology, University of Copenhagen, DK-2200 Copenhagen, Denmark c Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore 637551, Singapore b

AR T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

In an attempt to devise new antimicrobial treatments for biofilm infections, the bacterial cell-cell communication system termed quorum sensing has emerged as an attractive target. It has proven possible to intercept the communication system by synthetic non-native ligands and thereby lower the pathogenesis and antibiotic tolerance of a bacterial biofilm. To identify the structural elements important for antagonistic or agonistic activity against the Pseudomonas aeruginosa LasR protein, we report the synthesis and screening of new triazole-containing mimics of natural N-acyl homoserine lactones. A series of azide- and alkyne-containing homoserine lactone building blocks was used to prepare an expanded set of 123 homoserine lactone analogues through a combination of solution- and solid-phase synthesis methods. The resulting compounds were subjected to cell-based quorum sensing screening assays, thereby revealing several bioactive compounds, including 13 compounds with antagonistic activity and 9 compounds with agonistic activity.

Keywords: quorum sensing antibiotics 1,2,3-triazole homoserine lactone Pseudomonas aeruginosa

1. Introduction Chronic bacterial infections pose substantial challenges for the clinicians. In particular the formation of bacterial biofilms, which renders the infecting pathogens more resistant to conventional antibacterial treatments and the action of the host defence system, makes complete eradication of the resulting infection difficult. Bacterial biofilms are highly organized and encapsulated matrices, and their formation and development rely on intricate bacterial communication systems. In a process termed quorum sensing (QS), Gram-negative bacteria use N-acyl L-homoserine lactones (AHLs) as cell-to-cell signals to synchronize expressions of virulence factors in order to control temporal events that eventually lead to disease.1 The perturbation of this process by chemical interference with the QS system is emerging as a topic of increasing interest in the pharmaceutical industry and academia.1 The homoserine lactone ring is conserved in the AHL signal molecules whereas the acyl side chain varies in length and substitution pattern depending on the bacterial strain. A constant

——— ∗ Corresponding author. Tel. +45 45252419; e-mail: [email protected]

2009 Elsevier Ltd. All rights reserved.

basal level of AHL molecules is produced by the bacteria, which during population growth leads to an increased local AHL concentration. When a certain threshold level is reached, the expression of a range of target genes is induced. The AHL-mediated quorum sensor is primarily based on the luxIR homologues encoding the AHL syntethase and the luxR homologue AHL receptor that functions as a transcriptional activator of QS target genes. 2-5 In Pseudomonas aeruginosa the QS system is used to control pathogenicity and immune evasion.6-7 It has been estimated that

Figure 1. The two natural N-acyl L-homoserine lactone signal molecules of Pseudomonas aeruginosa, 3-oxo-C12-HSL and C4-HSL, and different QSIs from natural sources targeting the AHL-mediated QS system in P. aeruginosa.14-17

4-6% of the total number of genes are controlled by AHL QS. 8-10

and selective compounds.22

P. aeruginosa is an opportunistic pathogen that causes a number of nosocomial infections and primarily affects immunocompromised patients.11-12 Notably, P. aeruginosa is the most common airway pathogen in patients with the genetic hereditary disease cystic fibrosis.13

The aim of this study was to identify non-native AHLs synthesized from the basis of the AHL structure with agonistic or antagonistic activity towards the P. aeruginosa LasR protein. We have recently synthesized and evaluated a promising library of triazole-containing AHLs (compounds 1-51, Figure 2) in various reporter gene assays.23 In these studies, the QS reporter strain was Escherichia coli DH5α harboring the LasR expression vector pJN105L and a plasmid-borne lasI-lacZ fusion (pSC11), and the AbaR reporter strain was Acinetobacter baumannii abaI-lacZ.

The P. aeruginosa AHL-mediated QS system consists of a dual QS regulatory circuit encoded by the lasIR and rhlIR couples and their corresponding AHL signal molecules, N-(3oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL)18 and N-butanoyl L-homoserine lactone (C4-HSL),19 respectively. Previous studies have shown that it is possible to attenuate virulence and decrease the antibiotic tolerance of P. aeruginosa biofilms by antagonistic perturbation of the QS signaling system.8,14-15 Several antagonistic molecules and QS inhibitors (QSIs) have been identified using screening of natural or de novo synthetic compound libraries (Figure 1).20-21 Despite these efforts, it remains difficult to predict structure-activity relationships (SARs) necessary for the design of efficient QSIs and many different approaches have been applied in the search for potent

1 (n = 2 (n = 3 (n = 4 (n =

R

O N H

n

O N H

n

O

5 (n = 1) 6 (n = 3) 7 (n = 4) N N N

O

N H

O

N H

O

8

n

O O N H

N N

R

O

N H

O

9 (R = 4-OCH3) 10 (R = 4-NH2) 11 (R = 4-CH3) 12 (R = 3-CHO) 13 (R = 4-OH) 14 (R = 3-NH2)

n =1 n=3 n=4

31a 32a 33a

31b 32b 33b

34a

34b

35a 36a 37a 38a

35b 36b 37b 38b

39a

39b

40a

40b

41a

41b

42a

42b

R1: H 3,5-F 2 3,5-(OCH 3)2 4-OPh

R1 O

N N N

N H

H N

para

R:

27b 28b 29b 30b

O

O

N

meta

27a 28a 29a 30a

O

21

R

para

O

N N N

S

O

O

H 3CO

meta N

22a

22b

23a

23b

NH 2 24a OH 25a OCH 3 26a

24b 25b 26b

S

S R1: R1

O O N H

O

O

HN

O

O

N N N 43

NH

S

O

O O

O

H N

N

O

O O

44

HN

O

NH

O

N N N

N N

HN

45

O

O

O

N

N N N

O

O

O N

O

H N

O

N N N

O

H N 46

O

N N

N O

O

N

HN

O O

O

O

N N N

H N O

O

NH 47

N

O

N N

O

H N

N

N N N

O

NH

N N 51

N N N

NH O

48

O O N H

vii, viii

N H NHAc

R

55

N3

N N N

Phe-Gly-NH2 NHAc 59a (R = BB1) 59b (R = BB2) 59c (R = BB3) O

N N N

Phe-Gly-NH2 60a (R = BB1) 60b (R = BB2) 60c (R = BB3)

O

Figure 2. Known triazole-containing AHLs (included and screened in the present study).23

O

N3 vi

vii, viii

N H 56

R

O

O

O O

Phe-Gly-NH 2

O

63 (BB3-N3)

O

O

N H

O

58a (R = BB1) 58b (R = BB2) 58c (R = BB3)

O

50

O

N N

O iii, iv, v

N H 62 (BB2-N3)

N N O

O

O

R N

57a (R = BB1) 57b (R = BB2) 57c (R = BB3)

N

54

O O

N vii, viii

N H

N3

N H 61 (BB1 -N3)

O

NH

R N

O ii

O O

vii, viii 53

52 (H-Phe-Gly-Rink-PEGA800)

O O

49

O

NH

O

N

H2N

O

O O

N N

N N N

NH

O

Phe-Gly-NH2

N H

i

O

N H

N N N

O

O

O

N N N O

O

O

2 3 4 5

n= n= n= n=

n

O

20 O

R:

O

N N N

2.1. Solid-phase synthesis of triazole-containing AHLs

With readily available azido-functionalized AHL building blocks 61-63 at hand (Scheme 1), our attention turned to the development of a solid-phase synthesis strategy to access their triazole counterparts. We initially followed a strategy relying on the base-labile 4-hydroxymethylbenzoic acid (HMBA) linker, which is compatible with a range of chemical transformations on solid support.24-28 Compounds immobilized through the corresponding ester linkages are typically liberated using aqueous base, such as 0.1 M NaOH (aq). However, we noted how such cleavage reactions were frequently accompanied by undesired opening of the homoserine lactone ring, and our attention instead turned to the application of the Rink amide linker. The Fmoc-modified linker was easily coupled to the amino-functionalized PEGA800 (polyethylene glycol dimethyl acrylamide) resin using TBTU (N-[(1H-benzotriazol-1yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide), deprotected with 20% piperidine in DMF, and subjected to similar rounds of amide bond forming and deprotection reactions to give the aminofunctionalized solid-supported peptide 52, which was further

O N H

N

R

O

19

H 3CO S

N H

N N

O

N N N

O

N N N

2. Results and discussion

O

N H O 15 (R = H) 16 (R = 4-OPh) 17 (R = 3,5-F2) 18 (3,5-(OCH3) 2)

O

2) 3) 4) 5)

O

N N N

O

N N N

Herein we now wish to report the synthesis of new analogues and the biological evaluation of our expanded set of triazolecontaining AHLs in cell-based QSI screens based on expression of green fluorescence protein (GFP) in both P. aeruginosa (lasBgfp; rhlA-gfp) and E. coli (luxI-gfp). In total, 123 triazolecontaining AHLs and close analogues were evaluated for agonistic and antagonistic activities in these assays.

O

Scheme 1. Solid-phase synthesis of triazole-containing AHLs: (i) 4ethynylbenzoic acid, TBTU, NEM, DMF, 2 h; (ii) 3-ethynylbenzoic acid, TBTU, NEM, DMF, 2 h; (iii) Fmoc-Pra-OH, TBTU, NEM, DMF, 2 h; (iv) 20% piperidine (DMF), 30 min; (v) 20% Ac2O (DMF), 1 h; (vi) 4pentynoic acid, TBTU, NEM, DMF, 2 h; (vii) 61-63, CuI, sodium ascorbate, 2,6-lutidine, NMP/H2 O (4:1), 16 h; (viii) 95% TFA, 16 h. NEM = N-ethylmorpholine, Fmoc = fluorenylmethyloxycarbonyl, Pra = Propargylglycine.

3

modified by coupling with various alkynyl carboxylic acids leading to alkyne-containing peptides 53-56. The alkynyl peptides were subjected to copper(I)-mediated azide-alkyne cycloaddition (CuAAC)29-30 reactions with three different azido-functionalized AHLs (61-63) using a procedure optimized for the solid support.31 The peptido-triazole AHLs were released from the solid support by treatment with 95% TFA (aq), thereby providing compounds 57a-c - 60a-c in excellent purity (generally > 95%) and good yields (typically > 50%). The high purity of the resulting compounds thus demonstrates the applicability of the presented solid-phase approach and the results are promising for the generation of larger libraries of AHL conjugates. 2.2. Synthesis of sulfur-modified triazole-containing AHLs With the recent discovery of aryl thioether 42a (Scheme 1) as one of the strongest reported LasR antagonists (IC50 = 2.64 uM), 23 we set out to synthesize a series of close analogues of this compound (Scheme 2). It was of general concern to keep compounds sufficiently soluble while retaining high antagonistic activity, thus methoxy substituents were incorporated into the aromatic moiety of the compounds.

2.3. Synthesis of 1,4- and 1,5-disubstituted triazole-containing lactones In our pursuit of novel triazole-containing AHL analogues, we decided to investigate the replacement of the AHL amido group with a 1,2,3-triazole moiety, which has previously been suggested as an effective isoster to the amide functionality.32-33 The required (S)-azido lactone 82 was conveniently synthesized from L-homoserine lactone (Scheme 3). Two different procedures were investigated for the synthesis of this azide. Even though azidotriflate, when freshly prepared, was highly effective for the reaction, the trifluoromethane sulfonamide side-product proved difficult to remove by flash column chromatography. By changing the azidation reagent to imidazole-1-sulfonyl azide hydrochloride substrate conversion was still high and all sideproducts were easily removed. The latter approach was also more attractive since imidazole-1-sulfonyl azide hydrochloride is both crystalline and shelf-stable. Table 1. Synthesis of 1,4- and 1,5-disubstituted triazoles. a O O

N N N

O R

i

O N3

O

ii

O

N N N R

83a-l

82

84a-l

1,4-disubstituted triazole, yield (%)b

1,5-disubstituted triazole, yield (%)b

1

83a, 75

84a, 63c

2

83b, 79

84b, 62

3

83c, 84

84c, 66c

4

83d, 95

84d, 75

5

83e, 79

84e, 79

6

83f, 72

84f, 85

7

83g, 80

84g, 95

8

83h, 79

84h, 57c

9

83i, 83

84i, 62

Scheme 2. Synthesis of sulfur-containing AHLs. (i) 62, 64-66 or 67-69, CuI, DIPEA, MeCN, rt, 16 h; (ii) 73-75, H2O2, H2 O/1,4-dioxane (1:1), 70 ˚C, 2-5 days; (iii) 73-75, m-CPBA, H2 O/1,4-dioxane (1:1), 70 ˚C, 2-3 days.

10

83j, 69

84j, 66

11

83k, 95

84k, 41

Both sulfonamides 70-72 and sulfides 73-75 could be accessed in excellent yields and purities using CuAAC reactions of azide building block 62 and the alkyne building blocks 64-69, which were readily available from the corresponding sulfonyl chlorides and sulfides by reaction with propargyl amine and bromide, respectively. The sulfides 73-75 were subsequently oxidized to form 1:1 diastereomeric mixtures of sulfoxides 76-78 with H2O2 and sulfones 79-81 with m-CPBA. Although these reactions were relatively clean, preparative HPLC was generally needed for efficient purification, and the compounds were isolated in 20-35% yield.

12

83l, 80

84l, 70

Entry O

R1

O S

R2

R2 N N N O

O H N

O

R1

64 (R1 = H, R 2 = OMe) 65 (R1 = OMe, R 2 = H) 66 (R1 = OMe, R 2 = OMe) O H i N O

N H

R

H N S O O

70 (R1 = H, R2 = OMe) 71 (R1 = OMe, R 2 = H) 72 (R1 = OMe, R 2 = OMe)

N3 O

62

H N

O R3

73 (R 3 = H, R 4 = OMe) 74 (R 3 = OMe, R 4 = H) 75 (R 3 = OMe, R 4 = OMe)

67 (R 3 = H, R 4 = OMe) 68 (R 3 = OMe, R 4 = H) 69 (R 3 = OMe, R 4 = OMe)

R4 ii

O

iii O S

N N N

O H N

R3 R4

N N N

O O

H N

O R4

(R 3

76 = H, = OMe) 77 (R3 = OMe, R 4 = H) 78 (R3 = OMe, R 4 = OMe)

S

O

R4

R4

79 = H, = OMe) 80 (R 3 = OMe, R 4 = H) 81 (R 3 = OMe, R 4 = OMe)

O O

O

R3 O

(R3

S R4

O

S

R3

N N N

O

i

O NH 2.HBr

i or ii

N3

O 82

Scheme 3. Synthesis of (S)-azido lactone: (i) TfN3 (2 equiv), K2 CO3 (2.6 equiv), CuSO4 (0.9 mol%), CH2Cl2, rt, 18 h (61%); or (ii) imidazole-1sulfonyl azide hydrochloride (1.2 equiv), K2CO3 (2.7 equiv), CuSO4.5H2 O (10 mol%), MeOH, rt, 18 h (54%). Tf = trifluoromethylsulfonyl.

a Reagents and conditions: (i) alkyne (1.5 equiv), CuI (15 mol%), DIPEA (3 equiv), MeCN, rt, 18 h; (ii) alkyne (1.5 equiv), Cp*Ru(PPh3)2Cl (3 mol%), THF, 65 °C, 18 h; b isolated yield after flash column chromatography; c after 18 h of reaction time, 3 mol% of catalyst was added and the reaction mixture was further stirred for 24 h. Cp* = pentamethylcyclopentadienyl.

With 82 in hand, we set out to synthesize a library of 1,4- and 1,5-disubstituted 1,2,3-triazoles. The CuAAC reaction of 82 was conducted under conditions previously reported,23 with a set of commercially available alkynes (1.5 equiv), 15 mol% copper(I) iodide and DIPEA (3 equiv). Formation of product was indicated by a green/yellow precipitate. However, product isolation by hot filtration in methanol and subsequent precipitation with Et2O resulted in low yields of impure products, containing high amounts of copper salts. Despite displaying a relatively low solubility in various organic solvents, particularly compounds with aromatic residues, flash column chromatography afforded the desired 1,4-disubstituted 1,2,3-triazole products 83a-l in good to excellent yields, generally as white or pale brown solids (Table 1). The corresponding 1,5-disubstituted 1,2,3-triazoles were

synthesized through ruthenium-catalyzed cycloaddition of 82 with a set of commercially available alkynes using 3 mol% Cp*Ru(PPh3)2Cl in THF at 65 °C.34 For some reactions, incomplete conversion of the azide was observed after 18 h of reaction time, and further amounts of catalyst (3 mol%) were added, consistently bringing about a full conversion of the azide after another 24 h. Generally, the isolated yields of 1,5disubstituted triazoles 84a-l in the ruthenium-catalyzed reactions were lower than the yields of the corresponding 1,4-disubstituted triazoles 83a-l obtained in the copper-catalyzed reactions, although ethynylcyclohexane and 3-ethynylthiophene (Table 1, entries 6 and 7) were exceptions to this general observation. 2.4. Biological activity In total, 123 compounds were tested for antagonistic activity with the following cell-based biological QSI screens; lasB-gfp,35 rhlA-gfp, 36 and luxI-gfp36 and for agonistic activity with the lasBgfp in an E. coli background.37 In our previous studies, we assessed molecular interactions with the LasR protein using an E. Coli K-12 strain harboring a LasR expression vector and a LasR β-galactosidase reporter. It was therefore interesting to also test the triazole-containing AHLs of our initial study alongside the novel compounds reported herein in cell-based QSI screens in P. aeruginosa (lasB-gfp; rhlA-gfp). The reporter strains, lasB-gfp and rhlA-gfp contain fusions of the QS-controlled lasB promoter or rhlA promoter to gfp(ASV) encoding an unstable GFP variant. The QS reporter system, luxI-gfp in an E. coli background contains a luxR gene and the promoter region of the luxI fused to gfp(ASV). Table 2. Selected IC50 values of compounds showing antagonistic activity tested with the following monitor screens; lasB-gfp, rhlA-gfp and/or luxI-gfp Compound 1 2 4 5 6 7 12 15 20 21 24a 61 62

IC50 for indicated strain (µM) lasB-gfp

rhlA-gfp

luxI-gfp

678 No activity 425 499 610 889 381 No activity 692 582 No activity No activity 1097

927 No activity No activity 799 898 No activity No activity No activity No activity 1027 No activity No activity No activity

71 751 578 88 50 342 5 192 602 55 95 223 244

In the reporter systems, induction of the QS system can be measured as increasing fluorescence, which means that the presence of an antagonist will result in a decrease in the GFP expression. Growth of the reporter strains was monitored to make sure that the test compounds were not affecting the growth rate. All compounds showing activity in preliminary screens with either of the monitor strains were subsequently tested at different concentrations to establish IC50 and EC50 values. IC50 and EC50 values were calculated from the curves expressing the specific fluorescence (GFP expression/cell density, see Figures 3 and 4 for selected examples), and the results for compounds showing antagonistic activity (below 1 mM) in any one of the QSI screens are shown in Table 2. EC50 values of selected compounds showing agonistic activity below 100 µM are shown in Table 3. Out of the 123 tested compounds, a total of 13 compounds showed antagonistic activity in at least one of the QSI screens.

Unfortunately, none of the novel compounds reported herein showed any noteworthy activities in any of the QSI screens. Interestingly, while the 13 compounds noted above showed activity in the luxI-gfp screen, activities in the lasB-gfp and rhlAgfp screens seemed to differ substantially (Table 2). In general the compounds displayed higher antagonistic activity when tested towards the Lux QS system compared to the Las and Rhl QS systems. This difference, besides a generally higher binding affinity for the Lux receptor protein, might also be explained by the use of different background strains in the monitor screens, namely E. coli for the Lux monitor screen and P. aeruginosa for the Las and Rhl monitor screens. Table 3. Selected EC50 values of compounds showing agonistic activity in concentrations below 100 µM of the QS system with the lasB-gfp (E. coli) reporter Compound 2 3 4 6 7 8 12 14 20

EC50 lasB-gfp (µM) 57 12 3 44 71 2 1 8 2

We note here that the high affinity of compound 12 for the Lux receptor places this compound among the most potent antagonists known for this protein with an IC50 value of 5 µM. Nineteen compounds displayed agonistic activity on the QS system with EC50 values ranging from 1 µM to almost 10 mM, 9 of them with an EC50 value below 100 µM (Table 3).

Figure 3. The half-maximal inhibitory concentration (IC50) for compounds 1 and 6 tested with the following monitor strains: lasB-gfp (A), rhlA-gfp (B) and luxI-gfp (C). IC50 values were calculated with the bioinformatics software PRISM (GraphPad®).

Interestingly, several compounds showed both antagonistic and agonistic activity, as exemplified by compounds 2, 4, 6, 7, 12 and 20. This could indicate that the compounds act as partial

5

agonists, which compete with the natural ligands for binding to LasR, thus leading to an overall inhibition of the QS system in the P. aeruginosa QSI assay. In the E. coli QS screening assay the compounds activate the QS system because no competition with the natural signal molecules is taking place. Another explanation for these results could be the use of different background strains, which can be of importance when screening for modulation of activity, and also suggests that slightly different mechanisms of action can be at play in different organisms.

Figure 4. The half-maximal effective concentration (EC50 ) for compound 12 and compound 20 tested with the lasB-gfp (E. coli) monitor strain. EC50 values were calculated with the bioinformatics software PRISM (GraphPad®).

The present study could not confirm the high potency of the previously reported aryl thioether 42a as a strong LasR antagonist in our P. aeruginosa lasB-gfp screen. The close sulfide, sulfoxide, sulfone and sulfonamide analogues (70-81) also proved completely inactive. One major reason for the discrepancy between the two studies could be the different background strains used to measure the QS modulating effects. Based on the presented biological results, clear SAR trends are difficult to extract. Nevertheless, it appears that the introduction of a triazole as an amide bond isostere is detrimental to any type of activity, and that it is of importance to maintain low molecular weights and rather apolar AHL side chains, as exemplified by the complete failure of compounds 57a-60c to display biological activity. 3. Conclusion In conclusion, we have synthesized a range of new triazolecontaining AHL analogues using efficient solution- and solidphase approaches. These methods enabled the rapid replacement and decoration of key structural elements of natural AHLs. Pooled with a previously synthesized library of triazolecontaining AHL analogues, an expanded set of 123 AHL analogues was tested for both agonistic and antagonistic activity in clinically relevant bacterial strains expressing GFP depending on the biological perturbation. Several compounds showed antagonistic and agonistic activities comparable to those of previously reported triazole-containing AHL analogues identified in E. coli strains harbouring the lacZ gene. The observed discrepancies in the activity of single compounds, when tested in reporter strains of different backgrounds, emphasize the challenges in comparing biological data obtained in different research laboratories, and suggest that times may be ripe for setting up common biological guidelines for the evaluation of compounds within the QS research community. The results presented herein only underlign the complexity of the task in finding non-natural AHL analogues capable of potently modulating the bacterial quorum sensing system of pathogenic bacteria, and much work remains in order to identify clear structure-activity relationships. In a continuous effort to expand our investigations on the homoserine lactone scaffold, and based on the developed solid-phase methodologies, we are currently focusing on the conjugation of various types of HSLs with more complex peptide chains and other types of organic synthons.

4. Experimental 4.1. Biological assays The reporter strains lasB-gfp (P. aeruginosa), rhlA-gfp (P. aeruginosa), luxI-gfp (E. coli) and lasB-gfp (E. coli) were used for measuring modulation of QS. The strains were grown in BT minimal medium (B medium plus 2.5 mg thiamine l-1 ) supplemented with 10% A10, 0.5% (wt/vol) glucose, 0.5% (wt/vol) Casamino Acids for 16 h at 30 °C with shaking at 180 rpm. The biological assays were conducted in 96-well microtiter dishes (Black Isoplate®, Perkin Elmer®, Waltham Massachusetts, USA) according to Hentzer er al.35 The corresponding signal molecule 3-oxo-C6-HSL was added in a final concentration of 100 nM to the screen with the reporter strain, luxI-gfp. Growth and green fluorescent protein (GFP) expression was monitored using Victor™ X4 multilabel plate reader (Perkin Elmer®, Waltham Massachusetts, USA) set at a constant temperature of 34 °C measuring every 15 minutes over a time course of 14 h. GFP expression was measured as fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. 4.2. General chemical methods and instrumentation Unless otherwise stated, all reactions were run under an argon atmosphere. Glassware was dried over a Bunsen flame under vacuum before contact with any of the reactants or solvents. All flasks were equipped with a rubber septum, through which transport of chemicals, from or to the flask, was performed by use of a syringe equipped with a needle. All solvents were of RPHPLC quality, and commercially available reagents were used without further purification. Thin layer chromatography (TLC) was performed on aluminum plates precoated with silica gel (Merck 25, 20x20 cm, 60 F254). The plates were either visualized under UV-light or stained with a solution of KMnO4 (3 g) and K2CO3 (20 g) in water (300 mL) mixed with 5% NaOH (5 mL). Flash column chromatography was performed using silica gel (FLUKA 60758 silica gel 60, particle size 0.035-0.070 mm, 220440 mesh ASTM) in various sizes of glass columns. New compounds were characterized by 1H NMR, RP-HPLC, and HRMS/LC-MS (ESI). For selected compounds 13C NMR was also recorded. 1 H NMR and 13 C NMR spectra were obtained on a Bruker Aspect-3000 spectrometer (operating at 200 MHz for proton and 50 MHz for carbon), a Varian Mercury-300 spectrometer (operating at 300 MHz for proton and 75 MHz for carbon), or a Varian Unity Inova-500 spectrometer (operating at 500 MHz for proton). The chemical shifts (δ) are reported in parts per million (ppm) and the coupling constants (J) in Hz. DMSO-d6 was used as the solvent, and signal positions were measured relative to the signal for DMSO (δ 2.50 ppm for 1H NMR and δ 39.43 for 13C NMR). Analytical RP-HPLC analysis was performed on a Waters Alliance 2695 RP-HPLC system using a Symmetry 60 Å C18 column (d 3.5 µm, 4.6 x 75 mm; column temp: 25 °C; flow: 1 mL/min) with detection at 215 nm and 254 nm. Eluents A (0.1% TFA in H2O) and B (0.1% TFA in acetonitrile) were used in a linear gradient (100% A to 100% B) in a total run time of 13 min. Analytical LC-MS (ESI) analysis was performed on a Waters AQUITY RP-UPLC system equipped with a diode array detector using an AQUITY UPLC BEH C18 column (d 1.7 µm, 2.1× 50 mm; column temp: 65 °C; flow: 0.6 mL/min). Eluents A (0.1% HCO2H in H2O) and B (0.1% HCO2H in acetonitrile) were used in a linear gradient (5% B to 100% B) in a total runtime of 2.6 min. The LC system was coupled to a SQD mass spectrometer. Analytical LC-HRMS (ESI) analysis was performed on an Agilent 1100 RP-LC system equipped with a diode array detector using a Phenomenex Luna C18 column (d 3 µm, 2.1 × 50 mm; column temp: 40 °C; flow:

6

0.4 mL/min). Eluents A (0.1% HCO2 H in H2O) and B (0.1% HCO2 H in acetonitrile) were used in a linear gradient (20% B to 100% B) in a total run time of 15 min. The LC system was coupled to a Micromass LCT orthogonal time-of-flight mass spectrometer equipped with a Lock Mass probe operating in positive electrospray mode. Preparative RP-HPLC was performed on a Waters Alliance reverse-phase HPLC system consisting of a Waters 2545 Binary Gradient Module equipped with a xBridgeTM Prep BEH130 C18 column OBDTM (5µm, 19 x 100 mm, column temp 25 °C, flow rate 20 mL/min), a Waters Photodiode Array Detector (detecting at 210-600 nm), a Waters UV Fraction Manager and a Waters 2767 Sample Manager. Elution was carried out in a linear reversed-phase gradient fashion combining water and acetonitrile buffered with 0.1% TFA.

4 . 2. 1. 2. P ep t i d o-tri a z ol eH SL 5 7b

Solid-phase synthesis was carried out using plastic-syringe technique. Flat-bottomed PE-syringes were fitted with polycarbonate filters, Teflon tubing and Teflon valves, which allow suction to be applied to the syringes. Acid-mediated release of products was carried out in Glass Luer tip style glass syringes fitted with polycarbonate filters. Material sufficient for LCMS analysis was obtained by cleaving a small resin sample (< 5mg) while material for NMR analysis was obtained from cleavage of a larger resin sample (typically 100 mg).

4 . 2. 1. 3. P ep t i d o-tri a z ol e H S L 5 7c

4 . 2. 1. So li d -p h as e s ynt h es is of p ep ti d o-tr ia zol e H S Ls 5 7 a-c - 6 0a- c Attachment of the Rink amide linker to the amino functionalized PEGA800 resin (0.4 mmol/g) was carried out by premixing TBTU (2.88 equiv), NEM (4 equiv) and the Rink amide linker (3 equiv) for 5 min. in DMF. The solution was then added to the preswollen resin in DMF and allowed to react for 2 hours, followed by washing with 6 x DMF. Peptide synthesis was carried out as described for the attachment of the Rink amide linker to PEGA800. Fmoc-deprotection was accomplished by addition of 20% piperidine in DMF for 2 min. and then 18 min, followed by washing with 6 x DMF. Formation of the resinbound 1,4-disubstituted 1,2,3-triazole was accomplished by premixing 2,6-lutidine (2 equiv), azide 61-63 (2 equiv), CuI (1 equiv) and sodium ascorbate (1 equiv) in NMP/H2O 2:1 for 5 min. The solution was then added to the preswollen resin in NMP/H2O 4:1 and allowed to react for 24 hours at room temperature followed by washing of the resin with 6 x pyridine, 6 x DMF and 6 x CH2Cl2. Release of the products was achieved by treatment with 95 % TFA in H2O for 16 hours, followed by washing with 4 x MeCN and 2 x CH2Cl2. All characterization analyses were performed on the crude compounds. 4 . 2. 1. 1. P ep t i d o-tri a z ol e H S L 5 7a Yield: 81% (19 mg). 1 H NMR (500 MHz, DMSO-d6) δ 9.5 (1H, s), 9.26 (1H, d, J = 8.0 Hz), 8.74 (1H, d, J = 8.0 Hz), 8.42 (1H, s), 8.35 (1H, t, J = 5.7 Hz), 8.16 (1H, dd, J = 8.0, 1.3 Hz), 8.04 (2H, d, J = 8.3 Hz), 8.00, (1H, d, J = 7.8 Hz), 7.94 (2H, d, J = 8.3 Hz), 7.79 (1H, t, J = 7.9 Hz), 7.36 (2H, d, J = 7.4 Hz), 7.27 (2H, t, J = 7.6 Hz), 7.22 – 7.10 (3H, m), 4.89 – 4.82 (1H, m), 4.75 – 4.69 (1H, m), 4.47 – 4.42 (1H, m), 4.34 – 4.28 (1H, m), 3.73 (1H, dd, J = 16.8, 6.0 Hz), 3.63 (1H, dd, J = 16.8, 5.6 Hz), 3.19 (1H, dd, J = 13.8, 4.1 H), 3.03 (1H, dd, J = 13.7, 10.8 Hz), 2.55 – 2.49 (1H, m), 2.43 – 2.33 (1H, m); 13C NMR (50 MHz, DMSO-d6) δ 175.1, 171.5, 170.8, 166.0, 164.9, 146.7, 138.4, 136.7, 135.2, 133.5, 132.8, 130.3, 129.1 (2C), 128.2 (2C), 128.1 (2C), 127.4, 126.2, 125.0 (2C), 123.0, 120.7, 118.9, 65.4, 55.2, 48.6, 42.0, 36.9, 28.0; HRMS (ESI) calcd for C31H30N7O6 [M+H]+ 596.2252, found 596.2245.

Yield: 47% (11 mg). 1H NMR (300 MHz, DMSO-d6) δ 9.53 (1H, s,), 9.19 (1H d, J = 8.0 Hz), 8.75 (1H, d, J = 8.0 Hz), 8.36 (1H, t, J = 5.7 Hz), 8.12 (4H, s,), 8.06 – 8.00 (2H, m), 7.97 – 7.91 (2H, m), 7.39 – 7.33 (2H, m), 7.30 – 7.10 (5H, m), 4.89 – 4.77 (1H, m), 4.77 – 4.69 (1H, m), 4.45 (1H, dt, J = 8.5, 1.6 Hz), 4.30 (1H, ddd, J = 10.2, 8.7, 6.6 Hz), 3.73 (1H, dd, J = 16.8, 5.9 Hz), 3.63 (1H, dd, J = 16.8, 5.6 Hz), 3.19 (1H, dd, J = 13.7, 4.2 Hz), 3.08 – 2.93 (2H, m), 2.53 – 2.29 (2H, m); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 171.5, 170.8, 166.0, 164.9, 146.7, 138.6, 138.4, 133.5, 133.3, 132.8, 129.1 (3C), 128.7, 128.2 (2C), 128.1 (2C), 126.2, 125.0 (2C), 120.5, 119.7 (2C), 65.4, 55.1, 48.5, 42.0, 36.9, 28.0; UPLC/MS (ESI) calcd for C31H30N7 O6 [M+H]+ 596.2, found 596.5. Yield: 85% (18 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.94 (1H, d, J = 7.8 Hz), 8.73 (1H, d, J = 8.0 Hz), 8.65 (1H, s), 8.35 (1H, t, J = 5.6 Hz), 7.95 (2H, d, J = 8.4 Hz), 7.88 (2H, d, J = 8.4 Hz), 7.35 (2H, d, J = 7.2 Hz), 7.30 – 7.10 (5H, m), 5.26 (2H, s), 4.74 – 4.62 (2H, m), 4.40 – 4.32 (1H, m), 4.28 – 4.17 (1H, m), 3.72 (1H, dd, J = 16.8, 5.9 Hz), 3.62 (1H, dd, J = 16.7, 5.6 Hz), 3.18 (1H, dd, J = 13.6, 4.0 Hz), 3.02 (1H, dd, J = 13.6, 10.7 Hz), 2.52 – 2.40 (1H, m), 2.26 – 2.11 (1H, m); 13C NMR (50 MHz, DMSO-d6) δ 174.8, 171.6, 170.8, 166.1, 165.6, 145.4, 138.4, 133.4, 133.0, 129.1 (2C), 128.1 (2C), 128.1 (2C), 126.2, 124.7 (2C), 123.8, 65.4, 55.2, 48.2, 42.2, 40.8, 40.3, 36.8, 28.2; HRMS (ESI) calcd for C26H28N7O6 [M+H]+ 534.2096, found 534.2087. 4 . 2. 1. 4. P ep t i d o-tri a z ol e H S L 5 8a Yield: 76% (18 mg). 1H NMR (500 MHz, DMSO-d6) δ 9.45 (1H, s), 9.26 (1H, d, J = 7.9 Hz), 8.82 (1H, d, J = 8.0 Hz), 8.47 (1H, s), 8.42 (1H, s), 8.35 (1H, t, J = 5.7 Hz), 8.17 (1H, d, J = 8.1 Hz), 8.09 (1H, d, J = 7.7 Hz), 8.00 (1H, d, J = 7.8 Hz), 7.83 – 7.76 (2H, m), 7.59 (1H, t, J = 7.7 Hz), 7.36 (2H, d, J = 7.7 Hz), 7.26 (2H, t, J = 7.5 Hz), 7.23 – 7.10 (3H, m), 4.85 (1H, dd J = 18.5, 9.4 Hz), 4.78 – 4.72 (1H, m), 4.45 (1H, t, J = 8.6 Hz), 4.34 – 4.28 (1H, m), 3.74 (1H, dd, J = 16.8, 5.9 Hz), 3.63 (1H, dd, J = 16.8, 5.5 Hz), 3.20 (1H, dd, J = 13.8, 4.1 Hz), 3.05 (1H, dd, J = 13.5, 10.9 Hz), 2.55 – 2.47(1H, m), 2.43 – 2.33 (1H, m); 13C NMR (50 MHz, DMSO-d6) δ 175.1, 171.5, 170.8, 166.2, 164.9, 146.9, 138.4, 136.7, 135.2, 134.8, 130.3, 130.2, 129.1 (2C), 128.9, 128.1 (2C), 127.4, 127.2, 126.2, 124.5, 123.0, 120.2, 118.9, 65.4, 55.1, 48.6, 42.0, 36.9, 28.0; HRMS (ESI) calcd for C31H30N7 O6 [M+H]+ 596.2252, found 596.2234. 4 . 2. 1. 5. P ep t i d o-tri a z ol e H S L 5 8b Yield: 66% (16 mg). 1H NMR (300 MHz, DMSO-d6) δ 9.48 (1H, s), 9.19 (1H, d, J = 8.0 Hz), 8.83 (1H, d, J = 8.1 Hz), 8.42 (1H, s), 8.37 (1H, t, J = 5.8 Hz), 8.18 – 8.02 (5H, m), 7.82 (1H,d, J = 7.9 Hz), 7.60 (1H, t, J = 7.8 Hz), 7.37 (2H, d, J = 7.1 Hz), 7.31 – 7.11 (5H, m), 4.89 – 4.70 (2H, m), 4.45 (1H, dt, J = 8.6, 1.5 Hz), 4.30 (1H, ddd, J = 10.1, 8.8, 6.6 Hz), 3.74 (1H, dd, J = 16.8, 5.9 Hz), 3.63 (1H, dd, J = 16.8, 5.6 Hz), 3.20 (1H, dd, J = 13.8, 4.2 Hz), 3.10 – 2.94 (2H, m), 2.53 – 2.29 (2H, m); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 171.4, 170.8, 166.2, 164.9, 147.0, 138.6, 138,4, 134.8, 133.3, 130.1, 129.1 (4C), 128.9, 128.1 (3C), 127.2, 126.2, 124.5, 120.1 119,6 (2C), 65.4, 55.1, 48.5, 42.0, 36.9, 28.0; UPLC/MS (ESI) calcd for C31H30N7O6 [M+H]+ 596.2, found 596.3. 4 . 2. 1. 6. P ep t i d o-tri a z ol e H S L 5 8c Yield: 84% (18 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.95 (1H, d, J = 7.9 Hz), 8.81 (1H, d, J = 8.0 Hz), 8.58 (1H, s), 8.36 (1H, t, J = 5.7 Hz), 8.30 (1H, s), 8.00 (1H, d, J = 7.7 Hz), 7.75 (1H, d, J = 7.8 Hz), 7.53 (1H, t, J = 7.7 Hz), 7.36 (2H, d, J = 7.1

7

Hz), 7.30 –7.10 (5H, m), 5.28 (2H, s), 4.77 – 4.62 (2H, m), 4.40 – 4.32 (1H, m), 4.28 – 4.18 (1H, m), 3.73 (1H, dd, J = 16.8, 6.0 Hz), 3.62 (1H, dd, J = 16.8, 5.6 Hz), 3.19 (1H, dd, J = 13.6, 4.2 Hz), 3.02 (1H, dd, J = 13.6, 10.8 Hz), 2.52 – 2.41 (1H, m), 2.67 – 2.11 (1H, m); 13C NMR (50 MHz, DMSO-d6) δ 174.8, 171.5, 170.8, 166.2, 165.6, 145.6, 138.4, 134.6, 130.6, 129.1 (2C), 128.8, 128.0 (2C), 127.8, 126.8, 126.2, 124.1, 123.3, 65.3, 55.5, 48.2, 42.0, 36.8, 28.2; HRMS (ESI) calcd for C26H28N7O6 [M+H]+ 534.2096, found 534.2105.

(1H, d, J = 8.0 Hz), 8.53 (1H, s), 8.38 –8.31 (2H, m), 8.10 – 8.00 (4H, m), 7.27 –7.08 (7H, m), 4.87 – 4.76 (1H, m), 4.53 – 4.40 (3H, m), 4.30 (1H, ddd, J = 10.2, 8.7, 6.6 Hz), 3.71 (1H, dd, J = 16.8, 6.0 Hz), 3.58 (1H, dd, J = 16.8, 5.6 Hz), 3.09 – 2.72 (4H, m), 2.54 – 2.28 (4H, m); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 171.6, 171.4, 170.8, 165.0, 147.4, 138.8, 138.0, 132.9, 129.0 (4C), 128.6, 128.0 (2C), 126.2, 120.3, 119.4 (2C), 65.4, 54.4, 48.5, 41.9, 37.1, 34.3, 28.0, 21.1; UPLC/MS (ESI) calcd for C27H30N7 O6 [M+H]+ 548.2, found 548.5.

4 . 2. 1. 7. P ep t i d o-tri a z ol e H S L 5 9a

4 . 2. 1. 1 2. Pep t i do -tri az ol e H SL 60 c

1

Yield: 91% (22 mg). H NMR (300 MHz, DMSO-d6) δ 9.25 (1H, d, J = 8.0 Hz), 8.51 (1H, s), 8.37 – 8.29 (3H, m), 8.13 (1H, d, J = 8.0 Hz), 8.04 (1H, d, J = 8.2 Hz), 7.96 (1H, d, J =7.7 Hz), 7.73 (1H, t, J = 7.9 Hz), 7.26 – 7.11 (7H, m), 4.89 – 4.78 (1H, m), 4-64 – 4.53 (1H, m), 4.52 – 4.40 (2H, m), 4.34 – 4.25 (1H, m), 3.73 (1H, dd, J = 16.8, 6.1 Hz), 3.56 (1H, dd, J = 16.8, 5.6 Hz), 2.55 – 2.31 (2H, m), 1.80 (3H, s); 13C NMR (50 MHz, DMSO-d6) δ 175.1,171.5, 171.1, 170.8, 169.4, 164.9, 144.2, 137.8, 136.8, 135.0, 130.2, 129.1 (2C) 128.0 (2C), 127.1, 126.2, 122.9, 121.4, 118.7, 65.4, 54.3, 52.2, 48.6, 42.0, 36.9, 28.0, 22.5; HRMS (ESI) calcd for C29H33 N8O7 [M+H]+ 605.2467, found 605.2477. 4 . 2. 1. 8. P ep t i d o-tri a z ol e H S L 5 9b Yield: 85% (21 mg). 1H NMR (300 MHz, DMSO-d6) δ 9.15 (1H, d, J = 8.0 Hz), 8.56 (1H, s), 8.37 – 8.30 (2H, m), 8.13 – 7.99 (4H, m), 7.27 – 7.10 (7H, m), 4.87 – 4.76 (1H, m), 4.63 – 4.39 (3H, m), 4.30 (1H, ddd, J = 10.3, 8.7, 6.6 Hz), 3.73 /1H, dd, J = 16.8, 6.2 Hz), 3.56 (1H, dd, J = 16.8, 5.5 Hz), 3.15 – 3.05 (2H, m), 3.00 (1H, dd, J = 14.9, 7.3 Hz), 2.84 (1H, dd, J = 13.8, 9.5 Hz), 2.52 – 2.29 (2H, m), 1.80 (3H, s); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 171.1, 170.8, 169.3, 165.0, 144.2, 138.7, 137.7, 133.0, 129.1 (4C), 128.6, 128.0 (2C), 126.2, 121.3, 119.4 (2C), 65.4, 54.3 52.1, 48.5, 42.0, 36.9, 27.9, 22.4 (2C); UPLC/MS (ESI) calcd for C29H32N8O7 [M+H]+ 605.3, found 605.5. 4 . 2. 1. 9. P ep t i d o-tri a z ol e H S L 5 9c Yield: 94% (20 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.81 (1H, d, J = 7.8 Hz), 8.34 – 8.22 (2H, m), 8.12 (1H, d, J = 7.9 Hz), 7.76 (1H, s), 7.34 – 7.08 (7H, m), 5.13 (2H, d, J = 2.8 Hz), 4.72 – 5.59 (1H, m), 4.53 – 4.41 (1H, m) 4.39 – 4.17 (3H, m), 3.71 (1H, dd, J = 16.7, 6.1 Hz), 3.56 (1H, dd, J = 16.8, 5.5 Hz), 3.15 – 2.95 (2H, m), 2.82, (2H, dd, J = 13.8, 9.6 Hz), 2.52 – 2.37 (1H, m), 2.23 – 2.08 (1H, m), 1.77 (3H, s); 13C NMR (50 MHz, DMSO-d6) δ 174.8, 171.0, 170.8, 169.4, 165.7, 137.8, 129.1 (2C), 128.0 (2C), 126.2, 124.4, 65.3, 54.1, 52.2, 51.2, 48.1, 42.0, 36.9, 28.2, 27.7, 22.4 (2C); HRMS (ESI) calcd for C24H31N8O7 [M+H]+ 543.2310, found 543.2322.

Yield: 79% (15 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.85 (1H, d, J = 8.0 Hz), 8.33 – 8.27 (2H, m), 7.71 (1H, m), 7.34 – 7.06 (7H, m), 5.09 (2H, m), 4.72 – 4.58 (1H, m), 4.51 – 4.41 (1H, m), 4.35 (1H, t, J = 8.7 Hz), 4.27 – 4.17 (1H, m), 3.68 (1H, dd, J = 16.8, 5.9 Hz), 3.58 (1H, dd, J = 16.7, 5.5 Hz), 3.06 (1H, dd, J = 13.8, 4.5 Hz), 2.82 – 2.71 (3H, m), 2.48 – 2.34 (3H, m), 2.24 – 2.08 (1H, m); 13C NMR (50 MHz, DMSO-d6) δ 174.9, 171.6, 170.9, 165.7, 138.1, 129.1 (2C), 128.0 (2C), 126.2, 123.5, 65.4, 54.3, 51.3, 48.2, 42.0, 37.1, 34.6, 28.2, 22.5, 21.1; HRMS (ESI) calcd for C22H28N7O6 [M+H]+ 486.2096, found 486.2118. 4 . 2. 2. Sy nt h esi s of p r op a rg yls ulf o ne a mi des 64- 6 6 Methoxy sulfonyl chloride (2.5 mmol, 1 equiv) was dissolved in dry CH2Cl2, followed by addition of triethylamine (2.5 mmol, 1 equiv) and propargylamine (2.5 mmol, 1 equiv). The reaction mixture was stirred at RT overnight at which point full conversion was observed. The formed ammonium salt was filtered off and the mixture evaporated in vacuo. The crude solid was recrystallized from MeCN and Et 2O, yielding the title compounds as light brown fluffy solids. These were used as such in subsequent reactions. The presence of small amounts of Et3N.HCl salt could still be detected, as evident from the corresponding 1H NMR spectra. 4 . 2. 2. 1. S ul f on ami d e buil di ng bl oc k 64

38

Yield: 91% (514 mg). Mp: 70-72˚C; 1H NMR (300 MHz, DMSO-d6) δ 7.82 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 4.7 (bs, 1H), 3.9 (s, 3H), 3.81 (d, J = 2.5 Hz, 2H), 2.10 (t, J = 2.5 Hz, 1H); 13C NMR (50 MHz, DMSO-d6) δ 163.1, 131.0, 129.6 (2C), 114.2 (2C), 78.1, 72.9, 55.6, 32.8; HRMS (ESI) calcd for C10H12NO3S [M+H]+ 226.0538, found 226.0531. 4 . 2. 2. 2. S ul f on ami d e buil di ng bl oc k 65 Yield: 87% (492 mg). Mp: 49-51˚C; 1H NMR (300 MHz, DMSO-d6) δ 7.50-7.38 (m, 3H), 7.11 (ddd, J = 7.9, 2.6, 1.3 Hz, 1H), 4.84 (t, J = 6.2 Hz, 1H), 3.87-3.83 (m, 5H) 2.12 (t, J = 2.6 Hz, 1H); 13C NMR (50 MHz, DMSO-d6) δ 159.9, 140.6, 130.1, 119.5 (2C), 111.9, 77.6, 73.0, 55.7, 32.9; HRMS (ESI) calcd for C10H12NO3S [M+H]+ 226.0538, found 226.0539.

4 . 2. 1. 10. Pep t i do -tri az ol e H SL 60 a

4 . 2. 2. 3. S ul f on ami d e buil di ng bl oc k 66

Yield: 75% (16 mg). 1H NMR (300 MHz, DMSO-d6) δ 9.23 (1H, d, J = 8.1 Hz), 8.48 (1H, s), 8.39 –8.31 (3H, m), 8.08 – 8.02 (1H, m), 7.95 (1H, d, J = 7.7 Hz), 7.72 (1H, t, J = 7.9 Hz), 7.25 – 7.07 (7H, m), 4.89 – 4.78 (1H, m), 4.53 – 4.39 (2H, m), 4.35 – 4.25 (1H, m), 3.70 (1H, dd, J = 16.8, 5.8 Hz), 3.59 (1H, dd, J = 16.9, 5.6 Hz), 3.05 (1H, dd, J = 13.8, 4.3 Hz), 2.91 – 2.71 (3H, m), 2.59 – 2.29 (4H, m); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 171.6, 171.5, 170.9, 165.0, 147.4, 138.0, 136.9, 135.0, 130.2, 129.1 (2C), 128.0 (2C), 127.1, 126.2, 122.8, 120.4, 118.6, 65.4, 54.4, 48.6, 42.0, 37.1, 34.4, 28.0, 21.1; HRMS (ESI) calcd for C27H30N7 O6 [M+H]+ 548.2252, found 548.2241.

Yield: 90% (576 mg). Mp: 111-113 ˚C; 1H NMR (300 MHz, DMSO-d6δ 7.51 (dd, J = 8.5, 2.2 Hz, 1H), 7.35 (d, J = 2.1 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 4.76 (s, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.83 (d, J = 2.0 Hz, 2H), 2.12 (t, J = 2.5 Hz, 1H); 13C NMR (300 MHz, DMSO-d6) δ 152.8, 149.1, 131.0, 121.4, 110.5, 109.8, 78.1, 72.9, 56.2 (2C), 32.9; HRMS (ESI) calcd for C11H14NO4S [M+H]+ 256.0644, found 256.0639.

4 . 2. 1. 11. Pep t i do -tri az ol e H SL 60 b Yield: 75% (16 mg). 1H NMR (300 MHz, DMSO-d6) δ 9.15

4 . 2. 3. Sy nt h esi s of p r op a rg yls ulfi d es 67- 69. Methoxy thiol (3.0 mmol, 1 equiv) was dissolved in DMF (10 mL). Propargyl bromide (3.6 mmol, 1.2 equiv) was added followed by potassium carbonate (4.2 mmol, 1.4 equiv). The slurry was allowed to stir at RT for two days at which point full conversion was observed on TLC. The mixture was transferred to

8

a separatory funnel containing water (20 mL) and Et 2O (20 mL). The aqueous phase was extracted twice with Et2O and the combined organic layers were washed with water (5 x 20 mL) and brine (20 mL). The organic layer was then dried over anhydrous MgSO4 and concentrated in vacuo to give a brown oil. The compounds were purified by flash column chromatography on silica gel (10% EtOAc in heptane) to give the title compounds as light brown oils. 4 . 2. 3. 1. S u lfi de b uil di n g b l ock 6 7

39

Yield: 76% (408 mg). 1H NMR (300 MHz, CDCl3) δ 7.48 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 8.9 Hz, 2H), 3.81 (s, 3H), 3.49 (d, J = 2.6 Hz, 2H), 2.22 (t, J = 2.6, 1H); 13C NMR (50 MHz, CDCl3 ) δ 159.8, 134.5 (2C), 125.0, 114.6 (2C), 80.2, 71.6, 55.3, 24.6; HRMS (ESI) calcd for [M+H]+ 179.0531, found 179.0526. 4 . 2. 3. 2. S u lfi de b uil di n g b l ock 6 8 Yield: 79% (424 mg). 1H NMR (300 MHz, CDCl3) δ 7.277.20 (m, 1H), 7.05-6.99 (m, 2H), 6.81-6.76 (m, 1H), 3.81 (s, 3H), 3.62 (dd, J = 2.6, 0.5 Hz, 2H), 2.25 (dt, J = 2.6, 0.5 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 159.8, 136.3, 129.8, 121.8, 115.0, 112.7, 79.8, 71.6, 55.3, 22.3; HRMS (ESI) calcd for [M+H]+ 179.0531, found 179.0526. 4 . 2. 3. 3. S u lfi de b uil di n g b l ock 6 9 Yield: 80% (502 mg). 1H NMR (300 MHz, CDCl3) δ 7.147.08 (m, 1H), 6.83 (d, J = 8.2 Hz, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 3.52 (d, J = 2.6 Hz, 1H), 2.24 (t, J = 2.6 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 149.2, 149.0, 125.4 (2C), 115.8, 111.5, 80.3, 71.6, 55.9, 24.5; HRMS (ESI) calcd for C11H13O2S [M+H]+ 209.0636, found 209.0633. 4 . 2. 4. Sy n t hesi s of sulf o nam i de H S Ls a nd s u lfi de H S Ls 7 0 -7 5 by C u AA C Azido HSL 62 (0.60 mmol, 1 equiv) was dissolved in MeCN (0.075 M). CuI (0.090 mmol, 0.15 equiv) was added followed by either 64-66 or 67-69 (0.75 mmol, 1.25 equiv). DIPEA (1.80 mmol, 3. equiv) was added and precipitation of product was observed immediately. The reaction was stirred overnight at RT at which point full conversion was observed by LCMS. The precipitate was filtered off and washed with cold MeCN. The filtrate was concentrated in vacuo, redissolved in boiling AcOH, and subjected to hot filtration. The AcOH filtrate was concentrated in vacuo and the solid was recrystallized either from MeOH/Et2O (73-74), MeCN/Et2O (71-72) or CH2Cl2 (70, 75). The two sets of precipitates for each compound were combined. The HSL sulfides were isolated as yellow solids and the HSL sulfonamides as brown solids. 4 . 2. 4. 1. H S L Sulf on a mi de 7 0 Yield: 90% (255 mg). Mp: 180-183 ˚C; 1H NMR (300 MHz, DMSO-d6) δ 9.17 (d, J = 8.0 Hz, 1H), 8.58 (s, 1H), 8.14-8.03 (m, 2H), 7.98 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.9 Hz, 2H), 7.05 (d, J = 8.9 Hz, 2H), 4.88-4.76 (m, 1H), 4.42 (t, J = 8.6 Hz, 1H), 4.354.24 (m, 1H), 4.12 (d, J = 5.5 Hz, 1H), 3.75 (s, 3H), 2.55-2.13 (m, 2H); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 164.9, 162.1, 144.7, 138.5, 133.1, 131.9, 129.0 (2C), 128.8 (2C), 121.6, 119.5 (2C), 114.1 (2C), 65.4, 55.5, 48.5, 38.0, 27.9; HRMS (ESI) calcd for C21H21 N5O6S [M+H]+ 472.1291, found 472.1281. 4 . 2. 4. 2. H S L Sulf on a mi de 7 1 Yield: 87% (246 mg). Mp: 155-157 ˚C; 1H NMR (300 MHz, DMSO-d6) δ 9.18 (d, J = 7.9 Hz, 1H), 8.62 (s, 1H), 8.29 (t, J = 6.0 Hz, 1H), 8.06 (d, J = 8.7 Hz, 2H), 7.97 (d, J = 8.7 Hz, 2H), 7.46 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.29-7.26 (s, 1H), 7.12 (dd, J = 7.8, 2.2 Hz, 1H), 4.86-4.78 (m, 1H), 4.44 (t, J

= 8.8 Hz, 1H), 4.34-4.24 (m, 1H), 4.16 (d, J = 6.0 Hz, 2H), 3.77 (s, 3H), 2.53-2.26 (m, 2H); 13C NMR (50 MHz, DMSO-d6 ) δ 175.2, 164.9, 159.3, 144.6, 141.6, 138.5, 133.1, 130.2, 129.0 (2C), 121.7, 119.5 (2C), 118.7, 118.3, 111.5, 65.4, 55.5, 48.5, 38.0, 28.0; HRMS (ESI) calcd for C21 H21N5O6 S [M+H]+ 472.1291, found 472.1269. 4 . 2. 4. 3. H S L S ulf on am i de 7 2 Yield: 91% (274 mg). Mp: 216-219 ˚C; 1H NMR (300 MHz, DMSO-d6) δ 9.17 (d, J = 8.0 Hz, 1H), 8.56 (s, 1H), 8.14-8.02 (m, 3H), 7.95 (d, J = 8.6 Hz, 2H), 7.36 (dd, J = 8.3, 1.7 Hz, 1H), 7.25 (d, J = 1.8 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 4.88-4.78 (m, 1H), 4.44 (t, J = 8.6 Hz, 1H), 4.35-4.24 (m, 1H), 4.13 (d, J = 5.9 Hz, 2H), 3.78 (s, 3H), 3.73 (s, 3H), 2.52-2.28 (m, 2H); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 164.9, 151.8, 148.5, 144.7, 138.5, 133.1, 131.9, 129.0 (2C), 121.6, 120.4, 119.5 (2C), 110.9, 109.5, 65.4, 55.7 (2C), 48.5, 38.0, 28.0; HRMS (ESI) calcd for C22H23N5 O7S [M+H]+ 502.1396, found 502.1397. 4 . 2. 4. 4. H S L S ulfi de 7 3 Yield: 83% (211 mg). Mp: 184-186 ˚C; 1H NMR (300 MHz, DMSO-d6) δ 9.17 (d, J = 7.9 Hz, 1H), 8,72 (s, 1H), 8.06 (d, J = 9.0 Hz, 2H), 8.02 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 4.48-4.39 (m, 1H), 4.44 (dt, J = 8.5, 1.32 Hz, 1H), 4.34-4.22 (m, 3H), 3.73 (s, 3H) 2.53-2.26 (m, 2H); 13 C NMR (50 MHz, DMSO-d6) δ 175.2, 164.9, 158.7, 145.4, 138.6, 133.1, 132.6 (2C), 129.0 (2C), 125.2, 121.4, 119.5 (2C), 114.7 (2C), 65.4, 55.2, 48.5, 29.4, 28.0; HRMS (ESI) calcd for C21H21N4 O4S [M+H]+ 425.1284, found 425.1302. 4 . 2. 4. 5. H S L S ulfi de 7 4 Yield: 87% (221 mg). Mp: 168-171 ˚C; 1H NMR (300 MHz, DMSO-d6) δ 9.17 (d, J = 7.91 Hz, 1H), 8.82 (s, 1H), 8.10-7.99 (m, 4H), 7.23 (t, J = 8.2 Hz, 1H), 6.99-6.90 (m, 2H), 6.79-6.73 (m, 1H), 4.88-4.73 (m, 1H), 4.50-4.35 (m, 3H), 4.36-4.21 (m, 1H), 3.72 (s, 3H), 2.46-2.23 (m, 2H); 13C NMR (50 MHz, DMSO-d6) δ 175.2, 164.9, 159.7, 145.2, 138.6, 136.9, 133.1, 129.9, 129.0 (2C), 121.5, 120.2, 119.5 (2C), 113.3, 111.9, 65.4, 55.1, 48.5, 28.0, 27.0; HRMS (ESI) calcd for C21H21N4O4S [M+H]+ 425.1284, found 425.1275. 4 . 2. 4. 6. H S L S ulfi de 7 5 Yield: 75% (204 mg). Mp: 106-108 ˚C; 1H NMR (300 MHz, DMSO-d6) δ 9.16 (d, J = 8.0 Hz, 1H), 8.73 (s, 1H), 8.05 (s, 4H), 7.00-6.87 (m, 3H), 4.86-4.75 (m, 1H), 4.44 (t, J = 8.9 Hz, 1H), 4.28 (s, 3H), 3.72 (s, 6H), 2.53-2.26 (m, 2H); 13C NMR (50 MHz, DMSO-d6) δ 180.6, 170.3, 153.7, 150.8, 144.0, 138.4, 134.4 (2C), 130.9, 128.9, 126.8, 124.9 (2C), 120.0, 117.6, 70.8, 60.9 (2C), 53.9, 34.6, 33.3; HRMS (ESI) calcd for C22H22N4O5S [M+H]+ 455.1389, found 455.1368. 4 . 2. 5. Sy nt h esi s of H S L su lf oxi d es 76- 78 b y o x i dati o n of s ul fi des HSL sulfides 73-75 (0.06 mmol, 1 equiv) were dissolved in a 1:1 mixture of H2O and 1,4-dioxane (10 mL) at 70 ˚C. H2O2 (0.60 mmol, 10 equiv) was added (3.5 % w/w in H2O) and the reaction was stirred for 3 days. At this point, HSL sulfides 73 and 74 were fully converted whereas the HSL sulfide 75 was stirred for another 5 days with an additional 10 equiv of H2O2. The reaction mixtures were worked up by extracting the mixture with EtOAc (3 x 10 ml). The combined organic layers were washed with brine (10 mL) and water (10 mL), dried over anhydrous MgSO4 and concentrated in vacuo. The resulting solids were purified by preparative HPLC (yields ranging from 25 to 35% (710 mg)).

9

4 . 2. 5. 1. H S L Sulf oxi d e 76 Purity: 79% (RP-HPLC); 1H NMR (300 MHz, DMSO-d6 ) δ 9.16 (d, J = 8.0 Hz, 1H), 8.70 (s, 1H), 8.01-8.11 (m, 4H), 7.51 (d, J = 8.7 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H), 4.88-4.76 (m, 1H), 4.37-4.48 (m, 2H), 4.23-4.34 (m, 2H), 3.80 (s, 3H), 2.26-2.46 (m, 2H); HRMS (ESI) calcd for C21H20 N4O5 S [M+H]+ 441.1233, found 441.1232. 4 . 2. 5. 2. H S L Sulf oxi d e 77 Purity: 82% (RP-HPLC); 1H NMR (300 MHz, DMSO-d6 ) δ 9.17 (d, J = 8.0 Hz, 1H), 8.71 (s, 1H), 8.02-8.10 (m, 4H), 7.45 (t, J = 7.8, 1H), 7.10 (m, 3H), 4.76-4.88 (m, 1H), 4.38-4.54 (m, 2H), 4.34-4.24 (m, 2H), 3.75 (s, 3H), 2.53-2.26 (m, 2H); HRMS (ESI) calcd for C21H20N4 O5S [M+H]+ 441.1233, found 441.1240. 4 . 2. 5. 3. H S L Sulf oxi d e 78 Purity: 78% (RP-HPLC); 1H NMR (300 MHz, DMSO-d6 ) δ 9.17 (d, J = 8.00 Hz, 1H), 8.70 (s, 1H), 8.22-7.87 (m, 4H), 7.10 (s, 3H), 5.10-4.67 (m, 1H), 4.44 (m, 2H), 4.29 (m, 2H), 2.442.28 (m, 2H); HRMS (ESI) calcd for C22H22N4 O6S [M+H]+ 471.1338, found 471.1331. 4 . 2. 6. Sy n t hesi s of H SL sulf o nes 7 9- 8 1 b y oxi d ati on o f s ulfi des. HSL sulfides 73-75 (0.06 mmol, 1 equiv) were dissolved in a 1:1 mixture of H2O and 1,4-dioxane (5 mL) at 70 ˚C. m-CPBA (0.18 mmol, 3 equiv) was added and the reaction was stirred for 2 days. At this point, HSL sulfides 73 and 74 were fully converted whereas the HSL sulfide 75 was stirred for another day with an addition of 2 equiv of m-CPBA. The reaction mixtures were worked up by extracting the H2O/dioxane mixture with EtOAc (3 x 10 mL). The combined organic phases were washed with brine (10 ml) and water (10 ml), dried over anhydrous MgSO4 and concentrated in vacuo. The resulting solids were purified by preparative HPLC (yields ranging from 20 to 35% (5-10 mg)). 4 . 2. 6. 1. H S L Sulf on e 79 Purity: 95% (RP-HPLC); 1H NMR (300 MHz, DMSO-d6 ) δ 9.17 (d, J = 8.00 Hz, 1H), 8.87 (s, 1H), 8.07 (s, 4H), 7.13 (d, J = 9.0 Hz, 2H), 7.71 (d, J = 9.0 Hz, 1H), 5.08-4.63 (m, 3H), 4.44 (dt, J = 8.7, 1.7 Hz, 1H), 4.36-4.23 (m, 1H), 3.85 (s, 3H), 2.482.22 (m, 2H); HRMS (ESI) calcd for C21H20N4 O6S [M+H]+ 457.1182, found 457.1176. 4 . 2. 6. 2. H S L Sulf on e 80 Purity: 93% (RP-HPLC); 1H NMR (300 MHz, DMSO-d6 ) δ 9.17 (d, J = 7.89 Hz, 1H), 8.88 (s, 1H), 8.07 (s, 4H), 7.54 (m, 1H), 7.40-7.26 (m, 3H), 4.95 (s, 2H), 4.90-4.74 (m, 1H), 4.494.38 (m, 1H), 4.35-4.24 (m, 1H), 3.81 (s, 3H), 2.45-2.29 (m, 2H); HRMS (ESI) calcd for C21H20N4O6S [M+H]+ 457.1182, found 457.1183. Purity: >95% (RP-HPLC); 1H NMR (300 MHz, DMSO-d6) δ 9.17 (d, J = 7.9 Hz, 1H) 8.86 (s, 1H), 8.07 (s, 4H), 7.34 (dd, J = 8.5, 2.0 Hz, 1H), 7.25 (d, J = 2.0 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 4.90-4.76 (m, 3H), 4.44 (dt, J = 8.6, 8.6, 1.5 Hz, 1H), 4.374.23 (m, 1H), 3.85 (s, 3H), 3.77 (s, 3H), 2.48-2.26 (m, 2H); HRMS (ESI) calcd for C21H20N4O6S [M+H]+ 487.1287, found 487.1287. L-Homoserine

4 . 2. 8. Sy nt h esi s of t ri az olyl d i hy d r of ur an on es 83a-l b y C uA AC Azidodihydrofuranone 82 (0.79 mmol, 1 equiv) and alkyne (1.18 mmol, 1.5 equiv) were dissolved in MeCN (1 mL) in a 3.5 mL vial. A solution of Cu(I) (0.12 mmol, 0.15 equiv) in MeCN (0.5 mL) was added, followed by DIPEA (2.37 mmol, 3 equiv). The mixture was stirred at RT overnight. The solvent was removed in vacuo, and the residue was purified by flash column chromatography (EtOAc:heptane) to give the title compounds. 4 . 2. 8. 1. 1 , 4- Tri az ol e l act o ne 8 3a Yield: 75% (115 mg). NMR (300 MHz, DMSO-d6) δ 8.07 (s, 1H), 5.92 – 5.81 (m, 1H), 4.63-4.52 (m, 1H), 4.49 – 4.36 (m, 1H), 2.91 – 2.68 (m, 2H), 2.61 (t, J = 7.5 Hz, 2H), 1.69 – 1.55 (m, 2H), 0.92 (td, J = 7.3, 1.4 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 147.0, 122.1, 66.0, 57.4, 29.2, 27.0, 22.1, 13.6; HRMS (ESI) calcd for C9H13N3 O2 [M+H]+ 196.1081, found 196.1076. 4 . 2. 8. 2. 1 , 4- Tri az ol e l act o ne 8 3b Yield: 79% (130 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.06 (s, 1H), 5.85 (dd, J = 10.8, 9.1 Hz, 1H), 4.57 (td, J = 8.5, 2.5 Hz, 1H), 4.42 (ddd, J = 9.9, 8.7, 6.5 Hz, 1H), 2.92 – 2.68 (m, 2H), 2.63 (t, J = 7.6 Hz, 2H), 1.66 – 1.51 (m, 2H), 1.42 – 1.25 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.5, 147.3, 122.1, 66.1, 57.4, 31.0, 29.3, 24.7, 21.7, 13.7; 210.3; HRMS (ESI) calcd for C10H15N3O2 [M+H]+ 210.1237, found 210.1236. 4 . 2. 8. 3. 1 , 4- Tri az ol e l act o ne 8 3c Yield: 93% (164 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.06 (s, 1H), 5.86 (dd, J = 10.8, 9.1 Hz, 1H), 4.57 (td, J = 8.6, 2.5 Hz, 1H), 4.42 (ddd, J = 9.8, 8.8, 6.5 Hz, 1H), 2.91 – 2.71 (m, 2H), 2.62 (t, J = 7.6 Hz, 2H), 1.68 – 1.50 (m, 2H), 1.37 – 1.23 (m, 4H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 147.2, 122.0, 66.0, 57.4, 30.8, 29.2, 28.5, 24.9, 21.8, 13.8; HRMS (ESI) calcd for C11H17N3 O2 [M+H]+ 224.1394, found 224.1395. 4 . 2. 8. 4. 1 , 4- Tri az ol e l act o ne 8 3d

4 . 2. 6. 3. H S L Sulf on e 81

4 . 2. 7. Sy n t hesi s of azi d odi hy d rof u ran on e 82

CuSO4.5H2O (50 mg, 0.2 mmol) were added. The resulting mixture was stirred at RT overnight. The solvent was removed in vacuo and the residue was redissolved in H2O (300 mL). The solution was acidified with aqueous 1M HCl to pH = 2, and extracted with EtOAc (3 x 200 mL). The organic layers were combined, dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc:heptane) to afford the title compound as a yellow oil. Yield: 54% (1.37 g). 1H NMR (300 MHz, DMSO-d6 ) δ 4.75 (dd, J = 10.2, 8.7 Hz, 1H), 4.36 (td, J = 8.8, 2.2 Hz, 1H), 4.27 – 4.17 (m, 1H), 2.60 – 2.47 (m, 1H), 2.13 – 1.97 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 174.35, 65.93, 56.46, 28.25.

Yield: 95% (178 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.06 (s, 1H), 5.86 (dd, J = 10.8, 9.1 Hz, 1H), 4.56 (td, J = 8.5, 2.5 Hz, 1H), 4.42 (ddd, J = 9.8, 8.7, 6.5 Hz, 1H), 2.91 – 2.67 (m, 2H), 2.62 (t, J = 7.6 Hz, 2H), 1.67 – 1.49 (m, 2H), 1.42 – 1.14 (m, 6H), 0.86 (t, J = 6.8 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 147.2, 122.0, 66.0, 57.4, 31.0, 29.2, 28.8, 28.2, 24.9, 22.0, 13.9; HRMS (ESI) calcd for C12H19 N3O2 [M+H]+ 238.1550, found 238.1554.

40

lactone hydrobromide salt (3.64 g, 20 mmol) was dissolved in MeOH (100 mL). Imidazole-1-sulfonyl azide hydrochloride (5.0 g, 24 mmol), K2CO3 (7.4 g, 54 mmol), and

4 . 2. 8. 5. 1 , 4- Tri az ol e l act o ne 8 3e Yield: 79% (120 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.04 (s, 1H), 5.83 (dd, J = 10.8, 9.1 Hz, 1H), 4.56 (td, J = 8.6, 2.4 Hz, 1H), 4.41 (ddd, J = 9.7, 8.9, 6.5 Hz, 1H), 2.90 – 2.65 (m, 2H),

10

1.96 (tt, J = 8.4, 5.0 Hz), 0.96 – 0.87 (m, 2H), 0.76 – 0.68 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 149.2, 120.9, 66.0, 57.4, 29.2, 7.6, 7.6 (2C); HRMS (ESI) calcd for C9H11N3O2 [M+H]+ 194.0924, found 194.0925. 4 . 2. 8. 6. 1 , 4- Tri az ol e l act o ne 8 3f Yield: 72% (134 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.05 (s, 1H), 5.85 (dd, J = 10.8, 9.2 Hz, 1H), 4.56 (td, J = 8.6, 2.5 Hz, 1H), 4.49 – 4.36 (m, 1H), 2.92 – 2.60 (m, 3H), 2.06 – 1.85 (m, 2H), 1.84 – 1.61 (m, 3H), 1.50 – 1.11 (m, 5H); 13C NMR (75 MHz, DMSO-d6) δ 172.5, 152.5, 120.9, 66.1, 57.5, 34.6, 32.5 (2C), 29.3, 25.6, 25.6 (2C); HRMS (ESI) calcd for C12H17N3O2 [M+H]+ 236.1394, found 236.1390. 4 . 2. 8. 7. 1 , 4- Tri az ol e l act o ne 8 3g Yield: 80% (149 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.66 (s, 1H), 7.89 (dd, J = 2.9, 1.2 Hz), 7.67 (dd, J = 5.0, 2.9 Hz, 1H), 7.52 (dd, J = 5.0, 1.3 Hz, 1H), 5.96 (dd, J = 10.9, 9.1 Hz, 1H), 4.61 (td, J = 8.6, 2.4 Hz, 1H), 4.46 (ddd, J = 15.3, 8.8, 6.5 Hz, 1H), 2.98 – 2.74 (m, 2H); HRMS (ESI) calcd for C10H9N3O2S [M+H]+ 236.0488, found 236.0484.

4 . 2. 8. 1 2. 1, 4- T ri a z ol e l a ct on e 83l Yield: 80% (203 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.42 (t, J = 7.9 Hz, 2H), 7.13 (dt, J = 16.0, 7.5 Hz, 5H), 5.97 (dd, J = 10.8, 9.2 Hz, 1H), 4.62 (td, J = 8.6, 2.2 Hz, 1H), 4.55 – 4.37 (m, 1H), 3.11 – 2.65 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 156.7, 156.3, 146.3, 130.2, 127.0, 125.6, 123.8, 121.3, 119.0, 118.9, 66.2, 57.9, 29.3; HRMS (ESI) calcd for C18H15 N3O3 [M+H]+ 322.1186, found 322.1189. 4 . 2. 9. Sy nt h esi s of t ri az olyl d i hy d r of ur an on es 84a-l b y Ru AAC Azidodihydrofuranone 82 (0.44 mmol, 1 equiv), alkyne (0.66 mmol, 1.5 equiv) and Cp*Ru(PPh3)2Cl (0.013 mmol, 3 mol%) were dissolved in THF (3 mL). The mixture was stirred at 65 °C overnight. For the synthesis of 84a, 84c, and 84h, incomplete conversion of the azide was observed, and an additional 3 mol% of Cp*Ru(PPh3) 2Cl was added. After another 16 h at 65 °C, the solvent was removed in vacuo and the residue was purified by flash column chromatography on silica gel (EtOAc:heptane) to give the title compounds. 4 . 2. 9. 1. 1 , 5- Tri az ol e l act o ne 8 4a

4 . 2. 8. 8. 1 , 4- Tri az ol e l act o ne 8 3h Yield: 79% (143 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.82 (s, 1H), 7.87 (d, J = 7.4 Hz, 2H), 7.54 – 7.43 (m, 2H), 7.41 – 7.29 (m, 1H), 5.99 (t, J = 10.0 Hz, 1H), 4.63 (td, J = 8.5, 2.1 Hz, 1H), 4.47 (td, J = 9.1, 6.9 Hz, 1H), 3.03 – 2.77 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 146.7, 130.4, 129.0, 128.2, 125.2, 121.7, 66.2, 57.9, 29.3; HRMS (ESI) calcd for C12H11N3O2 [M+H]+ 230.0924, found 230.0926.

Yield: 63% (54 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.60 (s, 1H), 5.78 (t, J = 9.6 Hz, 1H), 4.69 – 4.55 (m, 1H), 4.43 (dd, J = 16.6, 8.6 Hz, 1H), 3.00 – 2.77 (m, 2H), 2.73 – 2.58 (m, 2H), 1.63 (sixt, J = 7.3 Hz, 2H), 0.94 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 138.8, 131.7, 66.3, 55.0, 29.0, 24.0, 21.3, 13.5; HRMS (ESI) calcd for C9H13N3O2 [M+H]+ 196.1081, found 196.1082. 4 . 2. 9. 2. 1 , 5- Tri az ol e l act o ne 8 4b

4 . 2. 8. 9. 1 , 4- Tri az ol e l act o ne 8 3i 1

Yield: 83% (162 mg). H NMR (300 MHz, DMSO-d6) δ 8.80 (d, J = 1.2 Hz, 1H), 7.99 – 7.85 (m, 2H), 7.37 – 7.24 (m, 2H), 6.06 – 5.92 (m, 1H), 4.63 (td, J = 8.5, 1.3 Hz, 1H), 4.55 – 4.39 (m, 1H), 3.02 – 2.59 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 172.4, 161.9 (d, J = 244.7 Hz), 145.9, 127.3 (d, J = 8.3 Hz), 126.9 (d, J = 3.0 Hz), 121.6, 116.0 (d, J = 21.7 Hz), 66.2, 57.9, 29.3; HRMS (ESI) calcd for C12H10 FN3O2 [M+H]+ 248.0830, found 248.0829. 4 . 2. 8. 10. 1 , 4- T ri a z ol e l a ct on e 83j Yield: 69% (141 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.69 (s, 1H), 7.94 – 7.73 (m, 2H), 7.13 – 6.99 (m, 2H), 5.96 (dd, J = 10.8, 9.2 Hz, 1H), 4.62 (td, J = 8.5, 2.4 Hz, 1H), 4.54 – 4.38 (m, 1H), 3.79 (s, 3H), 3.00 – 2.64 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 159.2, 146.6, 126.6, 122.9, 120.7, 114.4, 66.2, 57.8, 55.2, 29.3; HRMS (ESI) calcd for C13H13N3O3 [M+H]+ 260.1030, found 260.1036. 4 . 2. 8. 11. 1 , 4- T ri a z ol e l a ct on e 83 k Yield: 95% (223 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.87 (dd, J = 8.4, 2.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 6.00 (dd, J = 10.9, 9.1 Hz, 1H), 4.63 (td, J = 8.6, 2.2 Hz, 1H), 4.47 (ddd, J = 15.2, 8.8, 6.4 Hz, 1H), 3.02 – 2.56 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.2, 144.5, 131.8, 131.3, 131.0, 130.4, 126.8, 125.3, 122.8, 66.2, 58.00, 29.3; HRMS (ESI) calcd for C12H9Cl2N3O2 [M+H]+ 298.0145, found 298.0143.

Yield: 62% (57 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.60 (s, 1H), 5.77 (t, J = 9.6 Hz, 1H), 4.62 (ddd, J = 8.7, 7.2, 4.0 Hz, 1H), 4.51 – 4.32 (m, 1H), 2.94 – 2.75 (m, 2H), 2.69 (dd, J = 8.5, 6.9 Hz, 2H), 1.68 – 1.47 (m, 2H), 1.35 (sixt, J = 7.5 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 139.0, 131.7, 66.3, 55.0, 29.8, 28.9, 21.8, 21.7, 13.6; UP-LC/MS (ESI) calcd for C10 H15N3O2 [M+H]+ 209.1, found 209.2. 4 . 2. 9. 3. 1 , 5- Tri az ol e l act o ne 8 4c Yield: 67% (66 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.60 (s, 1H), 5.77 (t, J = 9.6 Hz, 1H), 4.69 – 4.54 (m, 1H), 4.44 (dd, J = 16.5, 8.6 Hz, 1H), 3.01 – 2.76 (m, 2H), 2.68 (dd, J = 8.5, 7.0 Hz, 2H), 1.89 – 1.36 (m, 2H), 1.36 – 1.17 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 139.0, 131.7, 66.3, 55.0, 30.7, 28.9, 27.4, 22.0, 21.8, 13.9; HRMS (ESI) calcd for C11H17N3 O2 [M+H]+ 224.1394, found 224.1394. 4 . 2. 9. 4. 1 , 5- Tri az ol e l act o ne 8 4d Yield: 75% (78 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.60 (s, 1H), 5.77 (t, J = 9.6 Hz, 1H), 4.68 – 4.55 (m, 1H), 4.51 – 4.36 (m, 1H), 3.01 – 2.77 (m, 2H), 2.68 (dd, J = 8.5, 7.0 Hz, 2H), 1.79 – 1.44 (m, 2H), 1.44 – 1.07 (m, 6H), 0.86 (t, J = 6.7 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 139.0, 66.3, 55.0, 49.3, 30.9, 29.0, 28.2, 27.7, 22.1, 22.0, 13.9; HRMS (ESI) calcd for C12H19N3 O2 [M+H]+ 238.1550, 238.1546. 4 . 2. 9. 5. 1 , 5- Tri az ol e l act o ne 8 4e

11

Yield: 79% (67 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.48 (s, 1H), 5.95 (t, J = 9.7 Hz, 1H), 4.63 (dt, J = 8.7, 5.7 Hz, 1H), 4.47 (q, J = 8.4 Hz, 1H), 2.88 (dt, J = 8.6, 5.7 Hz, 2H), 2.02 – 1.76 (m, 1H), 1.14 – 0.90 (m, 2H), 0.85 – 0.60 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.4, 141.1, 130.3, 66.3, 55.4, 28.7, 7.1, 7.0, 3.3; HRMS (ESI) calcd for C9 H11N3O2 [M+H]+ 194.0924, found 194.0924. 4 . 2. 9. 6. 1 , 5- Tri az ol e l act o ne 8 4f Yield: 85% (88 mg). 1H NMR (300 MHz, DMSO-d6) δ ppm 1.13-1.50 (m, 1H), 1.61-2.00 (m, 1H), 2.67-2.96 (m, 3H), 4.43 (q, J = 8.59 Hz, 1H), 4.63 (dt, J = 8.70, 5.85 Hz, 1H), 5.81 (t, J = 9.63 Hz, 1H), 7.60 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 172.5, 144.0, 130.0, 66.3, 54.9, 32.4, 32.1 (2C), 29.3, 25.6, 25.6, 25.2; HRMS (ESI) calcd for C12H17 N3O2 [M+H]+ 236.1394, found 236.1395. 4 . 2. 9. 7. 1 , 5- Tri az ol e l act o ne 8 4g Yield: 95% (98 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.94 (dd, J = 2.8, 1.2 Hz, 1H), 7.82 (dd, J = 5.0, 2.9 Hz, 1H), 7.40 (dd, J = 5.0, 1.2 Hz, 1H), 5.82 (t, J = 9.5 Hz, 1H), 4.69 – 4.58 (m, 1H), 4.47 (q, J = 8.5 Hz, 1H), 3.09 – 2.83 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.6, 134.6, 132.6, 128.2, 127.7, 126.4, 125.6, 66.5, 55.9, 29.3; HRMS (ESI) calcd for C10H9N3O2S [M+H]+ 236.0488, found 236.0498. 4 . 2. 9. 8. 1 , 5- Tri az ol e l act o ne 8 4h

129.3, 126.5, 66.5, 55.8, 29.2; HRMS (ESI) calcd for C12H9Cl2N3O2 [M+H]+ 298.0145, found 298.0146. 4 . 2. 9. 1 2. 1, 5- T ri a z ol e l a ct on e 84l Yield: 70% (99 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.65 – 7.38 (m, 4H), 7.31 – 7.04 (m, 5H), 5.71 (t, J = 9.6 Hz, 1H), 4.63 (td, J = 8.3, 2.7 Hz, 1H), 4.45 (dd, J = 16.1, 8.7 Hz, 1H), 3.08 – 2.80 (m, 2H); 13 C NMR (75 MHz, DMSO-d6) δ 172.7, 158.3, 155.7, 138.7, 132.6, 130.9, 130.3, 124.3, 120.5, 119.5, 118.6, 66.4, 55.6, 29.4; HRMS (ESI) calcd for C18H15N3O3 [M+H]+ 322.1186, found 322.1190.

Acknowledgments This work was supported by the DSF Center for Antimicrobial Research, Technical University of Denmark, and University of Copenhagen. References and notes 1. 2. 3. 4. 5.

Yield: 57% (57 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.97 (s, 1H), 7.68-7.48 (m, 5H), 5.72 (t, J = 9.6 Hz, 1H), 4.62 (td, J = 8.3, 3.1 Hz, 1H), 4.45 (dd, J = 16.1, 8.9 Hz, 1H), 3.07 – 2.82 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.70, 139.2, 132.7, 129.8, 129.3, 129.0, 125.9, 66.4, 55.7, 29.5. 23.2; HRMS (ESI) calcd for C12H11N3 O2 [M+H]+ 230.0924, found 230.0928.

6.

4 . 2. 9. 9. 1 , 5- Tri az ol e l act o ne 8 4i

9.

1

Yield: 62% (67 mg). H NMR (300 MHz, DMSO-d6) δ 7.97 (s, 1H), 7.68 – 7.55 (m, 2H), 7.53 – 7.37 (m, 2H), 5.69 (t, J = 9.6 Hz, 1H), 4.63 (td, J = 8.5, 2.6 Hz, 1H), 4.44 (dt, J = 15.9, 7.9 Hz, 1H), 3.14 – 2.81 (m, 2H); 13 C NMR (75 MHz, DMSO-d6) δ 172.7, 164.6, 161.3, 138.3, 132.8, 131.5, 131.4, 122.38, 122.34, 116.50, 116.21, 66.47, 55.65, 29.4; HRMS (ESI) calcd for C12H10FN3O2 [M+H]+ 248.0830, found 248.0832.

7. 8.

10. 11. 12. 13. 14.

4 . 2. 9. 10. 1 , 5- T ri a z ol e l a ct on e 84j Yield: 66% (75 mg). 1H NMR (300 MHz, DMSO-d6) δ 7.89 (d, J = 1.1 Hz, 1H), 7.53 – 7.44 (m, 2H), 7.19 – 7.10 (m, 2H), 5.68 (t, J = 9.5 Hz, 1H), 4.62 (td, J = 8.2, 2.0 Hz, 1H), 4.44 (dd, J = 16.5, 8.3 Hz, 1H), 3.83 (s, 3H), 3.04 – 2.82 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.8, 160.4, 139.0, 132.3, 130.5, 117.9, 114.7, 66.4, 55.5, 55.4, 29.4; HRMS (ESI) calcd for C13H13N3O3 [M+H]+ 260.1030, found 260.1032. 4 . 2. 9. 11. 1 , 5- T ri a z ol e l a ct on e 84 k Yield: 41% (54 mg). 1H NMR (300 MHz, DMSO-d6) δ 8.05 (s, 1H), 7.91 – 7.82 (m, 2H), 7.55 (dd, J = 8.3, 2.0 Hz, 1H), 5.74 (t, J = 9.6 Hz, 1H), 4.64 (td, J = 8.6, 2.4 Hz, 1H), 4.43 (td, J = 9.1, 6.8 Hz, 1H), 3.13 – 2.84 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 172.7, 137.1, 133.4, 132.8, 132.0, 131.4, 130.9,

15.

16. 17.

18. 19. 20. 21.

Jakobsen, T. H.; Bjarnsholt, T.; Jensen, P. O.; Givskov, M.; Hoiby, N. Future Microbiol 2013, 8, 901-921. Fuqua, W. C.; Winans, S. C.; Greenberg, E. P. J. Bacteriol. 1994, 176, 269-275. Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001, 55, 165-199. Fuqua, C.; Greenberg, E. P. Nat. Rev. Mol. Cell Biol. 2002, 3, 685-695. Fuqua, C.; Parsek, M. R.; Greenberg, E. P. Annu. Rev. Genet. 2001, 35, 439-468. Alhede, M.; Bjarnsholt, T.; Jensen, P. O.; Phipps, R. K.; Moser, C.; Christophersen, L.; Christensen, L. D.; van Gennip, M.; Parsek, M.; Høiby, N.; Rasmussen, T. B.; Givskov, M. Microbiology 2009, 155, 3500-3508. de Kievit, T. R.; Iglewski, B. H. Infect. Immun. 2000, 68, 4839-4849. Hentzer, M.; Wu, H; Andersen, J. B.; Riedel, K.; Rasmussen, T. B.; Bagge, N.; Kumar, N.; Schembri, M. A.; Song, Z.; Kristoffersen, P.; Manefield, M.; Costerton, J. W.; Molin, S.; Eberl, L.; Steinberg, P.; Kjelleberg, S.; Høiby, N.; Givskov, M. EMBO J. 2003, 22, 3803-3815. Schuster, M.; Lostroh, C. P.; Ogi, T.; Greenberg, E. P. J. Bacteriol. 2003, 185, 2066-2079. Wagner, V. E.; Bushnell, D.; Passador, L.; Brooks, A. I.; Iglewski, B. H. J. Bacteriol. 2003, 185, 2080-2095. Lyczak, J. B.; Cannon, C. L.; Pier, G. B. Microbes Infect. 2000, 2, 10511060. Obritsch, M. D.; Fish, D. N.; MacLaren, R.; Jung, R. Pharmacotherapy 2005, 25, 1353-1364. Woodward, T. C.; Brown, R.; Sacco, P.; Zhang, J. J. Med. Econ. 2010, 13, 492-499. Jakobsen, T. H.; van Gennip, M.; Phipps, R. K.; Shanmugham, M. S.; Christensen, L. D.; Alhede, M.; Skindersøe, M. E.; Rasmussen, T. B.; Friedrich, K.; Uthe, F.; Jensen, P. O.; Moser, C.; Nielsen, K. F.; Eberl, L.; Larsen, T. O.; Tanner, D.; Høiby, N.; Bjarnsholt, T.; Givskov, M. Antimicrob. Agents Chemother. 2012, 56, 2314-2325. Jakobsen, T. H.; Bragason, S. K.; Phipps, R. K.; Christensen, L. D.; van Gennip, M.; Alhede, M.; Skindersøe, M.; Larsen, T. O.; Høiby, N.; Bjarnsholt, T.; Givskov, M. Appl. Environ. Microbiol. 2012, 78, 24102421. Ganin, H.; Rayo, J.; Amara, N.; Levy, N.; Krief, P.; Meijler, M. M. Med. Chem. Commun. 2013, 4, 175-179. Rasmussen, T. B.; Skindersøe, M. E.; Bjarnsholt, T.; Phipps, R. K.; Christensen, K. B.; Jensen, P. O.; Andersen, J. B.; Koch, B.; Larsen, T. O.; Hentzer, M.; Eberl, L.; Høiby, N.; Givskov, M. Microbiology 2005, 151, 1325-1340. Pearson, J. P.; Gray, K. M.; Passador, L.; Tucker, K. D.; Eberhard, A.; Iglewski, B. H.; Greenberg, E. P. Proc. Natl. Acad. Sci. USA 1994, 91, 197-201. Pearson, J. P.; Passador, L.; Iglewski, B. H.; Greenberg, E. P. Proc. Natl. Acad. Sci. USA 1995, 92, 1490-1494. Galloway, W. R. J. D.; Hodgkinson, J. T.; Bowden, S. D.; Welch, M.; Spring, D. R. Chem. Rev. 2011, 111, 28-67. Mattmann, M. E.; Blackwell, H. E. J. Org. Chem. 2010, 75, 6737-6746.

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35. Hentzer, M.; Riedel, K.; Rasmussen, T. B.; Heydorn, A.; Andersen, J. B.; Parsek, M. R.; Rice, S. A.; Eberl, L.; Molin, S.; Høiby, N.; Kjelleberg, S.; Givskov, M. Microbiology 2002, 148, 87-102. 36. Yang, L.; Rybtke, M. T.; Jakobsen, T. H.; Hentzer, M.; Bjarnsholt, T.; Givskov, M.; Tolker-Nielsen, T. Antimicrob. Agents Chemother. 2009, 53, 2432-2443. 37. Andersen, J. B.; Heydorn, A.; Hentzer, M.; Eberl, L.; Geisenberger, O.; Christensen, B. B.; Molin, S.; Givskov, M. Appl. Environ. Microbiol. 2001, 67, 575-585. 38. Peng, H. M; Zhao, J.; Li, X.; Advanced Synthesis & Catalysis 2009, 351, 1371-1377. 39. The compound is reported in the literature, but sufficient data is not available from any of the references (see Pourcelot, G.; Cadiot, P.; Georgoulis, C. Tetrahedron 1982, 38, 2123 – 2128). 40. The compound is reported in the literature and was previously synthesized using a different procedure (see Tennyson, R. L.; Cortez, G. S.; Galicia, H. J.; Kreiman, C. R.; Thompson, C. M.; Romo, D. Org. Lett. 2002, 4, 533-536).

Supplementary Material Supplementary data, including characterization data for all new compounds, associated with this article can be found, in the online version, at xxxx

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