Chemical Synthesis of (RP)- and (SP)-[16O,17O ... - ACS Publications

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Chemical Synthesis of (RP)- and (SP)‑[16O,17O,18O]Phosphoenol Pyruvate Petra Malová Križková,† Susanne Prechelmacher,† Alexander Roller,‡ and Friedrich Hammerschmidt*,† †

Institute of Organic Chemistry, University of Vienna, Währingerstrasse 38, A-1090 Vienna, Austria Institute of Inorganic Chemistry, University of Vienna, Währingerstrasse 42, 1090 Vienna, Austria

‡

S Supporting Information *

ABSTRACT: Enzymes and chirality are intimately associated. For their mechanisms to be studied, chiral substrates are needed as probes. Here, we report a concise synthesis of (RP)- and (SP)[16O,17O,18O]phosphoenol pyruvate starting from enantiomerically pure (R)-2-chloro-1-phenylethanol, which was transformed into 18 O-labeled 3-methyl-1-phenylbutane-1,3-diol. The diol was reacted with tris(dimethylamino)phosphane and consecutively with H217O to yield a mixture of cyclic H-phosphonates labeled with 17O and 18 O. They were silylated and subjected to a Perkow reaction with ethyl 3-chloropyruvate. Two protected-[16O,17O,18O]phosphoenol pyruvates were formed and finally globally deprotected. Their configuration was reassessed by a known enzymatic test in combination with conversion of the formed D-glucose-6-phosphate into mixtures of labeled methyl D-glucose-4,6-phosphates, which were analyzed by 31P NMR spectroscopy. The enzymatic test supported the configuration assigned to labeled stereogenic phosphorus atoms on the basis of synthesis.

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[16O,17O,18O]phosphoenol pyruvate as intermediates in a reaction cascade by rearranging [16O,17O,18O]phosphonopyruvate utilizing phosphoenol pyruvate (PEP) mutase.12 To date, no direct chemical synthesis of P-chiral [16O,17O,18O]phosphoenol pyruvate has been disclosed, which would allow the easy preparation of (RP)- and (SP)-[γ-16O,17O,18O]ATP from ADP.

INTRODUCTION Enzymes catalyze stereospecific chemical transformations and discriminate not only between enantiomers but also between ligands at pro-chiral and pro-pro-chiral centers.1 The methyl and phosphoryl group displaying pro-pro chirality of biochemical importance can become chiral by virtue of the hydrogen isotopes H, D, and T and the oxygen isotopes 16O, 17O and 18 O, respectively. Substrates containing these chirally labeled groups are important tools for studying the cryptic stereochemistry of enzymes. The chiral methyl group realized in the form of chiral acetic acid was first prepared, analyzed, and employed to elucidate enzyme mechanisms by the groups of Arigoni2 and Cornforth.3 Enantiomerically pure (R)- and (S)[H,D,T]methanol, the smallest chiral molecules, were recently prepared directly (i.e., not through cleavage of a C−C bond).4 Phosphorus is a pro-pro-pro-chiral center in the phosphate anion, a pro-pro-chiral center in phosphomonoesters, and a prochiral center in phosphodiesters. These various centers can be made chiral by substituting 16O by S, 17O, and 18O atoms or a combination thereof. Initially, Eckstein pioneered the preparation and application of nucleoside phosphorothioates in part also containing oxygen isotopes for delineating the stereochemistry of nucleotidyl and phosphoryl transferases.5,6 Tsai and Chang prepared the enantiomers of chiral [16O,17O,18O]thiophosphate and converted them to ATPβS.7,8 Lowe et al. exploited the chirality of mandelic acid to construct P-chiral [16O,17O,18O]phosphate monoesters and [γ-16O,17O,18O]ATP. They used a method previously developed7 by Tsai and Chang to analyze the chirality of their species by 31P NMR spectroscopy.9−11 Knowles et al. prepared the enantiomers of © 2017 American Chemical Society

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RESULTS AND DISCUSSION We reasoned that a direct chemical synthesis of (RP)- and (SP)[16O,17O,18O]PEP [1b; for assigning (R)- and (S)-configuration to the phosphorus atom, its oxygen atoms are assumed to be ionized] would be a rewarding synthetic puzzle that had not yet been solved. Furthermore, this highly reactive phosphate donor allows for the easy enzyme-catalyzed preparation of P-chiral [γ-16O,17O,18O]ATP and other biochemically important phosphate-containing metabolites. They serve as biochemical tools to address mechanisms of phosphoryl transfers recently investigated by using a variety of metal fluorides as phosphate mimics.13 Our approach built on the Perkow reaction14−16 of phosphites with alkyl 3-halopyruvates yielding protected PEPs17 (Scheme 1). We reasoned that cyclic monosilyl phosphite 4 (R3 = TMS) derived from an enantiomerically pure 1,ω-diol 6 could be added to the carbonyl carbon atom of the carbonyl group of 3-halopyruvate 5 to give 3 as an intermediate on the pathway to protected phosphoenol pyruvate 2 (for mechanistic details, see below). Global Received: July 18, 2017 Published: September 8, 2017 10310

DOI: 10.1021/acs.joc.7b01783 J. Org. Chem. 2017, 82, 10310−10318

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The Journal of Organic Chemistry Scheme 1. Retrosynthetic Analysis for the Synthesis of (Chirally Labeled) PEP

Scheme 2. Conversion of Racemic 1,3-Diol 7 into a cis/transMixture of Protected Phosphoenol Pyruvates 13

deprotection would furnish PEP (1a). Not surprisingly, isotopomers of 4 containing one 17O and 18O atom each in place of two 16O atoms with opposite configuration at the phosphorus atom would deliver (R P )- and (S P )[16O,17O,18O]PEP. We anticipated the formation of two diastereomeric enol phosphates 2 because the enol pyruvyl substituent on phosphorus might be either cis or trans to R2 in the phosphorus-containing ring depending on the configuration of starting cyclic phosphite 4. To evaluate the feasibility of the sequence and optimize the reaction conditions, we first performed the transformations with unlabeled racemic, then enantiomeric, and finally with 17 O- and 18O-labeled enantiomeric compounds. To begin, 1,3diol (±)-7 was converted to a cis/trans-mixture (1:1) of cyclic H-phosphonates (±)-8 by a known procedure18 using bis(3,3,3-trifluoroethyl) H-phosphonate (Scheme 2). We assign cis configuration to that isomer of cyclic H-phosphonates (±)-8, trimethylsilylated phosphites (±)-9, or enol phosphates (±)-13 with the phenyl ring and the substituent on phosphorus (oxygen atom, trimethylsiloxy, or enol pyruvyl group) occupying equatorial positions. However, in the trans isomers, the substituents on phosphorus are axially oriented. These cyclic H-phosphonates were immediately silylated19 in dry toluene with N,N-diethyl-trimethylsilylamine at 50 °C for 40 h to furnish a cis/trans-mixture of silyl phosphites (±)-9 as monitored by 31P NMR spectroscopy of a sample taken from the reaction mixture. The following Perkow reaction with ethyl 3-chloropyruvate was a smooth transformation in dry toluene at room temperature.20 Addition of the silyl phosphites to the carbonyl group of ethyl chloropyruvate generated phosphonium intermediates (±)-10 and (±)-11, which underwent migration of phosphorus with its substituents from the carbon to the oxygen atom with expulsion of chloride. The silyloxyphosphonium chlorides trans- and cis-(±)-12 decomposed and gave enol phosphates trans- and cis-(±)-13 at a reaction time of 2 h at room temperature. Luckily, this mixture could be easily separated by flash chromatography. The less polar trans- and the more polar cis-enol phosphate were obtained in 38 and 35% overall yields, respectively, starting from the cis/trans-mixture of H-phosphonates (±)-8. The configuration of trans-(±)-13 was secured by an X-ray structure analysis. Consequently, the more polar enol phosphate must have the phenyl and enol pyruvyl groups equatorially

positioned, meaning it is cis-configured. Global deprotection should pose no problems and was therefore postponed. To address stereochemical questions arising from the conversion of H-phosphonates (±)-8 to silyl phosphites (±)-9 and then to enol phosphates (±)-13, the cis- and trans-isomers of (±)-8 obtained by flash chromatography17 were silylated individually with N,N-diethyl-trimethylsilylamine and reacted with ethyl 3-chloropyruvate as before. The less polar cis-H-phosphonate delivered homogeneous enol phosphate trans-(±)-13 in 56% yield. The trans-H-phosphonate gave a mixture of cis-(±)-13 in admixture with 7% of trans(±)-13. However, isomerization was minimized (≤2%, 67% yield) with N,N-dimethyl-trimethylsilylamine as silylating agent19 and solvent because of the much shorter reaction time of 1.5 h at 50 °C compared to 40 h at the same temperature with N,N-diethyl-trimethylsilylamine used only as a reagent. These findings suggested that H-phosphonate trans(±)-8 is configurationally less stable than the respective cisisomer and therefore partly epimerized under the reaction 10311


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The Journal of Organic Chemistry

labile benzylic position. Additionally, elimination interfered with nucleophilic substitution, although the reaction was started at −45 °C in a mixture of THF and toluene. Therefore, we were forced to opt for a new approach. To begin with the second approach, commercially available chlorohydrin (R)-19 (ee >99%) was converted to (S)-benzoate (92%) again using the Mitsunobu protocol with PhCO2H or PhC18O2H in the labeled series as acid. Importantly, we hoped that the chlorine atom would interfere with the formation of a carbenium ion at the benzylic position (Scheme 4). Benzoate

conditions for silylation. It is known that 1,3-substituted cyclic cis-H-phosphonates are thermodynamically more stable than the trans-isomers.21 Epimerization of phosphite 9 is unlikely, as similar ones are thermodynamically quite stable, the trans even ́ and Borisenko23 found that cyclic more than the cis.22 Nifantev H-phosphonate trans-4-methyl-2-oxo-2H-1,3,2-dioxa-phosphorinane isomerized to the cis-isomer by inversion of configuration at the phosphorus atom upon heating. As the silyl phosphites were exclusively converted to enol phosphates cis- and trans(±)-13, respectively, the Perkow reaction is stereospecific and follows a retentive course. This sequence demonstrated that just one enantiomer of diol 7 labeled with 16O and 18O and a 17 O-labeled H-phosphonate would suffice to access both enantiomers of P-chiral [16O,17O,18O]PEP. With this result in hand, the synthesis of one enantiomer of diol 7 was started before embarking on the preparation of the corresponding labeled isotopomer. We had to select reagents later readily accessible with oxygen isotopes as well and reaction steps ensuring stereochemical integrity. The first and unsuccessful approach to diol (S)-7 is given in Scheme 3.

Scheme 4. Successful Synthesis of 1,3-Diol 7 of High ee and 18 O-Labeled

Scheme 3. Attempted Synthesis of 1,3-Diol 7 of High ee

(S)-20 was reductively deprotected with DIBAH to afford chlorohydrin (S)-19 in 68% yield and of the same ee (by GC on chiral stationary phase) as that of the starting chlorohydrin. Next, the 1-phenyl-substituted C2 moiety had to be coupled with acetone, which was the critical step of the sequence. For this to be achieved, chlorohydrin (S)-19 was transformed first with n-BuLi at −78 °C into the corresponding lithium alkoxide, whose chlorine atom was exchanged for lithium by means of lithium sand in combination with di-tert-butyl-biphenyl (DTBB).27,28 The respective β-oxidoalkyl lithium formed as intermediate was finally added to acetone to give 1,3-diol (R)-7 in 57% yield. This coupling step delivered an acceptable yield, which varied significantly. Satisfyingly, the ee was >99% and indicated that the Mitsunobu reaction followed a stereospecific course, an SN2 reaction as expected. Replacing DTBB by naphthaline did not improve the yield.29,30 Reaction of 1,3-diol (R)-7 with tris(dimethylamino)phosphine22 in CH3CN under microwave irradiation at 110 °C for 1 h delivered cyclic phosphoramidites as intermediates (Scheme 5). Their ratio in the reaction reaction mixture was 1:6 based on the signals at 139.6 and 139.3 ppm in the 31P NMR spectrum, respectively, probably with the cis-isomer predominating.31 These moisture-sensitive compounds32,33 were not purified but were treated with water and TMSCl generating HCl to convert them into the corresponding Hphosphonates (R,RP)- and (R,SP)-8 in 88% yield in a ratio of 1.0:1.5 in favor of the more polar trans-isomer. This result is the consequence of acid-catalyzed substitution of the dimethylamino group31 with inversion of configuration followed by equilibration of the H-phosphonates.21 The flash chromatographed mixture was silylated with N,N-dimethyl-trimethylsilylamine and subjected to the Perkow reaction with ethyl 3chloropyruvate as before. The two enantiomerically pure diastereomeric enol phosphates (R,SP)- and (R,RP)-13 were obtained in a combined yield of 52%.

Known hydroxyketone24 14 was silylated with TBDMSOTf/ 2,6-lutidine in 93% yield and then enantioselectively reduced to (+)-16 in 95% yield by the Corey, Bakshi, Shibata protocol25 using (S)-(−)-2-methyl-CBS-oxazaborolidine. The reducing agent BH3·THF had to be slowly added with a syringe pump to obtain a high ee. To determine the ee and configuration of (+)-16, it was deprotected with Bu4NF. The diol (+)-7 formed was analyzed by HPLC using a chiral column. The ee was found to be 91%, and the configuration was (R) based on an authentic sample of (R)-(+)-7 prepared in the course of the next and successful approach. Consequently, monoprotected 1,3-diol (+)-16 was assigned (R)-configuration. Mitsunobu reaction26 of (+)-16 with Ph3P/DIAD/4-nitrobenzoic acid (or 4-nitro-[carboxy-18O2]benzoic acid for the labeled species) furnished the corresponding 4-nitrobenzoate (S)-17 in 66% yield and alkene 18 in 25%. Global deprotection of the 4nitrobenzoate under mild conditions (K2CO3/MeOH, 84% yield) followed by desilylation (Bu4NF, 93%) furnished levorotary 1,3-diol 7 (93% yield, ee 49%) having the (S)configuration as expected. Evidently, partial racemization occurred during the Mitsunobu reaction at the configurationally 10312


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carboxylic ester cleavage, delivered unlabeled and (RP)- and (SP)-[16O,17O,18O1]PEP [(RP)- and (SP)-(1b)], respectively. The chemoenzymatic analysis of the P-chiral PEPs was performed according to the method developed by Lowe et al.11 and already used by Knowles et al.12 In short, the reaction mixture contained D-glucose, (RP)- or (SP)-[16O,17O,18O1]PEP, catalytic ADP, pyruvate kinase, and hexokinase. The phosphoryl group is transferred by pyruvate kinase from phosphoenol pyruvate to ADP with inversion of the configuration36,37 and from there to the C-6 hydroxyl group of D-glucose, again with inversion of configuration38 by hexokinase. Glucose-6-phosphate was isolated by ion exchange chromatography and then cyclized with KOtBu after activation with diphenyl phosphorochloridate, following an invertive process. The cyclic 4,6-phosphate salt was methylated with methyl iodide to give methyl cyclic 4,6-phosphates (Scheme 6; for detailed interpretation and analysis of 31P NMR spectra, see Schemes S1−S4), purified by flash chromatography, and investigated by 31P NMR spectroscopy. Each sample of our labeled PEPs consisted mainly of (R)- and (S)-configured species in admixture with PEP containing one 18O atom, as the starting diol was enantiomerically pure and contained 98% 18O at the benzylic position. Furthermore, the [17O]H2O (70% 17O, 28% 16O, 2% 18O) contained virtually no 18O and gave Hphosphonates (R,RP)- and (R,SP)-[17O,18O]8 with 64% 17O and 36% 18O at the phosphorus atom, neglecting for the sake of simplicity the 2% of 16O at the benzylic position and the 2% of 18 O in [17O]H2O. However, Lowe et al. and Knowles et al. used 17 [ O]H2O containing less than 50% of 17O and 53% and 31% of 18O, respectively. Not surprisingly, the samples of P-chiral compounds of these authors contained additionally more than 30% of [18O2]species, which complicated the stereochemical analysis and influenced the line intensities of their 31P NMR spectra. The lower number of resonances and the clearly visible differences in their intensities in our spectra are noteworthy.

Scheme 5. Conversion of H-Phosphonates 7 to PEP and (RP)- and (SP)-[16O,17O,18O]PEP

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CONCLUSIONS In summary, we have reported a concise synthesis of the enantiomers of P-chiral [16O,17O,18O]phosphoenol pyruvate starting from commercially available (R)-2-chloro-1-phenylethanol of >99% ee, which is coupled with acetone to give 18Olabeled 1,3-diol. It was converted to cyclic 17O- and 18O-labeled silyl phosphites subjected to a Perkow reaction to give two enol phosphates separable by flash chromatography. They were easily deprotected to afford the respective P-chiral PEPs. Their stereochemistry was ascertained by a known chemoenzymatic test comprising transfer of the phosphate group to the C-6 hydroxyl of D-glucose, cyclization to a 4,6-diphosphate, and a final methylation. The obtained cyclic methyl phosphates were analyzed by 31P NMR spectroscopy. Our synthesis allows rapid access to P-chiral PEPs, a pivotal precursor for ATP, and a variety of P-chiral phosphate esters of biological importance.

The sequence in the optically active series was repeated by substituting benzoic acid with [carboxy-18O2]benzoic acid prepared from H218O with 98.3% 18O in the Mitsunobu reaction to generate benzoate (S)-[18O2]20 (Scheme 4). Subsequently, the benzoyl group was removed as before to recover chlorohydrin (S)-[18O1]19 (89% yield, >97% ee), which delivered 18O-labeled 1,3-diol (R)-[18O]7 upon coupling with acetone. The phosphoramidites were formed and hydrolyzed with H217O (70% 17O, 28% 16O, 2% 18O) as before. The mixture of H-phosphonates (R,SP)- and (R,RP)[17O,18O1]8 (cis/trans = 1.0:1.2) was again silylated and subjected to the Perkow reaction to finish the synthesis of the protected enantiomers of P-chiral phosphoenol pyruvate (R,SP)- and (R,RP)-[17O,18O1]13. They were separated by flash chromatography and obtained in 36 and 39% yields, respectively. Each contained 64% 17O and 99% 18O as determined by HRMS. Global deprotection of the three enol phosphates, the unlabeled one and both labeled ones, with TMSBr/allylTMS34,35 for phosphorus and aqueous KOH for

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EXPERIMENTAL SECTION

General Information. 1H, 13C (J-modulated), and 31P NMR spectra were recorded in CDCl3 or D2O on a Bruker Avance AV III 400 (1H: 400.27 MHz, 13C: 100.65 MHz, 31P: 162.03 MHz), AV 400 (1H: 400.13 MHz, 13C: 100.61 MHz, 31P: 161.98 MHz), and AV III 600 (1H: 600.25 MHz, 13C: 150.93 MHz, 31P: 242.94 MHz) at 25 °C. Chemical shifts (δ) are reported in parts per million (ppm) relative to internal residual CHCl3 (δH 7.24; δC 77.00), HOD (δH 4.80), and external H3PO4 (85%; δP 0.00), and coupling constants (J) are in Hz. Data for 1H NMR spectra are reported as follows: chemical shift, 10313


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comprising components LC-20AT, SIL-20A HT, CTO-20AC, SPD20A, and CMB-20A; column: Chiralpak IC (250 × 4.6 mm, particle size 5 μm, solvent: heptanes + 0.1% 92:8 2-propanol/2-propanol; 0.7 mL/min), LC solutions. GC-MS: Agilent Technologies 78908 GC System and MS system 5975C VL MSD with Triple-Axis Detector; column: MEGA-DEX DMP Beta (dimethyl-pentyl β-cyclodextrin); F.T., 0.25 μm; I.D., 0.25 μm; L., 25 m; He, 1 mL/min. Anhydrous THF was refluxed over potassium and distilled prior to use. Pyridine was dried by refluxing over powdered CaH2, then distilled and stored over molecular sieves (4 Å). All other solvents, also dry ones, and chemicals were used as purchased from Sigma-Aldrich, Acros, Fluka, or Merck. Flash column chromatography was performed over Silica 60 M (particle size, 0.040−0.063 mm) from MachereyNagel. Reactions were monitored by analytical thin layer chromatography (TLC) using precoated silica gel plates from Macherey-Nagel (TLC Silica gel 60 F254, 250 μm thickness). Spots were visualized by UV and/or dipping into a solution of (NH4)6Mo7O24·4H2O (25.0 g) and Ce(SO4)2·4H2O (1.0 g) in 10% aqueous H2SO4 (500 mL) followed by heating with a heat gun. Ethyl 2-(((2R*,6R*)-4,4-Dimethyl-2-oxido-6-phenyl-1,3,2-dioxaphosphinan-2-yl)oxy)-acrylate [trans-(±)-13] and Ethyl 2(((2S*,6R*)-4,4-Dimethyl-2-oxido-6-phenyl-1,3,2-dioxaphosphinan-2-yl)-oxy)acrylate [cis-(±)-13]. A mixture18 of racemic 3methyl-1-phenylbutane-1,3-diol [(±)-7] (360 mg, 2.9 mmol) and bis(trifluoroethyl) H-phosphonate (541 mg, 0.35 mL, 2.2 equiv, 90%) in dry pyridine (10 mL) was stirred for 1 h at rt under an argon atmosphere. The solution was concentrated under reduced pressure. The residue was twice dissolved in dry toluene (8 mL each time) and again concentrated under reduced pressure. Finally, the residue was dried at 0.5 mbar/20 °C. N,N-Diethyl-trimethylsilylamine (872 mg, 1.14 mL, 6.0 mmol, 3.0 equiv) and dry toluene (10 mL) were added to the cyclic H-phosphonate. The mixture was heated and stirred at 50 °C under argon for 40 h and then concentrated under reduced pressure. The oily residue was dried at 0.5 mbar/20 °C before a solution of ethyl 3-chloropyruvate39 (362 mg, 2.4 mmol, 1.2 equiv) in dry toluene (10 mL) was added under an argon atmosphere. After stirring the reaction mixture for 2 h at ambient temperature, it was concentrated under reduced pressure. The residue was purified by flash chromatography (2:1 heptanes/EtOAc, v/v; trans-(±)-13: Rf = 0.42; cis-(±)-13: Rf = 0.21) to give trans-(±)-13 (258 mg, 38%) and cis-(±)-13, which was flash chromatographed (3:1 heptanes/EtOAc, v/v) a second time to give a homogeneous product (238 mg, 35%). Both compounds were crystallized by allowing solutions in heptanes/ CH2Cl2 to slowly concentrate at room temperature; trans-(±)-13: colorless crystals, mp 78−79 °C; cis-(±)-13: colorless crystals, mp 76− 77 °C. Similarly, H-phosphonate cis-(±)-8 (277 mg, 1.22 mmol) was converted to homogeneous enol-phosphate trans-(±)-13 (235 mg, 56%), and H-phosphonate trans-(±)-8 (376 mg, 1.66 mmol) was converted to enol phosphate cis-(±)-13 (299 mg, 53%) containing 7% of trans-(±)-13. Similarly, H-phosphonate trans-(±)-8 (124 mg, 0.55 mmol) was converted to enolmphosphate cis-(±)-13 (125 mg, 67%) containing ≤2% of trans-(±)-13, except that for silylation a solution in N,N-dimethyl-trimethylsilylamine (3 mL) was stirred under an argon atmosphere at 50 °C for 1.5 h. trans-(±)-13: 1H NMR (400.27 MHz, CDCl3): δ 7.39−7.29 (m, 5H), 5.93 (t, J = 2.4 Hz, 1H), 5.82 (t, J = 2.4 Hz, 1H), 5.77 (ddd, J = 11.9, 2.4, 1.6 Hz, 1H), 4.23 (AB system, coupling with CH3, JAB = 10.9 Hz, J = 7.1 Hz, 2H), 2.20 (dd, J = 14.7, 12.0 Hz, 1H), 1.92 (td, J = 14.7, 2.4 Hz, 1H), 1.73 (s, 3H), 1.50 (d, J = 2.8 Hz, 3H), 1.28 (t, J = 7.1 Hz, 3H). 13C NMR (100.65 MHz, CDCl3): δ 161.9 (d, J = 8.1 Hz), 143.1 (d, J = 7.4 Hz), 138.7 (d, J = 10.3 Hz), 128.65 (2C), 128.62, 125.6 (2C), 110.9 (d, J = 3.9 Hz), 84.9 (d, J = 8.1 Hz), 77.7 (d, J = 5.9 Hz), 61.8, 45.3 (d, J = 5.0 Hz), 31.5 (d, J = 9.6 Hz), 25.5, 14.0. 31P NMR (162.04 MHz, CDCl3): δ −14.02; IR (ATR): ν 2984, 1720, 1627, 1292, 1178, 1158, 1141, 1030, 991, 927 cm−1; Anal. Calcd for C16H21O6P: C, 56.47; H, 6.22; O, 28.21. Found: C, 56.45; H, 6.02; O, 28.31. cis-(±)-13: 1H NMR (400.27 MHz, CDCl3): δ 7.41 (m, 5H), 5.94 (t, J = 2.4 Hz, 1H), 5.74 (t, J = 2.4 Hz, 1H), 5.62 (ddd, J = 12.4, 4.6, 2.6 Hz, 1H), 4.23 (AB system, coupling with CH3, JAB = 11.4 Hz, J = 7.1 Hz, 2H), 2.63 (dd, J = 15.1, 12.4 Hz, 1H), 1.98 (td, J = 15.1, 2.6

Scheme 6. Preparation of P-Chiral D-glucose-6-phosphates 21, Their Cyclization and Methylation, and 31P NMR Spectra (242.94 MHz, 1:1 DMSO-d6/MeOH-d4) of the Respective Mixtures of Cyclic Methyl Phosphates 22 and 23a

a

(A,B) 31P NMR parameters: sweep width, 1000 Hz; acquisition time, 5 s; number of scans, 10375; Gaussian multiplication (0.3) and line broadening (−0.7) in 128 K. (A) Cyclic methyl phosphates 22 with equatorial MeO; (B) cyclic methyl phosphates 23 with axial MeO; derived from (RP)-15. (C,D) 31P NMR parameters: sweep width, 2430 Hz; acquisition time, 5 s; number of scans, 9423; Gaussian multiplication (0.02) and line broadening (−1.4) in 128 K. (C) Cyclic methyl phosphates 22 with equatorial MeO; (D) axial methyl phosphates 23 with axial MeO; derived from (SP)-15. multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = mutliplet), coupling constants, and integration. IR spectra were recorded on a Bruker VERTEX 70 IR spectrometer as ATR spectra. Melting points were measured on a Leica Galen III Thermovar instrument and are uncorrected. Optical rotations were measured on a PerkinElmer 341 polarimeter in a 1 dm quartz cell. High-resolution mass spectra (HRMS) were obtained using a Bruker Maxis Q-TOF mass spectrometer (ESI) and a Finnigan MAT, Bremen, MAT 95S mass spectrometer (EI). Analytical HPLC: Shimadzu system 10314


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The Journal of Organic Chemistry Hz, 1H), 1.64 (s, 3H), 1.51 (d, J = 1.1 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H). 3C NMR (100.65 MHz, CDCl3): δ 161.7 (d, J = 8.2 Hz), 143.6 (d, J = 7.8 Hz), 138.7 (d, J = 6.5 Hz), 128.72 (CHAr), 128.7 (3C), 125.8 (2C), 110.2 (d, J = 4.2 Hz), 83.9 (d, J = 7.2 Hz), 78.8 (d, J = 6.0 Hz), 61.8, 43.4 (d, J = 9.8 Hz), 30.2 (d, J = 3.6 Hz), 27.4 (d, J = 4.1 Hz), 14.1. 31P NMR (162.03 MHz, CDCl3): δ −13.49. IR (ATR): ν 2983, 1725, 1647, 1294, 1003, 986, 967 cm−1. Anal. Calcd for C16H21O6P: C, 56.47; H, 6.22; O, 28.21. Found: C, 56.54; H, 5.84; O, 28.32. 3-(tert-Butyldimethylsilyl)oxy-3-methyl-1-phenylbutan-1one (15). 2,6-Lutidine (2.164 g, 2.35 mL, 20.2 mmol, 2 equiv) and tert-butyldimethylsilyl triflate (TBDMSOTf) (3.47 g, 3.02 mL, 13.13 mmol, 1.3 equiv) were slowly added dropwise to a stirred solution of β-hydroxyketone24 14 (1.80 g, 10.1 mmol) in dry CH2Cl2 (30 mL) at 0 °C under an argon atmosphere. The reaction mixture was allowed to stir for 18 h while warming to rt. Water (30 mL) was added, and the phases were separated. The aqueous phase was extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed with water (20 mL), HCl (1 M, 20 mL), water (20 mL), and a saturated aqueous solution of NaHCO3 (20 mL), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by flash chromatography (1:1 heptanes/EtOAc, v/v; Rf = 0.44) to yield βsilyloxyketone 15 (2.734 g, 93%) as a colorless oil. 1 H NMR (600.25 MHz, CDCl3): δ 7.97−7.92 (m, 2H), 7.53−7.49 (m, 1H), 7.44−7.39 (m, 2H), 3.08 (s, 2H), 1.39 (s, 6H), 0.71 (s, 9H), 0.01 (s, 6H). 13C NMR (150.93 MHz, CDCl3): δ 199.4, 138.5, 132.7, 128.7 (2C), 128.3 (2C), 73.7, 51.8, 30.4 (2C), 25.7 (3C), 17.9, −2.1 (2C). IR (ATR): 2955, 2929, 2855, 1677, 1250, 1148, cm−1. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C17H28O2SiNa, 315.1751; found, 315.1760. Anal. Calcd for C17H28O2Si: C, 69.81; H, 9.65. Found: C, 69.92; H, 9.61. (+)- and (±)-3-(tert-Butyldimethylsilyl)oxy-3-methyl-1-phenylbutan-1-ol [(+)- and (±)-16]. BH3·THF (2.45 mL, 2.45 mmol, 1.1 equiv, 1 M solution in THF) was added to a solution of (S)-(−)-2methyl-CBS-oxazaborolidine [(S)-B-Me-425] (62 mg, 0.223 mmol, 0.1 equiv) in dry THF (2 mL) under an argon atmosphere at ambient temperature. The mixture was cooled at −25 °C and stirred, and silyloxyketone 15 (653 mg, 2.23 mmol) dissolved in dry THF (4 mL) was added with a syringe pump within 90 min. The reaction mixture was allowed to slowly warm in the bath to room temperature within 3 h. EtOH (2 mL) was added slowly with cooling (exothermic reaction with evolution of hydrogen!) and was then concentrated under reduced pressure. Water (20 mL) and pentaerythritol (411 mg, 3 mmol) were added to the residue. After stirring for 15 min, the mixture was extracted with CH2Cl2 (3 × 15 mL). The combined organic layers were dried (MgSO4) and concentrated under reduced pressure. The residue was flash chromatographed 1:1 heptanes/ CH2Cl2 (v/v; Rf = 0.33) to furnish silyloxyalcohol (+)-16 (623 mg, 95%) as colorless oil; [α]D20 +28.8 (c 2.49, acetone); 91% ee by HPLC of 1,3-diol on chiral column. A sample of (±)-16 needed as standard for HPLC was prepared by reduction of β-silyloxyketone 15 (50 mg, 0.17 mmol) with NaBH4 (16 mg, 0.43 mmol, 2.5 equiv) in dry EtOH (1 mL) for 15 h rt. A solution of pethaerythritol (70 mg, 0.51 mmol) in water (10 mL) was added. After stirring for 2 h, the mixture was extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with water (3 × 3 mL), dried (MgSO4), and concentrated under reduced pressure. The residue was flash chromatographed as above to give silyloxyalcohol (±)-16 (41 mg, 82%) as a colorless oil. The spectroscopic data are identical to those of (+)-16. The enantiomers of (±)-16 could not be separated on the Chiralpak IC column by HPLC. 1 H NMR (600.25 MHz, CDCl3): δ 7.38−7.34 (m, 2H), 7.33−7.28 (m, 2H), 7.24−7.20 (m, 1H), 5.05 (dd, J = 10.8, 1.6 Hz, 1H), 4.61 (s, 1H), 1.95 (dd, J = 14.5, 10.8 Hz, 1H), 1.62 (dd, J = 14.5, 1.6 Hz, 1H), 1.47 (s, 3H), 1.32 (s, 3H), 0.91 (s, 9H), 0.182 (s, 3H), 0.175 (s, 3H). 13 C NMR (150.93 MHz, CDCl3): δ 145.1, 128.2 (2C), 127.0, 125.7 (2C), 76.0, 71.6, 52.9, 31.8, 28.0, 25.8 (3C), 17.9, −1.9, −2.0. IR (ATR): ν 3478, 2955, 2930, 2857, 1253, 1141, 996 cm−1. HRMS (ESITOF) m/z: [M + Na]+ calcd for C17H30O2SiNa, 317.1907; found,

317.1919. Anal. Calcd for C17H30O2Si: C, 69.33; H, 10.27. Found: C, 69.21; H, 10.31. Desilylation of (+)-3-(tert-Butyldimethylsilyl)oxy-3-methyl1-phenylbutan-1-ol [(+)-16]−(R)-(+)-3-methyl-1-phenylbutane-1,3-diol [(R)-7]. A solution of silyloxyalcohol (+)-16 (60 mg, 0.205 mmol) in dry THF (2 mL) and Bu4NF (0.55 mL, 0.55 mmol, 1 M in THF) were combined at rt and stirred for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was flash chromatographed (1:1 heptanes/EtOAc, v/v; Rf = 0.24) to furnish diol (R)-718,40 (35 mg, 95%) as a crystalline product; [α]D20 +49.0 (c 0.94, acetone), 91% ee by HPLC. (S)-(+)- and (±)-3-(tert-Butyldimethylsilyl)oxy-3-methyl-1phenylbutyl 4-nitrobenzoate [(S)-(+)- and (±)-17] and (E)tert-Butyldimethyl-((2-methyl-4-phenylbut-3-en-2-yl)oxy)silane (18). A mixture of alcohol (R)-16 (483 mg, 1.65 mmol, ee 91%), triphenylphosphane (606 mg, 2.31 mmol, 1.4 equiv), and 4nitrobenzoic acid (386 mg, 2.31 mmol, 1.4 equiv) was dissolved in dry THF/toluene (12 and 3 mL) under an argon atmosphere. The reaction mixture was cooled to −45 °C, and diisopropyl azodicarboxylate (DIAD, 467 mg, 0.46 mL, 2.31 mmol, 1.4 equiv) was added slowly. After stirring for 15 h (during which time the temperature of the cooling bath was allowed to rise slowly to +8 °C) a few drops of EtOH were added. After stirring for 15 min, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (1:1 heptanes/CH2Cl2, v/v; alkene: Rf = 0.92; nitrobenzoate: Rf = 0.46) to give alkene 18 (900 mg, 25%) and 4nitrobenzoate (S)-17 (481 mg, 66%), both as colorless oils. An analytical sample of (±)-17 for HPLC was prepared by esterification of (±)-16 (80 mg, 0.27 mmol) with 4-nitrobenzoyl chloride (86 mg, 0.55 mmol, 2 equiv) in dry pyridine (2 mL) at room temperature. Aqueous extractive work up of the reaction mixture gave a residue that was flash chromatographed (1:1 heptanes/CH2Cl2, v/v; Rf = 0.46) to yield (±)-17 (112 mg, 92%) as a colorless, viscous oil. The spectra were identical to those of (S)-17. The enantiomers of (±)-17 could not be separated by HPLC on the Chiralpak IC column. (S)-17: [α]D22 +20.1 (c 1.25, acetone); 1H NMR (400.27 MHz, CDCl3): δ 8.28−8.23 (m, 2H), 8.22−8.18 (m, 2H), 7.41−7.36 (m, 2H), 7.36−7.29 (m, 2H), 7.29−7.23 (m, 1H), 6.31 (dd, J = 8.7, 2.9 Hz, 1H), 2.31 (dd, J = 14.8, 8.7 Hz, 1H), 2.01 (dd, J = 14.8, 2.9 Hz, 1H), 1.28 (s, 3H), 1.24 (s, 3H), 0.89 (s, 9H), 0.07 (s, 6H). 13C NMR (100.65 MHz, CDCl3): δ 163.7, 150.5, 141.6, 136.0, 130.7 (2C), 128.7 (2C), 128.0, 126.5 (2C), 123.5 (2C), 75.2, 72.6, 51.2, 31.4, 29.6, 25.9 (3C), 18.1, −2.1 (2C). IR (ATR): ν 2954, 2928, 2855, 1724, 1527, 1270 1035,cm−1. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C24H33NO5SiNa, 466.2016; found, 466.2020. Anal. Calcd for C24H33NO5Si: C, 64.98; H, 7.50; N, 3.16. Found: C, 65.02; H, 7.44; N, 3.13. 18: 1H NMR (400.27 MHz, CDCl3): δ 7.38−7.32 (m, 2H), 7.32− 7.26 (m, 2H), 7.22−7.19 (m, 1H), 6.51 (d, J = 16.0 Hz, 1H), 6.26 (d, J = 16.0 Hz, 1H), 1.38 (s, 6H), 0.90 (s, 9H), 0.08 (s, 6H). 13C NMR (100.65 MHz, CDCl3): δ 138.7, 137.4, 128.5 (2C), 127.1, 126.3 (2C), 125.9, 73.2, 30.6 (2C), 25.9 (3C), 18.1, −2.1 (2C). IR (ATR): ν 2955, 2928, 2855, 1252, 1143, 1035 cm−1. HRMS (EI) m/z: M+ calcd for C17H28OSi, 276.1904; found, 276.1897. (S)-(−)-3-(tert-Butyldimethylsilyl)oxy-3-methyl-1-phenylbutan-1-ol [(S)-16] and (S)-(−)-3-Methyl-1-phenylbutane-1,3-diol [(S)-7]. A mixture of K2CO3 (124 mg, 1.24 mmol, 1.5 equiv) and 4nitrobenzoate (S)-17 (367 mg, 0.827 mmol) in dry MeOH (3 mL) was stirred for 2 h at room temperature and then concentrated under reduced pressure. The residue was purified by flash chromatography (10:1 heptanes/EtOAc, v/v; TLC: Rf = 0.44) to give alcohol (S)-16 (210 mg, 87%) as a colorless oil; [α]D22 −14.8 (c 2.21, acetone). The spectra (1H NMR and IR) were superimposable to those of (+)-16. Part of this monosilylated diol (S)-16 (95 mg, 0.325 mmol) was deprotected by the procedure used for (+)-16 to give (S)-7 (55 mg, 93%); [α]D20 −28.8 (c 1.14, acetone); 49% ee by HPLC. (S)-(−)-2-Chloro-1-phenylethyl benzoate [(S)-20] and (S)(−)-2-Chloro-1-phenylethyl[ 18 O 2 -carboxy]benzoate {(S)[18O2]20}. DIAD (480 mg, 2.38 mmol, 0.47 mL, 1.2 equiv) was added to a mixture of commercially available (R)-(−)-2-chloro-1phenylethanol [(R)-19, 310 mg, 1.98 mmol, 0.26 mL, >99% ee by 10315


Article


H NMR (400.27 MHz, CDCl3): δ 7.38−7.30 (m, 4H), 7.27−7.22 (m, 1H), 5.07 (dd, J = 11.0, 2.2 Hz, 1H), 3.10 (br s, 1H), 1.83 (AB system, JAB = 14.7 Hz, JAX = 11.0 Hz, JBX = 2.2 Hz, 2H), 1.43 (s, 3H), 1.27 (s, 3H). 13C NMR (100.66 MHz, CDCl3): δ 144.7, 128.4 (2C), 127.4, 125.6 (2C), 72.3, 71.6, 50.4, 31.8, 27.6. The NMR spectra were identical to those found in the literature42 for (±)-7. Similarly, labeled chlorohydrin (S)-[18O1]19 (890 mg, 5.61 mmol) was converted to labeled 1,3-diol (R)-[18O1]7 (540 mg, 53%) as colorless crystals; mp 67−68 °C (hexanes/CH2Cl2); [α]D28 +54.2 (c 1.12, acetone). The NMR spectra were identical to those of (R)-7. IR (ATR): ν 2806, 2526, 1650, 1579, 1453, 1415, 1321, 1272, 1184, 1113, 928 cm −1 . HRMS (ESI-TOF) m/z: [M + Na] + calcd for C11H16O18ONa, 205.1085; found, 205.1085; 98.6% 18O. (2S,6R)-(−)- and (2R,6R)-(−)-4,4-Dimethyl-6-phenyl-1,3,2-dioxaphosphinane-2-oxide [(R,SP)- and (R,RP)-8]; (2S,6R)-(−)- and (2R,6R)-(−)-4,4-Dimethyl-6-phenyl-1,3,2-[1- 18O1]-dioxaphosphinane-2-[2-17O]oxides {(R,SP)- and (R,RP)-[17O,18O1]8}. A mixture of diol (R)-7 (901 mg, 5.0 mmol) and tris(dimethylamino)phosphane22 (1.142 g, 7.0 mmol, 1.27 mL, 1.4 equiv) in dry CH3CN (17 mL) was stirred in a microwave oven at 110 °C for 1 h. The solution was concentrated under reduced pressure, and the crude mixture of dioxaphosphinanes [31P NMR (162.03 MHz, CDCl3): ratio of signals at 139.8:139.4 ppm, 1:6] were reacted with H2O (180 mg, 10.0 mmol, 0.18 mL) and TMSCl (760 mg, 7.0 mmol, 0.89 mL) in dry THF (15 mL) under an argon atmosphere for 10 min at 50 °C. After the addition of hexamethyldisilazane (888 mg, 5.5 mmol, 1.15 mL), the mixture was concentrated in vacuo, and the crude product was purified by flash chromatography (EtOAc, Rf = 0.69) to yield a mixture of cyclic H-phosphonates [990 mg, combined yield 88%, (R,SP)-8: (R,RP)-8 = 1:1.5]. The NMR spectra were identical to those of (±)-8,18 except for the different intensities of the resonances. Similarly, (R)-[18O1]7 (911 mg, 5.0 mmol) was converted via cyclic phosphoramidites (1:3 ratio by 31P NMR) with H217O (200 μL, 10.0 mmol, 2 equiv, 70% 17O, 28% 16O and 2% 18O) to a mixture (779 mg, 68%) of cyclic H-phosphonates [(R,SP)-[17O,18O1]8:(R,RP)-[17O,18O1] 8 = 1:1.2] as a colorless oil; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C11H15O218ONa, 251.0694; found, 251.0685; [M + Na]+ calcd for C11H15O17O18ONa, 252.0736; found, 252.0726; 98.6% 18O, 64.3% 17 O. The 1H NMR spectra were identical to those of the unlabeled species except for the different intensities of the resonances. The 31P NMR spectrum of the compounds not containing 17O was identical to the spectrum for the racemic unlabeled compounds. The 17Ocontaining molecules gave very much broadened signals (see Supporting Information). Ethyl 2-{(2R,6R)-((4,4-Dimethyl-2-oxido-6-phenyl-1,3,2-dioxaphosphinan-2-yl)oxy}acrylate and Ethyl 2-{(2S,6R)-((4,4-Dimethyl-2-oxido-6-phenyl-1,3,2-dioxaphosphinan-2-yl)oxy}acrylate [(R,RP)- and (R,SP)-13]; Ethyl 2-{(2R,6R)-((4,4-Dimethyl2-[17O]oxido-6-phenyl-1,3,2-[1- 18O1]dioxaphosphinan-2-yl)oxy}acrylate and Ethyl 2-{(2S,6R)-((4,4-Dimethyl-2-[17O]oxido6-phenyl-1,3,2-[1- 18 O 1 ]dioxaphosphinan-2-yl)oxy}acrylate {(R,RP)-[17O,18O1]13 and (R,SP)-[17O,18O1]13}. This conversion was performed in analogy to the procedure given for the preparation of trans- and cis-(±)-13. A mixture of cyclic H-phosphonates (R,SP)- and (R,RP)-8 (165 mg, 0.73 mmol) was silylated with N,N-dimethyltrimethylsilane (3 mL, 90 min, monitored by 31P NMR) and then reacted with ethyl 3-chloropyruvate39 (132 mg, 0.88 mmol, 1.2 equiv) to a mixture of enol phosphates. Flash chromatography gave (R,RP)-13 (64 mg, 26%, after a second chromatography) and (R,SP)-13 (64 mg, 26%). (R,RP)-13 was crystallized from hexanes/CH2Cl2 to yield colorless needles, mp 98.5−99.5 °C, [α]D20 −24.3 (c 1.13, acetone). The (R,SP)-13 (cis) was also crystallized from hexanes/CH2Cl2 to yield colorless crystals, mp 100−101 °C, [α]D20 −17.1 (c 0.89, acetone). The NMR spectra of (R,SP)-13 and (R,RP)-13 were identical to those of cis- and trans-(±)-13, respectively. Similarly, a mixture of labeled H-phosphonates (R,SP)- and (R,RP)[17O,18O1]8 (579 mg, 2.53 mmol, ratio: 1:1.2) was silylated with N,Ndimethyl-trimethylsilylamine (4 mL, 90 min, 50 °C, see 31P NMR spectrum) and reacted with ethyl 3-chloropyruvate39 to a mixture of labeled enol phosphates. Flash chromatography furnished isomers (R,RP)-[17O,18O1]13 (315 mg, 36%) and (R,SP)-[17O,18O1]13 (343 1

GC], benzoic acid (291 mg, 2.38 mmol, 1.2 equiv), and Ph3P (624 mg, 2.4 mmol, 1.2 equiv) in dry toluene (5 mL) under an argon atmosphere at 0 °C. After stirring for 1.5 h at room temperature, a few drops of water were added, and the mixture was concentrated under reduced pressure. The crude product was purified by flash chromatography (10:1 hexanes/EtOAc, v/v; Rf = 0.60) to yield benzoate (S)-20 (480 mg, 93%) as a colorless oil; [α]D20 −38.6 (c 1.31, acetone). 1 H NMR (400.27 MHz, CDCl3): δ 8.13−8.07 (m, 2H), 7.59−7.54 (m, 1H), 7.48−7.42 (m, 4H), 7.39−7.30 (m, 3H), 6.19 (dd, J = 7.8, 4.5 Hz, 1H), 3.89 (AB system, JAB = 11.7 Hz, JAX = 7.8 Hz, JBX = 4.5 Hz, 2H). 13C NMR (100.62 MHz, CDCl3): δ 165.4, 137.2, 133.2, 129.79 (2C), 129.75, 128.8, 128.7 (2C), 128.4 (2C), 126.6 (2C), 75.6, 46.7. IR (ATR): ν 1719, 1452, 1315, 1265, 1108, 1069, 1026 cm−1. Anal. Calcd for C15H13ClO2: C, 69.10; H, 5.03; O, 12.27. Found: C, 69.07; H, 4.99; O, 12.32. Similarly, [18O2-carboxy]benzoic acid (1.94 g, 15.39 mmol, 1.1 equiv), prepared41 from H218O (98.3% 18O), Ph3P (4.039 g, 15.4 mmol, 1.1 equiv), and DIAD (3.114 g, 3.03 mL, 1.1 equiv), were used to convert (R)-(−)-2-chloro-1-phenylethanol [(R)-19, 2.193 g, 14.0 mmol, 1.85 mL, ee 97%] to (S)-[18O2]20 (3.32 g, 90%), [α]D28 −37.3 (c 1.17, acetone). The NMR spectra were identical to those of the unlabeled species. IR (ATR): ν 1693, 1494, 1451, 1315, 1267, 1088, 1067, 1025 cm−1. (S)-(+)-2-Chloro-1-phenylethanol [(S)-19] and (S)-(+)-2Chloro-1-phenyl-[1-18O1]ethanol {(S)-[18O1]19}. DIBAL (5.51 mmol, 5.51 mL, 1.0 M solution in toluene) was added to a stirred solution of benzoate (S)-20 (440 mg, 1.69 mmol) in dry THF (4 mL) under an argon atmosphere at −78 °C, and the mixture was slowly warmed to rt in the bath overnight (16 h). Water (2 mL) and HCl (5 mL, 2 M) were added. The aqueous phase was extracted with CH2Cl2 (3 × 5 mL), and the combined organic phases were washed with water (5 mL) and a saturated aqueous solution of NaHCO3 (5 mL), dried (Na2SO4), and concentrated under reduced pressure. The crude product was purified by flash chromatography (5:1 hexanes/EtOAc, v/ v; Rf = 0.32) to yield chlorohydrin (S)-19 (180 mg, 68%) as a colorless oil, [α]D20 +51.41 (c 1.025, acetone), bought (R)-(−)-2-chloro-1phenylethanol: [α]D20 −50.4 (c 1.1, acetone); >99% ee by GC-MS. 1 H NMR (400.27 MHz, CDCl3): δ 7.39−7.28 (m, 5H), 4.89 (td, J = 8.8, 3.3 Hz, 1H), 3.69 (AB system, JAB = 11.2 Hz, JAX = 8.8 Hz, JBX = 3.3 Hz, 2H), 2.61 (d, J = 3.3 Hz, 1H). Similarly, (S)-[18O2]20 (2.01 g, 7.59 mmol) was converted to chlorohydrin (S)-[18O1]19 (1.10 g, 91%), [α]D20 +45.9 (c 1.00, acetone); >99% ee by GC-MS. 1 H NMR spectrum of (S)-[18O2]19 was identical to that of the unlabeled species. 13C NMR (100.62 MHz, CDCl3): δ 139.9, 128.7 (2C), 128.5, 126.0 (2C), 74.1 (this resonance is shifted by 2 Hz to higher field relative to unlabeled species), 50.9. IR (ATR): ν 3376, 1494, 1454, 1426, 1248, 1200, 1056, 1007 cm−1. (R)-(+)-3-Methyl-1-phenylbutane-1,3-diol [(R)-7] and (R)(+)-3-Methyl-1-phenylbutane-1,3-[1-18O1]diol {(R)-[18O1]7}. nBuLi (2.2 mL, 5.5 mmol, 2.5 M in hexanes) was added to a stirred solution of chlorohydrin (S)-19 (783 mg, 5.0 mmol, ee 97%) in dry THF (7.0 mL) under an argon atmosphere at −78 °C. After 15 min, a dark blue mixture of the radical anion of 4,4′-di-tert-butylbiphenyl, prepared by stirring a mixture of 4,4′-di-tert-butylbiphenyl (DTBB) (3.996 g, 15.0 mmol) and lithium (0.11 g, 15.0 mmol, from new! bottle, granular, 4−10 mesh particle size, high sodium, 99%) in dry THF (17 mL) under an argon atmosphere at room temperature for 4 h was added over a period of 30 min. Stirring was continued for 4 h at −78 °C before acetone (871 mg, 15.0 mmol, 1.10 mL) was added. After 16 h at −78 °C, water (10 mL) was added followed by neutralization with concentrated HCl (pH paper) an hour later. The mixture was extracted with CH2Cl2 (3 × 15 mL), and the combined organic phases were washed with water (10 mL), dried (Na2SO4), and concentrated under reduced pressure. The crude product was purified by flash chromatography (3:1 hexanes/EtOAc,v/v; Rf = 0.18) to yield 1,3-diol (R)-7 (510 mg, 57%) as colorless crystals; 98% ee by HPLC, mp 69−70 °C (hexanes/CH2Cl2); [α]D20 +55.6 (c 1.1, acetone). 10316



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mg, 39%). (R,RP)-[17O,18O1]13: mp 95−98 °C (hexanes/CH2Cl2); [α]D28 −20.0 (c 1.27, acetone); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C16H21O518OPNa, 365.1010; found, 365.1000; [M + Na]+ calcd for C16H21O417O18OPNa, 366.1053; found, 366.1040; > 99% 18 O, 63.4% 17O. (R,SP)-[17O,18O1]13: mp 100−102 °C (hexanes/ CH2Cl2); [α]D20 −19.7 (c 1.15, acetone); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C16H21O518OPNa, 365.1010; found, 365.1000; [M + Na]+ calcd for C16H21O417O18OPNa, 366.1053; found 366.1041; 98.7% 18O, 64.5% 17O. The 1H and 13C NMR spectra of (R,RP)- and (R,SP)-[17O,18O1]13 were identical to those of the unlabeled species trans- and cis-(±)-13. The 31P NMR signals for the 17O-containing species were very much broadened (see spectra). Phosphoenol Pyruvate (PEP, 1a), (R P )- and (S P )[16O,17O,18O1]Phosphoenol Pyruvate {(RP)-[16O,17O,18O]PEP [(RP)-1b] and (SP)-[16O,17O,18O]PEP [(SP)-1b]}. A mixture of protected enol phosphate (R,RP)-13 (416 mg, 1.22 mmol), TMSBr (1.122 g, 7.33 mmol, 0.97 mL, 6.0 equiv), and allylTMS35 (419 mg, 3.67 mmol, 0.58 mL, 3.0 equiv) in dry 1,2-dichloroethane (5 mL) was stirred at room temperature under an argon atmosphere for 1.5 h. Then, it was concentrated under reduced pressure (0.5 mbar). The residue was again dissolved in dichloroethane, and the solution was concentrated. After the addition of aqueous KOH (10.3 mL, 0.4 M), the solution was immediately extracted with CH2Cl2 (2 × 3 mL). The CH2Cl2 dissolved in the aqueous layer was removed on the rotary evaporator. The aqueous phase was left at 0 °C for 5 h until the deprotection of the carboxylic ester was finished (monitored by 31P NMR). The mixture was brought to pH 7.4 with CO2 and lyophilized to yield PEP (1a) (1.06 mmol, 86%; the amount was determined by 31 P NMR with racemic 2-amino-1-hydroxyethylphosphonic acid as internal standard) as a potassium salt. 1 H NMR (400.27 MHz, D2O): δ 5.41 (1H), 5.24 (1H)..13C NMR (150.93 MHz): δ 171.9 (d, J = 8.7 Hz,), 149.8 (d, J = 7.4 Hz), 99.9 (d, J = 3.3 Hz). 31P NMR (162.03 MHz, D2O): δ −0.43. Similarly, labeled protected phosphoenol pyruvate (R,RP )[ 1 7 O, 1 8 O 1 ]13 (219 mg, 0.64 mmol) was converted to [(RP)-16O,17O,18O]PEP [(RP)-1b] (0.491 mmol, 77%; the amount was determined by 31P NMR with racemic 2-amino-1-hydroxyethylphosphonic acid as internal standard) as potassium salt. Similarly, labeled protected phosphoenol pyruvate (R,SP)-[17O,18O1]13 (265 mg, 0.77 mmol) was converted to (SP)-[16O,17O,18O]PEP [(SP)-1b] (0.57 mmol, 73%; the amount was determined by 31P NMR with racemic 2amino-1-hydroxyethylphosphonic acid as internal standard) as potassium salt. NMR spectra of the labeled compounds were identical to those of the unlabeled species except for the very much broadened signals in the 31P NMR spectra for the 17O-containing species. Enzymatic Transfer of Phosphate Group from P-Chiral PEP to ADP and from There to the C-6 Hydroxyl Group of DGlucose Followed by Cyclization to 4,6-Phosphate and Esterification to Cyclic Methyl Phosphates Following Literature Procedures.. 1 1 , 1 2 The potassium salt of (R P )[16O,17O,18O]PEP used for the test was prepared from protected phosphoenol pyruvate (R,RP)-[17O,18O1]13 (219 mg, 0.64 mmol). The potassium salt of (SP)-[16O,17O,18O]PEP used for the test was prepared from protected phosphoenol pyruvate (R,SP)-[17O,18O1]13 (303 mg, 0.886 mmol). In each case, a few mg of cyclic methyl phosphate was obtained and analyzed by 31P NMR spectroscopy.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Friedrich Hammerschmidt: 0000-0003-2193-1405 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Elena Micoratti for ee determinations, P. Unteregger for recording mass spectra, S. Felsinger and H.-P. Kählig for recording NMR spectra, and J. Theiner for combustion analyses.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b01783. Detailed analysis for conversion of (RP)- and (SP)[16O,17O,18O]PEP to methyl D-glucose-4,6-phosphates 22 and 23; chiral HPLC and chiral GC-MS for ee determination; 1H, 13C, and 31P NMR spectra of all new compounds; and ORTEP representation of compound trans-(±)-13 (PDF) Crystallographic data for trans-(±)-13 (CIF) 10317


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Chemical Synthesis of (RP)- and (SP)-[16O,17O ... - ACS Publications

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