Enzymatic Regioselective Levulinylation of 2′‐Deoxyribonucleosides and 2′‐O‐Methylribonucleosides

Iván Lavandera1, Javier García1, Susana Fernández1, Miguel Ferrero1, Vicente Gotor1, Yogesh S. Sanghvi2

1 Universidad de Oviedo, Oviedo, 2 Rasayan, Inc., Encinitas, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.11
DOI:  10.1002/0471142700.nc0211s21
Online Posting Date:  July, 2005
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Abstract

The levulinyl‐protected nucleosides are key building blocks for the solution‐phase synthesis of oligonucleotides. Short and efficient syntheses of 3′‐ and 5′‐O‐levulinylated 2′‐deoxyribonucleosides and 2′‐O‐methylribonucleosides have been developed from the corresponding nucleosides by enzyme‐catalyzed regioselective acylation in organic solvents or from 3′,5′‐di‐O‐levulinyl derivatives by regioselective enzymatic hydrolysis. Lipase‐mediated levulinylation of various nucleosides has been accomplished with acetonoxime levulinate as an acyl donor. Use of immobilized Pseudomonas cepacia lipase (PSL‐C) has furnished 3′‐O‐levulinylated 2′‐deoxyribonucleosides in excellent yields. Similarly, Candida antarctica lipase B (CAL‐B) has provided 5′‐O‐levulinylated nucleosides in high yields. 3′‐O‐Levulinylated 2′‐deoxyribonucleosides and 2′‐O‐methylribonucleosides were prepared via selective hydrolysis of 3′,5′‐di‐O‐levulinate esters using CAL‐B.

Keywords: lipases; enzymatic synthesis; enzymatic hydrolysis; enzymatic acylation; levulinylation; 3′‐O‐levulinyl‐2′‐deoxyribonucleosides; 3′‐O‐levulinyl‐2′‐O‐methylribonucleosides; 5′‐O‐levulinyl‐2′‐deoxyribonucleosides; 5′‐O‐levulinyl‐2′‐O‐methylribonucleosides

     
 
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Table of Contents

  • Synthesis of 3′‐O‐Levulinyl‐2′‐Deoxyribonucleosides
  • Basic Protocol 1: Preparation of 3′‐O‐Levulinylthymidine via Regioselective Enzymatic Acylation with PSL‐C
  • Support Protocol 1: Preparation of O‐Levulinyl Acetonoxime from Levulinic Acid and Acetonoxime
  • Basic Protocol 2: Preparation of N4‐Benzoyl‐3′‐O‐Levulinyl‐2′‐Deoxycytidine via Regioselective Enzymatic Acylation with PSL‐C
  • Basic Protocol 3: Preparation of N6‐Benzoyl‐3′‐O‐Levulinyl‐2′‐Deoxyadenosine via Regioselective Enzymatic Acylation with PSL‐C
  • Basic Protocol 4: Preparation of N2‐Isobutyryl‐3′‐O‐Levulinyl‐2′‐Deoxyguanosine via Regioselective Enzymatic Hydrolysis with CAL‐B
  • Support Protocol 2: Preparation of N2‐Isobutyryl‐3′,5′‐Di‐O‐Levulinyl‐2′‐Deoxyguanosine
  • Synthesis of 5′‐O‐Levulinyl‐2′‐Deoxyribonucleosides
  • Basic Protocol 5: Preparation of 5′‐O‐Levulinylthymidine via Regioselective Enzymatic Acylation with CAL‐B
  • Basic Protocol 6: Preparation of N4‐Benzoyl‐5′‐O‐Levulinyl‐2′‐Deoxycytidine via Regioselective Enzymatic Acylation with CAL‐B
  • Basic Protocol 7: Preparation of N6‐Benzoyl‐5′‐O‐Levulinyl‐2′‐Deoxyadenosine via Regioselective Enzymatic Acylation with CAL‐B
  • Basic Protocol 8: Preparation of N2‐Isobutyryl‐5′‐O‐Levulinyl‐2′‐Deoxyguanosine via Regioselective Enzymatic Acylation with CAL‐B
  • Synthesis of 3′‐O‐Levulinyl‐2′‐O‐Methylribonucleosides
  • Basic Protocol 9: Preparation of 3′‐O‐Levulinyl‐2′‐O‐Methyluridine via Regioselective Enzymatic Hydrolysis with CAL‐B
  • Basic Protocol 10: Preparation of N4‐Benzoyl‐3′‐O‐Levulinyl‐2′‐O‐Methylcytidine via Regioselective Enzymatic Hydrolysis with CAL‐B
  • Basic Protocol 11: Preparation of N6‐Benzoyl‐3′‐O‐Levulinyl‐2′‐O‐Methyladenosine via Regioselective Enzymatic Hydrolysis with CAL‐B
  • Basic Protocol 12: Preparation of N2‐Isobutyryl‐3′‐O‐Levulinyl‐2′‐O‐Methylguanosine via Regioselective Enzymatic Hydrolysis with CAL‐B
  • Support Protocol 3: Preparation of 3′,5′‐Di‐O‐Levulinyl‐2′‐O‐Methylribonucleosides
  • Synthesis of 5′‐O‐Levulinyl‐2′‐Methylribonucleosides
  • Basic Protocol 13: Preparation of 5′‐O‐Levulinyl‐2′‐O‐Methyluridine via Regioselective Enzymatic Acylation with CAL‐B
  • Basic Protocol 14: Preparation of N4‐Benzoyl‐5′‐O‐Levulinyl‐2′‐O‐Methylcytidine via Regioselective Enzymatic Acylation with CAL‐B
  • Basic Protocol 15: Preparation of N6‐Benzoyl‐5′‐O‐Levulinyl‐2′‐O‐Methyladenosine via Regioselective Enzymatic Acylation with CAL‐B
  • Basic Protocol 16: Preparation of N2‐Isobutyryl‐5′‐O‐Levulinyl‐2′‐O‐Methylguanosine via Regioselective Enzymatic Acylation with CAL‐B
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 3′‐O‐Levulinylthymidine via Regioselective Enzymatic Acylation with PSL‐C

  Materials
  • Thymidine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Pseudomonas cepacia lipase (PSL‐C; 904 propyl laurate units per gram [PLU/g]; Amano Enzyme)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled from sodium and benzophenone
  • Methanol (MeOH)
  • Dichloromethane (CH 2Cl 2)
  • p‐Anisaldehyde solution (see recipe)
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • Sodium sulfate (Na 2SO 4)
  • Diethyl ether (Et 2O), cold (4°C)
  • Silica gel
  • Ethyl acetate (EtOAc)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 30°C
  • Microcentrifuge tubes
  • Capillary tubes
  • Silica gel 60 F 254 aluminum‐backed TLC plates
  • UV light source
  • Heat gun
  • Buchner funnels with filter paper circles
  • 250‐ and 500‐mL round‐bottom flasks
  • Rotary evaporator equipped with a cooling condenser
  • 500‐mL separatory funnel
  • 250‐mL Erlenmeyer flasks
  • 1‐L filter flask
  • Glass funnel with Whatman no. 1 filter paper
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Support Protocol 1: Preparation of O‐Levulinyl Acetonoxime from Levulinic Acid and Acetonoxime

  • Acetone oxime (Aldrich)
  • Levulinic acid (Aldrich)
  • 1,3‐Dicyclohexylcarbodiimide (DCC; Aldrich)
  • 25‐L three‐neck round‐bottom flask equipped
  • Overhead stirrer
  • Thermometer
  • Addition funnel
  • Cooling bath

Basic Protocol 2: Preparation of N4‐Benzoyl‐3′‐O‐Levulinyl‐2′‐Deoxycytidine via Regioselective Enzymatic Acylation with PSL‐C

  Materials
  • N4‐Benzoyl‐2′‐deoxycytidine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Pseudomonas cepacia lipase (PSL‐C; 904 propyl laurate units per gram; Amano Enzyme)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled from sodium and benzophenone
  • Methanol (MeOH), hot (65°C)
  • Diethyl ether (Et 2O), cold (4°C)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 40°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 3: Preparation of N6‐Benzoyl‐3′‐O‐Levulinyl‐2′‐Deoxyadenosine via Regioselective Enzymatic Acylation with PSL‐C

  Materials
  • N6‐Benzoyl‐2′‐deoxyadenosine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Pseudomonas cepacia lipase (PSL‐C; 904 propyl laurate units per gram; Amano Enzyme)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled from sodium and benzophenone
  • Dichloromethane (CH 2Cl 2)
  • Diethyl ether (Et 2O), cold (4°C)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 40°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 4: Preparation of N2‐Isobutyryl‐3′‐O‐Levulinyl‐2′‐Deoxyguanosine via Regioselective Enzymatic Hydrolysis with CAL‐B

  Materials
  • N2‐Isobutyryl‐3′,5′‐di‐O‐levulinyl‐2′‐deoxyguanosine ( S.4d; see protocol 6)
  • 1,4‐Dioxane
  • 0.15 M KH 2PO 4 buffer (see recipe)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram [PLU/g]; Novo Nordisk)
  • Dichloromethane (CH 2Cl 2)
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • Chloroform (CHCl 3)
  • Sodium sulfate (Na 2SO 4)
  • Diethyl ether (Et 2O)
  • 500‐mL Erlenmeyer flask with rubber septum
  • Orbital shaker, 40°C
  • Buchner funnel with filter paper circles
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • 500‐mL separatory funnel
  • 250‐mL Erlenmeyer flasks
  • Glass funnel with Whatman no. 1 filter paper
  • Additional reagents and equipment for TLC (see protocol 1)

Support Protocol 2: Preparation of N2‐Isobutyryl‐3′,5′‐Di‐O‐Levulinyl‐2′‐Deoxyguanosine

  • N2‐Isobutyryl‐2′‐deoxyguanosine (Rasayan)
  • Triethylamine (Et 3N)
  • Anhydrous 1,4‐dioxane, freshly distilled from sodium and benzophenone
  • Levulinic acid
  • 1,3‐Dicyclohexylcarbodiimide (DCC)
  • 4‐(Dimethylamino)pyridine (DMAP; Aldrich)

Basic Protocol 5: Preparation of 5′‐O‐Levulinylthymidine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • Thymidine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled from sodium and benzophenone
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 10°C
  • Additional reagents and equipment for analysis and work up of regioselectively levulinated thymidine (see protocol 1)

Basic Protocol 6: Preparation of N4‐Benzoyl‐5′‐O‐Levulinyl‐2′‐Deoxycytidine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • N4‐Benzoyl‐2′‐deoxycytidine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled over sodium and benzophenone
  • Dichloromethane (CH 2Cl 2)
  • Methanol (MeOH)
  • Diethyl ether (Et 2O), cold (4°C)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 30°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flask
  • Rotary evaporator
  • 1‐L filter flask
  • 250‐mL Erlenmeyer flask
  • Horst heating mantle
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 7: Preparation of N6‐Benzoyl‐5′‐O‐Levulinyl‐2′‐Deoxyadenosine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • N6‐Benzoyl‐2′‐deoxyadenosine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled over sodium and benzophenone
  • Methanol (MeOH), hot (65°C)
  • Diethyl ether (Et 2O), cold (4°C)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 30°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flask
  • Rotary evaporator
  • 1‐L filter flask
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 8: Preparation of N2‐Isobutyryl‐5′‐O‐Levulinyl‐2′‐Deoxyguanosine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • N2‐Isobutyryl‐2′‐deoxyguanosine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled over sodium and benzophenone
  • Methanol (MeOH)
  • Dichloromethane (CH 2Cl 2)
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • Chloroform (CHCl 3)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel
  • Ethyl acetate (EtOAc)
  • Diethyl ether (Et 2O)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 40°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • 500‐mL separatory funnel
  • Glass funnel with Whatman no. 1 filter paper
  • 1‐L filter flask
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 9: Preparation of 3′‐O‐Levulinyl‐2′‐O‐Methyluridine via Regioselective Enzymatic Hydrolysis with CAL‐B

  Materials
  • 3′,5′‐Di‐O‐levulinyl‐2′‐O‐methyluridine ( S.6a; see protocol 15)
  • 1,4‐Dioxane
  • 0.15 M KH 2PO 4 buffer (see recipe)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • 500‐mL Erlenmeyer flask
  • Rubber septum
  • Orbital shaker, 30°C
  • Additional reagents and equipment for TLC (see protocol 1) and for workup and purification of the product (see protocol 5)

Basic Protocol 10: Preparation of N4‐Benzoyl‐3′‐O‐Levulinyl‐2′‐O‐Methylcytidine via Regioselective Enzymatic Hydrolysis with CAL‐B

  Materials
  • N4‐Benzoyl‐3′,5′‐di‐O‐levulinyl‐2′‐O‐methylcytidine ( S.6b; see protocol 15)
  • 1,4‐Dioxane
  • 0.15 M KH 2PO 4 buffer (see recipe)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • 500‐mL Erlenmeyer flask
  • Rubber septum
  • Orbital shaker, 30°C
  • Additional reagents and equipment for TLC (see protocol 1) and for workup and purification of the product (see protocol 5)

Basic Protocol 11: Preparation of N6‐Benzoyl‐3′‐O‐Levulinyl‐2′‐O‐Methyladenosine via Regioselective Enzymatic Hydrolysis with CAL‐B

  Materials
  • N6‐Benzoyl‐3′,5′‐di‐O‐levulinyl‐2′‐O‐methyladenosine ( S.6c; see protocol 15)
  • 1,4‐Dioxane
  • 0.15 M KH 2PO 4 buffer (see recipe)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Dichloromethane (CH 2Cl 2)
  • Silica gel
  • Acetone
  • Diethyl ether (Et 2O)
  • 500‐mL Erlenmeyer flask
  • Rubber septum
  • Orbital shaker, 30°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • 1‐L filter flask
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 12: Preparation of N2‐Isobutyryl‐3′‐O‐Levulinyl‐2′‐O‐Methylguanosine via Regioselective Enzymatic Hydrolysis with CAL‐B

  Materials
  • N2‐Isobutyryl‐3′,5′‐di‐O‐levulinyl‐2′‐O‐methylguanosine ( S.6d; see protocol 15)
  • 1,4‐Dioxane
  • 0.15 M KH 2PO 4 buffer (see recipe)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • 500‐mL Erlenmeyer flask
  • Rubber septum
  • Orbital shaker, 30°C
  • Additional reagents and equipment for TLC (see protocol 1) and for workup and purification of the product (see protocol 5)

Support Protocol 3: Preparation of 3′,5′‐Di‐O‐Levulinyl‐2′‐O‐Methylribonucleosides

  • 2′‐O‐Methylribonucleoside (Rasayan): 2′‐O‐methyluridine, N4‐benzoyl‐2′‐O‐methylcytidine, N6‐benzoyl‐2′‐O‐methyladenosine, or N2‐isobutyryl‐2′‐O‐methylguanosine
  • Triethylamine (Et 3N)
  • Anhydrous 1,4‐dioxane, freshly distilled from sodium and benzophenone
  • Levulinic acid
  • Dicyclohexylcarbodiimide (DCC)
  • 4‐(Dimethylamino)pyridine (DMAP)
  • 200‐mL round‐bottom flask equipped with a three‐way glass stopcock

Basic Protocol 13: Preparation of 5′‐O‐Levulinyl‐2′‐O‐Methyluridine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • 2′‐O‐Methyluridine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled over sodium and benzophenone
  • Dichloromethane (CH 2Cl 2)
  • Diethyl ether (Et 2O), cold (4°C)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 40°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flask
  • Rotary evaporator
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 14: Preparation of N4‐Benzoyl‐5′‐O‐Levulinyl‐2′‐O‐Methylcytidine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • N4‐Benzoyl‐2′‐O‐methylcytidine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled over sodium and benzophenone
  • Dichloromethane (CH 2Cl 2)
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • Sodium sulfate (Na 2SO 4)
  • Diethyl ether (Et 2O), cold (4°C)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 40°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • 250‐mL separatory funnel
  • Glass funnel with Whatman no. 1 filter paper
  • Filter flask
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 15: Preparation of N6‐Benzoyl‐5′‐O‐Levulinyl‐2′‐O‐Methyladenosine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • N6‐Benzoyl‐2′‐O‐methyladenosine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled over sodium and benzophenone
  • Dichloromethane (CH 2Cl 2)
  • Silica gel
  • Acetone
  • Diethyl ether (Et 2O), cold (4°C)
  • 1‐L Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 40°C
  • Buchner funnels with filter paper circles
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • 1‐L filter flask
  • Additional reagents and equipment for TLC (see protocol 1)

Basic Protocol 16: Preparation of N2‐Isobutyryl‐5′‐O‐Levulinyl‐2′‐O‐Methylguanosine via Regioselective Enzymatic Acylation with CAL‐B

  Materials
  • N2‐Isobutyryl‐2′‐O‐methylguanosine (Rasayan)
  • O‐Levulinyl acetonoxime (see protocol 2)
  • Immobilized Candida antarctica lipase B (CAL‐B, under the trade name Novozyme 435; 10,000 propyl laurate units per gram; Novo Nordisk)
  • Anhydrous nitrogen
  • Anhydrous tetrahydrofuran (THF), freshly distilled over sodium and benzophenone
  • Dichloromethane (CH 2Cl 2)
  • Silica gel
  • Ethyl acetate (EtOAc)
  • Diethyl ether (Et 2O)
  • Methanol (MeOH)
  • 500‐mL Erlenmeyer flask equipped with a three‐way glass stopcock
  • Rubber septum
  • Orbital shaker, 40°C
  • Buchner funnels with filter paper circles
  • 250‐ and 500‐mL round‐bottom flasks
  • Rotary evaporator
  • 1‐L filter flask
  • Additional reagents and equipment for TLC (see protocol 1)
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Literature Cited

Literature Cited
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