Chemistry & Biology
Biocatalysis with Modular PKS Ketoreductases
(Bu2BOTf) (2.83 ml of a 1 M solution in DCM, 2.83 mmol, 1.18 eq.) was supplied
via syringe. Diisopropylethylamine (0.56 ml, 3.19 mmol, 1.33 eq.) was then
added dropwise over 2 min, and the solution was cooled to ꢂ78ꢀC. After acet-
aldehyde (0.151 ml, 270 mmol, 1.12 eq.) was added, the solution was stirred at
ꢂ78ꢀC for 30 min, allowed to warm to 0ꢀC, and further stirred for 2 hr. A solution
of 3.3 ml Na2HPO4 (1 M, pH 7.4) and 8.3 ml MeOH was added to the reaction,
followed by the dropwise addition of 8.5 ml 2:1 MeOH:30% H2O2 (aq.). After
1.5 hr, organic solvents were evaporated in vacuo and the remaining material
was resuspended in 10 ml 5% NaHCO3 (aq.). The product was extracted in
DCM (3 3 20 ml), washed with 20 ml 5% NaHCO3 (aq.) and then 20 ml brine,
dried with MgSO4, filtered, and concentrated in vacuo to yield a clear oil
(350 mg, 53% after flash chromatography). Flash chromatography was per-
formed with a 15%–30% EtOAc in hexanes gradient. Rf 0.38 (50% EtOAc in
hexanes).
(4S,20S,30R)-3-(30-Hydroxy-20-Methylhexanoyl)-4-Benzyl-2-Oxazolidinone (8).
The synthesis for 7 was followed with the exception that butyrylaldehyde
(0.213 ml, 2.41 mmol, 1.12 eq.) was substituted for acetaldehyde. Flash chro-
matography was performed in the same manner, yielding 241 mg (33%) of the
desired product. Rf 0.67 (50% EtOAc in hexanes).
(4S,20R,30R)-3-(30-Hydroxy-20-Methylbutanoyl)-4-Benzyl-2-Oxazolidinone (9)
and (4S,20R,30S)-3-(30-Hydroxy-20-Methylbutanoyl)-4-Benzyl-2-Oxazolidinone
(10). Synthesis followed that of 7, with the exception that two equivalents of
Bu2BOTf were used (2.4 ml of a 1 M solution in DCM, 2.4 mmol, 2 eq.).
Rf 0.56 and 0.60 (50% EtOAc in hexanes).
tem equipped with an electrospray-ionization source. A 5%–95% B gradient
over 12 min at a flow rate of 0.7 ml/min was run in which the mobile
phases were (A) H2O with 0.1% formic acid and (B) acetonitrile with 0.1% formic
acid.
3-Oxopentanoyl-N-Acetylcysteamine Thioester (1). 1H NMR (400 MHz,
CDCl3) d 1.08 (t, 3H, J = 7.4 Hz, CH3), 1.96 (s, 3H, CH3-C=O), 2.55 (q, 2H,
J = 7.4 Hz C-CH2-C=O), 3.00–3.10 (m, 2H, S-CH2), 3.4–3.5 (m, 2H, N-CH2),
3.7 (s, 2H, O=C-CH2-C=O), 5.88 (br s, 1H, NH). ESI-MS expected mass:
218.3; observed mass: 218.2.
3-Oxobutanoyl-N-Acetylcysteamine Thioester. 1H NMR (400 MHz, CDCl3)
d 1.92 (s, 3H, CH3-C(=O)-N), 2.20 (s, 3H, CH3-C=O), 2.93–3.16 (m, 2H,
S-CH2), 3.36–3.42 (m, 2H, N-CH2), 3.62 (s, 2H, O=C-CH2-C=O), 5.99 (br s,
1H, NH).
(2RS)-Methyl-3-Oxobutanoyl-N-Acetylcysteamine Thioester (2). 1H NMR
(400 MHz, CDCl3) d 1.33 (d, 3H, J = 7.5 Hz, CH3-C-(C=O)2), 1.90 (s, 3H,
CH3-C(=O)-N), 2.19 (s, 3H, CH3-C=O), 2.95–3.08 (m, 2H, S-CH2), 3.30–3.46
(m, 2H, N-CH2), 3.71 (q, 1H, J = 7.5 Hz, O=C-CH-C=O), 5.87 (br s, 1H, NH).
ESI-MS expected mass: 218.3; observed mass: 218.4.
(2RS)-Methyl-3-Oxopentanoyl-N-Acetylcysteamine Thioester (3). 1H NMR
(400 MHz, CDCl3) d 1.08 (t, 3H, J = 7.5 Hz, CH3), 1.38 (d, 3H, J = 7.5 Hz,
CH3-C-(C=O)2), 1.96 (s, 3H, CH3-C=O), 2.48–2.68 (m, 2H, C-CH2-C=O),
3.00–3.14 (m, 2H, S-CH2), 3.36–3.52 (m, 2H, N-CH2), 3.79 (q, 1H, J = 7.5 Hz,
O=C-CH-C=O), 5.92 (br s, 1H, NH). ESI-MS expected mass: 232.3; observed
mass: 232.4.
3-Oxohexanoyl-N-Acetylcysteamine Thioester. 1H NMR (400 MHz, CDCl3)
d 0.90–1.00 (m, 3H, CH3), 1.55–1.70 (m, 2H, C-CH2-C), 1.96 (s, 3H, CH3-C=O),
2.48–2.60 (m, 2H, C-CH2-C=O), 3.00–3.14 (m, 2H, S-CH2), 3.40–3.52 (m, 2H,
N-CH2), 3.70 (s, 2H, O=C-CH2-C=O), 5.92 (br s, 1H, NH).
(2RS)-Methyl-3-Oxohexanoyl-N-Acetylcysteamine Thioester (4). 1H NMR
(400 MHz, CDCl3) d 0.85 (t, 3H, J = 7.6 Hz, CH3), 1.33 (d, 3H, J = 7.9 Hz,
CH3-C-(C=O)2), 1.50–1.60 (m, 2H, C-CH2-C), 1.98 (s, 3H, CH3-C=O), 2.45
(m, 2H, C-CH2-C=O), 2.95–3.12 (m, 2H, S-CH2), 3.34–3.52 (m, 2H, N-CH2),
3.74 (q, 1H, J = 7.9 Hz, O=C-CH-C=O), 5.92 (br s, 1H, NH). ESI-MS expected
mass: 246.3; observed mass: 246.4.
(2RS)-Ethyl-3-Oxopentanoyl-N-Acetylcysteamine Thioester (5). 1H NMR
(400 MHz, CDCl3) d 0.94 (t, 3H, J = 7.3 Hz, CH3), 1.17 (t, 3H, J = 8.4 Hz,
CH3-C-C=O), 1.88–1.98 (m, 2H, C-CH2-C-(C=O)2), 2.15 (s, 3H, CH3-C=O),
2.48–2.66 (m, 2H, C-CH2-C=O), 3.01–3.20 (m, 2H, S-CH2), 3.41–3.58 (m, 2H,
N-CH2), 3.68–3.74 (m, 1H, O=C-CH-C=O), 6.27 (br s, 1H, NH). ESI-MS
expected mass: 246.3; observed mass: 246.4.
(4S)-4-Benzyl-3-Propyl-2-Oxazolidinone (6). 1H NMR (400 MHz, CDCl3)
d 1.22 (t, 3H, J = 8.0 Hz, CH3), 2.76–2.82 (m, 1H, one of CH2-Ph), 2.90–3.05
(m, 2H, CH2-C=O), 3.31 (dd, 1H, J = 15.2 Hz, J = 5.5 Hz, one of CH2-Ph),
4.16–4.23 (m, 2H, CH2-O), 4.66–4.70 (m, 1H, CH-N).
(4S,20S,30R)-3-(30-Hydroxy-20-Methylbutanoyl)-4-Benzyl-2-Oxazolidinone (7).
1H NMR (400 MHz, CDCl3) d 1.23 (d, 3H, J = 7.4 Hz, CH3), 1.28 (d, 3H, J = 6.1Hz,
CH3-C-C=O), 2.76–2.82 (m, 1H, one of CH2-Ph), 2.93 (br s, 1H, OH), 3.26 (dd,
1H, J = 12.3 Hz, J = 3.3 Hz, one of CH2-Ph), 3.75 (dq, 1H, J = 9.8 Hz,
J = 3.1 Hz, CH), 4.16–4.23 (m, 2H, CH2-O), 4.65–4.69 (m, 1H, CH-N).
(4S,20S,30R)-3-(30-Hydroxy-20-Methylhexanoyl)-4-Benzyl-2-Oxazolidinone(8).
1H NMR (400 MHz, CDCl3) d 0.94 (t, 3H, J = 7.2 Hz, CH3), 1.30 (d, 3H, CH3-C-
C=O), 1.35–1.45 (m, 1H, one of C-CH2-C), 1.45–1.58 (m, 1H one of C-CH2-
C), 2.75–2.81 (m, 1H, one of CH2-Ph), 2.84 (d, 1H, OH), 3.26 (dd, 1H,
J = 13.7 Hz, J = 3.1 Hz, one of CH2-Ph), 3.76 (dq, 1H, J = 7.2 Hz, J = 2.6 Hz,
CH), 3.95–4.00 (m, 1H, CH-C=O), 4.17–4.27 (m, 2H, CH2-O), 4.68–4.74
(m, 1H, CH-N).
(4S,20R,30R)-3-(30-Hydroxy-20-Methylbutanoyl)-4-Benzyl-2-Oxazolidinone(9).
1H NMR (400 MHz, CDCl3) d 1.22 (d, 3H, J = 5.6 Hz, CH3), 1.32 (d, 3H, J = 6.2Hz,
CH3-C-C=O), 2.56 (d, 1H, J = 7.4 Hz one of CH2-Ph), 2.82 (br s, 1H, OH),
3.28–3.35 (m, 1H, one of CH2-Ph), 3.90–3.95 (m, 1H, CH), 3.96–4.00 (m, 1H,
CH-C=O), 4.15–4.25 (m, 2H, CH2-O), 4.67–4.72 (m, 1H, CH-N).
(4S,20R,30R)-3-(30-Hydroxy-20-Methylhexanoyl)-4-Benzyl-2-Oxazolidinone (11).
1H NMR (400 MHz, CDCl3) d 0.94 (t, 3H, CH3), 1.20 (d, 3H, J = 7.2 Hz, CH3-C-
C=O), 1.35–1.45 (m, 1H, one of C-CH2-C), 1.45–1.58 (m, 1H one of C-CH2-
C), 2.75–2.82 (m, 1H, one of CH2-Ph), 2.91 (d, 1H, J = 7.2 Hz, OH), 3.31 (dd,
1H, J = 7.9 Hz, J = 3.1 Hz, one of CH2-Ph), 3.85 (dq, 1H, J = 7.2 Hz, J =
2.7 Hz, CH), 3.95–4.04 (m, 1H, CH-C=O), 4.15–4.26 (m, 2H, CH2-O), 4.66–
4.74 (m, 1H, CH-N).
(4S,20R,30R)-3-(30-Hydroxy-20-Methylhexanoyl)-4-Benzyl-2-Oxazolidinone(11)
and (4S,20R,30S)-3-(30-Hydroxy-20-Methylhexanoyl)-4-Benzyl-2-Oxazolidinone
(12). Synthesis followed that of 8, with the exception that two equivalents of
Bu2BOTf was added (4.32 ml of a 1 M solution in DCM, 4.32 mmol, 2 eq.). Rf
0.73 and 0.77 (50% EtOAc in hexanes).
(2S,3R)-3-Hydroxy-2-Methylbutanoyl-N-Acetylcysteamine Thioester (13).
Aldol product 7 (187 mg, 0.68 mmol) was dissolved in 3 ml of a 4:1 mixture of
THF:H2O and cooled to 0ꢀC. Slowly,
3 ml of 30% H2O2 was added,
followed by 1 ml saturated LiOH (0.5 g LiOH in 1 ml H2O). The reaction was
left to stir at 0ꢀC for 2 hr, at which point Na2SO3 was added until H2O2 was
quenched (bubbling ceased). The aqueous phase was washed with DCM
(2 3 15 ml), and the DCM layers were back-extracted with H2O (1 3 15 ml).
The combined aqueous layers were cooled to 0ꢀC and the pH was lowered
to 1 with 6 M HCl. The aldol acid was extracted into EtOAc (3 3 20 ml), dried
with MgSO4, filtered, and concentrated in vacuo (as the aldol acid is somewhat
volatile, the solution was only concentrated to 1 ml). 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide (211.1 mg, 1.36 mmol, 2 eq.) was added to the solution
under nitrogen at 0ꢀC. NAC (96.8 mg, 0.81 mmol, 1.2 eq.) and a few crystals
of 4-dimethylaminopyridine were added. After 4 hr at 0ꢀC, the reaction
was quenched with 5 ml H2O. Product was extracted into EtOAc (3 3 15 ml),
washed with 5 ml brine, dried with MgSO4, and filtered. A yellowish oil was
produced after concentration in vacuo (50 mg, 47%). Rf 0.20 (EtOAc).
(2S,3R)-3-Hydroxy-2-Methylhexanoyl-N-Acetylcysteamine Thioester (14).
14 was synthesized in the same manner as 13 from 8 (81%). Rf 0.35 (in EtOAc).
(2R,3R)-3-Hydroxy-2-Methylbutanoyl-N-Acetylcysteamine Thioester (15)
and (2R,3S)-3-Hydroxy-2-Methylbutanoyl-N-Acetylcysteamine Thioester (16).
15 and 16 were synthesized in the same manner as 13 from a mixture of 9
and 10 (33%, 15 + 16). They were then separated by reverse-phase HPLC
on a semipreparative C18 column (BondClone, 300 3 7.8 mm; Phenomenex)
connected to a Waters 1525 HPLC system through a gradient of 15%–
100% B over 30 min, where the mobile phases were (A) H2O with 0.1% TFA
and (B) MeOH with 0.1% TFA, pumped at a flow rate of 2 ml/min. Rf 0.15
and 0.20 (in EtOAc).
(2R,3R)-3-Hydroxy-2-Methylhexanoyl-N-Acetylcysteamine Thioester (17)
and (2R,3S)-3-Hydroxy-2-Methylhexanoyl-N-Acetylcysteamine Thioester (18).
17 and 18 were synthesized in the same manner as 13 from a mixture of 11
and 12 (85%, 17 + 18). They were then separated as described for 15 and
16. Rf 0.30 and 0.35 (in EtOAc).
Characterization via NMR and LC-MS
NMR was performed either on a Varian Mercury 400 MHz or a Varian INOVA 500
MHz instrument. LC-MS was performed on an Agilent Technologies 1200
Series HPLC with a Gemini C18 column (5 mm, 2 3 50 mm; Phenomenex)
coupled to an Agilent Technologies 6130 quadrupole mass spectrometer sys-
1338 Chemistry & Biology 18, 1331–1340, October 28, 2011 ª2011 Elsevier Ltd All rights reserved