New Routes to Lipophilic Amino Acids: Synthesis of Alkynyl and Fluoro-Containing Alanine Derivatives

Abstract

Branched α-amino acids incorporating an alkynyl group have been prepared by copper-catalysed reaction of a serine-derived organozinc reagent with bromoallenes. Substantial improvements to our previously reported Negishi cross-coupling of the serine-­derived organozinc reagent with cycloalkenyl triflates are possible using a combination of LiCl and SPhos as ligand. Finally, we report a preliminary example of addition of HF to cycloalkenyl alanine ­derivatives, leading to the corresponding tertiary fluoride.

α-Amino acids with lipophilic side chains are useful components of enzyme inhibitors. We have previously reported that enantiomerically pure cycloalkenyl alanine derivatives may be prepared either by copper-catalysed reaction of the serine-derived organozinc reagent 1 with cycloalk-1-en-3-yl phosphates 2, or by Negishi cross-coupling of the same organozinc reagent 1 with cycloalkenyl triflates 3. [¹] Subsequent hydrogenation gave the corresponding saturated derivatives. These cycloalkyl alanine derivatives continue to be of interest as components of enzyme inhibitors, and recent methods for their preparation rely on resolution by diastereoisomer separation, highlighting the need for stereoselective routes to these compounds. [²] In order to improve both the activity and the metabolic stability of pseudopeptides containing such highly lipophilic residues, efficient routes to new classes of α-amino acid incorporating either an alkynyl group, 4, or a tertiary fluoride 5 were desirable (Figure  [¹] ).

The proposed route to alkynes 4 involved disconnection at the branch point, and is illustrated in Scheme  [¹] . The two obvious synthetic equivalents for the propargylic cation 6 were the dicobalt hexacarbonyl cation 7 [³] and the allenic bromide 8. The latter was selected for initial investigation, since reactions of the zinc reagent 1 with allylic electrophiles generally proceed selectively in an S_N2′ manner. [4]

The allenic bromides 8a-c were prepared by the Landor method, [5] as applied by Eaton [6] and Backvall. [7] Initial optimisation of the copper(I)-catalysed reaction of the organozinc reagent 1, prepared from iodide 9 in DMF, with allenic bromide 8a established that the reaction was best conducted at 0 ˚C for an extended period, giving the desired alkyne 4a, along with small amounts of the allene 10a. Copper(I)-catalysed reaction of the organozinc reagent 1 with allenic bromides 8b and 8c was optimally carried out at -10 ˚C, giving satisfactory yields of the desired products 4b and 4c (Scheme  [²] , Table  [¹] ), along with the corresponding allenes 10b and 10c. The terminal alkyne in products 4 provides a site for further functionalisation, so a range of more complex compounds is potentially accessible.

It has recently been established that the yields in Negishi cross-coupling reactions can often be improved by the use of biarylphosphine ligands. [8] Although most reports have concentrated on the use of aryl halides as cross-coupling partners, [9] there are a number of reports in which the use of biarylphosphine ligands has also been shown to be effective in Negishi cross-coupling with vinyl halides and triflates. [¹0] In our previously published method for the Negishi coupling of cycloalkenyl triflates with organozinc reagent 1, [¹] we had employed (Ph₃P)₂PdCl₂ as the catalyst, obtaining yields in the range 41-46% (and 58% in a larger scale coupling with cycloheptenyl triflate). Given these modest yields, we have therefore investigated the use of the biarylphosphine ligand SPhos 11 in cross-coupling of organozinc reagent 1 with cycloalkenyl triflates. Application of the standard conditions that we had previously reported for the coupling of the organozinc reagent 1 with aryl iodides, namely Pd₂(dba)₃ (2.5 mol%) and SPhos 11 (5 mol%) at room temperature, [¹¹] but using cyclopentenyl triflate, [¹²] resulted in a significantly improved yield of the desired adduct 12a (70%). These conditions were also effective for coupling with cyclohexenyl triflate, but the yields for coupling with cycloheptenyl and cyclooctenyl triflate were lower (Scheme  [³] , Table  [²] ). It has been known for some time that addition of lithium halides improves yields in Stille cross-coupling of aryl triflates, [¹³] and also that the addition of LiCl can increase the reactivity of alkylzinc halides by the proposed formation of higher order organozincate intermediates. [¹4] [¹5] We were pleased to observe that addition of LiCl (1.8 equiv) to the cross-coupling of zinc reagent 1 with both cycloheptenyl and cyclooctenyl triflate resulted in a consistent increase in isolated yield of the desired products 12c and 12d. In contrast, the use of either tetrabutylammonium chloride or tetrabutylammonium bromide reduced the isolated yields substantially. It is not possible to be sure whether the increase in yield using LiCl is due to stabilisation of the catalyst, or to an increase in the reactivity of the alkylzinc iodide 1, or to a combination of both factors. Nevertheless, it is clear that the combined use of SPhos as ligand, and the addition of LiCl, has allowed a significant improvement in the efficiency of the formation of the cycloalkenyl alanine derivatives 12a-d (and therefore, the corresponding cycloalkyl alanine derivatives formed by subsequent hydrogenation [¹] ).

In earlier work, we have reported the synthesis of the ­methylated cyclopentylalanine derivative 13, [¹] for use as a component of an enzyme inhibitor. Subsequent work established that incorporation of the analogous fluorinated derivative 5 rendered the inhibitor less susceptible to ­metabolic degradation, but synthetic access to this compound was inefficient and required resolution. [¹6] It has been reported that simple tertiary fluorides can be prepared by addition of HF to cyclic alkenes using HF˙pyridine, [¹7] which suggested that a very simple route to enantiomerically pure 5 might be possible through HF addition to the alkene ent-12a; the compatibility of this reagent with protected amino acid functionality was an obvious concern. A preliminary trial was conducted on ent-12a, prepared in an analogous way to 12a, but using protected iodoalanine ent-9 derived from d-serine. In the event, after screening a variety of solvents, use of toluene gave the best results for HF addition. Rapid removal of the Boc group was observed, to give the HF salt of the deprotected amine. This salt was evidently sufficiently soluble to allow HF addition, although the reaction did not proceed to completion. Boc-reprotection allowed the isolation of the desired product 5 (30%, 44% based on recovered ent-12a), along with recovered starting material ent-12a (32%). Although the overall yield was not high, the route is short and simple. Hydrolysis of methyl ester 5 proceeded uneventfully to give the free acid 14 (93%), illustrating the stability of the tertiary fluoride to these basic conditions (Scheme  [4] ). While this process has yet to be optimised, it provides a very simple and direct route to the fluorocyclopentylalanine derivative 14.

In conclusion, we have reported simple methods [¹8] for the synthesis of a range of new α-amino acids with lipophilic side chains.

Acknowledgment

We thank Dr Salem Abuhanak for preliminary experiments.

References and Notes

• 1 Carrillo-Marquez T. Caggiano L. Jackson RFW. Grabowska U. Rae A. Tozer MJ. Org. Biomol. Chem.  2005,  3:  4117 
  Search in Google Scholar
• 2 Kerns JK. Nie H. Bondinell W. Widdowson KL. Yamashita DS. Rahman A. Podolin PL. Carpenter DC. Jin Q. Riflade B. Dong X. Nevins N. Keller PM. Mitchell L. Tomaszek T. Bioorg. Med. Chem. Lett.  2011,  21:  4409 
  Search in Google Scholar
• 3 Nicholas KM. Acc. Chem. Res.  1987,  20:  207 
  Search in Google Scholar
• 4 Dunn MJ. Jackson RFW. Pietruszka J. Turner D. J. Org. Chem.  1995,  60:  2210 
  Search in Google Scholar
• 5 Landor SR. Patel AN. Whiter PF. Greaves PM.
  J. Chem. Soc. C  1966,  1223 
• 6 Sigman MS. Eaton BE. J. Am. Chem. Soc.  1996,  118:  11783 
  Search in Google Scholar
• 7 Loefstedt J. Franzen J. Baeckvall J.-E. J. Org. Chem.  2001,  66:  8015 
  Search in Google Scholar
• 8 Milne JE. Buchwald SL. J. Am. Chem. Soc.  2004,  126:  13028 
  Search in Google Scholar
• 9a Manolikakes G. Dong ZB. Mayr H. Li JS. Knochel P. Chem. Eur. J.  2009,  15:  1324 
  Search in Google Scholar
• 9b Han C. Buchwald SL. J. Am. Chem. Soc.  2009,  131:  7532 
  Search in Google Scholar
• 10a Albrecht D. Bach T. Synlett  2007,  1557 
• 10b Manolikakes G. Hernandez CM. Schade MA. Metzger A. Knochel P. J. Org. Chem.  2008,  73:  8422 
  Search in Google Scholar
• 11 Ross AJ. Lang HL. Jackson RFW. J. Org. Chem.  2010,  75:  245 
  Search in Google Scholar
• 12 Stanislawski PC. Willis AC. Banwell MG. Org. Lett.  2006,  8:  2143 
  Search in Google Scholar
• 13 Echavarren AM. Stille JK. J. Am. Chem. Soc.  1987,  109:  5478 
  Search in Google Scholar
• 14a Achonduh GT. Hadei N. Valente C. Avola S. O’Brien CJ. Organ MG. Chem. Commun.  2010,  46:  4109 
  Search in Google Scholar
• 14b Hunter HN. Hadei N. Blagojevic V. Patschinski P. Achonduh GT. Avola S. Bohme DK. Organ MG. Chem. Eur. J.  2011,  17:  7845 
  Search in Google Scholar
• 15a Koszinowski K. Bohrer P. Organometallics  2009,  28:  100 
  Search in Google Scholar
• 15b Koszinowski K. Bohrer P. Organometallics  2009,  28:  771 
  Search in Google Scholar
• 16 Hardick D, Tozer M, Canfield J, Wilson M, Rae A, Fallon P, Classon B, Lindquist C, and Ayesa S. inventors; WO Patent  WO2006064286 (A1).  (Medivir UK Ltd. / UK; Peptimmune, Inc)
• 17 Grimes RM, Hartley CD, Mordaunt JE, Shah P, Slater MJ, and White GV. inventors; WO Patent  2008059042 (A1).  (Smithkline Beecham Corporation, USA)

General Method for the Preparation of Alkynes 4:
The organozinc reagent 1 was prepared from protected iodoalanine 9 (329 mg, 1 mmol) as a ca. 1 M solution in DMF according to our previously reported method.^¹¹ CuBr˙DMS (20.6 mg, 0.1 mmol) was gently heated under vacuum in a dry flask until the powder turned light green, and was then allowed to cool under nitrogen. DMF (0.6 mL) and the bromoallene 8 (1.1 mmol) were added. The reaction mixture was cooled to -10 ˚C. After 2 min stirring at -10 ˚C, a solution of the organozinc reagent 1 (1 mL of a 1 M solution in DMF, 1 mmol) was added dropwise via syringe over 10 min. The reaction mixture was stirred for the time indicated at either -10 ˚C or 0 ˚C. The reaction mixture was applied directly to a silica gel column, which was eluted (5% Et₂O in toluene) to give the alkynes 4 ^¹9-²¹ and the allenes 10 as light yellow oils.
General Method for the Preparation of Alkenes 12: The cycloalken-1-yl triflate 3 (1.3 mmol, 1.3 equiv), Pd₂(dba)₃ (22 mg, 2.5 mol%), SPhos (21 mg, 5 mol%) and LiCl (1.8 mmol, 1.8 equiv), if required, were added at r.t. to the organozinc reagent 1 (1 mL of a ca. 1 M solution in DMF). The reaction mixture was stirred at r.t. overnight under a positive pressure of nitrogen, and then applied directly to a silica gel column, which was eluted (10% EtOAc in petroleum ether) to give the cross-coupled product 12.
( R )-2-Amino-3-(1-fluorocyclopentyl)propionic Acid Methyl Ester (5): Alkene ent-12a (492 mg, 1.83 mmol) was dissolved in toluene (4 mL) in a Teflon bottle, cooled to 0 ˚C and stirred vigorously. HF-pyridine (70% HF, 3.2 mL, CAUTION: very toxic) was added and the bottle was sealed. After 2 h, the reaction mixture was carefully transferred into a slurry of CaCO₃ (12.8 g) in H₂O (50 mL) and CH₂Cl₂ (35 mL) cooled to 0 ˚C. The mixture was stirred while the pH was adjusted to ca. 10 by addition of sat. aq Na₂CO₃, followed by stirring for a further 30 min. Celite (6.4 g) was washed with Na₂CO₃ solution, H₂O, EtOH, EtOAc and CH₂Cl₂ in sequence and added to the slurry. The suspension was filtered and the filter cake was washed with CH₂Cl₂ (50 mL in portions) and H₂O (25 mL). The phases were separated and the aqueous layer was extracted with CH₂Cl₂ (2 × 20 mL). The combined organic phases were washed with sat. aq NaHCO₃ (20 mL) and evaporated. The crude mixture of amino esters (0.253 g) was dissolved in 1,4-dioxane (4 mL), aq sat. NaHCO₃ (6 mL) was added and the mixture was cooled to 0 ˚C with stirring. Boc₂O (0.323 g, 1.48 mmol) in 1,4-dioxane (4 mL) was added to the reaction mixture. The reaction mixture was allowed to reach r.t. and stirred for 1 h. The reaction mixture was diluted with Et₂O (10 mL) and H₂O (10 mL) and the layers were separated. The aqueous phase was extracted with Et₂O (2 × 10 mL). The combined organic phases were washed with H₂O and brine (10 mL each) and evaporated. The product was purified by gradient column chromatography [Column 40 mm i.d. × 82 mm, loaded with 50 g Biotage KP-Sil (silica gel) 2-17% EtOAc in iso-hexane] to give 5 ^²² (158 mg, 30% over two steps) and recovered ent-12a (160 mg, 32%).

Characterisation data for alkyne 4a: [α]_D ^²¹ +9.8 (c = 1.0, CHCl₃). ^¹H NMR (500 MHz, CDCl₃): δ = 1.26 (s, 3 H), 1.27 (s, 3 H), 1.42 (s, 9 H), 1.74 (dd, J = 9.1, 14.0 Hz, 1 H), 1.90 (dd, J = 4.3, 14.2 Hz, 1 H), 2.15 (s, 1 H), 3.71 (s, 3 H), 4.43-4.48 (m, 1 H), 5.17 (d, J = 7.7 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 28.4, 28.9, 29.8, 29.8, 44.3, 51.8, 52.2, 69.2, 79.9, 90.5, 155.2, 173.5. IR (ATR): 3293, 2973, 1744, 1705, 1508, 1436, 1391, 1366, 1279, 1246, 1209, 1160, 1048, 1028, 868, 779, 629 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₄H₂₄NO₄: 270.1705; found: 270.1716.

Characterisation data for alkyne 4b: [α]_D ^²¹ +2.9 (c = 10.2, CHCl₃). ^¹H NMR (500 MHz, CDCl₃): δ = 1.42 (s, 9 H), 1.45-1.54 (m, 2 H), 1.55-1.72 (m, 2 H), 1.73-1.89 (m, 3 H), 1.90-2.00 (m, 2 H), 2.04 (dd, J = 4.3, 14.2 Hz, 1 H), 2.18 (s, 1 H), 3.71 (s, 3 H), 4.43-4.56 (m, 1 H), 5.18 (d, J = 7.7 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 23.4, 23.8, 28.4, 40.2, 40.5, 41.1, 41.9, 52.2, 52.5, 70.1, 79.8, 89.8, 155.2, 173.5. IR (ATR): 3294, 2954, 2874, 1709, 1507, 1437, 1391, 1366, 1248, 1214, 1162, 1049, 1019, 868, 778, 630 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₆H₂₆NO₄: 296.1862; found: 296.1864.

Characterisation data for alkyne 4c: mp 56-57 ˚C; [α]_D ^²¹ +2.0 (c = 7.0, CHCl₃). ^¹H NMR (500 MHz, CDCl₃): δ = 1.04-1.27 (m, 3 H), 1.42 (s, 9 H), 1.56-1.70 (m, 5 H), 1.70-1.85 (m, 3 H), 1.89 (dd, J = 4.1, 14.1 Hz, 1 H), 2.33 (s, 1 H), 3.70 (s, 3 H), 4.45-4.51 (m, 1 H), 5.21 (d, J = 7.6 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 22.5, 22.6, 25.8, 28.4, 35.2, 37.0, 38.0, 44.3, 51.2, 52.2, 71.9, 79.8, 88.5, 155.2, 173.7. IR (ATR): 3306, 2930, 2857, 2360, 1744, 1712, 1506, 1449, 1391, 1366, 1252, 1206, 1165, 1048, 1023, 866, 776, 632 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₇H₂₈NO₄: 310.2018; found: 310.2017.

Characterisation data for 5: mp 47-51 ˚C; [α]_D ^²¹ -5.2 (c = 1.0, CHCl₃). ^¹H NMR (400 MHz, CDCl₃): δ = 1.43 (s, 9 H), 1.50-1.70 (m, 4 H), 1.73-1.85 (m, 2 H), 1.93-2.07 (m, 2 H), 2.10-2.32 (m, 2 H), 3.72 (s, 3 H), 4.40-4.50 (m, 1 H), 5.21 (br d, J = 7.0 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 23.5, 23.6, 28.3, 37.65 (d, J = 13 Hz), 37.8 (d, J = 13 Hz), 39.9 (d, J = 23 Hz), 51.3, 52.3, 79.9, 105.9 (d, J = 172 Hz), 155.2, 173.1. ^¹9F NMR (376 MHz, CDCl₃): δ = -142.7.
IR (ATR): 3364, 2974, 1750, 1718, 1508, 1367, 1167 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₄H₂₅NO₄F: 290.1768 (base peak is 579 [M₂H⁺]); found: 290.1769.

References and Notes

• 1 Carrillo-Marquez T. Caggiano L. Jackson RFW. Grabowska U. Rae A. Tozer MJ. Org. Biomol. Chem.  2005,  3:  4117 
  Search in Google Scholar
• 2 Kerns JK. Nie H. Bondinell W. Widdowson KL. Yamashita DS. Rahman A. Podolin PL. Carpenter DC. Jin Q. Riflade B. Dong X. Nevins N. Keller PM. Mitchell L. Tomaszek T. Bioorg. Med. Chem. Lett.  2011,  21:  4409 
  Search in Google Scholar
• 3 Nicholas KM. Acc. Chem. Res.  1987,  20:  207 
  Search in Google Scholar
• 4 Dunn MJ. Jackson RFW. Pietruszka J. Turner D. J. Org. Chem.  1995,  60:  2210 
  Search in Google Scholar
• 5 Landor SR. Patel AN. Whiter PF. Greaves PM.
  J. Chem. Soc. C  1966,  1223 
• 6 Sigman MS. Eaton BE. J. Am. Chem. Soc.  1996,  118:  11783 
  Search in Google Scholar
• 7 Loefstedt J. Franzen J. Baeckvall J.-E. J. Org. Chem.  2001,  66:  8015 
  Search in Google Scholar
• 8 Milne JE. Buchwald SL. J. Am. Chem. Soc.  2004,  126:  13028 
  Search in Google Scholar
• 9a Manolikakes G. Dong ZB. Mayr H. Li JS. Knochel P. Chem. Eur. J.  2009,  15:  1324 
  Search in Google Scholar
• 9b Han C. Buchwald SL. J. Am. Chem. Soc.  2009,  131:  7532 
  Search in Google Scholar
• 10a Albrecht D. Bach T. Synlett  2007,  1557 
• 10b Manolikakes G. Hernandez CM. Schade MA. Metzger A. Knochel P. J. Org. Chem.  2008,  73:  8422 
  Search in Google Scholar
• 11 Ross AJ. Lang HL. Jackson RFW. J. Org. Chem.  2010,  75:  245 
  Search in Google Scholar
• 12 Stanislawski PC. Willis AC. Banwell MG. Org. Lett.  2006,  8:  2143 
  Search in Google Scholar
• 13 Echavarren AM. Stille JK. J. Am. Chem. Soc.  1987,  109:  5478 
  Search in Google Scholar
• 14a Achonduh GT. Hadei N. Valente C. Avola S. O’Brien CJ. Organ MG. Chem. Commun.  2010,  46:  4109 
  Search in Google Scholar
• 14b Hunter HN. Hadei N. Blagojevic V. Patschinski P. Achonduh GT. Avola S. Bohme DK. Organ MG. Chem. Eur. J.  2011,  17:  7845 
  Search in Google Scholar
• 15a Koszinowski K. Bohrer P. Organometallics  2009,  28:  100 
  Search in Google Scholar
• 15b Koszinowski K. Bohrer P. Organometallics  2009,  28:  771 
  Search in Google Scholar
• 16 Hardick D, Tozer M, Canfield J, Wilson M, Rae A, Fallon P, Classon B, Lindquist C, and Ayesa S. inventors; WO Patent  WO2006064286 (A1).  (Medivir UK Ltd. / UK; Peptimmune, Inc)
• 17 Grimes RM, Hartley CD, Mordaunt JE, Shah P, Slater MJ, and White GV. inventors; WO Patent  2008059042 (A1).  (Smithkline Beecham Corporation, USA)

General Method for the Preparation of Alkynes 4:
The organozinc reagent 1 was prepared from protected iodoalanine 9 (329 mg, 1 mmol) as a ca. 1 M solution in DMF according to our previously reported method.^¹¹ CuBr˙DMS (20.6 mg, 0.1 mmol) was gently heated under vacuum in a dry flask until the powder turned light green, and was then allowed to cool under nitrogen. DMF (0.6 mL) and the bromoallene 8 (1.1 mmol) were added. The reaction mixture was cooled to -10 ˚C. After 2 min stirring at -10 ˚C, a solution of the organozinc reagent 1 (1 mL of a 1 M solution in DMF, 1 mmol) was added dropwise via syringe over 10 min. The reaction mixture was stirred for the time indicated at either -10 ˚C or 0 ˚C. The reaction mixture was applied directly to a silica gel column, which was eluted (5% Et₂O in toluene) to give the alkynes 4 ^¹9-²¹ and the allenes 10 as light yellow oils.
General Method for the Preparation of Alkenes 12: The cycloalken-1-yl triflate 3 (1.3 mmol, 1.3 equiv), Pd₂(dba)₃ (22 mg, 2.5 mol%), SPhos (21 mg, 5 mol%) and LiCl (1.8 mmol, 1.8 equiv), if required, were added at r.t. to the organozinc reagent 1 (1 mL of a ca. 1 M solution in DMF). The reaction mixture was stirred at r.t. overnight under a positive pressure of nitrogen, and then applied directly to a silica gel column, which was eluted (10% EtOAc in petroleum ether) to give the cross-coupled product 12.
( R )-2-Amino-3-(1-fluorocyclopentyl)propionic Acid Methyl Ester (5): Alkene ent-12a (492 mg, 1.83 mmol) was dissolved in toluene (4 mL) in a Teflon bottle, cooled to 0 ˚C and stirred vigorously. HF-pyridine (70% HF, 3.2 mL, CAUTION: very toxic) was added and the bottle was sealed. After 2 h, the reaction mixture was carefully transferred into a slurry of CaCO₃ (12.8 g) in H₂O (50 mL) and CH₂Cl₂ (35 mL) cooled to 0 ˚C. The mixture was stirred while the pH was adjusted to ca. 10 by addition of sat. aq Na₂CO₃, followed by stirring for a further 30 min. Celite (6.4 g) was washed with Na₂CO₃ solution, H₂O, EtOH, EtOAc and CH₂Cl₂ in sequence and added to the slurry. The suspension was filtered and the filter cake was washed with CH₂Cl₂ (50 mL in portions) and H₂O (25 mL). The phases were separated and the aqueous layer was extracted with CH₂Cl₂ (2 × 20 mL). The combined organic phases were washed with sat. aq NaHCO₃ (20 mL) and evaporated. The crude mixture of amino esters (0.253 g) was dissolved in 1,4-dioxane (4 mL), aq sat. NaHCO₃ (6 mL) was added and the mixture was cooled to 0 ˚C with stirring. Boc₂O (0.323 g, 1.48 mmol) in 1,4-dioxane (4 mL) was added to the reaction mixture. The reaction mixture was allowed to reach r.t. and stirred for 1 h. The reaction mixture was diluted with Et₂O (10 mL) and H₂O (10 mL) and the layers were separated. The aqueous phase was extracted with Et₂O (2 × 10 mL). The combined organic phases were washed with H₂O and brine (10 mL each) and evaporated. The product was purified by gradient column chromatography [Column 40 mm i.d. × 82 mm, loaded with 50 g Biotage KP-Sil (silica gel) 2-17% EtOAc in iso-hexane] to give 5 ^²² (158 mg, 30% over two steps) and recovered ent-12a (160 mg, 32%).

Characterisation data for alkyne 4a: [α]_D ^²¹ +9.8 (c = 1.0, CHCl₃). ^¹H NMR (500 MHz, CDCl₃): δ = 1.26 (s, 3 H), 1.27 (s, 3 H), 1.42 (s, 9 H), 1.74 (dd, J = 9.1, 14.0 Hz, 1 H), 1.90 (dd, J = 4.3, 14.2 Hz, 1 H), 2.15 (s, 1 H), 3.71 (s, 3 H), 4.43-4.48 (m, 1 H), 5.17 (d, J = 7.7 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 28.4, 28.9, 29.8, 29.8, 44.3, 51.8, 52.2, 69.2, 79.9, 90.5, 155.2, 173.5. IR (ATR): 3293, 2973, 1744, 1705, 1508, 1436, 1391, 1366, 1279, 1246, 1209, 1160, 1048, 1028, 868, 779, 629 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₄H₂₄NO₄: 270.1705; found: 270.1716.

Characterisation data for alkyne 4b: [α]_D ^²¹ +2.9 (c = 10.2, CHCl₃). ^¹H NMR (500 MHz, CDCl₃): δ = 1.42 (s, 9 H), 1.45-1.54 (m, 2 H), 1.55-1.72 (m, 2 H), 1.73-1.89 (m, 3 H), 1.90-2.00 (m, 2 H), 2.04 (dd, J = 4.3, 14.2 Hz, 1 H), 2.18 (s, 1 H), 3.71 (s, 3 H), 4.43-4.56 (m, 1 H), 5.18 (d, J = 7.7 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 23.4, 23.8, 28.4, 40.2, 40.5, 41.1, 41.9, 52.2, 52.5, 70.1, 79.8, 89.8, 155.2, 173.5. IR (ATR): 3294, 2954, 2874, 1709, 1507, 1437, 1391, 1366, 1248, 1214, 1162, 1049, 1019, 868, 778, 630 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₆H₂₆NO₄: 296.1862; found: 296.1864.

Characterisation data for alkyne 4c: mp 56-57 ˚C; [α]_D ^²¹ +2.0 (c = 7.0, CHCl₃). ^¹H NMR (500 MHz, CDCl₃): δ = 1.04-1.27 (m, 3 H), 1.42 (s, 9 H), 1.56-1.70 (m, 5 H), 1.70-1.85 (m, 3 H), 1.89 (dd, J = 4.1, 14.1 Hz, 1 H), 2.33 (s, 1 H), 3.70 (s, 3 H), 4.45-4.51 (m, 1 H), 5.21 (d, J = 7.6 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 22.5, 22.6, 25.8, 28.4, 35.2, 37.0, 38.0, 44.3, 51.2, 52.2, 71.9, 79.8, 88.5, 155.2, 173.7. IR (ATR): 3306, 2930, 2857, 2360, 1744, 1712, 1506, 1449, 1391, 1366, 1252, 1206, 1165, 1048, 1023, 866, 776, 632 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₇H₂₈NO₄: 310.2018; found: 310.2017.

Characterisation data for 5: mp 47-51 ˚C; [α]_D ^²¹ -5.2 (c = 1.0, CHCl₃). ^¹H NMR (400 MHz, CDCl₃): δ = 1.43 (s, 9 H), 1.50-1.70 (m, 4 H), 1.73-1.85 (m, 2 H), 1.93-2.07 (m, 2 H), 2.10-2.32 (m, 2 H), 3.72 (s, 3 H), 4.40-4.50 (m, 1 H), 5.21 (br d, J = 7.0 Hz, 1 H). ^¹³C NMR (125 MHz, CDCl₃): δ = 23.5, 23.6, 28.3, 37.65 (d, J = 13 Hz), 37.8 (d, J = 13 Hz), 39.9 (d, J = 23 Hz), 51.3, 52.3, 79.9, 105.9 (d, J = 172 Hz), 155.2, 173.1. ^¹9F NMR (376 MHz, CDCl₃): δ = -142.7.
IR (ATR): 3364, 2974, 1750, 1718, 1508, 1367, 1167 cm^-¹. HRMS (ES): m/z [M + H⁺] calcd for C₁₄H₂₅NO₄F: 290.1768 (base peak is 579 [M₂H⁺]); found: 290.1769.