S1 Supporting Information For Beta-ketoacyl-ACP substrate mimics for 3-oxoacyl-ACP utilizing ...

81 Pages·2017·8.78 MB·English

Electronic Supplementary Material (ESI) for ChemComm.
This journal is © The Royal Society of Chemistry 2018
Supporting Information

For

Beta-ketoacyl-ACP substrate mimics for 3-oxoacyl-ACP utilizing AHL synthases

Contents
Scheme for synthesis of β-ketoacyl-ACP mimics……………………………………….S2
General information……………………………………………………………………...S3
Experimental procedures…………………………………………………..………..S4-S20
NMR characterization of compounds (1H and 13C NMR spectra)……………..…..S21-S38
HRMS spectra for 4-Succ – 14-Succ…………………………………………..…..S38-S43
HRMS spectra for 4-CoA – 15-CoA………………………………………………S44-S49
HRMS spectra for 1-ACP – 15-ACP…………………………………………..….S50-S57
EsaI wild-type and T140A mutant growth, expression, and purification………………..S58
Apo-ACP growth, expression, and purification……………………………………S59-S60
Sfp growth, expression, and purification…………………………………………..S60-S61
YspI growth, expression, and purification…………………………………………S61-S62
HPLC assay………………………………………………………………………..S62-S65
Substrate velocity curves…………………………………………………………..S66-S74
Tables for kinetic constants………………………………………………………..S75-S77
References…………………………………………………………………………..…..S78
S1
A
O O
O O O
O O O O O O O
+
R OH R OH
O R O R OR' R OR'
R: C3H7; C5H11 O O RR==CC35HH71:1 :4 5-A-Accidid
O
O O
O O n OH R O O R OH
n O
n=1: 6-Acid R=CH3: 8-Acid
n=2: 7-Acid R= C3H7: 9-Acid
B
O O O
O O O
CoA-SH apo-ACP
R' OH + HO N R' O N R' S-CoA Sfp R' S-ACP

O O
Scheme S1. Synthetic scheme for β-ketoacyl-ACP mimics. The numbers correspond to
substrates listed in Figure 2 (main text). A) Synthesis of carboxylic acid starting materials
used to synthesize substrates 4-9. Meldrum’s acid procedure was used to introduce the
beta-keto moiety in substrate 4 and 5. Ring opening of gamma and delta-lactones yielded
the corresponding carboxylic acids for substrates 6-9. B) General synthetic procedure for
acyl-ACP synthesis. The carboxylic acids were first activated to a succinimide ester or an
imidazole amide, which was coupled with CoA to synthesize acyl-CoA. Bacillus subtilis
sfp phosphopantetheinyl transferase was used to enzymatically couple acyl-CoAs and apo-
ACP to make the corresponding acyl-ACPs (1-ACP-15-ACP) described in this study.

S2
General Information
All chemical reagents and solvents were purchased from commercial sources and used
without further purification, except where indicated. The acyl-CoA compounds (1-CoA,
2-CoA and 3-CoA) were commercially available from Sigma-Aldrich. Ethyl-3-
oxohexanoate was purchased from Acros Organics and used to make 4-DM-Ester. Methyl
3-oxooctanoate was prepared according to the procedure described by D’Oca et al.[1] Ni+-
NTA resin was purchased from Qiagen. Silica gel 230-400 mesh from Fisher was used in
flash column chromatography. A Thermo Scientific Evolution 260 Bio UV-Vis
spectrophotometer was used to measure the concentration of analytes. HPLC data was
analyzed by Chromeleon 7.2 software on a Thermo Scientific Dionex UltiMate-3000
HPLC system. Thermo Scientific Hypersil Gold C18 reverse-phase analytical UHPLC
column (25002-054630) and preparative HPLC column (25005-159070) were used in acyl-
ACP and acyl-CoA syntheses, respectively. Deuterated chloroform and methanol solvent
were obtained commercially through Cambridge Isotope Laboratories, Inc. NMR spectra
were recorded at 298 K using BRUKER AVANCE III 300 MHz and 600 MHz
spectrometers. Chemical shifts were expressed in parts per million (ppm) and referenced
to residual solvent as the internal reference for 1H (CDCl : δ = 7.24 ppm or CD OD: δ =
3 3
3.31 ppm) and 13C (CDCl : δ = 77.16 ppm and CD OD: δ = 49.00 ppm). We are grateful
3 3
to the following PI’s for providing plasmids for the following proteins: Pantoea Stewartii
EsaI (Prof. Mair Churchill, University of Colorado-Denver), Bacillus subtilis Sfp (Prof.
Michael D. Burkart, University of California-San Diego), Yersinia pestis YspI (Prof. E. P.
Greenberg, University of Washington-Seattle) and Escherichia coli ACPg (Prof. John
Cronan, University of Illinois-Urbana Champaign).

S3
Experimental Procedures
General procedure for the preparation of 2,2-dimethyl β-ketoesters (4-DM-Ester and
5-DM-Ester). To a dry THF solution (60 mL) containing a suspension of NaH (1 equiv,
60% wt. dispersion in mineral oil) under a nitrogen atmosphere, was added the appropriate
β-ketoester (1 equiv). The solution was stirred at room temperature until hydrogen gas
evolution stopped (~30 minutes), then methyl iodide (1 equiv) was added and the solution
heated to reflux for 12 h. After cooling to room temperature, additional NaH (1 equiv, 60%
wt. dispersion in mineral oil) and methyl iodide (1 equiv) were added, and the solution
refluxed for another 12 h. The completion of the reaction was checked using TLC (5%
ethyl acetate: 95% hexane). To this solution, was added a saturated solution of ammonium
chloride until all the white precipitate dissolved, which was extracted with
dichloromethane (3 x 70 mL). The organic layer was separated, dried with anhydrous
sodium sulfate, filtered and the solvent removed under reduced pressure. The products
were purified by column chromatography on silica gel (99:1 hexanes/EtOAc).

Ethyl 2,2-dimethyl-3-oxohexanoate (4-DM-Ester).
O O
O

Yield from 2.0 g of ethyl-3-oxohexanoate: 2.13 g (91%). 1H NMR (600 MHz, CDCl ): δ
3
4.15 (q, J = 7.2 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 1.59 (sextet, J = 7.3 Hz, 2H), 1.33 (s,
6H), 1.23 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H).

S4
Methyl 2,2-dimethyl-3-oxooctanoate (5-DM-Ester).
O O
O

Yield from 0.714 g of methyl 3-oxooctanoate[1] : 0.6 g (72%). 1H NMR (600 MHz, CDCl ):
3
δ 3.69 (s, 3H), 2.40 (t, J = 7.3 Hz, 2H), 1.55 (quintet, J = 7.5 Hz, 2H), 1.34 (s, 6H), 1.27
(sextet, J = 7.5 Hz, 2H), 1.24-1.18 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H). 13C NMR (600 MHz,
CDCl ): δ 208.3, 174.5, 55.8, 52.6, 38.1, 31.5, 23.7, 22.7, 22.2, 14.1. The NMR spectra for
3
5-DM-Ester were identical to data obtained for the product by an alternate procedure.[2]

General procedure for the preparation of 2,2’-dimethyl β-ketoacids (4-Acid and 5-
Acid). The appropriate 2,2-dimethyl ketoester (1 equiv) was added to a 1 N NaOH (4.3
mL) solution and stirred overnight. The aqueous solution was then washed twice with ethyl
acetate (2 x 8 mL), and cooled to 0 ºC using an ice bath. 2 N HCl was added slowly to
adjust the pH to 2, resulting in the formation of a cloudy solution. The workup for acids 4-
Acid and 5-Acid differ and the details are discussed below.

2,2-Dimethyl-3-oxohexanoic acid (4-Acid).
O O
OH

For 4-Acid, the solution was then washed with petroleum ether (10 mL) and the aqueous
phase was collected and lyophilized to afford 4-Acid as a white solid. Yield from 0.2 g of
ethyl 2,2-dimethyl-3-oxohexanoate: 0.12 g (74%). 1H NMR (300 MHz, CD OD): δ 2.53
3
S5
(t, J = 7.2 Hz, 2H), 1.59 (sextet, J = 7.3 Hz, 2H), 1.26 (s, 6H), 0.90 (t, J = 7.4 Hz, 3H). 13C
NMR (300 MHz, CD OD): δ 209.1, 175.6, 55.1, 39.3, 21.0, 16.9, 12.5.
3

2,2-Dimethyl-3-oxooctanoic acid (5-Acid).
O O
OH

For 5-Acid, petroleum ether (10 mL) was used to extract the product from the aqueous
solution. The organic layer was collected and the solvent removed under reduced pressure
to afford 5-Acid as a white solid. Yield from 0.6 g of methyl 2,2-dimethyl-3-oxooctanoate:
0.54 g (97%). 1H NMR (600 MHz, CDCl ): δ 2.51 (t, J = 7.3 Hz, 2H), 1.57 (quintet, J =
3
7.4 Hz, 2H), 1.39 (s, 6H), 1.35-1.23 (m, 4H), 0.86 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz,
CDCl ): δ 208.4, 178.9, 55.6, 38.2, 31.5, 23.7, 22.7, 22.3, 14.1.
3

General procedure for preparation of 4-oxocarboxylic acids (6-Acid and 7-Acid).
Compounds 6-Acid and 7-Acid were prepared similar to the protocol described by
Ambrosio et al.[3] The appropriate alkyl-substituted γ-lactone (1 equiv) was hydrolyzed
overnight with 0.5 M NaOH in EtOH (2 equiv). The solvent was removed using reduced
pressure and the residue dissolved in ~40 mL of a phosphate buffer (5.5 g NaH PO and
2 4
1.6 g Na HPO ). Concentrated 10-15% NaOCl (2.4 equiv) was added and the solution
2 4
stirred for 20 h at room temperature. Concentrated HCl was added to adjust the pH to 3
before the product was extracted using diethyl ether (2 x 20 mL). The organic layer was
washed with brine (40 mL), dried with anhydrous MgSO , filtered and the solvent removed
4
to afford the 4-oxocarboxylic acid.
S6
4-Oxohexanoic acid (6-Acid).
O
OH
O
Yield from 0.24 g of γ-hexalactone: 0.21 g (81%). 1H NMR (600 MHz, CDCl ): δ 2.71 (t,
3
J = 6.4 Hz, 2H), 2.62 (t, J = 6.4 Hz, 2H), 2.46 (q, J = 7.4 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H).
The 13C NMR spectrum for 6-Acid was identical to data obtained for the product by
Ambrosio and co-workers.[3]

4-Oxooctanoic acid (7-Acid).
O
OH
O
Yield from 0.50 g of γ-octalactone: 0.26 g (48%). 1H NMR (300 MHz, CDCl ): δ 2.69 (t,
3
J = 6.2 Hz, 2H), 2.60 (t, J = 6.2 Hz, 2H), 2.42 (t, J = 7.5 Hz, 2H), 1.55 (quint, J = 7.5 Hz,
2H), 1.29 (sextet, J = 7.5 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (300 MHz, CDCl ):
3
δ 209.3, 178.8, 42.6, 36.9, 27.9, 26.1, 22.5, 14.0.

General procedure for preparation of 5-oxocarboxylic acids (8-Acid and 9-Acid).
Compounds 8-Acid and 9-Acid were prepared similar to the protocol described by
Ambrosio et al.[3] The appropriate alkyl-substituted δ-lactone (1 equiv) was hydrolyzed
overnight with 0.5 M NaOH in EtOH (2 equiv). The solvent was removed using reduced
pressure and the residue dissolved in ~40 mL of a phosphate buffer (5.5 g NaH PO and
2 4
S7
1.6 g Na HPO ). Concentrated 10-15% NaOCl (2.4 equiv) was added and the solution
2 4
stirred for 20 h at room temperature. Concentrated HCl was added to adjust the pH to 3
before the product was extracted using diethyl ether (2 x 20 mL). The organic layer was
washed with brine (40 mL), dried with anhydrous MgSO , filtered and the solvent removed
4
to afford the 5-oxocarboxylic acid.

5-Oxohexanoic acid (8-Acid).
O O
OH
Yield from 0.104 g of δ-hexalactone: 0.15 g (29%). 1H NMR (300 MHz, CDCl ): δ 2.51
3
(t, J = 7.2 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.13 (s, 3H), 1.88 (quint, J = 7.2 Hz, 2H). The
13C NMR spectrum for 8-Acid was identical to data obtained for the product by Ambrosio
and co-workers.[3]

5-Oxooctanoic acid (9-Acid).
O O
OH
Yield from 0.11 g, 0.79 mmol) of δ-octalactone: 0.084 g (67%). 1H NMR (600 MHz,
CDCl ): δ 2.47 (t, J = 7.2 Hz, 2H), 2.37 (t, J = 7.1 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H), 1.88
3
(quint, J = 7.2 Hz, 2H), 1.58 (sextet, J = 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR
(600 MHz, CDCl ): δ 210.5, 178.8, 45.0, 41.5, 33.1, 18.8, 17.5, 13.9.
3

S8
General Procedure for the preparation of N-hydroxysuccinimide esters (4-Succ – 15-
Succ). To a 1,4-dioxane (3 mL) solution of the appropriate carboxylic acid (1 equiv), N-
hydroxysuccinimide (1 equiv) and N,N ʹ-dicyclohexylcarbodiimide (1 equiv) were added
and stirred for 24 hours. Diethyl ether (2 mL) was added to the reaction mixture and the
white cloudy solution was filtered through Celite and the solvent removed under reduced
pressure. Warm methanol (2 mL) was used to dissolve the residue and, upon precipitation
of the urea by-product upon cooling to room temperature, the solution was filtered through
Celite and the solvent removed. The residue was then titurated with 1,4-dioxane (3 mL),
filtered with Celite and the filtrate collected and the solvent removed under reduced
pressure to give the product.

2,2-Dimethyl-3-oxohexanoyl N-hydroxysuccinimide ester (4-Succ).
O
O O
N
O
Me Me
O
Further purification by column chromatography on silica gel (1:1 hexanes/EtOAc) was
necessary. Yield from 0.15 g of 2,2-dimethyl-3-oxohexanoic acid: 0.062 g (26%). 1H NMR
(600 MHz, CDCl ): δ 2.81 (d, J = 6.5 Hz, 4H), 2.64 (t, J = 7.1 Hz, 2H), 1.63 (sextet, J =
3
7.3 Hz, 2H), 1.48 (s, 6H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (600 MHz, CDCl ): δ 205.5,
3
169.7, 168.9, 54.7, 40.3, 25.6, 22.1, 17.3, 13.6. HRMS (ESI-TOF) m/z calculated for
[M+Na]+: 278.0999, observed: 278.1107.

S9
2,2-Dimethyl-3-oxooctanoyl N-hydroxysuccinimide ester (5-Succ).
O
O O
N
O
Me Me O
Further purification by column chromatography on silica gel (1:1 hexanes/EtOAc) was
necessary. Yield from 0.027 g of 2,2-dimethyl-3-oxooctanoic acid: 0.035 g (83%). 1H
NMR (600 MHz, CDCl ): δ 2.81 (d, J = 6.1 Hz, 4H), 2.65 (t, J = 7.3 Hz, 2H), 1.59 (quint,
3
J = 7.4 Hz, 2H), 1.48 (s, 6H), 1.32-1.21 (m, 4H), 0.85 (t, J = 7.1 Hz, 3H). 13C NMR (600
MHz, CDCl ): δ 205.6, 169.7, 168.9, 54.7, 38.5, 31.3, 25.8, 23.5, 22.6, 22.2, 14.1. HRMS
3
(ESI-TOF) m/z calculated for [M+Na]+: 306.1312, observed: 306.1333.

4-Oxohexanoyl N-hydroxysuccinimide ester (6-Succ).
O
O
N
O
O O
Yield from 0.21 g of 4-oxohexanoic acid: 0.15 g (41%). 1H NMR (600 MHz, CDCl ): δ
3
2.89 (t, J = 6.4 Hz, 2H), 2.84 (t, J = 6.4 Hz, 2H), 2.82 (d, J = 6.5 Hz, 4H), 2.47 (q, J = 7.3
Hz, 2H), 1.07 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, CDCl ): δ 207.9, 169.2, 168.5,
3
36.4, 35.9, 25.8, 25.3, 7.9. HRMS (ESI-TOF) m/z calculated for [M+Na]+: 250.0686,
observed: 250.0700.

S10