Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of β-processing domains via diffusible, synthetic substrates

Natural and modified substrates coupled with LC-MS/MS analysis of products revealed the stereospecificity and stereoselectivity of a polyketide didomain.


S-(2-acetamidoethyl) (6S,7R,E)-7-hydroxy-4,6-dimethyl-3-oxonon-4-enethioate (4).
The crude silylether 25 (20.6 mg, 0.0437 mmol) was transferred as an acetonitrile solution (0.200 mL) to a polypropylene tube (15 mL, BD Falcon TM ) and cooled via icewater bath (0 °C). To the pre-chilled solution was added a 48% hydrofluoric acid solution diluted in acetonitrile (11:89, 2.50 mL). The reaction mixture was placed in refrigerator (4 °C) for 24 h. TLC analysis of the reaction mixture indicated incomplete conversion and an addition portion of hydrofluoric acid solution (freshly prepared and identical to above, 1 mL) was added to the reaction mixture at 0 °C. After an additional 12 h at 4 °C, the reaction mixture was neutralized at 0 °C via addition of aqueous, saturated sodium bicarbonate solution. The reaction mixture was extracted with ethyl acetate (4 x 25 mL). The combined organic fractions were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. Crude product was purified by flash column chromatography (5% MeOH/dichloromethane) yielding a slightly yellow oil (11.8 mg, 0.0374 mmol, 86% from 24). Scheme S1. Synthetic route towards L-alcohol substrate 6a. An asymmetric aldol reaction between key aldehyde 14 and Nagao's chiral auxiliary ent-15 and displacement of the thiazoldinone with N-acetylcysteamine furnished thioester S2. Final deprotection with hydrogen fluoride afforded the desired diol substrate 6a.

S13
(2 mL) was sequentially added imidazole (26.0 mg, 0.384 mmol, 3.00 equiv.) and Nacetylcysteamine (16.0 μL, 0.154 mmol, 1.20 equiv.). The transparent, yellow solution was vigorously stirred under argon atmosphere for 15 h at ambient temperature. The reaction was quenched upon addition of an aqueous, saturated ammonium chloride solution (3 mL) and the biphasic solution was separated. The aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated under vacuum and purified via silica column flash chromatography (5% MeOH/dichloromethane) using a small plug of copper(II) sulfate impregnated silica gel (1 cm on top) furnished the title compound as a colorless oil (59.0 mg, 0.125 mmol, 97%). Chemical synthesis of thioether substrate 7a. Aldol adduct S1 was sequentially converted to the corresponding weinreb amide (S3) followed by protection with triethylsilyl trifluromethanesulfonate providing disilylether S4. Grignard addition and deprotection followed by Michael addition afforded thioether 7a.

(4R,5R,E)-2,4-dimethyl-5-((triisopropylsilyl)oxy)hept-2-enal (S9).
A round bottom flask containing acyl oxazolidinone S8 (0.373 g, 0.848 mmol) dissolved in anhydrous dichloromethane (27 mL) under argon atmosphere was cooled to -78 °C (dry iceacetone). To the chilled reaction mixture was added a toluene solution of diisobutylaluminum hydride (1.49 M, 1.14 mL, 1.70 mmol, 2 equiv.) dropwise over two minutes. The reaction mixture was stirred at -78 °C for 13 min and quenched upon sequential addition of methanol (10 mL) and saturated, aqueous potassium sodium tartrate solution (10 mL). The biphasic solution was allowed to warm to ambient temperature and separated. The aqueous layer was extracted with dichloromethane (3 x 15 mL) and the combined organic fractions were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified via flash column  (4S,5S,E)-2,4-dimethyl-5-((triisopropylsilyl)oxy)hept-2-enal (ent-S9). The title compound was synthesized in an analogous matter to its enantiomer and was identical with respect to 1 H-NMR and 13 C-NMR spectra. and stirred for 30 min. To the reaction was slowly added freshly distilled diisopropylethylamine 1 (0.114 mL, 0.657 mmol, 1.80 equiv.). The blood red reaction mixture was stirred for 2 h and transferred to dry ice-acetone bath (-78 °C). A dichloromethane (1.3 mL) solution of aldehyde S9 (0.114 g, 0.365 mmol, 1.00 equiv.) was slowly added to the cooled solution over 12 min. After stirring for 4 h at -78 °C the reaction was quenched via addition of ammonium chloride (5 mL). The biphasic mixture was warmed to ambient temperature and separated. The aqueous layer was extracted with dichloromethane (3 x 10 mL). Combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting crude oil was purified via flash chromatography (20% EtOAc/hexanes) to give both the aldol adduct S10 as a thick, bright yellow oil (99.3 mg, 0.192 mmol, 53%) and the dehydration product S11 as a viscous, yellow oil (44.  Scheme S4. Synthesis of dehydration product S14. By utilizing the serendipitous byproduct S11, the result of dehydration during an aldol reaction, we were able to quickly produce the dehydration product S14. This dienethioate served as the product standard in LC-MS/MS for the enzymatic dehydration reaction of 27. Additionally, the same compound was used as the product standard for TylDH3-catalyzed dehydration of 6d, as it is the enantiomer of the presumed product.

S-(2-acetamidoethyl)
(2E,4E,6R,7R)-7-hydroxy-4,6dimethylnona-2,4-dienethioate (S14). A polypropylene tube (15.0 mL, BD Falcon TM ) containing silylether S13 (18.0 mg, 0.0395) dissolved in acetonitrile (4.15 mL) was equilibrated in ice-water bath (0 °C). To the chilled starting material was added a solution 48% hydrofluoric acid in acetonitrile (11:89, 5.0 mL). The resulting acidified solution was stored at 4 °C for 48 h and quenched via addition of sodium sulfate at 0 °C. The reaction mixture was extracted with ethyl acetate (4 x 15 mL) and the combined organics were dried over anhydrous sodium sulfate. Filtration and concentration under vacuum gave the crude product residue that was purified by flash column chromatography (5% MeOH/dichloromethane) providing the desired alcohol as a cloudy, colorless oil (8.1 mg, . Upon literature analysis we discovered that this compound had been synthesized through a different route by Cane, DE and co-workers. 5 Our analytical data match that of the original report in all respects. Scheme S5. Chemical preparation of substrate 6d. The enantiomer of aldehyde S9 (ent-S9) was submitted to an aldol reaction with Nagao's acyl chiral auxiliary 15 to afford aldol adduct S15 as the only product. Interestingly, by decreasing the reaction time from 4 h to 1 h 45 min, we avoided the formation of elimination product with this diastereomer. Thiazolidinethione displacement followed by deprotection furnished the TylDH3 substrate 6d.

Cloning and Expression of TylDH3-KR3 Construct
Initial efforts to recombinantly express the mono-domain TylKR3 were hampered by with poor expression levels and protein aggregation. Strategies to alleviate these issues included an increase of rare tRNA codons (Rosetta cell line), optimization of codon selection (synthetic TylKR3 gene), toxic protein-compatible expression hosts (pLysS cell line), appending a fusion protein (attempted with SUMO, mOCR, and GST), chaperone coexpression (GroEL-GroES) and truncations of both N-and C-termini. Disappointingly, these techniques failed to improve expression of soluble non-aggregated TylKR3 and forced us to abandon the expression of the mono-domain construct.
The tylGII region encoding TylDH3-KR3 didomain comprising residues 957-1682 was cloned into a pMCSG7 vector and transformed into E. coli BL21-AI cells containing the pRARE plasmid. A large TB media culture (0.5 L in 2.8 L Fernbach flask) was inoculated with as small amount of overnight culture (5 mL) and incubated at 37 °C, shaking at 250 RPM until OD600 = 1.00-1.20. The culture was cooled to 20 °C and incubated with shaking (250 RPM) for 1 h. Cells were induced upon addition of IPTG (0.100 mM) and L-arabinose (1.00 g) and allowed to shake (250 RPM) at 20 °C for 19 h. The cell pellet was collected after centrifugation (4 °C, 5,000 x G, 30 min) and resuspended in lysis buffer (50 mM tricine, 50 mM ammonium sulfate, 100 mM urea, pH 8.5, 4 mL/g of pellet). The cells were lysed (3 x 2 min, 50% duty cycle, 40 % power, 4 °C) and centrifuged (4 °C, 28,600 x G, 45 min). The soluble protein was purified by sequential metal-immobilized affinity chromatography and size exclusion chromatography to afford approximately 9 mg of purified protein (18 mg / L) that was greater than 90% pure as judged by SDS-PAGE ( Figure S1) and to be near the predicted calculated mass by mass spectrometry (Figure S2). Figure S1. SDS-PAGE image of TylDH3-KR3 purification. The ladder, cell lysate, insoluble pellet, soluble protein and serial nickel elution fractions are shown in lanes 1-9, respectively. The band corresponding to TylDH3-KR3 is boxed.
S27 Figure S2-Mass spectrometry analysis of TylDH3-KR3. The convoluted (raw) spectrum and deconvoluted are both displayed. TylDH3-KR3 was found to have a mass of 76,264 Da. The spectrum was obtained using 0.5 mg/mL of recombinant TylDH3-KR3 in 25% formic acid.

Analysis of TylDH3-KR3 Dehydratase Activity
Steady-State Analysis. The enzymatic reactions were carried out in a total volume of 50 µL under initial velocity conditions containing TylDH3-KR3 (1 µM), reaction buffer (50 mM Tris, 150 mM NaCl, pH 8.0) and substrates 6b or 7b at variable concentrations (0.5, 1, 2, 3, 4, 6, 8 mM). The final DMSO concentration was held constant at 4%. After incubation at 25 °C for 8 min (the reaction found to be linear up to 10 min), 5 µL of the reaction mixture was added to 495 µL of 1:1 MeCN-reaction buffer (100-fold dilution). The resulting solution was vortexed, centrifuged and analyzed by 60 µL of the diluted reaction solution was added to a HPLC vial with 10 µL of internal standard 3 (320 nM) and analyzed by LC-MS/MS (Table S4) employing a Kinetix reverse-phase C 18 column (50 mm × 2.1 mm, 2.6 µm, Phenomenex) operated at 0.4 mL min -1 with a gradient between mobile phase A (H 2 O) and mobile phase B (MeCN). The gradient program was 0 min, 5% B; 2 min, 5% B; 7 min, 55% B; 8 min, 70% B; 9 min, 70% B; 10.5 min, 5% B; 12 min, 5% B. Standard curves of enzymatic products 8 and 9 were generated by injecting the authentic standard at varying concentrations with a fixed concentration of an internal standard (8 for the standard curve of 9 and 9 for the standard curve of 8). The amount of enzymatic product formation at each time point was calculated by plotting the area ratio (analyte/internal standard) into the standard curve. Control reactions for each concentration of substrate were performed without the addition of enzyme. Each reaction was performed in duplicate. The specificity constants (K M /k cat ) were determined by fitting the normalized v 0 vs [S] plots to linear equations ( Figure S3 panels A and B).
Substrates 6c and 6d were analyzed in an analogous way using synthetic S14 as the product standard (Figure S3 panels C and D). The LC-MS/MS trace of S14 is provided in Figure S4. Figure S3. Linear regression analysis of TylDH3KR3 kinetic data with substrates 6b, 7b, 6c, and 6d. The kinetic plots of thioester 6b, thioether 7b, thioester 6c and thioester 6d are shown in panels A, B, C and D, respectively. Represented data is the result of duplicate LC-MS/MS data normalized with controls lacking enzyme. Specificity constants (k cat /K M ) for each substrate are displayed below the corresponding plot. Figure S4. LC-MS/MS trace of dienethiolate S14. The synthetic compound S14 was used to confirm the identity of the dehydration products arising from thioesters 6c and 6d. A standard curve for quantitative determination of specificity constants was generated from the synthetic compound.