Supplementary Information

For

N-Acyl-homoserine lactone analog modulators of the Pseudomonas aeruginosa RhlI

quorum signal synthase

Daniel Shin1,6, Christoph Gorgulla2,3, Michelle E. Boursier4,7, Neilson Rexrode1, Eric C. Brown1,
Haribabu Arthanari2,5, Helen E. Blackwell4, and Rajesh Nagarajan1*
1Department of Chemistry and Biochemistry, Boise State University, 1910 University Dr., Boise,
ID 83725, USA.
2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,

240 Longwood Ave, Boston, MA 02115, USA.

3Department of Physics, Harvard University, 17 Oxford Street, Cambridge, MA 02138, USA.
4Department of Chemistry, University of Wisconsin–Madison, 1101 University Ave, Madison,

WI 53706, USA.
5Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02115, USA.
6Current Address: Idaho College of Osteopathic Medicine, 1401 E Central Dr., Meridian, ID

83642, USA.
7Current Address: Promega Corporation, 2800 Woods Hollow Rd., Fitchburg, WI 53711, USA.

*Corresponding author: rajnagarajan@boisestate.edu

Contents
Protein purification and kinetics methods………………………………………………........S2-S7

Dose-response curves, inhibition plots, substrate-velocity curves and docking poses..…….S8-S22

Table S1. Literature sources for previously reported compounds……………………………….S23

General information for preparation of compounds…………………………………………….S24

Experimental procedures for preparation of new compounds………...…………..………..S25-S36

NMR characterization of new compounds……………………………………………...….S37-S66

HRMS spectra for new compounds………………………………………………………..S66-S71

Mass spectroscopy of alkyl-ACP and alkyl-CoA compounds………………………….....S72-S75

References…………………………………………………………………………………S76-S77

- S1 -
Materials. All chemicals used for protein purification, enzyme assays and HPLC solvents were

from Sigma Aldrich or Thermo Fisher Scientific. Acyl-CoAs were purchased from Sigma

Aldrich Chemical Company or Life Sciences Resources Inc., Milwaukee, WI. Amylose resin and

Factor Xa were from New England Biolabs. All UV-Vis spectrophotometric data was collected

with Thermo Scientific Evolution 260 Bio UV-Vis spectrophotometer. HPLC data was collected

using a Thermo Scientific Dionex UltiMate 3000 UHPLC instrument and analyzed using

Chromeleon 7 software. All mass spectrometry data was collected with a Bruker maXis

Quadrupole Time-of-Flight (QTOF) mass spectrometer and analyzed with the Bruker Compass

Data Analysis software. The plasmid carrying the genes for the Pseudomonas aeruginosa RhlI

and Escherichia coli DK574-pJT94 ACP, respectively, were obtained as a generous gift from

Drs. Peter Tipton from University of Missouri, Columbia and Peter Greenberg at the University

of Washington, Seattle. The plasmid carrying Bacillus subtilis Sfp phosphopantetheinyl

transferase was supplied by Dr. Michael Burkart at the University of California, San Diego.

Protein Purification and Kinetics Methods

RhlI assays. The enzymatic reaction catalyzed by RhlI was monitored using a colorimetric assay

that is sensitive to the free holo-ACP thiol generated at the acylation step in AHL synthesis.[1, 2]

A typical reaction contained 30 M dichlorophenolindophenol (DCPIP), 100 mM HEPES, pH 7.3,

300 M SAM and varying concentrations of acyl-ACP. RhlI was maintained at 0.3 M for C4-

ACP and 0.9 M for non-native acyl-ACPs. The reaction mixture was incubated for 10 minutes

prior to RhlI addition to initiate AHL synthesis. The decrease in DCPIP absorbance at 600 nm was

followed for 5 minutes and the progress curve obtained was used to calculate initial rates. The

- S2 -
initial rate was fitted to Michaelis Menten equation using Graphpad Prism 7 to estimate kinetic

constants. All experiments were done in triplicate to check for reproducibility and to estimate

errors.

Dose-Response curves. A typical assay mixture was composed of 30 M DCPIP, 300 M SAM,

14 M C4-ACP, 100 mM HEPES buffer (pH 7.3) and varying concentrations (10 M -2 mM) of

AHL analogs in 10% DMSO (by volume). The reaction was initiated using 0.3 M RhlI and the

reduction of DCPIP by holo-ACP was followed as a decrease in absorbance at 600 nm. Each run

was conducted in triplicate and all of the data was included to generate dose-response inhibition

or activation curves. Initial rate was fitted to a dose-response equation to determine IC50

(concentration of inhibitor required to attain 50% maximum inhibitory effect) or EC50 (half-

maximal effective concentration) using Graphpad prism 7.[3] To determine if inhibition is time-

dependent, RhlI was incubated with 0, 10, 30 and 60 minutes and the pre-incubated enzyme was

used to inititate enzyme reaction. The IC50 values of inhibitors were determined as described

above.

Inhibition Analysis. To determine the mode of inhibition and Ki value for compounds 60, 64, and

85-88, a total of 4 inhibitor concentrations, two below and two above IC50 values were chosen and

assayed against C4-ACP as the variable substrate. Each assay mixture contained RhlI (0.3 M),

DCPIP (300 M), SAM (300 M), HEPES buffer (100 mM, pH 7.3), and varying concentrations

of C4-ACP (2-20 M) at a specific inhibitor concentration (0-400 M). The change in DCPIP

absorbance at 600 nm was used to estimate initial rates in each assay. The initial rate was fitted to

uncompetitive inhibition (for compunds 60 and 64) or noncompetitive inhibition (for compounds

- S3 -
85-88) equations to estimate Ki values.[3, 4, 5] The inhibition models were confirmed using

Akieke’s Information Criterion (AIC).

RhlI purification. RhlI was purified via a previously described method with minor

modifications.[6] An isolated colony was used to inoculate 25 mL of Lennox broth with ampicillin

(100 g/mL) and incubated at 37°C with shaking (225 rpm) for 8-12 hours or until visible turbidity.

The “mini-growth” was then transferred over to 1 L of Lennox broth (20 g broth/L) with ampicillin

(100 g/mL). The broth was incubated with shaking (225 rpm) at 37 °C. When the OD600 value

reached 0.6-0.8, IPTG was added to 0.5 mM final concentration to induce protein expression.

After 3 hours of incubation at room temperature, the media was pelleted by spinning down at 5,000

x g at 4°C for 15 minutes. The cell pellet was resuspended in “Buffer A” composed of 50 mM

Tris-HCl, pH 7.5, 0.2 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM

phenylmethanesulfonyl fluoride (PMSF), 0.1 mM Nα-tosyl-L-lysine chloromethyl ketone

hydrochloride (TLCK), 0.4 M sucrose, and 2.5% (v/v) glycerol. The resuspended mixture was

then lysed via sonication at 15,000 psi. The lysate was spun down for 40 minutes at 10,000 x g

and at 4°C. Protamine sulfate was added to the supernatant to a final concentration of 6 mg/g of

cell pellet to precipitate nucleic acids that were removed by centrifugation for 20 minutes at 10,000

x g and at 4°C. The supernatant was then loaded onto an amylose column equilibrated with 5x

bed volume of buffer A. The column was washed with 5x bed volume with buffer A and eluted

out using buffer A with 10 mM maltose. The purity of RhlI was checked with SDS-PAGE gel

electrophoresis. The protein sample was concentrated using 10 kD MWCO spin filter, the

concentration checked via UV-Vis spectrophotometry (ε280= 107510 M-1cm-1), and stored in buffer

A with 20% glycerol at -80 °C until further use. When necessary, maltose binding protein tag was

- S4 -
removed using Factor Xa protease. A sample fusion tag cleavage assay included 120 g of RhlI-

MBP fusion protein, 120 g of Factor Xa in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl and 2 mM

CaCl2, incubated for 2 hours (New England Biolabs Protocol P8010S). The reaction mixture was

then run through the amylose column as described above to remove the cleaved MBP tag except

that the untagged RhlI eluted in the wash buffer without the added maltose. The untagged RhlI

was spun down, concentrated and stored as described above.

Apo-ACP purification. Purification of Escherichia coli apo-acyl carrier protein (apo-ACP) was

accomplished by minor modification of previously published protocols.[1, 3, 4, 7] Chemical lysis

of cell pellets was achieved with B-PER reagent. ACP was purified using a Whatman DE52

diaminoethyl cellulose resin. The ACP was precipitated by addition of 0.02% sodium

deoxycholate and 5% trichloroacetate and after 30 min, the pellet was collected by centrifugation

and resuspended in 0.5 M Tris-HCl, pH 8.0 buffer. Fractions containing pure protein by SDS-

PAGE were pooled and concentrated using a 3 kD MWCO spin filter column.

Alkyl-CoA synthesis. To a solution of coenzyme A (CoA-SH; 50 mg, 65 mol), alkyl bromide

(120 mol) in 2 mL of 1:1 water:DMF and potassium carbonate was added until a pH of 8-9 was

achieved. The reaction was stirred under nitrogen overnight. Reaction completion was checked for

complete consumption of limiting CoA-SH reagent either by DCPIP colorimetric reduction or by

nitroprusside test. The reaction mixture was then diluted with water to a final volume of 5 mL and

extracted with 5 mL of diethyl ether. The crude product was collected and filtered using a 0.22

m centrifugal filter and purified using a semi-prep C-18 reverse phase HPLC with a gradient

beginning at 95% aqueous solvent A (25 mM ammonium acetate, pH 5) and ending at 70% organic

- S5 -
solvent B (acetonitrile + 0.1% TFA) at a flow rate of 3 mL/min over 11 minutes. Organic solvent

was removed via evaporation by a gentle stream of nitrogen through the product solution and the

aqueous solution was lyophilized to yield alkyl-CoA powder.[4] The identity of butyl-, hexyl-,

octyl- and decyl-CoA was confirmed by ESI-TOF mass spectrometry. Butyl-Coenzyme A (89)

Expected m/z [M + H+] = 824.1851, observed [M + H+] = 824.1835; Hexyl-Coenzyme A (90)

Expected m/z [M + H+] = 852.2164, observed [M + H+] = 852.2147; Octyl-Coenzyme A (91).

Expected m/z [M + H+] = 880.2477, observed [M + H+] = 880.2444.

Alkyl-ACP/Acyl-ACP synthesis. Acyl-/alkyl-pantetheine was transferred from acyl-CoA/alkyl-

CoA to apo-ACP via phosphopanetheinyl transferase (Sfp) catalyzed reaction.[3] A typical

reaction mixture consisted of 50 mM Tris-HCl pH 6.8, 10 mM magnesium chloride, 600 M apo-

ACP, 10 µM Sfp and 750 M acyl-/alkyl-CoA. During the preparation of long-chain acyl-

ACPs/alkyl-ACPs (carbon chain lengths greater than eight), precipitates formed in the reaction

mixture prevented the reaction from proceeding to completion. To avoid precipitation, the

corresponding long-chain acyl-CoAs were incrementally added to the reaction mixture over fifteen

minute intervals. The reaction mixture was incubated at 37 °C and checked every 30 minutes

using C-18 reverse phase HPLC until the reaction was complete. The HPLC method included a

linear gradient beginning with 25% Solvent A (water + 0.1% TFA) and ending with 25% solvent

B (acetonitrile + 0.1% TFA) at a flow rate of 0.6 mL/min over a period of 10 minutes. Since the

six-carbon chain ACP overlapped with apo-ACP peak, a new shallow gradient was setup from

25% A to 25% B at a flow rate of 0.2 mL/min over 60 min to resolve the two overlapping peaks.

Ammonium sulfate was then added to the reaction to 75% saturation and kept at 4 °C for 1 hour

to precipitate Sfp that was subsequently pelleted by centrifugation at 15,000 x g for 15 minutes.

- S6 -
The supernatant was desalted using a 3kD MWCO spin filter column. The concentration of acyl-

/alkyl-ACPs were determined using UV-Vis spectrophotometry (ε280= 1490 M-1cm-1), and stored

in 10 mM MES pH 6.1 + 20 % glycerol at -80 °C until further use. Acyl-ACPs were characterized

as described elsewhere.[3, 4] The identity of alkyl-ACPs were confirmed using ESI-TOF mass

spectrometry: Butyl-ACP (85) expected m/z [M + H+] = 8904.6 Da, observed [M + H+] = 8904.3

Da; Hexyl-ACP (86) expected m/z [M + H+] = 8932.6 Da, observed [M + H+] = 8932.4 Da; Octyl-

ACP (87) expected m/z [M + H+] = 8960.7 Da, observed [M + H+] = 8960.4 Da; Decyl-ACP (88)

expected m/z [M + H+] = 8988.7 Da, observed [M + H+] = 8988.4 Da.

AHL analog synthesis. Synthetic protocols and characterization data for compounds 34, 36, 56-

64 are included below starting in section “Experimental procedures for preparation of new

compounds” (page S24 of this document). The methodology for the synthesis of the remainder of

AHL analogs are described elsewhere and referenced appropriately in Table S1 (page S23).

Docking Methods. The ligands were prepared (3D coordinate generation by energy

minimization, conversion into the PDBQT format) with Open Babel. RhlI homology model was

created using Phyre2. The receptor structure was prepared with Maestro and AutoDockTools.

The dockings were carried out with AutoDock Vina. The exhaustiveness was set to 8, and 10

replicas (independent docking runs) were carried out for each ligand. The receptor was held

rigid, and the docking box had the dimensions 26.25 x 18.0 x 16.5 Angstrom. Docked ligands

were analyzed using UCSF Chimera.

- S7 -
Supplementary Assay Data

Figure S1. Dose-response curves for head group variants. Compounds 5 and 12 are fitted
using [inhibitor] vs Response Variable Slope equation. The Hill slopes for 5 and 12, respectively,
are 3.9 and 1.9.

- S8 -
Figure S2. Dose-response curves for L-homoserine lactone analogs.

- S9 -
Figure S3. Dose-response curves for D-homoserine lactone analogs. Compounds 31 and 35
are fitted using [inhibitor] vs Response Variable Slope equation. Compound 34 is fitted to
[Agonist] vs Response variable slope equation. The Hill slopes for 31, 34 and 35, respectively,
are 0.8, 1.0 and 2.5.

- S10 -
Figure S4. Dose-response curves for acyl-sulfonamide homoserine lactone analogs. The Hill
slope for compound 37, fitted to [Inhibitor] vs Response variable slope equation is 3.0.

- S11 -
Figure S5. Dose-response curves for L-homocysteine thiolactone analogs. Only the 12-
carbon chain in the unsubstituted and 3-oxoacyl series inhibited RhlI. Hill slopes for 46 and 55,
respectively, are 1.6 and 1.4.

- S12 -
Figure S6. Dose-response curves for D-homocysteine thiolactone analogs. The shorter 3-
oxoacyl-chain analogs activated RhII. Like the L-homocysteine thiolactone series, the 12-carbon
acyl and 3-oxoacyl chains inhibited RhlI. Hill slopes for both agonists and antagoinsts varied
between 1-3.5. Inhibition of long-chain AHL analogs (for example, compounds 60, 64 etc) via
micelles is less likely because a) none of the L-HSL analogs (including the long-chains) inhibited
RhlI, b) inhibition is stereospecific preferring the ‘D’ enantiomer for homoserine lactone, c) both
‘L’ and ‘D’ stereoisomers of the 12-carbon chain in the thiolactone AHL analog inhibited the
enzyme highlighting differences in lactone vs thiolactone headgroup specificity (compounds 46,
55, 60 and 64), d) AHL analogs display the expected uncompetitive inhibition mode with the
RhlI (Figure S80), e) even the long-chain analogs of acyl-ACPs, alkyl-ACPs and alkyl-CoAs all
bind tighter to RhlI (see Tables 2 and 3 in the main text) and f) the range of inhibitor
concentrations used to determine IC50 for compound 60 and other related inhibitors are below the
critical micelle concentrations (usually in the hundreds of micromolar to millimolar range)
reported in the literature (Reference 39 in main text).

- S13 -
 100
100
100
% RhlI Activity

% RhlI Activity

% RhlI Activity

% RhlI Activity
100

50 50
50 50

0 0 0 0
0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500
[Compound 4] (M) [Compound 65] (µM) [Compound 66] (µM) [Compound 67] (µM)

150
100
100
% RhlI Activity

% RhlI Activity

% RhlI Activity
% RhlI Activity
100
100

50 50
50
50

0 0 0 0
0 500 1000 1500 2000 2500 0 200 400 600 0 200 400 600 0 500 1000 1500 2000 2500
[Compound 68] (µM) [Compound 69] (M) [Compound 70] (µM) [Compound 71] (µM)

200
150
100
150
% RhlI Activity

% RhlI Activity

% RhlI Activity

% RhlI Activity
100
100
100
50
50
50
50

0 0 0 0
0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500
[Compound 72] (µM) [Compound 73] (µM) [Compound 74] (µM) [Compound 75] (µM)

100
100
% RhlI Activity

% RhlI Activity

% RhlI Activity

% RhlI Activity
100 100

50 50
50 50

0 0 0 0
0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500
[Compound 76] (µM) [Compound 77] (µM) [Compound 78] (µM) [Compound 79] (µM)
% RhlI Activity

100

50

0
0 500 1000 1500
[Compound 74 and 79] (µM)

Figure S7. Dose-response curves for nonlactone analogs. No compound in this series inhibited
RhlI.

- S14 -
Figure S8. Double reciprocal plots for dodecanoyl-D-homocysteine thiolactone and 3-
oxododecanoyl-D-homocysteine thiolactones. Both compounds are uncompetitive inhibitors of
RhlI. The Ki values for 60 and 64, respectively, are 86  10 M and 432  31 M.

Figure S9. Substrate-velocity curves for butyryl-ACP with dodecanoyl-D-homocysteine

thiolactone and 3-oxododecanoyl-D-homocysteine thiolactone inhibitors. Panels A and B
represent respectively, substrate-velocity curves for butyryl-ACP as a function of increasing
concentrations of compounds 60 and 64. Both compounds are uncompetitive inhibitors of RhlI.
The Ki values for 60 and 64, respectively, are 86  10 M and 432  31 M.

- S15 -
Figure S10. Substrate-velocity curves for acyl-ACPs with maltose binding protein cleaved
RhlI. Panels A and B, respectively, represent substrate-velocity curves for butyryl-ACP and
octanoyl-ACP with fusion tag cleaved RhlI. The Km and kcat values for acyl-ACPs with MBP-
tagged RhlI are presented in Table 2 in the main text.

Figure S11. IC50 curves for alkyl-ACPs with maltose binding protein cleaved RhlI. Panels A
and B, respectively, represent dose-response inhibition curves for butyl-ACP and octyl-ACP
with RhlI. The IC50 values of butyl-ACP and octyl-ACP with MBP-tagged RhlI, respectively, are
9.9  4.0 M and 0.06  0.02 M.

- S16 -
 1 2 3

Figure S12. SDS-PAGE gel of Maltose Binding Protein cleaved RhlI. Lane 1: EZ prestained
protein ladder; Lane 2: Factor Xa MBP cleavage assay mixture before amylose column; Lane 3:
After elution from amylose column. The molecular weight of RhlI is 34 kD.

Figure S13. Substrate-velocity curves for acyl-ACPs with RhlI. Panels A-E represent
substrate-velocity curves for butyryl-ACP, hexanoyl-ACP, octanoyl-ACP, decanoyl-ACP and
dodecanoyl-ACP.

- S17 -
Figure S14. Dose-response curves and double reciprocal plots for alkyl-ACPs. Panels A, C,
E and G represent dose response curves for compounds 85-88. Panels B, D, F and H represents
double reciprocal plots for RhlI inhibited by alkyl-ACPs. All alkyl-ACPs displayed
noncompetitive mode of inhibition. IC50 and Ki values for 85 – 88 are presented in the main text.

- S18 -
Figure S15. Substrate-velocity curves for butyryl-ACP in presence of alkyl-ACPs. Panels A,
B, C and D represent, respectively, substrate-velocity curves for butyryl-ACP as a function of
increasing concentrations of compounds 85, 86, 87 and 88. IC50 and Ki values for 85 – 88 are
presented in the main text.

Figure S16. IC50 curves for alkyl-ACPs with RhlI pre-incubated for different times with
inhibitor. RhlI enzyme was pre-incubated with butyl-ACP (A) or octyl-ACP (B) for 0-60
minutes and the IC50 curve was recorded for each of the pre-incubated mixtures. No significant
changes in IC50 values were observed for RhlI preincubated with butyl-ACP and octyl-ACP.

- S19 -
Figure S17. Dose-response curves for alkyl-CoAs. Panels A, B and C represent dose-response
curves, respectively, for compounds 89, 90 and 91. Butyl-CoA did not inhibit RhlI up to 1 mM.
The lower inhibition potency for alkyl-CoAs relative to alkyl-ACPs highlights carrier protein’s
contribution to overall substrate binding energies.

Figure S18. Docked pose of compound 31 on RhlI. A. The Aromatic Wall. Phenyl ring in
compound 31 packs deep in to the aromatic wall (Y105, F120, F147 and F173). B. A 90 degree
counterclockwise rotated zoomed view of pose A. F120 stacks on top of phenyl ring while Y105
stabilizes the aliphatic linker in 31. C. A 180 degree rotation of pose A along the vertical axis.
The -electron cloud from F147 and F173 stabilizes the phenyl ring in 31.

Figure S19. Docked pose of compound 34 on RhlI. A. Inhibition mode of compound 34. The 3-
oxohexanoyl chain attempts to reach out to the aromatic wall in the pocket. The shorter chain
doesn’t go deep enough in to this pocket. B. Activation mode of compound 34. The 3-oxoacyl-
chain stacks on top of W34 in the activation pocket. Since this compound activates RhlI, the
energetics of activation binding mode should be more favorable than inhibition mode. The
inhibition pocket is larger and thus the shorter chains must have more room to move around in

- S20 -
this pocket. The increased flexibility of the acyl-chain should promote ligand dissocation from
this pocket and hence the suppression of inhibitory activity for this compound.

Figure S20. Docking pose of compound 35. A. Inhibition mode of compound 35. The 3-
oxooctanoyl-chain of 35 binds deeper than the 3-oxohexanoyl-chain of 34 in the inhibition
pocket. B. Activation mode of compound 35. Like 34, the acyl-chain in 35 stacks against the
indole ring of W34. The greater stabilization of the 3-oxooctanoyl-chain in the inhibition pocket
should promote inhibitory activity of compound 35. The rigid receptor docking model, however,
predicted a better vina score for the activation (-6.6 kcal/mol) vs the inhibition (-6.1 kcal/mol)
mode.

Figure S21. Comparison of inhibition poses of 31, 34 and 35. Panels A, B and C, respectively,
shows the inhibition binding mode of compounds 34, 35 and 31 in RhlI. The acyl-chain of
compound 31 packs deeper in to the aromatic wall of inhibition pocket than compound 34.
Compund 35 barely enters this cavity. The relative strengths of acyl-chain packing in the
inhibition pocket matches the IC50 trends among these compounds (31 > 35; 34 is an activator
and so no inhibition was observed).

- S21 -
Figure S22. RhlI activity as a function of DMSO concentration. No significant changes in
RhlI activity was observed up to 15% DMSO (by volume). RhlI inhibition assays described in
this study were conducted at or below 10% DMSO.

Figure S23. SDS-PAGE gel of RhlI protein isolated using amylose chromatography. Lane
1: EZ prestained protein ladder; Lane 2: RhlI column load run-through; Lane 3: load wash;
Lanes 4-8: Buffer A (50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 1 mM EDTA, 0.1 mM PMSF, 0.1
mM TLCK, 0.4 M sucrose, and 2.5% (v/v) glycerol) +10 mM maltose elution fractions 1-5. RhlI
+ MBP has a combined molecular weight of 65.5 kD. RhlI purification protocol is discussed in
the main text.

- S22 -
Table S1. Literature sources for previously reported compounds used in study.

Compounds Publication Title References

1, 20, 22, 23 Small molecule inhibitors of bacterial quorum sensing and biofilm [8]
formation
2, 4, 5, 6, 7, Structure-function analyses of the N-butanoyl L-homoserine [9]
8, 9, 10, 11, lactone quorum–sensing signal define features critical to activity
12, 29, 30, in RhlR.
41, 42
3 Stereoisomers of P. aeruginosa autoinducer analog to probe the [10]
regulator binding site
13, 14, 15, Comparative analyses of N-acylated homoserine lactones reveal [11]
16, 17, 18, unique structural features that dictate their ability to activate or
19, 22, 24, inhibit quorum sensing
31, 32, 33,
35, 37, 38,
39, 40

25, 26, 27 Chemical interrogation of LuxR-type quorum sensing receptors [12]

reveals new insights into receptor selectivity and the potential for
interspecies bacterial signaling
28 The quorum-quenching lactonase from Bacillus thuringiensis is a [13]
metalloprotein
43, 44, 45, Thiolactone modulators of quorum sensing revealed through [14]
46, 48, 49, library design and screening
50, 51, 52,
53, 54, 55,
65, 67, 68,
70
47, 66 Synthetic ligands that modulate the activity of the RhlR quorum [15]
sensing receptor
69 N-Acyl glycinates as acyl donors in serine protease-catalyzed [16]
kinetic resolution of amines. Improvement of selectivity and
reaction rate
36 Synthesis of Pseudomonas quorum-sensing autoinducer analogs [17]
and structural entities required for induction of apoptosis in
macrophages

- S23 -
General Information for Preparation of Compounds

All chemical reagents and solvents used for synthesis of compounds were purchased from

commercial sources and used without purification. Deuterated chloroform was obtained

commercially through Cambridge Isotope Laboratories, Inc. NMR spectra were recorded at 298

K using a BRUKER AVANCE III 600 MHz spectrometer. Chemical shifts were expressed in parts

per million (ppm) and referenced to residual solvent as the internal reference for 1H (CDCl3: δ =

7.24 ppm) and 13C (CDCl3: δ = 77.16 ppm). High resolution mass spectrometry (HRMS) data was

collected with a Bruker maXis Quadrupole Time-of-Flight (QTOF) mass spectrometer and

analyzed with the Bruker Compass Data Analysis software. The headgroups 71, 72, and 73 and

carboxylic acids 75-79 were commercially available from Sigma-Aldrich. The headgroup 74, D-

homocysteine thiolactone ∙ HCl, was prepared according to the procedure described by Shinohara

et al.[18] Selected details of the synthetic methods for the new compounds 12, 29, 30, 41-42, 34,

and 56-64 and their characterization data are provided below.

- S24 -
Experimental Procedures for Preparation of New Compounds

Preparation of D-Sulfonamides (12, 41 and 42).

The D-sulfonamides (12, 41 and 42) were prepared in an analogous manner to their L-isomers,

whose synthesis have been previously reported.[9]

(R)-N-(2-oxotetrahydrofuran-3-yl)propane-1-sulfonamide (12).

O O

S
N O
O H H

1H NMR (500 MHz, CDCl3) δ 4.75 (d, J = 6.1 Hz, 1H), 4.53 – 4.40 (m, 1H), 4.30 (dddd, J = 33.6,

11.5, 9.0, 6.1 Hz, 2H), 3.24 – 3.07 (m, 2H), 2.78 (dddd, J = 12.7, 8.5, 5.7, 0.9 Hz, 1H), 2.27 (qd, J

= 11.7, 8.8 Hz, 1H), 1.92 (hd, J = 7.4, 3.3 Hz, 2H), 1.56 (s, 2H), 1.08 (t, J = 7.4 Hz, 3H);13C NMR

(126 MHz, CDCl3) δ 174.66, 65.78, 56.17, 52.31, 31.52, 17.50, 13.04 Expected [M+H]+:

225.0904, observed: 225.0900.

(R)-N-(2-oxotetrahydrofuran-3-yl)hexane-1-sulfonamide (41).

O O

S
3 N O
O H H

1H NMR (500 MHz, CDCl3) δ 5.02 (d, J = 6.8 Hz, 1H), 4.44 (t, J = 9.0 Hz, 1H), 4.33 (ddd, J =

11.8, 8.5, 6.9 Hz, 1H), 4.26 (ddd, J = 11.3, 9.4, 5.7 Hz, 1H), 3.24 – 3.11 (m, 2H), 2.80 – 2.69 (m,

1H), 2.27 (qd, J = 11.7, 8.8 Hz, 1H), 1.94 – 1.77 (m, 2H), 1.43 (p, J = 7.3 Hz, 2H), 1.31 (dt, J =

7.4, 3.7 Hz, 4H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 174.84, 65.78, 54.61,

52.28, 31.35, 31.26, 28.02, 23.64, 22.47, 14.07; Expected [M+H]+: 267.1373, observed: 267.1370.

- S25 -
(R)-N-(2-oxotetrahydrofuran-3-yl)decane-1-sulfonamide (42).

O O

S
7 N O
O H H

1H NMR (500 MHz, CDCl3) δ 5.33 (d, J = 7.4 Hz, 1H), 4.41 (t, J = 9.0 Hz, 1H), 4.33 (dt, J = 11.7,

8.1 Hz, 1H), 4.24 (ddd, J = 11.1, 9.5, 5.8 Hz, 1H), 3.16 (p, J = 7.8 Hz, 2H), 2.70 (ddd, J = 13.5,

8.5, 5.9 Hz, 1H), 2.28 (qd, J = 11.8, 8.9 Hz, 1H), 1.83 (dp, J = 15.5, 7.6, 6.9 Hz, 2H), 1.40 (p, J =

7.2 Hz, 2H), 1.34 – 1.16 (m, 12H), 0.86 (t, J = 6.9 Hz, 3H);13C NMR (126 MHz, CDCl3) δ 175.08,

65.77, 54.63, 52.20, 31.93, 30.87, 29.56, 29.42, 29.34, 29.20, 28.33, 23.62, 22.73, 14.17; Expected

[M+H]+: 323.1999, observed: 323.1995.

Preparation of D-Homoserine Lactones (29 and 30).

The D-homoserine lactones (29 and 30) were prepared in an analogous manner to their L-isomers,

whose synthesis have been previously reported.[9]

N-Octanoyl-D-homoserine lactone (29).

O O

3 N O
H H

1H NMR (500 MHz, CDCl3) δ 6.16 (d, 1H), 4.56 (ddd, J = 11.6, 8.6, 6.0 Hz, 1H), 4.46 (t, J = 9.0

Hz, 1H), 4.27 (ddd, J = 11.2, 9.3, 5.9 Hz, 1H), 2.83 (ddd, J = 13.3, 8.5, 6.0 Hz, 1H), 2.31 – 2.19

(m, 2H), 2.13 (qd, J = 11.6, 8.9 Hz, 1H), 1.63 (p, J = 7.5 Hz, 2H), 1.28 (dd, J = 12.3, 5.4 Hz, 8H),

0.87 (t, J = 6.9 Hz, 3H).;13C NMR (126 MHz, CDCl3) δ 175.77, 173.92, 66.24, 49.33, 36.31, 31.77,

30.69, 29.29, 29.09, 25.57, 22.72, 14.18; Expected [M+H]+: 228.1594, observed: 228.1592.

- S26 -
N-Cyclobutanoyl-D-homoserine lactone (30).

O O

N O
H H

1H NMR (400 MHz, CDCl3) δ 6.25 (d, J = 5.0 Hz, 1H), 4.58 (ddd, J = 11.5, 8.7, 6.5 Hz, 1H),

4.44 (t, J = 8.7 Hz, 1H), 4.27 (ddd, J = 11.1, 9.3, 6.0 Hz, 1H), 3.06 (p, J = 8.5 Hz, 1H), 2.88 –

2.65 (m, 1H), 2.48 – 2.04 (m, 5H), 2.04 – 1.73 (m, 2H);13C NMR (126 MHz, CDCl3) δ 175.69,

175.66, 66.25, 49.38, 39.55, 30.92, 25.42, 25.39, 18.29; Expected [M+H]+: 184.0968, observed:

184.0968.

Preparation of N-(3-Oxohexanoyl)-D-Homoserine Lactone (34).

O O O

N O
H H

N-(3-Oxohexanoyl)-D-homoserine lactone (34) was prepared similar to the protocol

described by Bycroft et al.[19] for making N-(3-oxoalkanoyl)-L-homoserine lactones. To a

solution of butyric acid (360 mg, 4.1 mmol) dissolved in dichloromethane (15 mL) at room

temperature, was added 4-(dimethylamino)pyridine (549 mg, 4.5 mmol), N,N-

dicyclohexylcarbodiimide (1010 mg, 4.9 mmol), and Meldrum’s acid (589 mg, 4.1 mmol). The

mixture was stirred overnight at room temperature before being filtered and the solvent removed

under reduced pressure. The residue was dissolved in ethyl acetate (25 mL) and washed with 2 M

HCl (2 x 10 mL). The organic layer was dried with MgSO4, filtered and the solvent removed to

afford the acylated Meldrum’s acid (425 mg), which was used without further purification.

A portion of the crude acylated Meldrum’s acid (100 mg, 0.47 mmol) was dissolved in

acetonitrile (15 mL) and D-homoserine lactone hydrochloride (64 mg, 0.47 mmol) and

- S27 -
triethylamine (0.08 mL, 0.58 mmol) were added. The solution was stirred for 2 hours at room

temperature and then refluxed for 3 hours. Removal of the solvent left a residue that was dissolved

in ethyl acetate (15 mL) and washed with a saturated aqueous solution of sodium bicarbonate (25

mL), a 1 M potassium hydrogen sulfate solution (25 mL) and a brine solution (25 mL). The organic

layer was dried with MgSO4, filtered and the solvent removed to afford 34 as a solid that was

purified by column chromatography on silica gel (2.5:1 hexanes/EtOAc to straight EtOAc

gradient). Yield from 100 mg (0.47 mmol) of the C4-acylated Meldrum’s acid: 15.7 mg, 16%

yield. 1H NMR (600 MHz, CDCl3): δ 7.65 (NH, br s, 1H), 4.57 (CH-lac, ddd (apparent dt), J =

10.6, 8.0, 8.0 Hz, 1H), 4.46 (CH-lac, dd (apparent t), J = 9.4 Hz, 1H), 4.25 (CH-lac, ddd (apparent

td), J = 10.0, 10.0, 6.1 Hz, 1H), 3.45 (CH2, s, 2H), 2.78-2.70 (CH-lac, m, 1H), 2.49 (CH2, t, J =

7.4 Hz, 2H), 2.26-2.16 (CH-lac, m, 1H), 1.61 (CH2, sextet, J = 7.4 Hz, 2H), 0.91 (CH3, t, J = 7.3

Hz, 3H); 13C NMR (150 MHz, CDCl3): δ 206.7, 174.9, 166.5, 66.1, 49.3, 48.3, 46.0, 30.1, 17.1,

13.7; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C10H15NO4 214.1074; Found 214.1081.

Preparation of N-(3-Oxododecanoyl)-D-homoserine Lactone (36).

O O O

N O
5 H H

N-(3-Oxododecanoyl)-D-homoserine lactone (36) was prepared similar to the protocol

described by Bycroft et al.[199] for making N-(3-oxoalkanoyl)-L-homoserine lactones. To a

solution of decanoic acid (250 mg, 1.45 mmol) dissolved in dichloromethane (15 mL) at room

temperature, was added 4-(dimethylamino)pyridine (195 mg, 1.6 mmol), N,N-

dicyclohexylcarbodiimide (359 mg, 1.74 mmol), and Meldrum’s acid (209 mg, 1.45 mmol). The

mixture was stirred overnight at room temperature before being filtered and the solvent removed

- S28 -
under reduced pressure. The residue was dissolved in ethyl acetate (15 mL) and washed with 2 M

HCl (2 x 10 mL). The organic layer was dried with MgSO4, filtered and the solvent removed to

afford the acylated Meldrum’s acid, which was used without further purification.

A portion of the crude acylated Meldrum’s acid (150 mg, 0.5 mmol) was dissolved in

acetonitrile (15 mL) and D-homoserine lactone hydrochloride (69 mg, 0.5 mmol) and triethyl

amine (61 mg, 0.6 mmol) were added. The solution was stirred for 2 hours at room temperature

and then refluxed for 3 hours. Removal of the solvent left a residue that was dissolved in ethyl

acetate (25 mL) and washed with a saturated aqueous solution of sodium bicarbonate (25 mL), a

1 M potassium hydrogen sulfate solution (25 mL) and a brine solution (25 mL). The organic layer

was dried with MgSO4, filtered and the solvent removed to afford 36 as a solid that was purified

by column chromatography (alumina, 2.5:1 hexanes/EtOAc to straight EtOAc gradient). Yield

from 150 mg (0.5 mmol) of the C10-acylated Meldrum’s acid: 50 mg, 34% yield. 1H NMR (600

MHz, CDCl3): δ 7.66 (NH, br s, 1H), 4.61-4.53 (CH-lac, m, 1H), 4.45 (1H, dd (apparent t), J = 9.0

Hz, H-5), 4.29-4.22 (CH-lac, m, 1H), 3.44 (CH2, s, 2H), 2.77-2.70 (CH-lac, m, 1H), 2.50 (CH2, t,

J = 7.2 Hz, 2H), 2.26-2.16 (CH-lac, m, 1H), 1.60-1.50 (CH2, m, 2H), 1.30-1.19 (CH2, m, 12H),

0.85 (CH3, t, J = 7.0 Hz, 3H); 13C NMR (150 MHz, CDCl3): δ 206.7, 174.8, 166.4, 66.0, 49.2,

48.2, 44.1, 32.0, 30.0, 29.50, 29.46, 29.4, 29.1, 23.5, 22.8, 14.2; HRMS (ESI-TOF) m/z: [M + H]+

Calcd for C16H27NO4 298.2013; Found 298.2062.

General Procedure for the Preparation of N-Acylated-D-homocysteine Thiolactones.

To a solution of the appropriate carboxylic acid (0.163 mmol) in acetonitrile (10 mL), was

added N,N-dicyclohexylcarbodiimide (33.6 mg, 0.163 mmol), N-hydroxysuccinimide (18.7 mg,

0.163 mmol), D-homocysteine thiolactone hydrochloride [18] (25.0 mg, 0.163 mmol) and

triethylamine (0.73 mL, 0.33 mmol). The mixture was stirred overnight before being concentrated
- S29 -
(~2 mL) and stored overnight at 4 ºC to precipitate out the dicyclohexylurea by-product. The

mixture was filtered through celite, the filtrate collected and the solvent removed under reduced

pressure. The residue was dissolved in chloroform (10 mL) and washed with a 5% sodium

bicarbonate solution (3 x 4 mL), a 2 M HCl solution (3 x 4 mL) and a brine solution (3 x 4 mL).

The organic layer was dried with MgSO4, filtered and the solvent removed to afford the acylated

thiolactone products. Further purification (except for N-butanoyl-D-homocysteine thiolactone) by

column chromatography on silica gel (2:1 hexanes/EtOAc) was necessary.

N-Butanoyl-D-homocysteine Thiolactone (56).

O S

N O
H H

Yield from 14.3 mg of butyric acid: 14.9 mg (49%). 1H NMR (CDCl3, 600 MHz): δ 5.92 (NH, br

s, 1H), 4.49 (CH-lac, ddd (apparent dt), J = 12.9, 6.4, 6.4 Hz, 1H), 3.33 (CH-lac, ddd (apparent

td), J = 11.7, 11.7, 5.1 Hz, 1H), 3.23 (CH-lac, ddd, J = 11.4, 4.5, 1.0 Hz, 1H), 2.93 (CH-lac, dddd,

J = 12.6, 5.7, 5.7, 1.0 Hz, 1H), 2.20 (CH2, dt, J = 7.3, 3.9 Hz, 2H), 1.94-1.84 (CH-lac, m, 1H), 1.65

(CH2, hextet, J = 7.3 Hz, 2H), 0.93 (CH3, t, J = 7.3 Hz, 3H); 13C NMR (CDCl3, 150 MHz): δ 205.8,

173.7, 59.7, 38.5, 32.4, 27.8, 19.2, 13.9; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C8H14NO2S

188.0734; Found 188.0763.

- S30 -
N-Hexanoyl-D-homocysteine Thiolactone (57).

O S

N O
H H

Yield from 18.9 mg of hexanoic acid: 9.6 mg (27%). 1H NMR (CDCl3, 600 MHz): δ 5.83 (NH, br

s, 1H), 4.48 (CH-lac, ddd (apparent dt), J = 12.8, 6.4, 6.4 Hz, 1H), 3.34 (CH-lac, ddd (apparent

td), J = 11.7, 11.7, 5.3 Hz, 1H), 3.24 (CH-lac, ddd, J = 11.4, 6.9, 1.1 Hz, 1H), 3.00-2.93 (CH-lac,

m, 1H), 2.22 (CH2, dt, J = 7.5, 3.5 Hz, 2H), 1.93-1.82 (CH-lac, m, 1H), 1.66-1.59 (CH2, m, 2H),

1.34-1.25 (CH2, m, 4H), 0.87 (CH3, t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 150 MHz): δ 205.6,

173.7, 59.6, 36.4, 32.2, 31.4, 27.6, 25.2, 22.4, 13.9; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for

C8H14NO2S 216.1047; Found 216.1099.

N-Octanoyl-D-homocysteine Thiolactone (58).

O S

3 N O
H H

Yield from 23.5 mg of octanoic acid: 10.5 mg (27%). 1H NMR (CDCl3, 600 MHz): δ 5.82 (NH,

br s, 1H), 4.48 (CH-lac, ddd (apparent dt), J = 12.9, 6.4, 6.4 Hz, 1H), 3.34 (CH-lac, ddd (apparent

td), J = 11.7, 11.7, 5.2 Hz, 1H), 3.23 (CH-lac, ddd, J = 11.3, 4.4, 0.9 Hz, 1H), 2.96 (CH-lac, dddd,

J = 12.8, 5.8, 5.8, 0.9 Hz, 1H), 2.22 (CH2, dt, J = 7.5, 3.5 Hz, 2H), 1.92-1.83 (CH-lac, m, 1H),

1.65-1.58 (CH2, m, 2H), 1.32-1.19 (CH2, m, 8H), 0.86 (CH3, t, J = 6.5 Hz, 3H); 13C NMR (CDCl3,

150 MHz): δ 205.8, 173.9, 59.8, 36.7, 32.5, 31.9, 29.9, 29.4, 27.8, 25.7, 22.8, 14.3; HRMS (ESI-

TOF) m/z: [M + H]+ Calcd for C12H22NO2S 244.1360; Found 244.1433.

- S31 -
N-Decanoyl-D-homocysteine Thiolactone (59).

O S

5 N O
H H

Yield from 28.0 mg decanoic acid: 15.4 mg (35%). 1H NMR (CDCl3, 600 MHz): δ 5.86 (NH, br

s, 1H), 4.48 (CH-lac, ddd (apparent dt), J = 12.7, 6.3, 6.3 Hz, 1H), 3.34 (CH-lac, ddd (apparent

td), J = 11.5, 11.5, 5.1 Hz, 1H), 3.23 (CH-lac, ddd, J = 11.4, 4.5, 1.1 Hz, 1H), 2.99-2.92 (CH-lac,

m, 1H), 2.21 (CH2, dt, J = 7.4, 3.6 Hz, 2H), 1.92-1.82 (CH-lac, m, 1H), 1.64-1.58 (CH2, m, 2H),

1.31-1.19 (CH2, m, 12H), 0.85 (CH3, t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 150 MHz): δ 205.5,

173.6, 59.5, 36.4, 33.9, 32.2, 31.8, 29.4, 29.3, 29.2, 27.6, 25.5, 22.6, 14.0; HRMS (ESI-TOF) m/z:

[M + H]+ Calcd for C14H26NO2S 272.1673; Found 272.1398.

N-Dodecanoyl-D-homocysteine Thiolactone (60).

O S

7 N O
H H

Yield from 32.6 mg dodecanoic acid: 20.2 mg (41%). 1H NMR (CDCl3, 600 MHz): δ 5.81 (NH,

br s, 1H), 4.47 (CH-lac, ddd (apparent dt), J =12.8, 6.2, 6.2 Hz, 1H), 3.34 (CH-lac, ddd (apparent

td), J = 11.7, 11.7, 5.3 Hz, 1H), 3.23 (CH-lac, ddd, J = 11.2, 4.4, 0.8 Hz, 1H), 2.97 (CH-lac, dddd,

J = 12.4, 6.4, 5.7 0.8 Hz, 1H), 2.21 (CH2, dt, J = 7.5, 3.4 Hz, 2H), 1.92-1.83 (CH-lac, m, 1H), 1.64-

1.59 (CH2, m, 2H), 1.31-1.19 (CH2, m, 16H), 0.86 (CH3, t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 150

MHz): δ 205.5, 173.6, 59.6, 36.4, 33.9, 32.2, 31.9, 29.6, 29.4, 29.3, 29.2, 27.6, 25.5, 24.9, 22.6,

14.1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H30NO2S 300.1986; Found 300.2042.

- S32 -
General Procedure for the Preparation of N-(3-Oxoalkanoyl)-D-homocysteine Thiolactones.

Scheme S1

O DCC, O O H
O O 4-DMAP, O O O O

HO R CH2Cl2
O O R O R
O O O

S
H2N
H HCl,
O
NEt3, CH3CN

O H
S
O N O O
H S
O R N
O R H H
O O

The general synthesis of N-(3-oxoacyl)-D-homocysteine thiolactones (Scheme S1)

followed the same procedure developed by Bycroft et al. for preparing N-(3-oxododecanoyl)-L-

homocysteine thiolactone.[19] To a solution of the corresponding carboxylic acid (2.1 mmol)

dissolved in dichloromethane (10 mL) at room temperature, was added 4-(dimethylamino)pyridine

(279.7 mg, 2.3 mmol), N,N-dicyclohexylcarbodiimide (515.4 mg, 2.5 mmol), and Meldrum’s acid

(300 mg, 2.1 mmol). The mixture was stirred overnight at room temperature before being filtered

and the solvent removed under reduced pressure. The residue was dissolved in ethyl acetate (10

mL) and washed with 2 M HCl (3 x 10 mL). The organic layer was dried with MgSO4, filtered and

the solvent removed to afford the acylated Meldrum’s acid, which was used without further

purification.

A portion of the crude acylated Meldrum’s acid (0.2 mmol) was dissolved in acetonitrile

(15 mL) and D-homoserine thiolactone hydrochloride (30 mg, 0.2 mmol) [17] and trimethylamine

- S33 -
(32.6 μL, 0.23 mmol) were added. The solution was stirred at room temperature for 1 hr and then

refluxed overnight. The solvent was then removed under reduced pressure and the residue was

dissolved in ethyl acetate (10 mL) and washed with a saturated sodium bicarbonate solution (3 x

10 mL), a 1 M potassium hydrogen sulfate solution (3 x 10 mL) and a brine solution (3 x 10 mL).

The organic layer was dried with MgSO4, filtered and the solvent removed to afford the acylated-

3-oxo thiolactone products. The products were purified by column chromatography on silica gel

(straight hexanes to 7:3 hexanes/EtOAc gradient).

N-(3-Oxohexanoyl)-D-homocysteine Thiolactone (61).

O O S

N O
H H

Yield from 41.8 mg (0.2 mmol) of the C4-acylated Meldrum’s acid: 25.4 mg (57%); 1H NMR

(CDCl3, 600 MHz): δ 7.43 (NH, br s, 1H), 4.56 (CH-lac, ddd (apparent dt), J = 12.9, 6.8, 6.8 Hz,

1H), 3.43 (CH2, s, 2H), 3.33 (CH-lac, ddd (apparent td), J = 11.7, 11.7, 5.2 Hz, 1H), 3.24 (CH-lac,

ddd, J = 11.5, 6.9, 1.0 Hz, 1H), 2.83 (CH-lac, dddd, J = 12.4, 6.7, 5.2, 1.2 Hz, 1H), 2.49 (CH2, t,

J = 7.4 Hz, 2H), 2.04-1.94 (CH-lac, m, 1H), 1.61 (CH2, hextet, J = 7.4 Hz, 2H), 0.91 (CH3, t, J =

7.4 Hz, 3H); 13C NMR (CDCl3, 150 MHz): δ 206.6, 204.7, 166.4, 59.5, 48.6, 46.0, 31.8, 27.7, 17.1,

13.7; HRMS (ESI-TOF) m/z: [M + H+] Calcd for C10H15NO3S 230.0845; Found 230.0896.

- S34 -
N-(3-Oxooctanoyl)-D-homocysteine Thiolactone (62).

O O S

N O
H H

Yield from 47.3 mg (0.2 mmol) of the C6-acylated Meldrum’s acid: 31.6 mg (63%); 1H NMR

(CDCl3, 600 MHz): δ 7.45 (NH, br s, 1H), 4.56 (CH-lac, ddd (apparent dt), J = 13.0, 6.6, 6.6 Hz,

1H), 3.43 (CH2, s, 2H), 3.33 (CH-lac, ddd (apparent td), J = 11.7, 11.7, 5.2 Hz, 1H), 3.23 (CH-lac,

ddd, J = 11.4, 7.0, 1.1 Hz, 1H), 2.82 (CH-lac, dddd, J = 12.3, 6.8, 5.2, 1.1 Hz, 1H), 2.50 (CH2, t,

J = 7.4 Hz, 2H), 2.04-1.94 (CH-lac, m, 1H), 1.56 (CH2, pentet, J = 7.4 Hz, 2H), 1.34-1.18 (m, 4H),

0.86 (CH3, t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 150 MHz): δ 206.7, 204.7, 166.4, 59.5, 48.6,

44.1, 31.7, 31.3, 27.7, 23.3, 22.6, 14.0; HRMS (ESI-TOF) m/z: [M + H+] Calcd for C12H19NO3S

258.1158; Found 258.1162.

N-(3-Oxodecanoyl)-D-homocysteine Thiolactone (63).

O O S

N O
3 H H

Yield from 52.8 mg (0.2 mmol) of the C8-acylated Meldrum’s acid: 30.7 mg (55%); 1H NMR

(CDCl3, 600 MHz): δ 7.46 (NH, br s, 1H), 4.56 (CH-lac, ddd (apparent dt), J = 12.9, 6.6, 6.6 Hz,

1H), 3.42 (CH2, s, 2H), 3.32 (CH-lac, ddd (apparent td), J = 11.8, 11.8, 5.3 Hz, 1H), 3.23 (CH-lac,

ddd, J = 11.4, 7.1, 1.0 Hz, 1H), 2.81 (CH-lac, dddd, J = 12.1, 6.7, 5.3, 1.1 Hz, 1H), 2.49 (CH2, t,

J = 7.4 Hz, 2H), 2.04-1.94 (CH-lac, m, 1H), 1.55 (CH2, pentet, J = 7.3 Hz, 2H), 1.30-1.18 (m, 8H),

0.84 (CH3, t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 150 MHz): δ 206.6, 204.7, 166.5, 59.4, 48.6,

- S35 -
44.0, 31.8, 31.7, 29.17, 29.14, 27.7, 23.6, 22.8, 14.2; HRMS (ESI-TOF) m/z: [M + H+] Calcd for

C14H23NO3S 286.1471; Found 286.1472.

N-(3-Oxododecanoyl)-D-homocysteine Thiolactone (64).

O O S

N O
5 H H

Yield from 58.3 mg (0.2 mmol) of the C10-acylated Meldrum’s acid: 48.4 mg (79%); 1H NMR

(CDCl3, 600 MHz): δ 7.45 (NH, br s, 1H), 4.56 (CH-lac, ddd (apparent dt), J = 12.9, 6.7, 6.7 Hz,

1H), 3.43 (CH2, s, 2H), 3.33 (CH-lac, ddd (apparent td), J = 11.7, 11.7, 5.2 Hz, 1H), 3.24 (CH-lac,

ddd, J = 11.2, 6.9, 1.0 Hz, 1H), 2.83 (CH-lac, dddd, J = 12.4, 8.0, 5.4, 1.2 Hz, 1H), 2.50 (CH2, t,

J = 7.3 Hz, 2H), 2.04-1.94 (CH-lac, m, 1H), 1.56 (CH2, pentet, J = 6.9 Hz, 2H), 1.30-1.18 (m,

12H), 0.85 (CH3, t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 150 MHz): δ 206.7, 204.7, 166.4, 59.5,

48.5, 44.1, 32.1, 31.8, 29.6, 29.5, 29.4, 29.2, 27.7, 23.6, 22.9, 14.3; HRMS (ESI-TOF) m/z: [M +

H+] Calcd for C16H27NO3S 314.1784; Found 314.1791.

- S36 -
NMR Characterization of Compounds

Figure S24. 1H NMR of 12 in CDCl3.

Figure S25. 13C NMR of 12 in CDCl3.

- S37 -
Figure S26. 1H NMR of 41 in CDCl3.

Figure S27. 13C NMR of 41 in CDCl3.

- S38 -
Figure S28. 1H NMR of 42 in CDCl3.

Figure S29. 13C NMR of 42 in CDCl3.

- S39 -
Figure S30. 1H NMR of 29 in CDCl3.

Figure S31. 13C NMR of 29 in CDCl3.

- S40 -
Figure S32. 1H NMR of 30 in CDCl3.

Figure S33. 13C NMR of 30 in CDCl3.

- S41 -
Figure S34. 1H NMR of 34 in CDCl3.

Figure S35. COSY NMR of 34 in CDCl3.

- S42 -
Figure S36. HSQC NMR of 34 in CDCl3.

Figure S37. HMBC NMR of 34 in CDCl3.

- S43 -
 Figure S38. 13C NMR of 34 in CDCl3.

Figure S39. 1H NMR of 56 in CDCl3.

- S44 -
Figure S40. COSY NMR of 56 in CDCl3.

Figure S41. HSQC NMR of 56 in CDCl3.

- S45 -
Figure S42. HMBC NMR of 56 in CDCl3.

Figure S43. 13C NMR of 56 in CDCl3.

- S46 -
Figure S44. 1H NMR of 57 in CDCl3.

- S47 -
Figure S45. COSY NMR of 57 in CDCl3.

Figure S46. 13C NMR of 57 in CDCl3.

- S48 -
Figure S47. 1H NMR of 58 in CDCl3.

Figure S48. COSY NMR of 58 in CDCl3.

- S49 -
Figure S49. HSQC NMR of 58 in CDCl3.

Figure S50. HMBC NMR of 58 in CDCl3.

- S50 -
Figure S51. 13C NMR of 58 in CDCl3.

Figure S52. 1H NMR of 59 in CDCl3.

- S51 -
Figure S53. COSY NMR of 59 in CDCl3.

Figure S54. HSQC NMR of 59 in CDCl3.

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Figure S55. HMBC NMR of 59 in CDCl3.

Figure S56. 13C NMR of 59 in CDCl3.

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Figure S57. 1H NMR of 60 in CDCl3.

Figure S58. COSY NMR of 60 in CDCl3.

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Figure S59. HSQC NMR of 60 in CDCl3.

Figure S60. HMBC NMR of 60 in CDCl3.

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Figure S61. 13C NMR of 60 in CDCl3.

Figure S62. 1H NMR of 61 in CDCl3.

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Figure S63. COSY NMR of 61 in CDCl3.

Figure S64. HSQC NMR of 61 in CDCl3.

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Figure S65. HMBC NMR of 61 in CDCl3.

Figure S66. 13C NMR of 61 in CDCl3.

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Figure S67. 1H NMR of 62 in CDCl3.

Figure S68. COSY NMR of 62 in CDCl3.

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Figure S69. HSQC NMR of 62 in CDCl3.

Figure S70. HMBC NMR of 62 in CDCl3.

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Figure S71. 13C NMR of 62 in CDCl3.

Figure S72. 1H NMR of 63 in CDCl3.

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Figure S73. COSY NMR of 63 in CDCl3.

Figure S74. HSQC NMR of 63 in CDCl3.

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Figure S75. HMBC NMR of 63 in CDCl3.

Figure S76. 13C NMR of 64 in CDCl3.

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Figure S77. 1H NMR of 64 in CDCl3.

Figure S78. COSY NMR of 64 in CDCl3.

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Figure S79. HSQC NMR of 64 in CDCl3.

Figure S80. HMBC NMR of 64 in CDCl3.

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Figure S81. 13C NMR of 64 in CDCl3.

HRMS Spectra for Compounds

Figure S82. HRMS Spectrum of Compound 34.

C10H15NO4
+
Expected m/z [M + H ]: 214.1074, observed: 214.1081;
Expected m/z [M + Na+]: 236.0893, observed: 236.0911;
Expected m/z [M + K+]: 252.0633, observed: 252.0641.

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 Figure S83. HRMS Spectrum of Compound 36.
C16H27NO4
+
Expected m/z [M + H ]: 298.2013, observed: 298.2062;
Expected m/z [M + Na+]: 320.1832, observed: 320.1882;
Expected m/z [M + K+]: 336.1571, observed: 336.1607;
Expected m/z [2M + H+]: 595.3953, observed: 595.4011;
Expected m/z [M + Na+]: 617.3772, observed: 617.3833.

Figure S84. HRMS Spectrum of Compound 56

C8H13NO2S
+
Expected m/z [M + H ]: 188.0734, observed: 188.0763;
Expected m/z [M + Na+]: 210.0554, observed: 210.0589.

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 Figure S85. HRMS Spectrum of Compound 57
C10H17NO2S
Expected m/z [M + H+]: 216.1047, observed: 216.1099;
Expected m/z [M + Na+]: 238.0867, observed: 238.0942.

Figure S86. HRMS spectrum of Compound 58

C12H21NO2S
Expected m/z [M + H+]: 244.1360, observed: 244.1405;
Expected m/z [M + Na+]: 266.1180, observed: 266.1232;
Expected m/z [M + K+]: 282.0919, observed: 282.0962.

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 Figure S87. HRMS Spectrum of Compound 59
C14H25NO2S
Expected m/z [M + H+]: 272.1673, observed: 272.1720;
Expected m/z [M + Na+]: 294.1493, observed: 294.1555;
Expected m/z [M + K+]: 310.1291, observed: 310.1291.

Figure S88. HRMS Spectrum of Compound 60

C16H29NO2S
Expected m/z [M + H+]: 300.1986, observed: 300.2042;
Expected m/z [M + Na+]: 322.1806, observed: 322.1872;
Expected m/z [M + K+]: 338.1545, observed: 338.1605.

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 Figure S89. HRMS Spectrum of Compound 61
C10H15NO3S
Expected m/z [M + H+]: 230.0845, observed: 230.0896;
Expected m/z [M + Na+]: 252.0665, observed: 252.0729;
Expected m/z [M + K+]: 268.0404, observed: 268.0452.

Figure S90. HRMS Spectrum of Compound 62

C12H19NO3S
Expected m/z [M + H+]: 258.1158, observed: 258.1162;
Expected m/z [M + Na+]: 280.0978, observed: 280.1008;
Expected m/z [M + K+]: 296.0717, observed: 296.0723.

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 Figure S91. HRMS Spectrum of Compound 63
C14H23NO3S
Expected m/z [M + H+]: 286.1471, observed: 286.1472;
Expected m/z [M + Na+]: 308.1291, observed: 308.1320;
Expected m/z [M + K+]: 324.1030, observed: 324.1035.

Figure S92. HRMS Spectrum of Compound 64

C16H27NO3S
Expected m/z [M + H+]: 314.1784, observed: 314.1791;
Expected m/z [M + Na+]: 336.1604, observed: 336.1633;
Expected m/z [M + K+]: 352.1343, observed: 352.1354.

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Mass spectroscopy of alkyl-ACP and alkyl-CoA compounds

Figure S93. Mass Spectrum of Compound 88 (butyl-ACP)

Expected m/z [M + 5H+]: 1781.9, observed: 1781.7;
Expected m/z [M + 6H+]: 1485.1, observed: 1484.9;
Expected m/z [M + 7H+]: 1273.1, observed: 1273.1;
Expected m/z [M + 8H+]: 1114.1, observed: 1113.9;
Expected m/z [M + 9H+]: 990.4, observed: 990.3;
Expected m/z [M + 10H+]: 891.5, observed: 891.4.

Figure S94. Mass Spectrum of Compound 89 (hexyl-ACP)

Expected m/z [M + 5H+]: 1787.5, observed: 1787.5;
Expected m/z [M + 6H+]: 1489.8, observed: 1489.6;
Expected m/z [M + 7H+]: 1277.1, observed: 1277.1;
Expected m/z [M + 8H+]: 1117.6, observed: 1117.6;
Expected m/z [M + 9H+]: 993.5, observed: 993.5;
Expected m/z [M + 10H+]: 894.3, observed: 894.2.

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Figure S95. Mass Spectrum of Compound 90 (octyl-ACP)
Expected m/z [M + 5H+]: 1793.1, observed: 1792.9;
Expected m/z [M + 6H+]: 1494.5, observed: 1494.2;
Expected m/z [M + 7H+]: 1281.1, observed: 1281.1;
Expected m/z [M + 8H+]: 1121.1, observed: 1120.9;
Expected m/z [M + 9H+]: 996.6, observed: 996.5;
Expected m/z [M + 10H+]: 897.1, observed: 897.0.

Figure S96. Mass Spectrum of Compound 91 (decyl-ACP)

Expected m/z [M + 5H+]: 1798.7, observed: 1798.5;
Expected m/z [M + 6H+]: 1499.1, observed: 1499.1;
Expected m/z [M + 7H+]: 1285.1, observed: 1285.1;
Expected m/z [M + 8H+]: 1124.6, observed: 1124.6;
Expected m/z [M + 9H+]: 999.8, observed: 999.7;
Expected m/z [M + 10H+]: 899.9, observed: 899.8.

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Figure S97. HRMS Spectrum of Compound 92 (butyl-CoA)
C25H44N7O16P3S
Expected m/z [M + H+]: 824.1851, observed: 824.1835;
Expected m/z [M + Na+]: 846.1670, observed: 846.1657;
Expected m/z [M + 2Na+ - H+]: 868.1490, observed: 868.1472.

Figure S98. HRMS Spectrum of Compound 93 (hexyl-CoA)

C27H48N7O16P3S
Expected m/z [M + H+]: 852.2164, observed: 852.2147;
Expected m/z [M + Na+]: 874.1983, observed: 874.1953;
Expected m/z [M + 2H+]: 426.6118, observed: 426.6105.

- S74 -
Figure S99. HRMS Spectrum of Compound 94 (octyl-CoA)
C29H52N7O16P3S
Expected m/z [M + H+]: 880.2477, observed: 880.2444;
Expected m/z [M + K+]: 918.2036, observed: 918.1978;
Expected m/z [M + 2H+]: 440.6275, observed: 440.6259.

Figure S100. HRMS Spectrum of Compound 95 (decyl-CoA)

C31H56N7O16P3S
Expected m/z [M + H+]: 908.2790, observed: 908.2733;
Expected m/z [M + 2H+]: 454.6431, observed: 454.6401;
Expected m/z [M + H+ + Na+]: 465.6345, observed: 465.6300.

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References

1. Montebello, A. N., Brecht, R. M., Turner, R. D., Ghali, M., Pu, X, Nagarajan, R. (2014). Acyl-
ACP substrate recognition in Burkholderia mallei BmaI1 acyl-homoserine lactone synthase,
Biochemistry 53, 6231–6242.

2. Shin, D., Frane, N. D., Brecht, R. M., Keeler, J., Nagarajan, R. (2015). A Comparative
Analysis of Acyl-Homoserine Lactone Synthase Assays. Chembiochem 16, 2651–2659.

3. Christensen, Q. H., Grove, T. L., Booker, S. J., Greenberg, E. P. (2013). A high-throughput

screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases. Proc Natl
Acad Sci USA 110, 13815–13820.

4. Christensen, Q.H., Brecht, R.M., Dudekula, D., Greenberg, E.P., Nagarajan, R. (2014).
Evolution of acyl-substrate recognition by a family of acyl-homoserine lactone synthases. PLoS
ONE 9, e112464.

5. Copeland, R.A. (2005). Evaluation of enzyme inhibitors in drug discovery. A guide for
medicinal chemists and pharmacologists. Methods Biochem Anal 46, 1–265.

6. Raychaudhuri, A., Jerga, A., Tipton, P. A. (2005). Chemical mechanism and substrate
specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa.
Biochemistry 44, 2974–2981.

7. Rock, C. O., Cronan, J. E. (1980). Improved purification of acyl carrier protein. Anal Biochem
102, 362–364.

8. Geske, G.D.; Wezeman, R. J.; Siegel, A. P.; Blackwell, H. E. (2015) Small molecule inhibitors
of bacterial quorum sensing and biofilm formation, J. Am. Chem. Soc. 127, 12762-12763.

9. Boursier, M. E.; Moore, J. D.; Heitman, K. M.; Shepardson-Fungairino, S. P.; Combs, J. B.;
Koenig, L. C.; Shin, D.; Brown, E. C.; Nagarajan, R.; Blackwell, H. E. (2018) Structure-function
analyses of the N-butanoyl-L-homoserine lactone quorum–sensing signal define features critical
to activity in RhlR, ACS Chem. Biol. 13, 2655-2662.

10. Jog, G. J.; Igarashi, J.; Suga, H. (2006) Stereoisomers of P. aeruginosa autoinducer analog to
probe the regulator binding site, ACS Chem. Biol. 13, 123-128.

11. Geske, G. D., O’Neill, J. C., Miller, D. M., Wezeman, R. J., Mattmann, M. E., Lin, Q., &
Blackwell, H. E. (2008) Comparative analyses of N-acylated homoserine lactones reveal unique
structural features that dictate their ability to activate or inhibit quorum sensing, ChemBioChem,
9, 389–400.

12. Gerdt, J. P., Wittenwyler, D. M., Combs, J. B., Boursier, M. E., Brummond, J. W., Xu, H., &
Blackwell, H. E. (2017) Chemical interrogation of LuxR-type quorum sensing receptors reveals

- S76 -
new insights into receptor selectivity and the potential for interspecies bacterial signaling, ACS
Chem. Biol. 12, 2457–2464.

13. Thomas, P. W., Stone, E. M., Costello, A. L., Tierney, D. L., & Fast, W. (2005). The
quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein, Biochemistry 44,
7559–7569.

14. McInnis, C. E., & Blackwell, H. E. (2011). Thiolactone modulators of quorum sensing
revealed through library design and screening, Bioorganic & Medicinal Chemistry 19, 4820–
4828.

15. Blackwell, H.; Boursier, M. E.; Moore, J. D. “Synthetic ligands that modulate the activity of
the RhlR quorum sensing receptor.” World Intellectual Property Organization Patent
WO2017190116A1, Nov 2, 2017.

16. Nechab, M., El Blidi, L., Vanthuyne, N., Gastaldi, S., Bertrand, M. P., & Gil, G. (2008). N-
Acyl glycinates as acyl donors in serine protease-catalyzed kinetic resolution of amines.
Improvement of selectivity and reaction rate, Organic & Biomolecular Chemistry 6, 3917–3920.

17. Horikawa, M.; Tateda, K.; Tuzuki, E.; Ishii, Y.; Ueda, C.; Takabatake, T.; Miyairi, S.;
Yamaguchi, K; Ishiguro, M. (2006) Synthesis of Pseudomonas quorum-sensing autoinducer
analogs and structural entities required for induction of apoptosis in macrophages, Bioorg. Med.
Chem. Lett. 16, 2130-2133.

18. Shinohara, Y.; Hasegawa, H.; Hashimoto, T.; Ichida, K. (2010) Synthesis of optically active
deuterium-labeled homocysteine thiolactone, Journal of Labelled Compounds and
Radiopharmaceuticals 53, 552-555.

19. Chhabra, S. R.; Harty, C.; Hooi, D. S. W.; Daykin, M.; Williams, P.; Telford, G.; Pritchard,
D. I.; Bycroft, B. W. (2003) Synthetic analogues of the bacterial signal (quorum sensing)
molecule N-(3-oxododecanoyl)-l-homoserine lactone as immune modulators, J. Med. Chem. 46,
97-104.

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