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General Method for Purification of r-Amino acid-N-carboxyanhydrides Using Flash Chromatography Jessica R. Kramer† and Timothy J. Deming*,†,‡ Department of Chemistry and Biochemistry and Department of Bioengineering, University of California, Los Angeles, California 90095-1600, United States Received September 20, 2010; Revised Manuscript Received October 13, 2010
We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure R-amino acid-N-carboxyanhydride (NCA) monomers, the widely used precursors for the synthesis of polypeptides, without the need for recrystallization. This technique was effective at removing all common impurities from NCAs and was found to work for a variety of NCAs, including those synthesized using different routes, as well as those bearing either hydrophilic or hydrophobic side chains. All chromatographed NCAs required no further purification and could be used directly to form high molecular weight polypeptides. This procedure is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. This method solves many long-standing problems in NCA purification and provides rapid access to NCAs that were previously inaccessible in satisfactory quality for controlled polymerization. This method is also practical in that it requires less time than recrystallization and often gives NCAs in improved yields.
Introduction The controlled synthesis of polypeptides of defined chain lengths and complex architectures has been actively pursued for many years.1,2 Recently, much progress has been made in the development of polymerization initiators and reaction conditions to achieve this goal.3,4 However, less attention has been paid to the purification of the R-amino acid-N-carboxyanhydride (NCA) monomers, which in many cases is the limiting factor in controlled polypeptide synthesis and commercial development of synthetic polypeptides.5 The need to obtain NCAs in high purity has been challenging scientists for over 50 years,6 and many approaches have been taken to solve this problem. Special preparation methods or purification steps are often employed to obtain NCAs sufficiently pure so that they can be crystallized, where recrystallization is most often the ultimate step required to prepare pure NCAs.7 Here, we describe the use of flash column chromatography on silica gel as a rapid and general method to obtain pure NCAs without the need for recrystallization. While this procedure simplifies purification of a wide range of NCAs, it is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. NCAs were first reported over 100 years ago by Hermann Leuchs2 and have been widely used for the synthesis of polypeptides since the 1940s (Figure 1).1 Over 200 different NCAs have been synthesized,8 and a wide variety of polypeptides have been prepared that contain diverse structures and functionalities. NCAs are most often synthesized by either treatment of R-amino acids with phosgene (Fuchs-Farthing method)7,9,10 or the reaction of N-alkyloxycarbonyl-amino acids with halogenating agents (Leuchs method; Figure 2).11,12 Triphosgene,13 diphosgene,14 and di-tert-butyltricarbonate15 have also been used as phosgene substitutes, but these reagents have * To whom correspondence should be addressed. Fax: (+1) 310-7945956. E-mail:
[email protected]. † Department of Chemistry and Biochemistry. ‡ Department of Bioengineering.
Figure 1. Polypeptide synthesis by ring-opening polymerization of NCAs.
Figure 2. Preparation of NCAs and resulting byproducts: (a) phosgene and (b) various halogenating reagents (e.g., PBr3, PCl3, PCl5, SOCl2, R,R-dichloromethylmethyl ether).
low volatility and any excess must be later removed by purification. Di-tert-butyltricarbonate, aside from having low reactivity, also generates tert-butyl alcohol, which may interfere with NCA polymerization.15 NCAs synthesized with phosgene contain byproducts including HCl,6 HCl-amino acid salts,16 2-isocyanatoacyl chlorides,17 and N-chloroformyl amino acids.17 NCAs prepared via the Leuchs method contain byproducts including HCl, HBr, alkyl halides as well as any excess, and byproducts, of low volatility halogenating agents.6 For use in the controlled synthesis of polypeptides, NCAs must be free of these electrophilic contaminants that can quench or inhibit chain growth. Chloride ions can also act as polymerization initiators in dimethylformamide and must be removed.18 NCAs are most readily purified by repeated crystallization under anhydrous conditions.19 There have been reports of NCA purification by sublimation, but this procedure is limited to a few, select NCAs and is accompanied by low yields due to
10.1021/bm101123k 2010 American Chemical Society Published on Web 11/03/2010
Purification of R-Amino acid-N-carboxyanhydrides
thermal polymerization.20,21 Recrystallization is useful for purification of many common NCAs that are high-melting solids, yet multiple recrystallizations are usually required to remove traces of impurities. Oily impurities, such as residual diphosgene, alkyl halides, and 2-isocyanatoacyl chlorides hinder NCA crystallization, and some form of initial purification must be used before the NCA can be crystallized.22 A number of NCA purification procedures have been developed. R-Pinene or limonene has been added to NCA preparations to consume HCl,5 and although this greatly assists the scale-up of NCA synthesis, removal of the alkyl chloride that is formed can be an issue. Washing NCA reaction mixtures in ethyl acetate with water and aqueous bicarbonate at 0 °C has also been used to remove HCl and HCl-amino acid salts.23 This procedure has been found to work well for some amino acids. However, difficult to separate emulsions can form with long-chain γ-alkyl-glutamate NCAs,23 and this procedure can also introduce an initiator, water, into NCAs, leading to premature polymerization. A second phosgenation or “rephosgenation” of NCAs has been proposed to eliminate hard to remove HCl-amino acid salts from NCAs,16 yet excess phosgene can lead to 2-isocyanatoacyl chloride formation with aliphatic amino acids. Columns of activated charcoal,24 zeolites,25 or urea,26 with or without Ag2O as a chloride scavenger, have also been used to purify NCA solutions, yet NCA polymerization during purification limits this procedure. While these methods are able to remove some impurities found in NCA preparations, further purification, most often recrystallization, is required to obtain NCAs suitable for controlled polypeptide synthesis. NCA recrystallizations are often slow and tedious, where considerable expertise is needed to determine solvent mixtures and compositions that will encourage NCA crystallization and separation from impurities. While some common NCAs (e.g., Nε-carbobenzyloxy-L-lysine NCA, Z-lys NCA, or γ-benzyl-L-glutamate NCA, Bn-Glu NCA) can be sufficiently purified after two recrystallizations, many NCAs, typically those with more polar or complex functionality (e.g., Nε-2-[2-(2-methoxyethoxy)ethoxy]acetyl-L-lysine NCA, EG2-Lys NCA), require an arduous process of six or more recrystallizations that can lead to low yields.12,27 Furthermore, some NCAs can only be isolated as oils (e.g., γ-alkyl-Lglutamate NCAs with long n-akyl chains23) or have low melting points (e.g., L-methionine NCA, Met NCA) and either cannot be recrystallized or recrystallized only with great difficulty.14,28,29 As more complex NCA monomers are developed to create polypeptides with new functionalities, the need for a method to purify NCAs that does not rely on recrystallization has become increasingly urgent. Recently, many novel NCAs have been reported that contain alkyne groups for click chemistry reactions,30,31 carbohydrates,32 and oligoethylene glycol segments,33 yet many of these monomers could not be recrystallized or adequately purified. To obtain high purity, noncrystalline glycosylated NCAs in our own lab,34 we investigated the use of flash column chromatography on silica gel for NCA purification. Flash column chromatography is widely used by synthetic organic chemists to purify a desired product from a variety of impurities including salts, inorganic compounds, and other organic molecules.35,36 Chromatography has many advantages in that it is fast, separation can be optimized easily, the silica gel is reasonably inexpensive, and, most importantly, works well for purification of both crystalline and noncrystalline compounds. This method has not been explored much in the past and has rarely been used for the general purification of NCAs,
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likely due to concerns over NCA stability toward silica gel. We are aware of only one example where silica gel chromatography was used to isolate the NCAs of L-asparagine and 37 L-glutamine prior to purification by recrystallization. Here, chromatography was used because the lack of protecting groups on these amino acids led to many side products, leading to low yields of NCAs (ca. 30%). We have found that, under anhydrous conditions, a wide range of NCAs can be purified using silica gel chromatography, and were all subsequently capable of forming high molecular weight polypeptides without the need for additional purification (i.e., recrystallization).
Experimental Section Materials and Methods. Unless stated otherwise, reactions were conducted in oven-dried glassware under an atmosphere of nitrogen using anhydrous solvents. Hexanes, THF, and diethyl ether were purified by first purging with dry nitrogen, followed by passage through columns of activated alumina. EtOAc was freshly distilled from CaH2. All commercially obtained reagents were used as received without further purification unless otherwise stated. (PMe3)4Co was prepared according to literature procedures.38 Reaction temperatures were controlled using an IKA magnetic temperature modulator, and unless stated otherwise, reactions were performed at room temperature (RT, approximately 23 °C). NMR spectra were recorded on Bruker spectrometers at 500 MHz for 1H and at 125 MHz for 13C{1H} NMR. Fourier transform infrared spectroscopy (FTIR) samples were prepared as thin films on NaCl plates and spectra were recorded on a Perkin-Elmer RX1 FTIR spectrometer. Tandem gel permeation chromatography/light scattering (GPC/LS) was performed on a SSI Accuflow Series III liquid chromatograph pump equipped with a Wyatt DAWN EOS light scattering detector and Wyatt Optilab rEX refractive index (RI) detectors. Separations were achieved using 105, 104, and 103 Å Phenomenex Phenogel 5 mm columns using 0.10 M LiBr in DMF as the eluent at 60 °C. All GPC/LS samples were prepared at concentrations of 5 mg/mL. Inductively coupled plasma-mass spectrometry (ICP-MS) samples were run on an Agilent 7500ce instrument in helium collision gas mode. General Preparation of NCAs by Phosgenation of r-Amino Acids. To a solution of amino acid in dry THF (0.15 M) in a Schlenk flask was added a solution of phosgene in toluene (20% (w/v), 2 equiv) via syringe. Caution! Phosgene is extremely hazardous and all manipulations must be performed in a well-ventilated chemical fume hood with proper personal protection and necessary precautions taken to avoid exposure. The reaction was stirred under N2 at 50 °C for 3 h. The reaction was evaporated to dryness and transferred to a dinitrogen filled glovebox. The condensate in the vacuum traps was treated with 50 mL of concentrated aqueous NH4OH to neutralize residual phosgene. General Preparation of NCAs from N-Carbobenzyloxy r-Amino Acids. To a solution of N-carbobenzyloxy R-amino acid in dry CH2Cl2 (0.05 M) in a Schlenk flask under N2 was added R,R-dichloromethylmethyl ether (1.5 equiv), and the solution was refluxed for 36 h. The reaction was evaporated to dryness under reduced pressure and transferred to a dinitrogen filled glovebox. General Procedure for Silica Chromatography of NCAs. Selecto silica gel 60 (particle size 0.032-0.063 mm) was heated to 150 °C under vacuum for 48 h before use. Thin-layer chromatography (TLC) was conducted with EMD gel 60 F254 precoated plates (0.25 mm) and visualized using a combination of UV, potassium permanganate, and phosphomolybdic acid staining. Inside a dinitrogen filled glovebox, silica was slurry packed into a glass column fitted with a glass filter frit. NCA was dissolved in a minimal amount of solvent, loaded onto the column, and eluted with additional solvent (see Supporting Information for details). Fractions were collected and analyzed by TLC for the presence of NCA. All fractions containing NCA were combined and condensed under reduced pressure. General Procedure for Recrystallization of NCAs. Inside a dinitrogen filled glovebox, NCAs were dissolved in dry THF and
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Table 1. NCA Monomers Purified by Column Chromatography
Figure 3. Image of plate used for thin layer chromatography of Glc-Lys NCA before (Crude) and after (Purified) column purification. Image has been rotated so that solvent flow is from left to right. Origin is where compounds were initially spotted onto plate (marked with “X”). AA imp are amino acid containing impurities. Image has been cropped for clarity but shows all data. TLC plate was stained for visualization of compounds using 20% phosphomolybic acid in ethanol with heat.
a NCA prepared from the corresponding amino acid using COCl2 and THF at 50 °C. b NCA prepared from the corresponding N-carbobenzyloxy amino acid using Cl2CHOCH3 and CH2Cl2 at 45 °C.
recrystallized via solvent diffusion under a layer of dry hexanes at RT. After 16 h, the mother liquor was poured off and solids were dried under vacuum. General Procedure for Polymerization of NCAs. All polymerization reactions were performed in a dinitrogen filled glovebox. To a solution of NCA in dry THF (50 mg/mL) was rapidly added, via syringe, a solution of (PMe3)4Co in dry THF (20 mM). The reaction was stirred at RT and the polymerization progress was monitored by removing small aliquots that were analyzed by FTIR. Polymerization reactions were generally complete within 1 h. Aliquots were removed for GPC analysis immediately upon polymerization completion. Reactions were precipitated from THF into hexanes, and polymers were collected by centrifugation and dried under reduced pressure to yield white solids (91-99% yield). General Procedure for Preparation of Statistical Copolymers of Methionine and Z-Lysine. In the drybox, Met NCA, and Z-Lys NCA were mixed in equimolar amounts and dissolved in THF (100 mg/mL). General polymerization procedures were followed as described above. Reactions were precipitated from THF into hexanes, and polymers were collected by centrifugation and dried under reduced pressure to yield white solids (88-94% yield).
Results and Discussion Silica gel chromatography was used to purify NCAs prepared by both the phosgene method and by halogenation of Z-protected amino acids (Table 1). Bn-Glu, Z-Lys, and Met NCAs were prepared by treatment with phosgene. Z-EG2-Lys12 and Z-GlcLys34 were prepared as previously reported, and the corresponding NCAs were prepared by treatment of the Z-protected amino acids with R,R-dichloromethylmethyl ether.39 Each NCA was loaded onto a column of silica gel and eluted using a solvent mixture whose composition was varied based on compound polarity (see Supporting Information). Passage through a silica
gel column proved effective at separating the NCAs from side products such as benzyl chloride and other amino acid based compounds, as evidenced by TLC (Figure 3), FTIR, and 1H and 13C NMR (see Supporting Information). It is also notable that chromatographic purification was not associated with any loss in NCA or polypeptide yield and, in some cases, improved NCA yields were obtained compared to recrystallized monomers. The NCAs obtained by chromatography were also found to be stable upon storage at -20 °C under N2 for over 1 year. While NMR and FTIR spectroscopy were able to show that chromatographically purified NCAs had few impurities, these techniques are unable to detect small amounts (
