Introduction

The reagent 4-(N,N-dimethylamino)pyridine (DMAP) has become the most popular catalyst for various processes, such as acylation, alkylation, silylation, the Baylis-Hillman reaction and nucleophilic substitution of alcohols and amines¹. DMAP also affords the advantage of working with mild reaction conditions to avoid, for example, racemization problems, and it enhances the rate of reaction by several orders compared with the process in the absence of catalyst; moreover, a catalytic amount of catalyst is usually required and can be recovered at the end of the process. The development of chiral DMAP derivatives has received considerable attention, as they have been successfully used like chiral nucleophilic catalysts in a wide range of asymmetric synthetic processes².

Biocatalysis is a powerful tool for modifying a wide range of substrates in very mild reaction conditions. Enzymes, in particular hydrolases, are widely used in organic synthesis as environmentally 'friendly' catalysts with broad substrate specificity, show high stereoselctivity, are commercially available and do not require the use of cofactors³. Of the hydrolase group, lipases of the group EC 3.1.1.3 are the biocatalysts most often used in organic synthesis because of their broad application in chemoselective, 'regioselective' and stereoselective processes⁴. The interest in lipases in organic chemistry increased substantially in the past decade because of their importance in the production of pharmaceuticals, agrochemicals and fine chemicals⁵. In that way, the kinetic resolution of racemic compounds⁶ and the desymmetrization of meso or prochiral compounds has achieved particular relevance in the past two decades⁷.

The resolution of pyridyl-ethanols was first achieved by the enzymatic hydrolysis or esterification of the corresponding substrates using the lipase SAM II from Pseudomonas species⁸. The use of other acylating agents, the exploration of the complementary enzymatic hydrolysis reaction and the use of other biocatalysts have allowed optimization of the resolution of those compounds and other bipyridylethanols for their application in the synthesis of optically pure oligopyridines^{9, 10, 11, 12, 13, 14}.

Here we report a detailed protocol for the lipase-mediated kinetic resolution of various 4-chloro-2-(1-hydroxyalkyl)pyridines 1a–1d, as those compounds are useful intermediates for the production of chemical catalysts derived from DMAP. Those catalysts can be used in a wide range of asymmetric processes. In addition, kinetic resolution permits researchers to obtain derivatives of the opposite stereochemistry, which allows the possibility of stereoselective complementary processes¹⁵ (Fig. 1).

Figure 1: Enzymatic kinetic resolution of 4-chloro-2-(1-hydroxyalkyl)pyridines (1a–1d).

THF, tetrahydrofuran.

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We did an exhaustive study to optimize the reaction conditions for the stereoselective enzymatic transesterification of racemic alcohols 1a–1d, finding Pseudomonas cepacia lipase as the optimal biocatalyst; the results obtained using this enzyme are summarized in Table 1. Reactions were slower when the alkyl group of the 4-chloro-2-(1-hydroxyalkyl)pyridine derivative was bulkier. A process is considered enantioselective when enantioselectivity reaches values over 200; in all the cases here, enantioselectivity values above 200 were achieved, demonstrating that biocatalysis represents a very effective tool for selectively modifying one of the enantiomers of the racemic pyridine derivatives.

Table 1: Kinetic resolution of alcohols 1a–1d.

Full table

Materials

Reagents

• 4-Chloro-2-(1-hydroxyalkyl)pyridine
• Immobilized P. cepacia lipase (Amano Pharmaceutical, lot no. ILPSAX10525K (1091 U/g))
• Nitrogen gas
• Tetrahydrofuran (Prolabo, cat. no. 28551.321)
• Vinyl acetate (Sigma-Aldrich, cat. no. V1503)
• 2-Propanol for HPLC analysis (Romil, cat. no. H625)
• n-Hexane for HPLC analysis (Romil, cat H389)
• Dichloromethane (CH₂Cl₂; Scharlau, cat. no. CL0332025B).
  Caution Wear nitrile gloves.
• Silica gel (Merck, cat. no. 1.09385.2500)
  Caution Wear latex gloves.
• Ethyl acetate (Merck, cat. no. 1.09623.6025)
• n-Hexane for 'flash' chromatography separation (Merck, cat. no. 1.04374.60259)
• Silica-gel 60 F₂₅₄ aluminum-backed thin-layer chromatography (TLC) plates
• Potassium permanganate solution (KMnO₄; Aldrich, cat. no. 20,798-5; see REAGENT SETUP
• Sodium hydroxide pellets (NaOH; Prolabo, cat. no. 28245.298; see REAGENT SETUP
  Caution Wear latex gloves.
• Potassium carbonate (K₂CO₃; Prolabo, cat. no. 26726.297; see REAGENT SETUP

Equipment

• Erlenmeyer flasks, 100 ml and 1 liter
• Glass plate for weighing
• Rubber septum
• Vacuum line and pump (Edwards)
• Plastic adaptor
• Short disposable syringe needle
• Balloon
• Glass syringe, 50 ml (Fortuna Optima)
• Large syringe needle (18-gauge; Aldrich)
• Glass microliter syringes: 1 ml without stop (Hamilton, cat. no. 81320/01) and 100 l with stop (Hamilton, cat no. 81000/00)
• Orbital shaker (Certomat BS-1; Braun Biotech)
• Nylon HPLC filters (Scharlau, cat. no. NY13045200)
• HPLC glass vial
• HPLC chromatograph with UV detector (Agilent 1100 series)
• HPLC columns (normal phase), Daicel Chiralcel OD or OB-H (25 cm in length 0.46 cm in internal diameter), set up with the mobile phases and conditions in Table 2

  Table 2: Analytical separation by HPLC of alcohols 1a–d and esters 3a–d.

  

  
  Full table
  
  
• Buchner funnel with a filter paper circle
• Filter flask, 250 ml
• Round-bottomed flask, 100 ml
• Rotary evaporator equipped with a cooling condenser (Heidolph)
• Distillation apparatus for dry solvents
• Chromatography column (30 cm in length 3 cm in internal diameter) with a 250-ml reservoir
• Polypropylene powder funnel
• TLC chamber
• Heat gun
• Dual-bank manifold to produce vacuum and nitrogen atmosphere (see REAGENT SETUP

Reagent setup

• 4-Chloro-2-(1-hydroxyalkyl)pyridines These compounds can be easily obtained using published methods⁹, which are available from the authors on request. As an example, for 4-chloro-2-cyanopyridine, begin with a solution of 4-chloropyridine N-oxide (5.00 g; 38.6 mmol) in dry CH₂Cl₂ (70 ml) and successively add trimethylsilyl cyanide (4.92 ml; 39.3 mmol) and N,N-dimethylcarbamyl chloride in small portions. Stir the resulting solution for 9 d at 25 °C (room temperature), then evaporate the solvent at reduced pressure. Purify the crude product by 'flash' chromatography (20% (vol/vol) ethyl acetate in n-hexane), yielding 4.54 g of a white solid (85%). As another example, for ()-4-chloro-2-(1-hydroxyalkyl)pyridine, begin with a 3.0-M solution of an alkylmagnesium halogen in diethyl ether (9.64 ml; 28.94 mmol) at 0 °C and, under a nitrogen atmosphere, add a solution of 4-chloro-2-cyanopyridine (1.00 g, 7.24 mmol) in dry diethyl ether (25 ml). After completing the addition, stir the mixture for 4 h at room temperature, then pour the solution into a saturated ammonium chloride solution at 0 °C (23.9 ml) and add concentrated HCl until the pH reaches 1. Stir the resulting mixture at room temperature for an additional 14 h, then neutralize the solution with NH₃ (aqueous) and extract with diethyl ether (three times with 15 ml each). Evaporate the solvent by distillation under reduced pressure to yield the corresponding ketone, which is used in the next step without further purification. Dissolve the crude product in methanol (60 ml) and cool to 0 °C, then add NaBH₄ (1.36 g; 36.10 mmol) in small portions. Stir the clear solution for 2 h at room temperature, then evaporate the methanol and dissolve the resulting white solid in water and extract with CH₂Cl₂ (three times with 10 ml each). Combine the organic phases, dry over Na₂SO₄ and evaporate at reduced pressure. Purify the crude product of the reaction by 'flash' chromatography.
• Potassium permanganate Prepare the potassium permanganate solution carefully with the following steps, wearing latex gloves: (i) Add 10 g K₂CO₃ to a 1-liter Erlenmeyer flask and dissolve in 200 ml distilled water. (ii) While stirring, add NaOH (1.5 g) and KMnO₄ (2 g) successively and stir this mixture for an additional 15 min.

Procedure

1. Points from here (point 1) up to and including point 9 are related to Set up the enzymatic reactionTiming: 20 min
   Caution Wear latex gloves for Steps 1–8 to avoid contaminating the reagents and to prevent skin injury.Add 3.12 mmol of a 4-chloro-2-(1-hydroxyalkyl)pyridine to a 100-ml Erlenmeyer flask.
2. Weigh 500 mg of P. cepacia lipase in a glass plate and transfer it to the 100-ml Erlenmeyer flask.
3. Seal the flask with a rubber septum.
4. Evacuate the Erlenmeyer flask by connecting it to a vacuum line for 3 min and then gently flush the flask with nitrogen gas for 5 s. Use a plastic adaptor fitted with a short disposable syringe needle for this step. Repeat this procedure three times.
   Critical step Lipases are moisture-sensitive reagents. Ensuring a total nitrogen atmosphere is very important to avoid possible hydrolysis processes, which could decrease the conversion or enantiomeric-excess values.Troubleshooting
5. Insert through the rubber septum a balloon filled with nitrogen gas connected to a plastic adaptor fitted with a short disposable syringe needle to avoid pressure that is too high (Fig. 2).

   Figure 2: Vacuum line and Erlenmeyer flask with a balloon filled with nitrogen and fitted with a short disposable syringe needle.

   

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6. Add 31 ml of dry tetrahydrofuran using a 50-ml glass syringe fitted with a 18-gauge large needle.
7. Add 859 l (9.38 mmol) vinyl acetate using a 1-ml glass syringe fitted with a short disposable syringe needle.
   Critical step Accurate addition of the required amount of vinyl acetate is extremely important for reproduction of the data in Table 1. Varying this amount could lead to loss of the enantioselectivity of the process.
8. Remove the balloon system from the rubber septum.
9. Wrap the septum with a strip of Parafilm to avoid moisture entry.
10. Points from here (point 10) up to and including point 11 are related to Development of the enzymatic reactionTiming: Depends on selected substrate; check Table 1Place the flask in an orbital shaker at 30 °C and shake the reaction mixture at 250 r.p.m. for the time required for the selected substrate. The reaction mixture must be left shaking overnight at 30 °C.
11. Monitor the progress of the reaction by analytical HPLC by the following steps: (i) Obtain a 10-l sample of the reaction mixture solution under nitrogen using a 100-l microliter syringe without removing the rubber septum. (ii) Dispense the contents of the syringe into a microcentrifuge tube and evaporate the remaining tetrahydrofuran using nitrogen flow. (iii) Dissolve the mixture in 250 l of a solution of 10% (vol/vol) 2-propanol in n-hexane. (iv) Filter the solution through a nylon HPLC filter and collect the resulting solution in an HPLC glass vial. (v) Inject 20 l of the sample into the HPLC system setup using the solvents and conditions in Table 2 and analyze the corresponding results.

    For the enzymatic kinetic resolution of these and other substrates using P. cepacia lipase or other lipases, reactions are considered finished when one of the enantiomers of the alcohol is almost invisible. The reaction times are dependent of the alcohol structure.

12. Points from here (point 12) up to and including point 14 are related to Stopping the enzymatic reactionTiming: 1 h 15 minStop the enzymatic reaction when one of the enantiomers of the alcohol disappears in the HPLC analysis. To do this, remove the rubber septum and 'filter off' the enzyme using a Buchner funnel with a filter paper circle, a 250-ml filter flask and a vacuum pump.
13. Wash the enzyme with dichloromethane (three times with 10 ml each) to obtain a combined filtrate and transfer this to a 100-ml round-bottomed flask.
14. Remove the solvent under reduced pressure in a rotary evaporator at 25 °C (this could take around 5 min) and dry the residual oil using a vacuum line.Pause Point The crude mixture can be left overnight at 4 °C.
15. Points from here (point 15) up to and including point 23 are related to General procedure for separating and purifying the final productsTiming: 2 hDissolve the resulting crude material in 15 ml dichloromethane, add 2 g silica gel and evaporate the solvent by distillation under reduced pressure using a rotary evaporator and further using a vacuum pump.
16. Pack a chromatography column (30 cm in length 3 cm in internal diameter) with 20 g silica gel using 150 ml of a solution of 20% (vol/vol) ethyl acetate in hexane for reactions with compound 1a or 15% (vol/vol) ethyl acetate in hexane for reactions with compounds 1b–1d.
    Critical step Exact measurement of the proportion of the organic solvent is advisable for perfect separation of compounds 1 and 3 in the 'flash' chromatography separation.
17. Load the crude mixture adsorbed on silica gel on the top of the silica bed using a polypropylene powder funnel.
    Critical step A thin and compact column front is necessary for good separation of the reaction products.
18. Elute the compounds by 'flash' chromatography with 300 ml of the solution of 20% (vol/vol) ethyl acetate in hexane, collecting 20-ml fractions in test tubes.
19. Analyze the collected fractions by TLC analysis using a solution of 20% (vol/vol) ethyl acetate in hexane with the following steps: (i) Obtain a small fraction from a full test tube using a glass Pasteur pipette. (ii) Spot a small amount of the sample on a silica-gel 60 F₂₅₄ aluminum-backed plate. (iii) Develop the plate using a mixture of 60% (vol/vol) ethyl acetate in hexane as the eluent. (iv) Visualize spots first under UV light and then by dipping the plate into the potassium permanganate solution and warming the plate with a heat gun.
20. Collect the tubes containing the fractions with esters (R)-3a–3d in a 100-ml round-bottomed flask and evaporate the liquid solution in a rotary evaporator and then in a vacuum pump.
21. Once esters (R)-3a–3d have been eluted, increase the column polarity, adding 500 ml of a solution of 60% (vol/vol) ethyl acetate in hexane to elute the alcohols (S)-1a–1d.
22. Analyze the collected fractions by TLC analysis (60% (vol/vol) ethyl acetate in hexane).
23. Collect the tubes containing the fractions with alcohols (S)-1a–1d in a 100-ml round-bottomed flask and evaporate the liquid solution in a rotary evaporator and then in a vacuum pump.

Timing

Steps 1–3: 2 min
Step 4: 15 min
Steps 5–6: 1 min
Steps 7–8: 2 min
Steps 9–10: 1 min
Step 11: 14 h for 1a and 1b, 38 h for 1c and 60 h for 1d
Step 12: 1 h
Step 13: 5 min
Step 14: 10 min
Step 15: 10 min
Step 16: 10 min
Steps 17–18: 25 min
Step 19: 15 min
Step 20: 15 min
Steps 21–22: 30 min
Step 23: 15 min

Troubleshooting

In Step 4, loss of the nitrogen atmosphere may cause hydrolysis problems during the transesterification step, so before adding the solvent, be sure that you have a good nitrogen atmosphere. Do not proceed further if you have any doubt at this point, and repeat Step 4 completely.

Anticipated results