protocol
Asymmetric synthesis of amines using tert-butanesulfinamide Hai-Chao Xu1,2, Somenath Chowdhury1 & Jonathan A Ellman1 1Department of
Chemistry, Yale University, New Haven, Connecticut, USA. 2Department of Chemistry, Xiamen University, Xiamen, China. Correspondence should be addressed to J.A.E. ([email protected]).
© 2013 Nature America, Inc. All rights reserved.
Published online 24 October 2013; doi:10.1038/nprot.2013.134
Chiral amines are prevalent in many bioactive molecules, including amino acids and pharmaceutical agents. tert-Butanesulfinamide (tBS) is a chiral amine reagent that has enabled the reliable asymmetric synthesis of a very broad range of different amine structures from simple, readily available starting materials. Three steps are commonly applied to the asymmetric synthesis of amines: (i) condensation of tBS with a carbonyl compound, (ii) nucleophile addition and (iii) tert-butanesulfinyl group cleavage. Here we demonstrate these steps with the preparation of a propargylic tertiary carbinamine, one of a class of amines that have been used for many different biological purposes, including click chemistry applications, diversity-oriented synthesis, the preparation of peptide isosteres and the development of protease inhibitors as drug candidates and imaging agents. The process described here can be performed in 3–4 d.
INTRODUCTION (Fig. 1)9–14. The frequency with which the reagent is used can be Chiral amines are present in peptides, proteins and many natural products, and they are prevalent in drugs. For these reasons, quantitatively evaluated by the number of publications that cite the development of methods for the synthesis of enantiomeri- its use as determined by a SciFinder substructure search (Fig. 2). A marked increase in usage since 2000 is apparent. Also notable is cally pure compounds that contain amine functionality has featured prominently in organic synthesis1. Enantiomerically the rapidly increasing frequency of its use reported in patents, of pure amines traditionally were obtained by classical resolu- which the large majority are for pharmaceutical applications. A number of factors have led to the popularity of tBS. In addition of racemic amines with enantiomerically pure chiral acids. This approach continues to be a very important and widely used tion to the commercial availability of both enantiomers at a reamethod. HPLC using chiral packing material is also increasingly sonable cost, the synthetic steps used to prepare amines from tBS being used to separate enantiomeric amines present in racemic are typically robust, straightforward and broad in scope (Fig. 3). Three steps are typically carried out. (i) Imine preparation 15–19: material2. The transition metal–catalyzed asymmetric reduction of imines and enamides is extensively used particularly for the the direct condensation of tBS with a wide range of aldehydes and large-scale production of enantiomerically enriched drugs that ketones proceeds in high yields under mild conditions to give staincorporate the amine functionality 3. Enzymatic methods for ble N-tert-butanesulfinyl imines 13 that are much less hydrolytipreparing enantiomerically pure amines, including enantioselec- cally labile or prone to tautomerization than most N-substituted imines. (ii) Nucleophile addition: N-tert-butanesulfinyl imines 13, tive reductions, are an increasingly used alternative for industrial production4,5. Many other types of chemical reactions that rely despite their stability, are substantially more electrophilic than on chiral catalysts6,7, reagents and auxiliaries8 have been devel- typical N-alkyl or aryl imines. This enhanced electrophilicity enaoped for the asymmetric synthesis of chiral amines and have bles clean and high-yield additions of a very wide range of diverse proven to be particularly useful for natural product synthesis, nucleophiles9. Moreover, the chirality and metal-coordinating the optimization of drug leads and the preparation of chemical ability of the sulfinyl group provide high-addition diastereoselectivity9. (iii) Sulfinyl group removal: the tert-butanesulfinyl biology reagents. group in addition product 14 serves as a convenient protecting tBS is a commercially available and completely stable chiral amine reagent that provides one of the most extensively used group that parallels the reactivity of the tert-butyloxycarbonyl approaches for the asymmetric synthesis of amines. The tBS reagent can readily be NH2 NH2 NH2 NH2 NH2 R4 NH2 R3 used for the efficient asymmetric synthesis CO H 1 1 1 R4 3 2 1 of a very broad range of amine-containing 1 5 R R R R1 CO H R R R 2 R R2 R2 R3 R2 R2 compounds, including α-branched R2 R3 R3 R2 R4 R5 amines 1, α-amino acids 2, β-amino 1 2 3 4a 4b 5 acids 3, allylic amines 4a and homoallylic NH2 NH2 OH NH NH2 NH2 NH2 NH2 R1 amines 4b, propargylic amines 5, 1,2OH NH2 F 4 F 1 1 1 1 1 2 1 R R R R R CF3 R R R amino alcohols 6, 1,3-amino alcohols 7, R3 R4 R3 R2 R2 R 2 R3 R2 R2 R3 R4 R2 F R4 1,2-diamines 8, aziridines 9 and many 6 8 7 9 10 12 11 different types of amines incorporating fluorine groups, with a subset of deriva- Figure 1 | Representative classes of amines that can be prepared using tBS (R groups can be H, alkyl, tives represented by compounds 10–12 aromatic or heteroaromatic). nature protocols | VOL.8 NO.11 | 2013 | 2271
protocol 250
Publications Patents
100
1
R
O
N
2
S
R
1
R
tBS
R
Nu
S
HN
O R
2
13
HCl
O
*
H2N
150
S
NH3 Cl
*
O
200
1
1
Nu
R
R
2
14
Nu
R
2
15
Figure 3 | Three common steps in the asymmetric synthesis of amines with tBS. Asterisks (*) denote a chiral center. Nu, nucleophile.
50
12
11
20
10
20
08
09
20
20
07
06
20
20
05
04
20
20
03
20
02
20
00
20
20
20
01
0
© 2013 Nature America, Inc. All rights reserved.
Figure 2 | The number of publications and patents per year that cite the use of tBS or its derivatives.
(Boc)-protecting group and as such is stable to strong bases, nucleophiles and many transition metal–catalyzed processes20–27. However, simple treatment with methanolic HCl provides the desired amine hydrochloride products 15 after precipitation with ethereal solvents, often in nearly quantitative yields and in analytically pure form28,29. Experimental design To demonstrate the use of tBS in asymmetric amine synthesis, we have selected a four-step sequence for the preparation of propargylic amine 19 (ref. 29) (Fig. 4) because it demonstrates the key operations when using the tBS reagent: (i) sulfinyl imine preparation in the synthesis of 16, (ii) highly diastereoselective nucleo philic addition to obtain tertiary carbinamine 17, (iii) use of the sulfinyl group as a base-stable and nucleophile-stable amineprotecting group during silyl group deprotection to provide 18 and (iv) straightforward sulfinyl group removal with simple acid treatment to yield the final amine salt 19. In addition to generally demonstrating the key aspects of the tBS reagent, this synthesis in particular describes the asymmetric synthesis of the very useful class of propargylic tertiary carbinamines30–35. For example, the Ellman laboratory has used propargylic tertiary carbinamines as intermediates in the synthesis of potent inhibitors of multiple different cysteine proteases (Fig. 5). Indeed, amine 19 has been specifically incorporated in inhibitors of cruzain 20 (refs. 36,37), the essential protease of Trypanosoma cruzi that is the causative agent of Chagas disease, and in cathepsin S activity–based inhibitor probes 21 (ref. 38) for noninvasive optical imaging of tumor-associated macrophages. Analogs of 19 have also been incorporated in inhibitors of caspases 22 (ref. 39) relevant to the study of many biological processes such as Huntington’s disease, and in inhibitors of dipeptidyl amino peptidase 23 (ref. 40) encoded by the causative agent of malaria Plasmodium falciparum. Additionally, cathepsin S inhibitors41–43 such as 24 serve as drug leads to treat autoimmune disorders 44. Similar tBS chemistry has also been used in the asymmetric synthesis of propargylic amines for diversity-oriented synthesis45. The first step of the synthesis of 19 requires the condensation of tBS with 3-methyl-2-butanone using Ti(OEt)4 as a mild and inexpensive acid catalyst and water scavenger. The Ti(OEt)4 reagent is effective for most ketones and can also be used for more reactive aldehydes15,16. However, even more convenient methods 2272 | VOL.8 NO.11 | 2013 | nature protocols
are available for condensing tBS with aldehydes as is summarized in the review cited in ref. 1. The additions of nucleophiles to ketimines (prepared from ketones) are inherently more challenging than additions to aldimines (prepared from aldehydes) because ketimines are less electrophilic and are more sterically hindered. N-Sulfinyl ketimines can be activated for the addition of organometallic reagents by pre-complexation with trimethylaluminum as is used in this example29,46. With this activating agent, acetylide additions to a variety of ketimines have provided the desired propargylic amines in high yield and diastereoselectivity (Fig. 6)20. The reaction can be carried out on a wide range of scales with comparable efficiency. The specific addition reaction described in this protocol has been conducted on scales from 0.5 mmol to 60 mmol of ketimine 16 with no change in yield or diastereoselectivity. In terms of reaction scope, unbranched alkyl ketimines (Fig. 6, entry 9) and aromatic ketimines (entry 10) are less-effective substrates and afford lower yields, probably due to competitive deprotonation of the ketimine to form a metalloenamine that is unreactive toward addition. The current procedure for the addition reaction to form propargyl amine 17 has been modified from our original publication29. The acetylide addition reaction is performed at low temperature for a shorter period of time to enhance operational convenience. In addition, an aqueous solution of NaHSO4 instead of aqueous acetic acid is used to much more rapidly hydrolyze the small amount of leftover ketimine during the workup to greatly reduce the time required for this step. Trimethylaluminum is a reactive compound that readily catches fire when exposed to air and therefore must be handled with care using inert atmosphere techniques as is described in this procedure. Although trimethylaluminum can often be left out,
Ti(OEt)4
O H2N
S
S
AlMe3, then LiCCTMS
O
Toluene, −78 °C to rt
80−85%
Bu4NF
O
HN TMS
17
S
THF, 60 °C
O
tBS
HN
N
16
S
HCl O
–
NH3 Cl
CH3OH, rt
THF, rt
93%
81−87%
18
99%
19
Figure 4 | Four-step procedure for the asymmetric synthesis of propargylic tertiary carbinamine 19. rt, room temperature.
© 2013 Nature America, Inc. All rights reserved.
protocol for many N-sulfinyl ketimine and organolithium substrate combinations, this omission results in reduced yields and/or diastereoselectivities29,46,47. For additions of other types of nucleophiles to N-sulfinyl ketimines9, and for nucleophilic addition to N-sulfinyl aldimines45,48,49, trimethyl aluminum is not used. Because reaction conditions will depend on the type of nucleophile, the original literature should be investigated for nucleophiles other than lithium acetylide. Removal of the silyl group to provide terminal acetylide 18 is straightforward and proceeds in high yield. Similarly, the removal of the sulfinyl group with acid treatment to provide amine hydrochloride 19 is exceedingly operationally straightforward and can be successfully performed in high yield for a very wide range of N-sulfinyl amines.
N
N
H N
N
F
O
F
O
N Bu
O
O O
N 6 H
N O
O
4 NH
N
N
SO3– N
N
H N
F
O
F
O
N Bu
O
N
N
N N
O
CN
H
F 23
O
S
–O S 3
H2N
S
N
21
5
F
O N H
O
F
22
OH
O
N
F
O
O
O N
F
O
N
F
S N
N
O
F
20
H N
N
H N
S
F
24
Figure 5 | Examples of inhibitors synthesized from the analogs of 19 (the corresponding propargylic amine moieties are highlighted in red).
MATERIALS REAGENTS ! CAUTION All chemicals used in this protocol are potentially harmful. Hence, this protocol should be carried out in a well-vented chemical fume hood while wearing proper personal protective equipment (gloves, lab coat and eye protection). • (S)-(-)-2-Methyl-2-propanesulfinamide (Allychem, CAS no. 343338-28-3; Allychem has very competitive prices for bulk quantities of 1 or more kilograms. Advanced Asymmetrics has competitive prices for the 2.5–250 g range. Common suppliers such as Sigma-Aldrich and Strem also market the reagent) • Tetrahydrofuran, anhydrous (THF; Sigma-Aldrich, cat. no. 401757) • 3-Methyl-2-butanone (Alfa Aesar, cat. no. B24527) • Titanium (IV) ethoxide (Ti(OEt)4; Alfa Aesar, stock no. 44670) • Silicone oil (Acros Organics, cat. no. 163850025) • Brine (saturated sodium chloride aqueous solution) • Sand (Sigma-Aldrich, cat. no. 274739) • Silica gel, 40–63-µm particle size, 230–400 mesh (Sorbent Technologies, cat. no. 40930-25) • Celite 545 (EMD, cat. no. CX0574-1) • Diethyl ether, anhydrous (J.T. Baker, cat. no. 9237-03) • Sodium sulfate, anhydrous (Na2SO4, Fisher Scientific, cat. no. S415-1) • Saturated sodium sulfate aqueous solution • Sodium bisulfate aqueous solution, 1 N (NaHSO4) • Hexane, American Chemical Society (ACS) grade (EMD, cat. no. HX0299-3) • Ethyl acetate, ACS grade (EMD, cat. no. EX0240-3) • Dichloromethane (DCM; Fisher Scientific, cat. no. D37SK-4) • Toluene, anhydrous (Sigma-Aldrich, cat. no. 244511) • Trimethylaluminum solution, 2 M in toluene (Sigma-Aldrich, cat. no. 198048) ! CAUTION Trimethylaluminum ignites spontaneously in air and reacts violently with water. Proper training should be acquired before handling this reagent. • n-Butyllithium, 2.5 M in hexane (n-BuLi; Sigma-Aldrich, cat. no. 230707, store in a 4 °C refrigerator) ! CAUTION n-Butyllithium is sensitive to moisture and air. It reacts violently with water, liberating flammable gases. Wearing protective gloves during the transfer of solutions containing n-butyllithium is highly recommended. Proper training should be acquired before handling this reagent. • Trimethylsilylacetylene (Alfa Aesar, stock no. A12856)
• Tetrabutylammonium fluoride solution, 1.0 M in THF (Sigma-Aldrich, cat. no. 216143; store it in a 4 °C refrigerator) • 2-Propanol (J.T. Baker, cat. no. 9084-05) • Saturated aqueous ammonium chloride solution • Hydrogen chloride solution, 4.0 M in dioxane (Sigma-Aldrich, cat. no. 345547) • Methanol (J.T. Baker, cat. no. 9070-05) • Chloroform (J.T. Baker, cat. no. 9180-01) ! CAUTION Chloroform is suspected to cause cancer. EQUIPMENT • Disposable plastic syringes with Luer lock, 6 ml, 12 ml, 30 ml and 50 ml (NormJet) • Disposable needles, 18 G × 1 1/2 inches • Thermally controlled stirring plate • Stirring bars, Teflon coated, 25.4 mm × 12.7 mm, 39.1 mm × 7.9 mm • Three-necked round-bottomed flask, 250 ml, 24/40 joints • Y-shaped glass tubing connector, 1/4 inch inner diameter (i.d.) • Single-necked round-bottomed flasks, 250 ml, 500 ml and 1 liter • Pear-shaped flask, 100 ml, 14/20 joint • Condenser (13.5-mm i.d., 24/40 joints) • Sleeve rubber septa for 24/40 joints • Rotary evaporator (Büchi) • Oven maintained at 80 °C • Mass balance • Beaker (500 ml) • Thin-layer chromatography (TLC) plates (EMD, silica gel 60, F254) • Flash chromatography column, 6 cm × 40 cm, 4 cm × 50 cm • Test tubes, 18 × 150 mm • Cannula, 16 G, 50-cm long • Dewar flasks, low form, 850 ml, 13-cm i.d. × 7.5-cm depth • Glass dish, 150 mm (outer diameter (o.d.)) × 75 mm (height) • Graduated cylinder, 100 ml • Büchner funnel, fritted, porosity 40–60 µm (60 ml, 150 ml) • Separation funnel, 500 ml • Pasteur pipette • UV lamp • Ground-glass stopper for 24/40 joint • Erlenmeyer flask, 500 ml nature protocols | VOL.8 NO.11 | 2013 | 2273
protocol PROCEDURE Synthesis of (S)-N-(3-methyl-2-butylidene)2-methylpropanesulfinamide (16) ● TIMING 22 h 1| Weigh out 2.89 g (24.2 mmol) of (S)-tertbutanesulfinamide in a 250-ml three-necked round-bottomed flask (with 24/40 joints) equipped with a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm). 2| Equip the center neck of the three-necked flask with a condenser (inner tube i.d. 13.5 mm, 24/40 joints). Place a Claisen adaptor on the condenser and connect the Claisen adaptor to a nitrogen line via a Y-joint that is also connected to a bubbler (see Fig. 7 for full reaction setup). 3| Place a rubber septa on each of the two remaining necks of the flask.
AlMe3, then N R2
Entry
Ketimine
N
1
S
N
S
S
© 2013 Nature America, Inc. All rights reserved.
5| Transfer into the flask 80 ml of anhydrous THF with a 50-ml disposable plastic syringe, 2.8 ml (26.2 mmol) of 3-methyl-2-butanone with a 6-ml disposable plastic syringe and 10.0 ml (48.4 mmol) of Ti(OEt)4 with a 30-ml disposable plastic syringe.
S
N
6
HN
Li
Ph
Li
TMS
Li
TMS
S
HN
O
S
HN
96:4a
87c
n-Pr
95:5b
71c
Ph
>99:1b
74c
TMS
>99:1b
81c
>99:1b
73c
TMS
>99:1b
80c
TMS
97:3b
85c
90:10a
80c
95:5b
46c
86:14a
20d
O
O
O TMS
S
HN
O
S
O
TMS
H
S
Li
TMS
Li
TMS
S
O
Li
TMS
O
H
HN
O
8
S
HN
O
S
O TMS
S
HN
O
9
TMS
N
S
O
Li
TMS
HN
TMS
Yield (%)
O
S
O
dr
H
N
10
R1
O
S
HN
n-Pr
H
N
11| After 5 min of stirring, filter the suspension through a pad of Celite (~10 g) in a 150-ml fritted Büchner funnel with porosity 40–60 µm (Fig. 8).
S
HN
TMS
O
Li
8| Remove the oil bath and allow the reaction mixture to cool down to room temperature (22 °C) (~40 min).
10| Pour the cooled reaction mixture into the beaker. White slurry forms immediately.
Li
S
N
7
R2
Product
O
N
6| Flow water through the condenser.
9| To a 500-ml beaker, add 80 ml of brine using a 100-ml graduated cylinder. Add an octagon Teflon-coated stirring bar (39.1 mm × 7.9 mm) and begin stirring.
Li
O
S
N
7| Immerse the reaction flask in a silicone oil bath preheated to 60 °C (silicone oil in a glass dish with 150 mm (o.d.) × 75 mm (i.d.)), and leave it stirring overnight (15 h).
O
3
5
R1
S
HN
Toluene, −78 °C to rt
TMS
N
4| Insert an 18-G disposable needle vent in one of the septa and flush with nitrogen for 5 min. Remove the vent needle.
R3
Li O
Alkynyllithium
2
4
S
S
O TMS
Figure 6 | Reactions of sulfinyl ketimines with acetylides. aDiastereomeric ratio was determined by NMR analysis. bDiastereomeric ratio was determined by HPLC assay. cIsolated yield of diastereomerically and analytically pure material after chromatography. dYield determined by NMR analysis using hexamethylbenzene as an external standard.
12| Wash the filter cake with diethyl ether (3 × 50 ml). 13| Transfer the filtrate into a 500-ml separation funnel. 14| After separation of the layers, extract the brine layer with diethyl ether (2 × 50 ml) and combine the organic phases. 15| Dry the organic solution over anhydrous Na2SO4 (~25 g) for 0.5 h, filter the Na2SO4 using a 60-ml fritted Büchner funnel (porosity 40–60 µm) into a 500-ml round-bottomed flask and concentrate under vacuum with a rotatory evaporator (~35 torr, 25 °C). 2274 | VOL.8 NO.11 | 2013 | nature protocols
protocol 16| Dissolve the residue in diethyl ether (10 ml) and transfer it to a 100-ml pear-shaped flask. Wash the round-bottomed flask with diethyl ether (2 × 5 ml), combine the solution and concentrate it with a rotary evaporator (~35 torr, 25 °C). 17| Pack a chromatography column (6 × 40 cm) with a slurry of silica gel prepared by mixing 120 g of silica gel with 250 ml of hexane. Cover the column with ~1-cm layer of sand. 18| Transfer the crude product to the column with a Pasteur pipette. Wash the flask with 1 ml of DCM, and add it to the column.
© 2013 Nature America, Inc. All rights reserved.
19| Elute with hexane/diethyl ether (1:1) at ~40 ml min−1. 20| Collect 25-ml fractions in test tubes of 18 × 150 mm. Identify the fractions containing the product using TLC by developing the thin-layer silica gel plates with the eluent used in the previous step (Rf = 0.3), and visualize it with a UV lamp (254 nm). 21| Combine fractions 10 to 29 in a 1-liter round-bottomed flask, and then remove the solvent using a rotary evaporator (~35 torr, water bath temperature at 25 °C) to yield the product as a colorless oil. After removing most of the solvent, keep the flask on the rotary evaporator for a further 1 h to remove residual solvent. CRITICAL STEP The product is volatile. Avoid drying the product under high vacuum. PAUSE POINT The liquid obtained can be stored at −20 °C for months without obvious decomposition.
Figure 7 | Typical setup for the synthesis of imine.
Synthesis of (S)-N-(S)-3,4-dimethyl-1-(trimethylsilyl)pent-1-yn-3-yl)-2-methylpropane-2-sulfinamide (17) ● TIMING 12 h CRITICAL The reaction in this section is sensitive to moisture and air and should be carried out under anhydrous conditions and under an inert atmosphere. 22| Weigh out 2.27 g (12.0 mmol) of 16 in a 250-ml oven-dried round-bottomed flask (24/40 joints, flask A) equipped with a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm); seal the flask with a rubber septum. 23| Place a nitrogen inlet needle into the septum along with a disposable needle vent and flush the flask with nitrogen for 5 min. Remove the vent needle and keep the flask under positive nitrogen pressure. 24| Transfer 34 ml of dry toluene into the flask with a 50-ml disposable plastic syringe. 25| Cool the solution to −78 °C by immersing the reaction flask in a dry ice/acetone bath for 5 min. The cooling bath is prepared in a Dewar flask (13 cm i.d. × 7.5 cm depth). 26| With stirring, slowly add 7.2 ml (14.4 mmol) of trimethylaluminum (2.0 M in toluene) to flask A over ~2 min using a 30-ml plastic syringe equipped with an 18-G, 30.5-cm stainless steel needle. After the addition is complete, remove the needle and syringe from the reaction flask. Flush the needle and syringe three times with hexane to remove residual trimethylaluminum. The flushing solvent is added to 2-propanol to inactivate any residual trimethylaluminum. ! CAUTION Trimethylaluminum is extremely reactive and pyrophoric and should be handled in a chemical fume hood. Proper personal protective equipment (gloves, a lab coat and eye protection) should be worn.
nature protocols | VOL.8 NO.11 | 2013 | 2275
protocol 27| To a separate oven-dried 250-ml round-bottomed flask (24/40 joints, flask B), add a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm) and seal the flask with a rubber septum. 28| Place a nitrogen inlet needle into the septum of flask B along with a vent needle and flush the flask with nitrogen for 5 min. Remove the vent needle and keep the flask under positive nitrogen pressure. 29| To flask B, add 5.1 ml (36.0 mmol) of trimethylsilyl acetylene with a 12-ml disposable plastic syringe, and add 42 ml of anhydrous toluene using a 50-ml disposable plastic syringe.
© 2013 Nature America, Inc. All rights reserved.
30| With stirring, cool the solution by placing flask B in a −78 °C dry ice/acetone bath. 31| Once the solution has cooled (~5 min), add 10.6 ml (26.4 mmol) of n-butyllithium (2.5 M in hexanes) dropwise with stirring over 5 min using a 30-ml disposable plastic syringe equipped with an 18-G, 30.5-cm stainless steel needle. After the addition is complete, remove the needle and syringe from the reaction flask. Flush the needle and syringe three times with hexane to remove residual n-butyllithium. The flushing solvent is added to 2-propanol to inactivate any residual n-butyllithium. ! CAUTION n-Butyllithium is sensitive to moisture and air. Figure 8 | Filtration of reaction mixture through Celite. It reacts violently with water, liberating flammable gases. Proper training on handling reactive chemicals should be taken before using this reagent. Wear proper personal protective equipment such as gloves, a lab coat and eye protection while manipulating this reagent. CRITICAL STEP The solution becomes jelly-like during or after the addition of n-butyllithium and thus stirring may be difficult. 32| After complete addition, stir the reaction mixture for an additional 20 min at the same temperature (−78 °C). 33| Transfer the toluene solution of 16 and trimethylaluminum in flask A into the lithium trimethylsilylacetylide solution in flask B via a 50-cm-long, 16-G cannula dropwise, while keeping both flasks in − 78 °C cooling baths (Fig. 9). Insert one end of the cannula to the bottom of flask A and insert the other end into flask B but keep the needle tip above the solution. Remove the nitrogen inlet from flask B and insert a vent needle. Control the addition rate by adjusting the nitrogen gas flow such that the solution from flask A is transferred over a period of ~20 min. ? TROUBLESHOOTING 34| Upon complete addition, insert a nitrogen inlet needle into flask B and remove the cannula and vent needle. 35| Remove the cooling bath and stir the mixture for 3 h. The progress of the reaction can be monitored by 1H NMR spectroscopy. Withdraw 0.1 ml of the reaction mixture with a 1-ml disposable plastic syringe and add to a mixture of ether (0.5 ml) and saturated Na2SO4 solution (0.5 ml). Collect the organic layer, concentrate it and obtain the 1H NMR spectrum of the residue. ? TROUBLESHOOTING 36| Cool the reaction mixture with stirring in an ice-water bath. 37| After 5 min of stirring in the cooling bath, remove the rubber septum and add saturated aqueous Na2SO4 (~10 ml) dropwise using a Pasteur pipette until no gas evolves upon addition. ! CAUTION Trimethylaluminum reacts violently with water. Hence, add the first 2 ml of saturated Na2SO4 aqueous solution dropwise in 3 min. 2276 | VOL.8 NO.11 | 2013 | nature protocols
protocol 38| Remove the ice-water bath and stir the reaction mixture for 20 min. 39| Filter the slurry through a 60-ml fritted Büchner funnel (porosity 40–60 µm) into a 500-ml round-bottomed flask, and then wash the filtrate cake with ethyl acetate (3 × 20 ml).
Nitrogen inlet
Cannula
© 2013 Nature America, Inc. All rights reserved.
40| Add to the 500-ml round-bottomed flask a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm).
Venting needle
41| Add 50 ml of 1 N NaHSO4 solution with a 100-ml graduated cylinder, and then stir the biphasic mixture vigorously (800 r.p.m.) for 1 h to hydrolyze the small amount of unreacted imine 16. CRITICAL STEP A small amount (
Asymmetric synthesis of amines using tert-butanesulfinamide Hai-Chao Xu1,2, Somenath Chowdhury1 & Jonathan A Ellman1 1Department of
Chemistry, Yale University, New Haven, Connecticut, USA. 2Department of Chemistry, Xiamen University, Xiamen, China. Correspondence should be addressed to J.A.E. ([email protected]).
© 2013 Nature America, Inc. All rights reserved.
Published online 24 October 2013; doi:10.1038/nprot.2013.134
Chiral amines are prevalent in many bioactive molecules, including amino acids and pharmaceutical agents. tert-Butanesulfinamide (tBS) is a chiral amine reagent that has enabled the reliable asymmetric synthesis of a very broad range of different amine structures from simple, readily available starting materials. Three steps are commonly applied to the asymmetric synthesis of amines: (i) condensation of tBS with a carbonyl compound, (ii) nucleophile addition and (iii) tert-butanesulfinyl group cleavage. Here we demonstrate these steps with the preparation of a propargylic tertiary carbinamine, one of a class of amines that have been used for many different biological purposes, including click chemistry applications, diversity-oriented synthesis, the preparation of peptide isosteres and the development of protease inhibitors as drug candidates and imaging agents. The process described here can be performed in 3–4 d.
INTRODUCTION (Fig. 1)9–14. The frequency with which the reagent is used can be Chiral amines are present in peptides, proteins and many natural products, and they are prevalent in drugs. For these reasons, quantitatively evaluated by the number of publications that cite the development of methods for the synthesis of enantiomeri- its use as determined by a SciFinder substructure search (Fig. 2). A marked increase in usage since 2000 is apparent. Also notable is cally pure compounds that contain amine functionality has featured prominently in organic synthesis1. Enantiomerically the rapidly increasing frequency of its use reported in patents, of pure amines traditionally were obtained by classical resolu- which the large majority are for pharmaceutical applications. A number of factors have led to the popularity of tBS. In addition of racemic amines with enantiomerically pure chiral acids. This approach continues to be a very important and widely used tion to the commercial availability of both enantiomers at a reamethod. HPLC using chiral packing material is also increasingly sonable cost, the synthetic steps used to prepare amines from tBS being used to separate enantiomeric amines present in racemic are typically robust, straightforward and broad in scope (Fig. 3). Three steps are typically carried out. (i) Imine preparation 15–19: material2. The transition metal–catalyzed asymmetric reduction of imines and enamides is extensively used particularly for the the direct condensation of tBS with a wide range of aldehydes and large-scale production of enantiomerically enriched drugs that ketones proceeds in high yields under mild conditions to give staincorporate the amine functionality 3. Enzymatic methods for ble N-tert-butanesulfinyl imines 13 that are much less hydrolytipreparing enantiomerically pure amines, including enantioselec- cally labile or prone to tautomerization than most N-substituted imines. (ii) Nucleophile addition: N-tert-butanesulfinyl imines 13, tive reductions, are an increasingly used alternative for industrial production4,5. Many other types of chemical reactions that rely despite their stability, are substantially more electrophilic than on chiral catalysts6,7, reagents and auxiliaries8 have been devel- typical N-alkyl or aryl imines. This enhanced electrophilicity enaoped for the asymmetric synthesis of chiral amines and have bles clean and high-yield additions of a very wide range of diverse proven to be particularly useful for natural product synthesis, nucleophiles9. Moreover, the chirality and metal-coordinating the optimization of drug leads and the preparation of chemical ability of the sulfinyl group provide high-addition diastereoselectivity9. (iii) Sulfinyl group removal: the tert-butanesulfinyl biology reagents. group in addition product 14 serves as a convenient protecting tBS is a commercially available and completely stable chiral amine reagent that provides one of the most extensively used group that parallels the reactivity of the tert-butyloxycarbonyl approaches for the asymmetric synthesis of amines. The tBS reagent can readily be NH2 NH2 NH2 NH2 NH2 R4 NH2 R3 used for the efficient asymmetric synthesis CO H 1 1 1 R4 3 2 1 of a very broad range of amine-containing 1 5 R R R R1 CO H R R R 2 R R2 R2 R3 R2 R2 compounds, including α-branched R2 R3 R3 R2 R4 R5 amines 1, α-amino acids 2, β-amino 1 2 3 4a 4b 5 acids 3, allylic amines 4a and homoallylic NH2 NH2 OH NH NH2 NH2 NH2 NH2 R1 amines 4b, propargylic amines 5, 1,2OH NH2 F 4 F 1 1 1 1 1 2 1 R R R R R CF3 R R R amino alcohols 6, 1,3-amino alcohols 7, R3 R4 R3 R2 R2 R 2 R3 R2 R2 R3 R4 R2 F R4 1,2-diamines 8, aziridines 9 and many 6 8 7 9 10 12 11 different types of amines incorporating fluorine groups, with a subset of deriva- Figure 1 | Representative classes of amines that can be prepared using tBS (R groups can be H, alkyl, tives represented by compounds 10–12 aromatic or heteroaromatic). nature protocols | VOL.8 NO.11 | 2013 | 2271
protocol 250
Publications Patents
100
1
R
O
N
2
S
R
1
R
tBS
R
Nu
S
HN
O R
2
13
HCl
O
*
H2N
150
S
NH3 Cl
*
O
200
1
1
Nu
R
R
2
14
Nu
R
2
15
Figure 3 | Three common steps in the asymmetric synthesis of amines with tBS. Asterisks (*) denote a chiral center. Nu, nucleophile.
50
12
11
20
10
20
08
09
20
20
07
06
20
20
05
04
20
20
03
20
02
20
00
20
20
20
01
0
© 2013 Nature America, Inc. All rights reserved.
Figure 2 | The number of publications and patents per year that cite the use of tBS or its derivatives.
(Boc)-protecting group and as such is stable to strong bases, nucleophiles and many transition metal–catalyzed processes20–27. However, simple treatment with methanolic HCl provides the desired amine hydrochloride products 15 after precipitation with ethereal solvents, often in nearly quantitative yields and in analytically pure form28,29. Experimental design To demonstrate the use of tBS in asymmetric amine synthesis, we have selected a four-step sequence for the preparation of propargylic amine 19 (ref. 29) (Fig. 4) because it demonstrates the key operations when using the tBS reagent: (i) sulfinyl imine preparation in the synthesis of 16, (ii) highly diastereoselective nucleo philic addition to obtain tertiary carbinamine 17, (iii) use of the sulfinyl group as a base-stable and nucleophile-stable amineprotecting group during silyl group deprotection to provide 18 and (iv) straightforward sulfinyl group removal with simple acid treatment to yield the final amine salt 19. In addition to generally demonstrating the key aspects of the tBS reagent, this synthesis in particular describes the asymmetric synthesis of the very useful class of propargylic tertiary carbinamines30–35. For example, the Ellman laboratory has used propargylic tertiary carbinamines as intermediates in the synthesis of potent inhibitors of multiple different cysteine proteases (Fig. 5). Indeed, amine 19 has been specifically incorporated in inhibitors of cruzain 20 (refs. 36,37), the essential protease of Trypanosoma cruzi that is the causative agent of Chagas disease, and in cathepsin S activity–based inhibitor probes 21 (ref. 38) for noninvasive optical imaging of tumor-associated macrophages. Analogs of 19 have also been incorporated in inhibitors of caspases 22 (ref. 39) relevant to the study of many biological processes such as Huntington’s disease, and in inhibitors of dipeptidyl amino peptidase 23 (ref. 40) encoded by the causative agent of malaria Plasmodium falciparum. Additionally, cathepsin S inhibitors41–43 such as 24 serve as drug leads to treat autoimmune disorders 44. Similar tBS chemistry has also been used in the asymmetric synthesis of propargylic amines for diversity-oriented synthesis45. The first step of the synthesis of 19 requires the condensation of tBS with 3-methyl-2-butanone using Ti(OEt)4 as a mild and inexpensive acid catalyst and water scavenger. The Ti(OEt)4 reagent is effective for most ketones and can also be used for more reactive aldehydes15,16. However, even more convenient methods 2272 | VOL.8 NO.11 | 2013 | nature protocols
are available for condensing tBS with aldehydes as is summarized in the review cited in ref. 1. The additions of nucleophiles to ketimines (prepared from ketones) are inherently more challenging than additions to aldimines (prepared from aldehydes) because ketimines are less electrophilic and are more sterically hindered. N-Sulfinyl ketimines can be activated for the addition of organometallic reagents by pre-complexation with trimethylaluminum as is used in this example29,46. With this activating agent, acetylide additions to a variety of ketimines have provided the desired propargylic amines in high yield and diastereoselectivity (Fig. 6)20. The reaction can be carried out on a wide range of scales with comparable efficiency. The specific addition reaction described in this protocol has been conducted on scales from 0.5 mmol to 60 mmol of ketimine 16 with no change in yield or diastereoselectivity. In terms of reaction scope, unbranched alkyl ketimines (Fig. 6, entry 9) and aromatic ketimines (entry 10) are less-effective substrates and afford lower yields, probably due to competitive deprotonation of the ketimine to form a metalloenamine that is unreactive toward addition. The current procedure for the addition reaction to form propargyl amine 17 has been modified from our original publication29. The acetylide addition reaction is performed at low temperature for a shorter period of time to enhance operational convenience. In addition, an aqueous solution of NaHSO4 instead of aqueous acetic acid is used to much more rapidly hydrolyze the small amount of leftover ketimine during the workup to greatly reduce the time required for this step. Trimethylaluminum is a reactive compound that readily catches fire when exposed to air and therefore must be handled with care using inert atmosphere techniques as is described in this procedure. Although trimethylaluminum can often be left out,
Ti(OEt)4
O H2N
S
S
AlMe3, then LiCCTMS
O
Toluene, −78 °C to rt
80−85%
Bu4NF
O
HN TMS
17
S
THF, 60 °C
O
tBS
HN
N
16
S
HCl O
–
NH3 Cl
CH3OH, rt
THF, rt
93%
81−87%
18
99%
19
Figure 4 | Four-step procedure for the asymmetric synthesis of propargylic tertiary carbinamine 19. rt, room temperature.
© 2013 Nature America, Inc. All rights reserved.
protocol for many N-sulfinyl ketimine and organolithium substrate combinations, this omission results in reduced yields and/or diastereoselectivities29,46,47. For additions of other types of nucleophiles to N-sulfinyl ketimines9, and for nucleophilic addition to N-sulfinyl aldimines45,48,49, trimethyl aluminum is not used. Because reaction conditions will depend on the type of nucleophile, the original literature should be investigated for nucleophiles other than lithium acetylide. Removal of the silyl group to provide terminal acetylide 18 is straightforward and proceeds in high yield. Similarly, the removal of the sulfinyl group with acid treatment to provide amine hydrochloride 19 is exceedingly operationally straightforward and can be successfully performed in high yield for a very wide range of N-sulfinyl amines.
N
N
H N
N
F
O
F
O
N Bu
O
O O
N 6 H
N O
O
4 NH
N
N
SO3– N
N
H N
F
O
F
O
N Bu
O
N
N
N N
O
CN
H
F 23
O
S
–O S 3
H2N
S
N
21
5
F
O N H
O
F
22
OH
O
N
F
O
O
O N
F
O
N
F
S N
N
O
F
20
H N
N
H N
S
F
24
Figure 5 | Examples of inhibitors synthesized from the analogs of 19 (the corresponding propargylic amine moieties are highlighted in red).
MATERIALS REAGENTS ! CAUTION All chemicals used in this protocol are potentially harmful. Hence, this protocol should be carried out in a well-vented chemical fume hood while wearing proper personal protective equipment (gloves, lab coat and eye protection). • (S)-(-)-2-Methyl-2-propanesulfinamide (Allychem, CAS no. 343338-28-3; Allychem has very competitive prices for bulk quantities of 1 or more kilograms. Advanced Asymmetrics has competitive prices for the 2.5–250 g range. Common suppliers such as Sigma-Aldrich and Strem also market the reagent) • Tetrahydrofuran, anhydrous (THF; Sigma-Aldrich, cat. no. 401757) • 3-Methyl-2-butanone (Alfa Aesar, cat. no. B24527) • Titanium (IV) ethoxide (Ti(OEt)4; Alfa Aesar, stock no. 44670) • Silicone oil (Acros Organics, cat. no. 163850025) • Brine (saturated sodium chloride aqueous solution) • Sand (Sigma-Aldrich, cat. no. 274739) • Silica gel, 40–63-µm particle size, 230–400 mesh (Sorbent Technologies, cat. no. 40930-25) • Celite 545 (EMD, cat. no. CX0574-1) • Diethyl ether, anhydrous (J.T. Baker, cat. no. 9237-03) • Sodium sulfate, anhydrous (Na2SO4, Fisher Scientific, cat. no. S415-1) • Saturated sodium sulfate aqueous solution • Sodium bisulfate aqueous solution, 1 N (NaHSO4) • Hexane, American Chemical Society (ACS) grade (EMD, cat. no. HX0299-3) • Ethyl acetate, ACS grade (EMD, cat. no. EX0240-3) • Dichloromethane (DCM; Fisher Scientific, cat. no. D37SK-4) • Toluene, anhydrous (Sigma-Aldrich, cat. no. 244511) • Trimethylaluminum solution, 2 M in toluene (Sigma-Aldrich, cat. no. 198048) ! CAUTION Trimethylaluminum ignites spontaneously in air and reacts violently with water. Proper training should be acquired before handling this reagent. • n-Butyllithium, 2.5 M in hexane (n-BuLi; Sigma-Aldrich, cat. no. 230707, store in a 4 °C refrigerator) ! CAUTION n-Butyllithium is sensitive to moisture and air. It reacts violently with water, liberating flammable gases. Wearing protective gloves during the transfer of solutions containing n-butyllithium is highly recommended. Proper training should be acquired before handling this reagent. • Trimethylsilylacetylene (Alfa Aesar, stock no. A12856)
• Tetrabutylammonium fluoride solution, 1.0 M in THF (Sigma-Aldrich, cat. no. 216143; store it in a 4 °C refrigerator) • 2-Propanol (J.T. Baker, cat. no. 9084-05) • Saturated aqueous ammonium chloride solution • Hydrogen chloride solution, 4.0 M in dioxane (Sigma-Aldrich, cat. no. 345547) • Methanol (J.T. Baker, cat. no. 9070-05) • Chloroform (J.T. Baker, cat. no. 9180-01) ! CAUTION Chloroform is suspected to cause cancer. EQUIPMENT • Disposable plastic syringes with Luer lock, 6 ml, 12 ml, 30 ml and 50 ml (NormJet) • Disposable needles, 18 G × 1 1/2 inches • Thermally controlled stirring plate • Stirring bars, Teflon coated, 25.4 mm × 12.7 mm, 39.1 mm × 7.9 mm • Three-necked round-bottomed flask, 250 ml, 24/40 joints • Y-shaped glass tubing connector, 1/4 inch inner diameter (i.d.) • Single-necked round-bottomed flasks, 250 ml, 500 ml and 1 liter • Pear-shaped flask, 100 ml, 14/20 joint • Condenser (13.5-mm i.d., 24/40 joints) • Sleeve rubber septa for 24/40 joints • Rotary evaporator (Büchi) • Oven maintained at 80 °C • Mass balance • Beaker (500 ml) • Thin-layer chromatography (TLC) plates (EMD, silica gel 60, F254) • Flash chromatography column, 6 cm × 40 cm, 4 cm × 50 cm • Test tubes, 18 × 150 mm • Cannula, 16 G, 50-cm long • Dewar flasks, low form, 850 ml, 13-cm i.d. × 7.5-cm depth • Glass dish, 150 mm (outer diameter (o.d.)) × 75 mm (height) • Graduated cylinder, 100 ml • Büchner funnel, fritted, porosity 40–60 µm (60 ml, 150 ml) • Separation funnel, 500 ml • Pasteur pipette • UV lamp • Ground-glass stopper for 24/40 joint • Erlenmeyer flask, 500 ml nature protocols | VOL.8 NO.11 | 2013 | 2273
protocol PROCEDURE Synthesis of (S)-N-(3-methyl-2-butylidene)2-methylpropanesulfinamide (16) ● TIMING 22 h 1| Weigh out 2.89 g (24.2 mmol) of (S)-tertbutanesulfinamide in a 250-ml three-necked round-bottomed flask (with 24/40 joints) equipped with a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm). 2| Equip the center neck of the three-necked flask with a condenser (inner tube i.d. 13.5 mm, 24/40 joints). Place a Claisen adaptor on the condenser and connect the Claisen adaptor to a nitrogen line via a Y-joint that is also connected to a bubbler (see Fig. 7 for full reaction setup). 3| Place a rubber septa on each of the two remaining necks of the flask.
AlMe3, then N R2
Entry
Ketimine
N
1
S
N
S
S
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5| Transfer into the flask 80 ml of anhydrous THF with a 50-ml disposable plastic syringe, 2.8 ml (26.2 mmol) of 3-methyl-2-butanone with a 6-ml disposable plastic syringe and 10.0 ml (48.4 mmol) of Ti(OEt)4 with a 30-ml disposable plastic syringe.
S
N
6
HN
Li
Ph
Li
TMS
Li
TMS
S
HN
O
S
HN
96:4a
87c
n-Pr
95:5b
71c
Ph
>99:1b
74c
TMS
>99:1b
81c
>99:1b
73c
TMS
>99:1b
80c
TMS
97:3b
85c
90:10a
80c
95:5b
46c
86:14a
20d
O
O
O TMS
S
HN
O
S
O
TMS
H
S
Li
TMS
Li
TMS
S
O
Li
TMS
O
H
HN
O
8
S
HN
O
S
O TMS
S
HN
O
9
TMS
N
S
O
Li
TMS
HN
TMS
Yield (%)
O
S
O
dr
H
N
10
R1
O
S
HN
n-Pr
H
N
11| After 5 min of stirring, filter the suspension through a pad of Celite (~10 g) in a 150-ml fritted Büchner funnel with porosity 40–60 µm (Fig. 8).
S
HN
TMS
O
Li
8| Remove the oil bath and allow the reaction mixture to cool down to room temperature (22 °C) (~40 min).
10| Pour the cooled reaction mixture into the beaker. White slurry forms immediately.
Li
S
N
7
R2
Product
O
N
6| Flow water through the condenser.
9| To a 500-ml beaker, add 80 ml of brine using a 100-ml graduated cylinder. Add an octagon Teflon-coated stirring bar (39.1 mm × 7.9 mm) and begin stirring.
Li
O
S
N
7| Immerse the reaction flask in a silicone oil bath preheated to 60 °C (silicone oil in a glass dish with 150 mm (o.d.) × 75 mm (i.d.)), and leave it stirring overnight (15 h).
O
3
5
R1
S
HN
Toluene, −78 °C to rt
TMS
N
4| Insert an 18-G disposable needle vent in one of the septa and flush with nitrogen for 5 min. Remove the vent needle.
R3
Li O
Alkynyllithium
2
4
S
S
O TMS
Figure 6 | Reactions of sulfinyl ketimines with acetylides. aDiastereomeric ratio was determined by NMR analysis. bDiastereomeric ratio was determined by HPLC assay. cIsolated yield of diastereomerically and analytically pure material after chromatography. dYield determined by NMR analysis using hexamethylbenzene as an external standard.
12| Wash the filter cake with diethyl ether (3 × 50 ml). 13| Transfer the filtrate into a 500-ml separation funnel. 14| After separation of the layers, extract the brine layer with diethyl ether (2 × 50 ml) and combine the organic phases. 15| Dry the organic solution over anhydrous Na2SO4 (~25 g) for 0.5 h, filter the Na2SO4 using a 60-ml fritted Büchner funnel (porosity 40–60 µm) into a 500-ml round-bottomed flask and concentrate under vacuum with a rotatory evaporator (~35 torr, 25 °C). 2274 | VOL.8 NO.11 | 2013 | nature protocols
protocol 16| Dissolve the residue in diethyl ether (10 ml) and transfer it to a 100-ml pear-shaped flask. Wash the round-bottomed flask with diethyl ether (2 × 5 ml), combine the solution and concentrate it with a rotary evaporator (~35 torr, 25 °C). 17| Pack a chromatography column (6 × 40 cm) with a slurry of silica gel prepared by mixing 120 g of silica gel with 250 ml of hexane. Cover the column with ~1-cm layer of sand. 18| Transfer the crude product to the column with a Pasteur pipette. Wash the flask with 1 ml of DCM, and add it to the column.
© 2013 Nature America, Inc. All rights reserved.
19| Elute with hexane/diethyl ether (1:1) at ~40 ml min−1. 20| Collect 25-ml fractions in test tubes of 18 × 150 mm. Identify the fractions containing the product using TLC by developing the thin-layer silica gel plates with the eluent used in the previous step (Rf = 0.3), and visualize it with a UV lamp (254 nm). 21| Combine fractions 10 to 29 in a 1-liter round-bottomed flask, and then remove the solvent using a rotary evaporator (~35 torr, water bath temperature at 25 °C) to yield the product as a colorless oil. After removing most of the solvent, keep the flask on the rotary evaporator for a further 1 h to remove residual solvent. CRITICAL STEP The product is volatile. Avoid drying the product under high vacuum. PAUSE POINT The liquid obtained can be stored at −20 °C for months without obvious decomposition.
Figure 7 | Typical setup for the synthesis of imine.
Synthesis of (S)-N-(S)-3,4-dimethyl-1-(trimethylsilyl)pent-1-yn-3-yl)-2-methylpropane-2-sulfinamide (17) ● TIMING 12 h CRITICAL The reaction in this section is sensitive to moisture and air and should be carried out under anhydrous conditions and under an inert atmosphere. 22| Weigh out 2.27 g (12.0 mmol) of 16 in a 250-ml oven-dried round-bottomed flask (24/40 joints, flask A) equipped with a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm); seal the flask with a rubber septum. 23| Place a nitrogen inlet needle into the septum along with a disposable needle vent and flush the flask with nitrogen for 5 min. Remove the vent needle and keep the flask under positive nitrogen pressure. 24| Transfer 34 ml of dry toluene into the flask with a 50-ml disposable plastic syringe. 25| Cool the solution to −78 °C by immersing the reaction flask in a dry ice/acetone bath for 5 min. The cooling bath is prepared in a Dewar flask (13 cm i.d. × 7.5 cm depth). 26| With stirring, slowly add 7.2 ml (14.4 mmol) of trimethylaluminum (2.0 M in toluene) to flask A over ~2 min using a 30-ml plastic syringe equipped with an 18-G, 30.5-cm stainless steel needle. After the addition is complete, remove the needle and syringe from the reaction flask. Flush the needle and syringe three times with hexane to remove residual trimethylaluminum. The flushing solvent is added to 2-propanol to inactivate any residual trimethylaluminum. ! CAUTION Trimethylaluminum is extremely reactive and pyrophoric and should be handled in a chemical fume hood. Proper personal protective equipment (gloves, a lab coat and eye protection) should be worn.
nature protocols | VOL.8 NO.11 | 2013 | 2275
protocol 27| To a separate oven-dried 250-ml round-bottomed flask (24/40 joints, flask B), add a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm) and seal the flask with a rubber septum. 28| Place a nitrogen inlet needle into the septum of flask B along with a vent needle and flush the flask with nitrogen for 5 min. Remove the vent needle and keep the flask under positive nitrogen pressure. 29| To flask B, add 5.1 ml (36.0 mmol) of trimethylsilyl acetylene with a 12-ml disposable plastic syringe, and add 42 ml of anhydrous toluene using a 50-ml disposable plastic syringe.
© 2013 Nature America, Inc. All rights reserved.
30| With stirring, cool the solution by placing flask B in a −78 °C dry ice/acetone bath. 31| Once the solution has cooled (~5 min), add 10.6 ml (26.4 mmol) of n-butyllithium (2.5 M in hexanes) dropwise with stirring over 5 min using a 30-ml disposable plastic syringe equipped with an 18-G, 30.5-cm stainless steel needle. After the addition is complete, remove the needle and syringe from the reaction flask. Flush the needle and syringe three times with hexane to remove residual n-butyllithium. The flushing solvent is added to 2-propanol to inactivate any residual n-butyllithium. ! CAUTION n-Butyllithium is sensitive to moisture and air. Figure 8 | Filtration of reaction mixture through Celite. It reacts violently with water, liberating flammable gases. Proper training on handling reactive chemicals should be taken before using this reagent. Wear proper personal protective equipment such as gloves, a lab coat and eye protection while manipulating this reagent. CRITICAL STEP The solution becomes jelly-like during or after the addition of n-butyllithium and thus stirring may be difficult. 32| After complete addition, stir the reaction mixture for an additional 20 min at the same temperature (−78 °C). 33| Transfer the toluene solution of 16 and trimethylaluminum in flask A into the lithium trimethylsilylacetylide solution in flask B via a 50-cm-long, 16-G cannula dropwise, while keeping both flasks in − 78 °C cooling baths (Fig. 9). Insert one end of the cannula to the bottom of flask A and insert the other end into flask B but keep the needle tip above the solution. Remove the nitrogen inlet from flask B and insert a vent needle. Control the addition rate by adjusting the nitrogen gas flow such that the solution from flask A is transferred over a period of ~20 min. ? TROUBLESHOOTING 34| Upon complete addition, insert a nitrogen inlet needle into flask B and remove the cannula and vent needle. 35| Remove the cooling bath and stir the mixture for 3 h. The progress of the reaction can be monitored by 1H NMR spectroscopy. Withdraw 0.1 ml of the reaction mixture with a 1-ml disposable plastic syringe and add to a mixture of ether (0.5 ml) and saturated Na2SO4 solution (0.5 ml). Collect the organic layer, concentrate it and obtain the 1H NMR spectrum of the residue. ? TROUBLESHOOTING 36| Cool the reaction mixture with stirring in an ice-water bath. 37| After 5 min of stirring in the cooling bath, remove the rubber septum and add saturated aqueous Na2SO4 (~10 ml) dropwise using a Pasteur pipette until no gas evolves upon addition. ! CAUTION Trimethylaluminum reacts violently with water. Hence, add the first 2 ml of saturated Na2SO4 aqueous solution dropwise in 3 min. 2276 | VOL.8 NO.11 | 2013 | nature protocols
protocol 38| Remove the ice-water bath and stir the reaction mixture for 20 min. 39| Filter the slurry through a 60-ml fritted Büchner funnel (porosity 40–60 µm) into a 500-ml round-bottomed flask, and then wash the filtrate cake with ethyl acetate (3 × 20 ml).
Nitrogen inlet
Cannula
© 2013 Nature America, Inc. All rights reserved.
40| Add to the 500-ml round-bottomed flask a Teflon-coated stirring bar (egg-shaped, 25.4 mm × 12.7 mm).
Venting needle
41| Add 50 ml of 1 N NaHSO4 solution with a 100-ml graduated cylinder, and then stir the biphasic mixture vigorously (800 r.p.m.) for 1 h to hydrolyze the small amount of unreacted imine 16. CRITICAL STEP A small amount (
