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Novel chemo-enzymatic route to a key intermediate for the taxol side-chain through enantioselective O-acylation. Unexpected acyl migration
Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
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Novel chemo-enzymatic route to a key intermediate for the taxol side-chain through enantioselective O-acylation. Unexpected acyl migration Eniko˝ Forró a,∗ , Zsolt Galla a , Zala Nádasdi a , Judit Árva a , Ferenc Fülöp a,b,∗ a b
Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary Stereochemistry Research Group of the Hungarian Academy of Sciences, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
a r t i c l e
i n f o
Article history: Received 17 February 2015 Received in revised form 13 March 2015 Accepted 16 March 2015 Available online 23 March 2015 Keywords: O-Acylation Acyl migration Enzyme catalysis Remote stereocentre Taxol
a b s t r a c t A new enzymatic strategy has been devised for the preparation of a key intermediate for the taxol sidechain from N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one. Burkholderia cepacia (PS-IM)catalyzed acylation of the primary OH group with an excess of vinyl butyrate (10 equiv.) furnished the corresponding diester and unreacted monoester with excellent enantiomeric excess values (ee ≥ 98%). Traces of undesirable enantiomeric diacetylated product and diol due to intramolecular acyl migration (∼6% mole fractions) were also detected. Finally, (2R,3S)-3-phenylisoserine hydrochloride (ee = 99%), a key intermediate for the taxol side-chain, was prepared from the corresponding diester enantiomer through acidic hydrolysis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Enantiomeric ˇ-amino acids and their derivatives are of great interest from both pharmaceutically and chemical aspects [1–11]. Thus, in recent years, acyclic ˇ-amino acids have been recognized as an important class of compounds in the design and synthesis of potential pharmaceutical drugs; for example, they can serve as essential building blocks for medicinally important molecules such as Taxol® and its analogue Taxotère® , currently considered to be among the most efficient drugs in cancer chemotherapy [12,13]. Taxol, isolated initially from the bark of the Pacific yew (Taxus brevifolia), is a potent cytotoxic microtubule-stabilizing agent that is used efficaciously in the treatment of a number of human cancers, including ovarian, breast and non-small-cell lung cancer [12–14]. To satisfy taxol needs, chemists have been working on semisynthetic methods involving synthetic side-chain coupling to the C(13)–O of the more readily available baccatin III derivatives [from the needles of various Taxus species (e.g. Taxus baccata)]. Since a 3-phenylisoserine-derived side-chain is essential for the
∗ Corresponding authors at: Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary. Tel.: +36 62 545564; fax: +36 62 545705. E-mail addresses: [email protected] (E. Forró), [email protected] (F. Fülöp). http://dx.doi.org/10.1016/j.molcatb.2015.03.015 1381-1177/© 2015 Elsevier B.V. All rights reserved.
antitumour activity of taxol, there is a need for the development of efficient processes for the preparation of (2R,3S)-3-amino-3phenyl-2-hydroxypropionic acid or its direct enantiopure sources. A relatively large number of such syntheses have been developed [15–19], and new synthetic strategies appear continuously [20,21]. The total synthesis of taxol has also been achieved, but is too complex to serve as a commercial source of the drug [22,23]. We have also published some very efficient enzyme-catalyzed direct and indirect strategies for the preparation of (2R,3S)3-phenylisoserine, either through Burkholderia cepacia lipase (PS-IM)-catalyzed hydrolysis of the ester function of racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate with H2 O as nucleophile in t-BuOMe or iPr2 O at 50 or 60 ◦ C (E > 200) [24] or through Candida antarctica lipase B (CAL-B)-catalyzed ring cleavage of racemic cis3-hydroxy-4-phenylazetidin-2-one with added H2 O in t-BuOMe at 60 ◦ C (E > 200) [25]. A new type of enzymatic two-step cascade reaction has also been devised and used successfully for the synthesis of (2R,3S)-3-phenylisoserine. In this latest strategy, the hydrolysis of racemic cis-3-acetoxy-4-phenylazetidin-2-one was performed in the presence of CAL-B with H2 O in iPr2 O at 60 ◦ C, and the hydrolysis of the ester at C(3) (the first enzymatic step, with relatively low E) was followed by a rapid ring cleavage of the corresponding lactam enantiomer (the second, highly enantioselective enzymatic step). At 50% overall conversion of the starting racemate, there were two enantiopure products, cis-(3S,4R)-3-hydroxy-4-phenylazetidin-2one and (2R,3S)-3-phenylisoserine with ee ≥ 98% [25].
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Scheme 1. Enzymatic O-acylation of (3S*,4R*)-1.
Scheme 2. Enzymatic O-acetylation of (3S*,4R*)-1.
In view of the earlier results on enzymatic acylations of primary alcohols with a remote stereocentre [2,4,26,27], our aim in the present research work was to develop an enantioselective strategy for the preparation of (2R,3S)-3-phenylisoserine, through the enzymatic O-acylation of racemic N-hydroxymethylated cis-3acetoxy-4-phenylazetidin-2-one [(3S*,4R*)-1] (Scheme 1). 2. Results and discussion Racemic cis-3-acetoxy-4-phenylazetidin-2-one was prepared according to the literature [19], and then transformed with paraformaldehyde under sonication [28] into the Nhydroxymethylated lactam (3S*,4R*)-(±)-1. The hydrolysis of (3S*,4R*)-(±)-1 in MeOH with saturated NaHCO3 and Na2 CO3 [29] furnished racemic N-hydroxymethylated cis-3-hydroxy-4phenylazetidin-2-one [(3S*,4R*)-(±)-4]. On the basis of earlier results on the enzymatic acylations of cyclic [28] and acyclic [30] N-hydroxymethylated ˇ-lactams, we first carried out the enzymatic acylation of racemic N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2one [(3S*,4R*)-1] with vinyl acetate (VA) (1.2 equiv.) as acyl donor in the presence of PS-IM (30 mg mL−1 ) in iPr2 O at 50 ◦ C (Scheme 2). Due to an unexpected acyl migration, allowed by the closeness of the OH and O-acyl groups and any H2 O present in the reaction medium, a considerable amount of the undesirable diol 4 (∼4% mole fraction, ee = 46%) was formed besides to the desired diacetate (3R,4S)-2 (6% mole fraction, apparent ee = 45%) and unreacted (3S,4R)-1 (81% mole fraction, ee = 10%) after 24 h. The mole fractions were determined with n-heptadecane as internal standard, by using GC on an l-Val column (Supporting information, S1). When the concentration of (3S*,4R*)-1 (in iPr2 O at 50 ◦ C) was decreased from 0.015 to 0.007 M, and then increased to 0.03 M, practically no change was observed in the mole fractions of 1, 2, 4 and 5 (Scheme 3) after 20 min. The intramolecular character of the acyl migration was therefore assumed. The acyl migration in the presence of enzyme [31–33] (PS-IM in iPr2 O at 50 ◦ C) was also investigated, when 95% 1, 3% 2, 2% 4 and traces (<1%) of 5 were determined in the reaction mixture after 1 h. In view of our recent results on the enzyme-catalyzed acylation of ˇ-hydroxy esters with regard to the competition of acylation and hydrolysis [34], the acetylation of (3S*,4R*)-1 was next performed with 6 equiv. of VA, when only traces of enantiomerically enriched 4 were detected after 10 min, at a conversion of 53% (ee1 = 91% and apparent ee2 = 80%, apparent E = 28). When Et3 N was added to the reaction mixture under the same conditions, practically no changes were observed (conv. = 54%, ee1 = 95% and apparent ee2 = 80%, apparent E = 33). Besides PS-IM, the enzymes CAL-B, C. antarctica lipase A (CALA), Pseudomonas fluorescens (lipase AK) and Candida rugosa (lipase
Scheme 3. The intramolecular acyl migration.
AY) were also tested for the acetylation of (3S*,4R*)-1 under the same conditions (0.015 M substrate, 30 mg mL−1 enzyme, 6 equiv. of VA, iPr2 O, 50 ◦ C). CAL-B catalyzed the ring cleavage of ˇ-lactam, in good accordance with our earlier results [25,35–37], while all the other enzymes tested catalyzed only the acetylation reactions (Table 1), but enantiomerically enriched 4 (due to the acyl migration) appeared in all of the reaction mixtures. Thus, lipase PS-IM was selected for further preliminary acylations. In an attempt to suppress the acyl migration, several solvents, such as toluene, t-BuOMe and 2-methyltetrahydrofuran (2-MeTHF), were tested in the lipase PS-IM (30 mg mL−1 )-catalyzed acetylation of (3S*,4R*)-1 with 6 equiv. of VA at 25 ◦ C (Table 2). Unfortunately, no significant beneficial effect was observed (2–4% mole fractions of 4 were also detected). To avoid the inaccuracy in the determination of ee for (3R,4S)-2 as a result of enzymatic acetylation (due to the intramolecular acyl migration), two other acyl donors, vinyl butyrate (VB) and 2,2,2trifluoroethyl butyrate (2,2,2-TFB) (6 equiv.), were also tested for the acylation of racemic (3S*,4R*)-1 in the presence of lipase PS-IM (30 mg mL−1 ) in iPr2 O at 50 ◦ C (Scheme 4). Excellent E values were observed (>200) at around 50% conversion, but racemic 2 (∼4% mole fraction) and enantiomerically
Table 1 Enzyme screening for the acetylation of (3S*,4R*)-1.a Enzyme (30 mg mL−1 )
1 (mol%)
ee1 b (%)
2 (mol%)
ee2 b , c (%)
4 (mol%)
ee4 b (%)
CAL-A Lipase AK Lipase AY
79 46 91
16 91 1
14 48 7
40 80 17
7 6 2
42 67 15
a b c
0.015 M substrate, 6 equiv. of VA, iPr2 O, 50 ◦ C, after 30 min. According to GC (Section 4). Apparent values.
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
103
Scheme 4. Enzymatic O-butyrylation of (3S*,4R*)-1.
Table 2 Solvent screening for the acetylation of (3S*,4R*)-1.a Solvent
Conv.b (%)
ee1 c (%)
ee2 c , d (%)
Apparent E
Toluene t-BuOMe 2-Me-THF
53 56 40
93 90 50
81 70 76
32 17 12
0.015 M substrate, lipase PS-IM (30 mg mL−1 ), 6 equiv. of VA, in the solvent tested, 50 ◦ C, after 1 h. b Conversion values calcd. from ee1 (unreacted substrate) and ee2 (product) values through the equation c = ee1 /(ee1 + ee2 ). c According to GC (Section 4). d Apparent values. a
Table 3 Conversion and enantioselectivity of butyrylation of (3S*,4R*)-1.a Acyl donor (6 equiv.)
Conv.b (%)
ee1 c (%)
ee6 c (%)
E
VB 2,2,2-TFB
48 26
92 34
99 99
>200 >200
0.015 M substrate, 30 mg mL−1 PS-IM, iPr2 O, 50 ◦ C, after 5 min. Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) values. c According to GC (Section 4). a
b
Table 4 Solvent screening for the butyrylation of (3S*,4R*)-1.a Solvent
Conv.b (%)
ee1 c (%)
ee6 c (%)
E
Toluene t-BuOMe 2-Me-THF
26 48 23
34 87 28
99 93 96
>200 78 64
a 0.015 M substrate, lipase PS-IM (30 mg mL−1 ), 6 equiv. of VB, in the solvent tested, 50 ◦ C, after 5 min. b Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) values. c According to GC (Section 4).
enriched 4 (ee ≤ 54%) (∼3% mole fraction) were also detected in the reaction mixtures besides to the desired products [(3R,4S)-6 and (3S,4R)-1] (Table 3 and Supporting information, S2). In order to prolong the reaction (the time needed to reach 50% conversion), solvent screening (toluene, t-BuOMe and 2-Me-THF) was performed (Table 4). Racemic 2 and enantiomerically enriched 4 were also detected in the reaction mixtures (≤6% mole fractions).
Table 5 Effects of temperature on the conversion and enantioselectivity of butyrylation of (3S*,4R*)-1.a Temperature (◦ C)
Reaction time (min)
Conv.b (%)
ee1 c (%)
ee6 c (%)
E
50 25 4
5 10 30
49 46 41
92 86 70
99 99 99
>200 >200 >200
0.015 M substrate, lipase PS-IM (30 mg mL−1 ), 6 equiv. of VB, iPr2 O. Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) values. c According to GC (Section 4). a
b
In view of the best combination of enantioselectivity and reaction rate, iPr2 O was chosen as reaction medium for the preparative-scale acylation. When the PS-IM-catalyzed butyrylation of (3S*,4R*)-1 with VB was carried out at different temperatures, on lowering of the reaction temperature from 50 to 25 ◦ C and then to 4 ◦ C, a considerable decrease in reaction rate was observed, without a drop in E (Table 5). Racemic 2 and enantiomerically enriched 4 were also present in the reaction mixtures (≤7% mole fractions). Finally, 25 ◦ C was chosen as reaction temperature for the preparative-scale reaction. Since the small-scale butyrylation of (3S*,4R*)-1 at 25 ◦ C slowed considerably at around 47% conversion, and E also decreased slightly (ee1 = 92%, ee6 = 94%, conv. = 49%, after 40 min), increased amounts of acyl donor (10 and 20 vs. 6 equiv.) were tested (10 equiv.: ee1 = 95%, ee6 = 98%, conv. = 49%, E > 200, after 30 min; 20 equiv.: ee1 = 97%, ee6 = 97%, conv. = 50%, E > 200, after 20 min). Finally, 10 equiv. of VB was selected for the preparative-scale butyrylation. On the basis of the preliminary results, the preparative-scale resolutions of (3S*,4R*)-1 were performed under the optimized conditions (see footnote to Table 6); and the results are reported in Table 6 and the Section 4. Acidic hydrolysis of enantiomeric (3R,4S)-6 (ee = 99%) and (3S,4R)-1 (ee = 98%) (Scheme 5) resulted in the desired amino acid hydrochlorides [(2R,3S)-3·HCl and (2S,3R)-3·HCl], [(2R,3S)3-phenylisoserine, the key intermediate for the taxol side-chain [(2R,3S)-3·HCl, ee = 99%], being one of them.
Table 6 Lipase PS-IM-catalyzed preparative-scale acylations of (3S*,4R*)-(±)-1. Acyl donor
(3S*,4R*)-(±)-1c (3S*,4R*)-(±)-1i a b c d e f g h i j k
VA VB
Reaction time (min)
30 20
Conv.a (%)
48 (54d ) 50
E
nde >200
Product enantiomer
Unreacted enantiomer
Yield (%)
Isomer
eeb (%)
22 46
(3R,4S)-2 (3R,4S)-6
99f 99
Values calcd. from ee1 (unreacted substrate) and ee2 or ee6 (product) values. Determined by GC (Section 4 and Supporting information, S1 and S2). 6 equiv. of VA; lipase PS-IM (10 mg mL−1 ) in iPr2 O at 25 ◦ C. Overall conversion. Not determined. Recrystallized three times from Et2 O. c 0.30. c 0.26. 10 equiv. of VB; lipase PS-IM (30 mg mL−1 ) in iPr2 O at 25 ◦ C. c 0.30. c 1.325.
[␣]D 25 EtOH +27g +35j
Yield (%)
Isomer
eeb (%)
[␣]D 25 (EtOH)
38 46
(3S,4R)-1 (3S,4R)-1
91 98
−36h −46k
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E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
Optical rotations were measured with a Perkin-Elmer 341 polarimeter. 1 H NMR spectra were recorded on a Bruker Avance DRX 400 spectrometer (Supporting information, Figs. S4–S7). Melting points (m.p.s) were determined on a Kofler apparatus. 4.2. Gram-scale acetylation of racemic N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one [(3S*,4R*)-1]
Scheme 5. Preparation of (2R,3S)-3·HCl and (2S,3R)-3·HCl.
The absolute configurations were proved by comparing the ˛ values with the literature data; the lit [25] ˛ value for (2R,3S)3-phenylisoserine {[˛]D 25 = −15 (c 0.25, 6N HCl), ee > 98%} is in good accordance with the value measured for enantiomeric 3, prepared from diacetate 6 (Scheme 5) under the same conditions {[˛]D 25 = −15 (c 0.25, 6 N HCl), ee = 99%}. The lipase PS-IM catalyzed acylation of (3S*,4R*)-1 therefore displayed 4S selectivity. 3. Conclusions A new enzyme-catalyzed strategy has been devised for the synthesis of enantiomeric (ee = 99%) 3-amino-3-phenyl-2hydroxypropionic acid [(2R,3S)-3], a key intermediate for the taxol side-chain. B. cepacia lipase PS-IM catalyzed the S-selective acylation of (3S*,4R*)-1 with VB (10 equiv.) in iPr2 O at 25 ◦ C with excellent enantioselectivity (E > 200). It should be highlighted that the possibility of intramolecular acyl migration during an enzymatic transformation of substrates containing both OCOR and OH functions needs to be carefully investigated. 4. Experimental 4.1. Materials and methods Lipase PS-IM (immobilized on diatomaceous earth) was from Amano Enzyme Europe Ltd. CAL-B (lipase B from C. antarctica), produced by the submerged fermentation of a genetically modified Aspergillus oryzae microorganism and adsorbed on a macroporous resin (Catalogue No. L4777), was from Sigma-Aldrich. CAL-A (lipase A from C. antarctica) was purchased from Roche Diagnostics Corporation. Lipase AK (P. fluorescens) was from Amano Pharmaceuticals, and lipase AY (C. rugosa) was from Fluka. The solvents were of the highest analytical grade. In a typical small-scale experiment, racemic substrate (0.015 M solution) in an organic solvent (1 mL) was added to the lipase tested (30 mg mL−1 ). The acyl donor (1.2, 6, 10 or 20 equiv.) was next added. The mixture was shaken at 4, 25 or 50 ◦ C. The progress of the reaction was followed by taking samples from the reaction mixture at intervals and analysing them by GC on an l-Val column (25 m) [80 ◦ C for 5 min → 160 ◦ C for 40 min (temperature rise 10 ◦ C min−1 ) → 190 ◦ C (temperature rise 20 ◦ C min−1 ); 70 kPa; retention times (min), (3S,4R)-1: 40.73 (antipode: 39.07); (3R,4S)2: 30.84 (antipode: 29.88); (3S,4R)-4: 61.04 (antipode: 60.11); (3R*,4S*)-5: 35.25 (antipode: 35.93); (3R,4S)-6: 50.00 (antipode: 47.58)]. The ee values for the ˇ-amino acids prepared were determined by a GC method [38] on a Chrompack Chirasil-Dex CB column after a simple and rapid double derivatization with (i) CH2 N2 (caution! derivatization with diazomethane should be performed under a well-working hood); (ii) Ac2 O in the presence of 4-dimethylaminopyridine and pyridine [140 ◦ C for 7 min → 190 ◦ C (temperature rise 10 ◦ C min−1 ; 100 kPa; retention times (min), (2R,3S)-3: 19.01 (antipode: 18.70)] (Supporting information, S3).
(3S*,4R*)-1 (200 mg, 0.85 mmol) was dissolved in iPr2 O (40 mL). Lipase PS-IM (400 mg, 10 mg mL−1 ) and VA (487 L, 5.10 mmol) were added and the mixture was shaken in an incubator shaker at 25 ◦ C for 30 min. The reaction was stopped by filtering off the enzyme at 54% overall conversion. The solvent was then evaporated off, and the residue was subjected to column chromatography (EtOAc:n-hexane 1:1). The resulting diacetylated product (3R,4S)-2 was crystallized out and then recrystallized three times from Et2 O [49 mg, 22%; [˛]D 25 = +27 (c 0.30, EtOH); ee = 99%; m.p.: 75–77 ◦ C]. The unreacted (3S,4R)-1 was similarly crystallized out from Et2 O [75 mg, 38%; [˛]D 25 = −36 (c 0.26, EtOH); ee = 91%; m.p.: 61–63 ◦ C]. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3S,4R)-1 were similar to those for (3S*,4R*)-1: ı = 1.67 (s, 3H, CH3 ), 4.44–4.47 (d, J = 11.5 Hz, 1H, CHA H), 5.06–5.13 (d, J = 11.5 Hz, 1H, CHB H), 5.13–5.18 (d, J = 4.8 Hz, 1H, CH2 OH) 5.82–5.87 (d, J = 4.8 Hz, 1H, CH2 OH) 7.27–7.40 (m, 5H, C6 H5 ). Analysis: calcd. for C12 H13 NO4 : C, 61.27; H, 5.57; N, 5.95; found: C, 61.34; H, 5.54; N, 5.81. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3R,4S)2: ı = 1.68 (s, 3H, CH2 CH3 ), 1.99 (s, 3H, CHCH3 ), 4.94–4.99 (d, J = 11.36 Hz, 1H, CHA H), 5.05–5.09 (d, J = 4.76 Hz, 1H, CH2 OH), 5.38–5.43 (d, J = 10.60 Hz, 1H, CHB H), 5.84–5.88 (d, J = 4.88 Hz, 1H, CH2 OH), 7.26–7.40 (m, 5H, C6 H5 ). Analysis: calcd. for C14 H15 NO5 : C, 60.64; H, 5.45; N, 5.05; found: C, 60.66; H, 5.44; N, 5.08. 4.3. Gram-scale butyrylation of racemic N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one Via the procedure described above, the reaction of racemic (3S*,4R*)-1 (100 mg, 0.43 mmol) and VB (545 L, 4.30 mmol) in iPr2 O (10 mL) in the presence of Lipase PS-IM (300 mg, 30 mg mL−1 ) at 25 ◦ C afforded the diacylated product (3R,4S)-6 [59 mg, 46%; [˛]D 25 = +35 (c 0.30, EtOH); ee = 99%; m.p.: 61.5–63.5 ◦ C (crystallized out from Et2 O)] and unreacted (3S,4R)-1, [46 mg, 46%; [˛]D 25 = −46 (c 1.325, EtOH); ee = 99%; m.p.: 61–63 ◦ C (crystallized out from Et2 O)] after 20 min. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3S,4R)-1 in the butyrylation reaction were similar to those for (3S,4R)-1 in the acetylation reaction. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3R,4S)6: ı = 0.86–0.93 (t, J = 7.4 Hz, 3H, CH2 CH2 CH3 ), 1.50–1.63 (m, 2H, CH2 CH2 CH3 ), 1.67 (s, 3H, OCOCH3 ), 2.15–2.29 (m, 2H, CH2 CH2 CH3 ), 4.94–5.01 (d, J = 11.2 Hz, 1H, CHA H), 5.04–5.08 (d, J = 4.9 Hz, 1H, CH2 OCO), 5.37–5.43 (d, J = 11.24 Hz, 1H, CHB H), 5.83–5.87 (d, J = 4.89 Hz, 1H, CH2 OCO), 7.27–7.40 (m, 5H, C6 H5 ). Analysis: calcd. for C16 H19 NO5 : C, 62.94; H, 6.27; N, 4.59; found: C, 62.90; H, 6.30; N, 4.61. 4.4. Preparation of (2R,3S)- and (2S,3R)-3-phenylisoserine hydrochlorides [(2R,3S)-3·HCl and (2S,3R)-3·HCl] Enantiomeric (3R,4S)-6 (49 mg, 0.16 mmol) or (3S,4R)-1 (53 mg, 0.23 mmol) was dissolved in 18% HCl (20 mL) and refluxed for 5 h. The solvent was then evaporated off, and the product was recrystallized from EtOH and Et2 O, which afforded white crystals of (2R,3S)-3·HCl [33 mg, 95%, [˛]D 25 = −15 (c 0.25, 6 N HCl), m.p. 215–217 ◦ C, ee = 99%] or (2S,3R)-3·HCl [48 mg, 95%, [˛]D 25 = +14.6 (c 0.33, 6 N HCl), m.p. 215–217 ◦ C, ee = 99%].
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
The 1 H NMR (400 MHz, D2 O, 25 ◦ C, TMS) data for (2R,3S)-3·HCl were similar to those for (2S,3R)-3·HCl: ı = 4.36–4.37 [d, J = 5.9 Hz, 1H, CH(OH)(COOH)], 4.59–4.61 (d, J = 5.9 Hz, 1H, CHNH2 ), 7.43–7.58 (m, 5H, C6 H5 ). Analysis: calcd. for C9 H12 ClNO3 : C, 49.67; H, 5.56; N, 6.44; Analysis: found for (2R,3S)-3·HCl: C, 49.48; H, 5.61; N, 6.38. Analysis: found for (2S,3R)-3·HCl: C, 49.50; H, 5.59; N, 6.44. Acknowledgements We are grateful to the Hungarian Research Foundation (OTKA No. K108943) and TÁMOP-4.2.2.A-11/1/KONV-2012-0035 for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.molcatb.2015.03.015. References [1] F. Fülöp, Chem. Rev 101 (2001) 2181–2204. [2] E. Forró, F. Fülöp, Mini Rev. Org. Chem. 1 (2004) 93–102. [3] A. Kuhl, M.G. Hahn, M. Dumié, J. Mittendorf, Amino Acids 29 (2005) 89–100. [4] A. Liljeblad, L.T. Kanerva, Tetrahedron 62 (2006) 5831–5854. [5] B.P. Mowery, S.E. Lee, D.A. Kissounko, R.F. Epand, R.M. Epand, B. Weisblum, S.S. Stahl, S.H. Gellman, J. Am. Chem. Soc. 199 (2007) 15474–15476. [6] C.S. Stauffer, A. Datta, J. Org. Chem. 73 (2008) 4166–4174. [7] L. Kiss, E. Forró, F. Fülöp, in: A.B. Hughes (Ed.), Synthesis of Carbocyclic -Amino Acids in Amino Acids, Peptides and Proteins in Organic Chemistry, 1, Wiley, Weinheim, 2009, pp. 367–409. [8] D. Fernandez, E. Torres, F.X. Aviles, R.M. Ortuno, J. Vendrell, Bioorg. Med. Chem. 17 (2009) 3824–3828.
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Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Novel chemo-enzymatic route to a key intermediate for the taxol side-chain through enantioselective O-acylation. Unexpected acyl migration Eniko˝ Forró a,∗ , Zsolt Galla a , Zala Nádasdi a , Judit Árva a , Ferenc Fülöp a,b,∗ a b
Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary Stereochemistry Research Group of the Hungarian Academy of Sciences, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
a r t i c l e
i n f o
Article history: Received 17 February 2015 Received in revised form 13 March 2015 Accepted 16 March 2015 Available online 23 March 2015 Keywords: O-Acylation Acyl migration Enzyme catalysis Remote stereocentre Taxol
a b s t r a c t A new enzymatic strategy has been devised for the preparation of a key intermediate for the taxol sidechain from N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one. Burkholderia cepacia (PS-IM)catalyzed acylation of the primary OH group with an excess of vinyl butyrate (10 equiv.) furnished the corresponding diester and unreacted monoester with excellent enantiomeric excess values (ee ≥ 98%). Traces of undesirable enantiomeric diacetylated product and diol due to intramolecular acyl migration (∼6% mole fractions) were also detected. Finally, (2R,3S)-3-phenylisoserine hydrochloride (ee = 99%), a key intermediate for the taxol side-chain, was prepared from the corresponding diester enantiomer through acidic hydrolysis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Enantiomeric ˇ-amino acids and their derivatives are of great interest from both pharmaceutically and chemical aspects [1–11]. Thus, in recent years, acyclic ˇ-amino acids have been recognized as an important class of compounds in the design and synthesis of potential pharmaceutical drugs; for example, they can serve as essential building blocks for medicinally important molecules such as Taxol® and its analogue Taxotère® , currently considered to be among the most efficient drugs in cancer chemotherapy [12,13]. Taxol, isolated initially from the bark of the Pacific yew (Taxus brevifolia), is a potent cytotoxic microtubule-stabilizing agent that is used efficaciously in the treatment of a number of human cancers, including ovarian, breast and non-small-cell lung cancer [12–14]. To satisfy taxol needs, chemists have been working on semisynthetic methods involving synthetic side-chain coupling to the C(13)–O of the more readily available baccatin III derivatives [from the needles of various Taxus species (e.g. Taxus baccata)]. Since a 3-phenylisoserine-derived side-chain is essential for the
∗ Corresponding authors at: Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary. Tel.: +36 62 545564; fax: +36 62 545705. E-mail addresses: [email protected] (E. Forró), [email protected] (F. Fülöp). http://dx.doi.org/10.1016/j.molcatb.2015.03.015 1381-1177/© 2015 Elsevier B.V. All rights reserved.
antitumour activity of taxol, there is a need for the development of efficient processes for the preparation of (2R,3S)-3-amino-3phenyl-2-hydroxypropionic acid or its direct enantiopure sources. A relatively large number of such syntheses have been developed [15–19], and new synthetic strategies appear continuously [20,21]. The total synthesis of taxol has also been achieved, but is too complex to serve as a commercial source of the drug [22,23]. We have also published some very efficient enzyme-catalyzed direct and indirect strategies for the preparation of (2R,3S)3-phenylisoserine, either through Burkholderia cepacia lipase (PS-IM)-catalyzed hydrolysis of the ester function of racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate with H2 O as nucleophile in t-BuOMe or iPr2 O at 50 or 60 ◦ C (E > 200) [24] or through Candida antarctica lipase B (CAL-B)-catalyzed ring cleavage of racemic cis3-hydroxy-4-phenylazetidin-2-one with added H2 O in t-BuOMe at 60 ◦ C (E > 200) [25]. A new type of enzymatic two-step cascade reaction has also been devised and used successfully for the synthesis of (2R,3S)-3-phenylisoserine. In this latest strategy, the hydrolysis of racemic cis-3-acetoxy-4-phenylazetidin-2-one was performed in the presence of CAL-B with H2 O in iPr2 O at 60 ◦ C, and the hydrolysis of the ester at C(3) (the first enzymatic step, with relatively low E) was followed by a rapid ring cleavage of the corresponding lactam enantiomer (the second, highly enantioselective enzymatic step). At 50% overall conversion of the starting racemate, there were two enantiopure products, cis-(3S,4R)-3-hydroxy-4-phenylazetidin-2one and (2R,3S)-3-phenylisoserine with ee ≥ 98% [25].
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Scheme 1. Enzymatic O-acylation of (3S*,4R*)-1.
Scheme 2. Enzymatic O-acetylation of (3S*,4R*)-1.
In view of the earlier results on enzymatic acylations of primary alcohols with a remote stereocentre [2,4,26,27], our aim in the present research work was to develop an enantioselective strategy for the preparation of (2R,3S)-3-phenylisoserine, through the enzymatic O-acylation of racemic N-hydroxymethylated cis-3acetoxy-4-phenylazetidin-2-one [(3S*,4R*)-1] (Scheme 1). 2. Results and discussion Racemic cis-3-acetoxy-4-phenylazetidin-2-one was prepared according to the literature [19], and then transformed with paraformaldehyde under sonication [28] into the Nhydroxymethylated lactam (3S*,4R*)-(±)-1. The hydrolysis of (3S*,4R*)-(±)-1 in MeOH with saturated NaHCO3 and Na2 CO3 [29] furnished racemic N-hydroxymethylated cis-3-hydroxy-4phenylazetidin-2-one [(3S*,4R*)-(±)-4]. On the basis of earlier results on the enzymatic acylations of cyclic [28] and acyclic [30] N-hydroxymethylated ˇ-lactams, we first carried out the enzymatic acylation of racemic N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2one [(3S*,4R*)-1] with vinyl acetate (VA) (1.2 equiv.) as acyl donor in the presence of PS-IM (30 mg mL−1 ) in iPr2 O at 50 ◦ C (Scheme 2). Due to an unexpected acyl migration, allowed by the closeness of the OH and O-acyl groups and any H2 O present in the reaction medium, a considerable amount of the undesirable diol 4 (∼4% mole fraction, ee = 46%) was formed besides to the desired diacetate (3R,4S)-2 (6% mole fraction, apparent ee = 45%) and unreacted (3S,4R)-1 (81% mole fraction, ee = 10%) after 24 h. The mole fractions were determined with n-heptadecane as internal standard, by using GC on an l-Val column (Supporting information, S1). When the concentration of (3S*,4R*)-1 (in iPr2 O at 50 ◦ C) was decreased from 0.015 to 0.007 M, and then increased to 0.03 M, practically no change was observed in the mole fractions of 1, 2, 4 and 5 (Scheme 3) after 20 min. The intramolecular character of the acyl migration was therefore assumed. The acyl migration in the presence of enzyme [31–33] (PS-IM in iPr2 O at 50 ◦ C) was also investigated, when 95% 1, 3% 2, 2% 4 and traces (<1%) of 5 were determined in the reaction mixture after 1 h. In view of our recent results on the enzyme-catalyzed acylation of ˇ-hydroxy esters with regard to the competition of acylation and hydrolysis [34], the acetylation of (3S*,4R*)-1 was next performed with 6 equiv. of VA, when only traces of enantiomerically enriched 4 were detected after 10 min, at a conversion of 53% (ee1 = 91% and apparent ee2 = 80%, apparent E = 28). When Et3 N was added to the reaction mixture under the same conditions, practically no changes were observed (conv. = 54%, ee1 = 95% and apparent ee2 = 80%, apparent E = 33). Besides PS-IM, the enzymes CAL-B, C. antarctica lipase A (CALA), Pseudomonas fluorescens (lipase AK) and Candida rugosa (lipase
Scheme 3. The intramolecular acyl migration.
AY) were also tested for the acetylation of (3S*,4R*)-1 under the same conditions (0.015 M substrate, 30 mg mL−1 enzyme, 6 equiv. of VA, iPr2 O, 50 ◦ C). CAL-B catalyzed the ring cleavage of ˇ-lactam, in good accordance with our earlier results [25,35–37], while all the other enzymes tested catalyzed only the acetylation reactions (Table 1), but enantiomerically enriched 4 (due to the acyl migration) appeared in all of the reaction mixtures. Thus, lipase PS-IM was selected for further preliminary acylations. In an attempt to suppress the acyl migration, several solvents, such as toluene, t-BuOMe and 2-methyltetrahydrofuran (2-MeTHF), were tested in the lipase PS-IM (30 mg mL−1 )-catalyzed acetylation of (3S*,4R*)-1 with 6 equiv. of VA at 25 ◦ C (Table 2). Unfortunately, no significant beneficial effect was observed (2–4% mole fractions of 4 were also detected). To avoid the inaccuracy in the determination of ee for (3R,4S)-2 as a result of enzymatic acetylation (due to the intramolecular acyl migration), two other acyl donors, vinyl butyrate (VB) and 2,2,2trifluoroethyl butyrate (2,2,2-TFB) (6 equiv.), were also tested for the acylation of racemic (3S*,4R*)-1 in the presence of lipase PS-IM (30 mg mL−1 ) in iPr2 O at 50 ◦ C (Scheme 4). Excellent E values were observed (>200) at around 50% conversion, but racemic 2 (∼4% mole fraction) and enantiomerically
Table 1 Enzyme screening for the acetylation of (3S*,4R*)-1.a Enzyme (30 mg mL−1 )
1 (mol%)
ee1 b (%)
2 (mol%)
ee2 b , c (%)
4 (mol%)
ee4 b (%)
CAL-A Lipase AK Lipase AY
79 46 91
16 91 1
14 48 7
40 80 17
7 6 2
42 67 15
a b c
0.015 M substrate, 6 equiv. of VA, iPr2 O, 50 ◦ C, after 30 min. According to GC (Section 4). Apparent values.
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
103
Scheme 4. Enzymatic O-butyrylation of (3S*,4R*)-1.
Table 2 Solvent screening for the acetylation of (3S*,4R*)-1.a Solvent
Conv.b (%)
ee1 c (%)
ee2 c , d (%)
Apparent E
Toluene t-BuOMe 2-Me-THF
53 56 40
93 90 50
81 70 76
32 17 12
0.015 M substrate, lipase PS-IM (30 mg mL−1 ), 6 equiv. of VA, in the solvent tested, 50 ◦ C, after 1 h. b Conversion values calcd. from ee1 (unreacted substrate) and ee2 (product) values through the equation c = ee1 /(ee1 + ee2 ). c According to GC (Section 4). d Apparent values. a
Table 3 Conversion and enantioselectivity of butyrylation of (3S*,4R*)-1.a Acyl donor (6 equiv.)
Conv.b (%)
ee1 c (%)
ee6 c (%)
E
VB 2,2,2-TFB
48 26
92 34
99 99
>200 >200
0.015 M substrate, 30 mg mL−1 PS-IM, iPr2 O, 50 ◦ C, after 5 min. Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) values. c According to GC (Section 4). a
b
Table 4 Solvent screening for the butyrylation of (3S*,4R*)-1.a Solvent
Conv.b (%)
ee1 c (%)
ee6 c (%)
E
Toluene t-BuOMe 2-Me-THF
26 48 23
34 87 28
99 93 96
>200 78 64
a 0.015 M substrate, lipase PS-IM (30 mg mL−1 ), 6 equiv. of VB, in the solvent tested, 50 ◦ C, after 5 min. b Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) values. c According to GC (Section 4).
enriched 4 (ee ≤ 54%) (∼3% mole fraction) were also detected in the reaction mixtures besides to the desired products [(3R,4S)-6 and (3S,4R)-1] (Table 3 and Supporting information, S2). In order to prolong the reaction (the time needed to reach 50% conversion), solvent screening (toluene, t-BuOMe and 2-Me-THF) was performed (Table 4). Racemic 2 and enantiomerically enriched 4 were also detected in the reaction mixtures (≤6% mole fractions).
Table 5 Effects of temperature on the conversion and enantioselectivity of butyrylation of (3S*,4R*)-1.a Temperature (◦ C)
Reaction time (min)
Conv.b (%)
ee1 c (%)
ee6 c (%)
E
50 25 4
5 10 30
49 46 41
92 86 70
99 99 99
>200 >200 >200
0.015 M substrate, lipase PS-IM (30 mg mL−1 ), 6 equiv. of VB, iPr2 O. Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) values. c According to GC (Section 4). a
b
In view of the best combination of enantioselectivity and reaction rate, iPr2 O was chosen as reaction medium for the preparative-scale acylation. When the PS-IM-catalyzed butyrylation of (3S*,4R*)-1 with VB was carried out at different temperatures, on lowering of the reaction temperature from 50 to 25 ◦ C and then to 4 ◦ C, a considerable decrease in reaction rate was observed, without a drop in E (Table 5). Racemic 2 and enantiomerically enriched 4 were also present in the reaction mixtures (≤7% mole fractions). Finally, 25 ◦ C was chosen as reaction temperature for the preparative-scale reaction. Since the small-scale butyrylation of (3S*,4R*)-1 at 25 ◦ C slowed considerably at around 47% conversion, and E also decreased slightly (ee1 = 92%, ee6 = 94%, conv. = 49%, after 40 min), increased amounts of acyl donor (10 and 20 vs. 6 equiv.) were tested (10 equiv.: ee1 = 95%, ee6 = 98%, conv. = 49%, E > 200, after 30 min; 20 equiv.: ee1 = 97%, ee6 = 97%, conv. = 50%, E > 200, after 20 min). Finally, 10 equiv. of VB was selected for the preparative-scale butyrylation. On the basis of the preliminary results, the preparative-scale resolutions of (3S*,4R*)-1 were performed under the optimized conditions (see footnote to Table 6); and the results are reported in Table 6 and the Section 4. Acidic hydrolysis of enantiomeric (3R,4S)-6 (ee = 99%) and (3S,4R)-1 (ee = 98%) (Scheme 5) resulted in the desired amino acid hydrochlorides [(2R,3S)-3·HCl and (2S,3R)-3·HCl], [(2R,3S)3-phenylisoserine, the key intermediate for the taxol side-chain [(2R,3S)-3·HCl, ee = 99%], being one of them.
Table 6 Lipase PS-IM-catalyzed preparative-scale acylations of (3S*,4R*)-(±)-1. Acyl donor
(3S*,4R*)-(±)-1c (3S*,4R*)-(±)-1i a b c d e f g h i j k
VA VB
Reaction time (min)
30 20
Conv.a (%)
48 (54d ) 50
E
nde >200
Product enantiomer
Unreacted enantiomer
Yield (%)
Isomer
eeb (%)
22 46
(3R,4S)-2 (3R,4S)-6
99f 99
Values calcd. from ee1 (unreacted substrate) and ee2 or ee6 (product) values. Determined by GC (Section 4 and Supporting information, S1 and S2). 6 equiv. of VA; lipase PS-IM (10 mg mL−1 ) in iPr2 O at 25 ◦ C. Overall conversion. Not determined. Recrystallized three times from Et2 O. c 0.30. c 0.26. 10 equiv. of VB; lipase PS-IM (30 mg mL−1 ) in iPr2 O at 25 ◦ C. c 0.30. c 1.325.
[␣]D 25 EtOH +27g +35j
Yield (%)
Isomer
eeb (%)
[␣]D 25 (EtOH)
38 46
(3S,4R)-1 (3S,4R)-1
91 98
−36h −46k
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E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
Optical rotations were measured with a Perkin-Elmer 341 polarimeter. 1 H NMR spectra were recorded on a Bruker Avance DRX 400 spectrometer (Supporting information, Figs. S4–S7). Melting points (m.p.s) were determined on a Kofler apparatus. 4.2. Gram-scale acetylation of racemic N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one [(3S*,4R*)-1]
Scheme 5. Preparation of (2R,3S)-3·HCl and (2S,3R)-3·HCl.
The absolute configurations were proved by comparing the ˛ values with the literature data; the lit [25] ˛ value for (2R,3S)3-phenylisoserine {[˛]D 25 = −15 (c 0.25, 6N HCl), ee > 98%} is in good accordance with the value measured for enantiomeric 3, prepared from diacetate 6 (Scheme 5) under the same conditions {[˛]D 25 = −15 (c 0.25, 6 N HCl), ee = 99%}. The lipase PS-IM catalyzed acylation of (3S*,4R*)-1 therefore displayed 4S selectivity. 3. Conclusions A new enzyme-catalyzed strategy has been devised for the synthesis of enantiomeric (ee = 99%) 3-amino-3-phenyl-2hydroxypropionic acid [(2R,3S)-3], a key intermediate for the taxol side-chain. B. cepacia lipase PS-IM catalyzed the S-selective acylation of (3S*,4R*)-1 with VB (10 equiv.) in iPr2 O at 25 ◦ C with excellent enantioselectivity (E > 200). It should be highlighted that the possibility of intramolecular acyl migration during an enzymatic transformation of substrates containing both OCOR and OH functions needs to be carefully investigated. 4. Experimental 4.1. Materials and methods Lipase PS-IM (immobilized on diatomaceous earth) was from Amano Enzyme Europe Ltd. CAL-B (lipase B from C. antarctica), produced by the submerged fermentation of a genetically modified Aspergillus oryzae microorganism and adsorbed on a macroporous resin (Catalogue No. L4777), was from Sigma-Aldrich. CAL-A (lipase A from C. antarctica) was purchased from Roche Diagnostics Corporation. Lipase AK (P. fluorescens) was from Amano Pharmaceuticals, and lipase AY (C. rugosa) was from Fluka. The solvents were of the highest analytical grade. In a typical small-scale experiment, racemic substrate (0.015 M solution) in an organic solvent (1 mL) was added to the lipase tested (30 mg mL−1 ). The acyl donor (1.2, 6, 10 or 20 equiv.) was next added. The mixture was shaken at 4, 25 or 50 ◦ C. The progress of the reaction was followed by taking samples from the reaction mixture at intervals and analysing them by GC on an l-Val column (25 m) [80 ◦ C for 5 min → 160 ◦ C for 40 min (temperature rise 10 ◦ C min−1 ) → 190 ◦ C (temperature rise 20 ◦ C min−1 ); 70 kPa; retention times (min), (3S,4R)-1: 40.73 (antipode: 39.07); (3R,4S)2: 30.84 (antipode: 29.88); (3S,4R)-4: 61.04 (antipode: 60.11); (3R*,4S*)-5: 35.25 (antipode: 35.93); (3R,4S)-6: 50.00 (antipode: 47.58)]. The ee values for the ˇ-amino acids prepared were determined by a GC method [38] on a Chrompack Chirasil-Dex CB column after a simple and rapid double derivatization with (i) CH2 N2 (caution! derivatization with diazomethane should be performed under a well-working hood); (ii) Ac2 O in the presence of 4-dimethylaminopyridine and pyridine [140 ◦ C for 7 min → 190 ◦ C (temperature rise 10 ◦ C min−1 ; 100 kPa; retention times (min), (2R,3S)-3: 19.01 (antipode: 18.70)] (Supporting information, S3).
(3S*,4R*)-1 (200 mg, 0.85 mmol) was dissolved in iPr2 O (40 mL). Lipase PS-IM (400 mg, 10 mg mL−1 ) and VA (487 L, 5.10 mmol) were added and the mixture was shaken in an incubator shaker at 25 ◦ C for 30 min. The reaction was stopped by filtering off the enzyme at 54% overall conversion. The solvent was then evaporated off, and the residue was subjected to column chromatography (EtOAc:n-hexane 1:1). The resulting diacetylated product (3R,4S)-2 was crystallized out and then recrystallized three times from Et2 O [49 mg, 22%; [˛]D 25 = +27 (c 0.30, EtOH); ee = 99%; m.p.: 75–77 ◦ C]. The unreacted (3S,4R)-1 was similarly crystallized out from Et2 O [75 mg, 38%; [˛]D 25 = −36 (c 0.26, EtOH); ee = 91%; m.p.: 61–63 ◦ C]. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3S,4R)-1 were similar to those for (3S*,4R*)-1: ı = 1.67 (s, 3H, CH3 ), 4.44–4.47 (d, J = 11.5 Hz, 1H, CHA H), 5.06–5.13 (d, J = 11.5 Hz, 1H, CHB H), 5.13–5.18 (d, J = 4.8 Hz, 1H, CH2 OH) 5.82–5.87 (d, J = 4.8 Hz, 1H, CH2 OH) 7.27–7.40 (m, 5H, C6 H5 ). Analysis: calcd. for C12 H13 NO4 : C, 61.27; H, 5.57; N, 5.95; found: C, 61.34; H, 5.54; N, 5.81. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3R,4S)2: ı = 1.68 (s, 3H, CH2 CH3 ), 1.99 (s, 3H, CHCH3 ), 4.94–4.99 (d, J = 11.36 Hz, 1H, CHA H), 5.05–5.09 (d, J = 4.76 Hz, 1H, CH2 OH), 5.38–5.43 (d, J = 10.60 Hz, 1H, CHB H), 5.84–5.88 (d, J = 4.88 Hz, 1H, CH2 OH), 7.26–7.40 (m, 5H, C6 H5 ). Analysis: calcd. for C14 H15 NO5 : C, 60.64; H, 5.45; N, 5.05; found: C, 60.66; H, 5.44; N, 5.08. 4.3. Gram-scale butyrylation of racemic N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one Via the procedure described above, the reaction of racemic (3S*,4R*)-1 (100 mg, 0.43 mmol) and VB (545 L, 4.30 mmol) in iPr2 O (10 mL) in the presence of Lipase PS-IM (300 mg, 30 mg mL−1 ) at 25 ◦ C afforded the diacylated product (3R,4S)-6 [59 mg, 46%; [˛]D 25 = +35 (c 0.30, EtOH); ee = 99%; m.p.: 61.5–63.5 ◦ C (crystallized out from Et2 O)] and unreacted (3S,4R)-1, [46 mg, 46%; [˛]D 25 = −46 (c 1.325, EtOH); ee = 99%; m.p.: 61–63 ◦ C (crystallized out from Et2 O)] after 20 min. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3S,4R)-1 in the butyrylation reaction were similar to those for (3S,4R)-1 in the acetylation reaction. The 1 H NMR (400 MHz, CDCl3 , 25 ◦ C, TMS) data for (3R,4S)6: ı = 0.86–0.93 (t, J = 7.4 Hz, 3H, CH2 CH2 CH3 ), 1.50–1.63 (m, 2H, CH2 CH2 CH3 ), 1.67 (s, 3H, OCOCH3 ), 2.15–2.29 (m, 2H, CH2 CH2 CH3 ), 4.94–5.01 (d, J = 11.2 Hz, 1H, CHA H), 5.04–5.08 (d, J = 4.9 Hz, 1H, CH2 OCO), 5.37–5.43 (d, J = 11.24 Hz, 1H, CHB H), 5.83–5.87 (d, J = 4.89 Hz, 1H, CH2 OCO), 7.27–7.40 (m, 5H, C6 H5 ). Analysis: calcd. for C16 H19 NO5 : C, 62.94; H, 6.27; N, 4.59; found: C, 62.90; H, 6.30; N, 4.61. 4.4. Preparation of (2R,3S)- and (2S,3R)-3-phenylisoserine hydrochlorides [(2R,3S)-3·HCl and (2S,3R)-3·HCl] Enantiomeric (3R,4S)-6 (49 mg, 0.16 mmol) or (3S,4R)-1 (53 mg, 0.23 mmol) was dissolved in 18% HCl (20 mL) and refluxed for 5 h. The solvent was then evaporated off, and the product was recrystallized from EtOH and Et2 O, which afforded white crystals of (2R,3S)-3·HCl [33 mg, 95%, [˛]D 25 = −15 (c 0.25, 6 N HCl), m.p. 215–217 ◦ C, ee = 99%] or (2S,3R)-3·HCl [48 mg, 95%, [˛]D 25 = +14.6 (c 0.33, 6 N HCl), m.p. 215–217 ◦ C, ee = 99%].
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
The 1 H NMR (400 MHz, D2 O, 25 ◦ C, TMS) data for (2R,3S)-3·HCl were similar to those for (2S,3R)-3·HCl: ı = 4.36–4.37 [d, J = 5.9 Hz, 1H, CH(OH)(COOH)], 4.59–4.61 (d, J = 5.9 Hz, 1H, CHNH2 ), 7.43–7.58 (m, 5H, C6 H5 ). Analysis: calcd. for C9 H12 ClNO3 : C, 49.67; H, 5.56; N, 6.44; Analysis: found for (2R,3S)-3·HCl: C, 49.48; H, 5.61; N, 6.38. Analysis: found for (2S,3R)-3·HCl: C, 49.50; H, 5.59; N, 6.44. Acknowledgements We are grateful to the Hungarian Research Foundation (OTKA No. K108943) and TÁMOP-4.2.2.A-11/1/KONV-2012-0035 for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.molcatb.2015.03.015. References [1] F. Fülöp, Chem. Rev 101 (2001) 2181–2204. [2] E. Forró, F. Fülöp, Mini Rev. Org. Chem. 1 (2004) 93–102. [3] A. Kuhl, M.G. Hahn, M. Dumié, J. Mittendorf, Amino Acids 29 (2005) 89–100. [4] A. Liljeblad, L.T. Kanerva, Tetrahedron 62 (2006) 5831–5854. [5] B.P. Mowery, S.E. Lee, D.A. Kissounko, R.F. Epand, R.M. Epand, B. Weisblum, S.S. Stahl, S.H. Gellman, J. Am. Chem. Soc. 199 (2007) 15474–15476. [6] C.S. Stauffer, A. Datta, J. Org. Chem. 73 (2008) 4166–4174. [7] L. Kiss, E. Forró, F. Fülöp, in: A.B. Hughes (Ed.), Synthesis of Carbocyclic -Amino Acids in Amino Acids, Peptides and Proteins in Organic Chemistry, 1, Wiley, Weinheim, 2009, pp. 367–409. [8] D. Fernandez, E. Torres, F.X. Aviles, R.M. Ortuno, J. Vendrell, Bioorg. Med. Chem. 17 (2009) 3824–3828.
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