General method for purification of α-amino acid-N-carboxyanhydrides using flash chromatography

Article abstract of DOI:10.1021/bm101123k

We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure α-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.

Full text of DOI:10.1021/bm101123k

3668
Biomacromolecules 2010, 11, 3668–3672
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-carboxy-
anhydride (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 polypep-
tides 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
Figure 1. Polypeptide synthesis by ring-opening polymerization of
NCAs.
Figure 2. Preparation of NCAs and resulting byproducts: (a) phos-
gene 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
* To whom correspondence should be addressed. Fax: (+1) 310-794-
5956. E-mail: demingt@seas.ucla.edu.
† Department of Chemistry and Biochemistry.
‡ Department of Bioengineering.
10.1021/bm101123k
 2010 American Chemical Society
Published on Web 11/03/2010

Purification of R-Amino acid-N-carboxyanhydrides
Biomacromolecules, Vol. 11, No. 12, 2010 3669
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.
likely due to concerns over NCA stability toward silica gel.
We are aware of only one example where silica gel chroma-
tography was used to isolate the NCAs of L-asparagine and
L-glutamine prior to purification by recrystallization.37 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.
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-L-
glutamate 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 seg-
ments,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.
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.
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 com-
pounds. This method has not been explored much in the past
and has rarely been used for the general purification of NCAs,
General Procedure for Recrystallization of NCAs. Inside a
dinitrogen filled glovebox, NCAs were dissolved in dry THF and

3670 Biomacromolecules, Vol. 11, No. 12, 2010
Kramer and Deming
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 purifica-
tion. 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% phospho-
molybic acid in ethanol with heat.
gel column proved effective at separating the NCAs from side
products such as benzyl chloride and other amino acid based
1
compounds, as evidenced by TLC (Figure 3), FTIR, and H
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 mono-
mers. 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 (<1%) of
impurities that will severely hamper controlled NCA polymer-
ization. A classic method to evaluate the suitability of NCAs
for preparation of polypeptides is to attempt polymerization at
a high monomer to initiator (M:I) ratio (i.e., 200:1) under
anhydrous conditions using carefully purified solvents.6 The
complete consumption of NCA under these conditions indicates
that the amount of electrophilic impurities is sufficiently low
so that their termination of chains is insignificant. To compare
the quality of NCAs purified by chromatography versus recrys-
tallization, polymerizations of Bn-Glu and Z-Lys NCAs were
conducted at M:I ratios of 300 to 1 using (PMe3)4Co40 initiator
in THF at room temperature. This initiator was chosen because
it has been shown to promote living polymerization of NCAs
and we have extensive experience in its use.40 As impurities in
NCAs will adversely affect any known NCA initiator, the
(PMe3)4Co data here is relevant to research using other methods
for NCA polymerization. Both polymerizations of chromato-
graphed NCAs went to completion (as determined by FTIR and
polymer isolation), while both polymerizations of recrystallized
samples were quenched by impurities and reached only about
50-60% conversion. This study showed that the purity of the
chromatographed NCAs was significantly higher than recrystal-
lized samples. To better quantitate impurity levels, the Z-Lys
NCAs from above were analyzed for chloride impurities using
ICP-MS. Recrystallized Z-Lys NCA was found to contain 1.8
ppm chlorine, while chromatographed Z-Lys NCA was found
to contain only 0.35 ppm. Likewise, crude Met NCA oil was
found to contain 207 ppm chlorine, and after chromatography
this level dropped to 0.36 ppm. Although the values for
chromatographed NCAs fall below the instrumental limit for
accurate detection (0.7 ppm), they confirm the level of chloride
impurities in these samples is very low.
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 polymeri-
zation 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. Reac-
tions 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-Glc-
Lys34 were prepared as previously reported, and the correspond-
ing 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
Polymerization studies to compare chain lengths using
chromatographed NCAs versus recrystallized NCAs were also
performed (Table 2). Bn-Glu, Z-Lys, and EG2-Lys NCAs, each

Purification of R-Amino acid-N-carboxyanhydrides
Biomacromolecules, Vol. 11, No. 12, 2010 3671
Table 2. Polymerization Data for NCAs Purified by
Chromatography Compared to Recrystallization
c
d
yieldb
(%)
Mn
Mn
yield
(%)e
c
monomera
GPC/LS
calcd
Mw/Mn
50 Lysf
93
92
97
90
61
67
62
91
49660
109600
37800
62070
28770
53890
57260
13100
13100
10950
10950
14400
14400
25025
1.12
1.12
1.17
1.12
1.16
1.08
1.23
97
98
94
96
91
93
91
NDi
50 Lysg
50 Gluf
50 Glug
50 EG2-Lysf
50 EG2-Lysg
50 Glc-Lysf
50 Metf,h
a Number indicates equivalents of monomer per (PMe3)4Co. Polymer-
izations run in THF at room temperature under N2. b Isolated yield of NCA.
c Observed molecular weight and polydispersity index after polymerization
as determined by GPC/LS. d Theoretical polymer molecular weight. e Total
isolatedyieldofpolypeptide.f Monomerpurifiedbysilicagelchromatography.
g Monomer purified by two recrystallizations. h Polymer not characterized
by GPC due to insolubility in DMF. i ND ) not determined.
Figure 4. Molecular weight (Mn, () and polydispersity index (Mw/Mn,
9) of equimolar statistical copolymerizations of Met NCA and Z-Lys
NCA as functions of monomer to initiator ratio ([M]/[I]) using (PMe3)4Co
in THF at 20 °C.
tides that were soluble in DMF and could be analyzed by GPC/
LS. Because the polymerizations went to 100% monomer
conversion and did not aggregate in nonsolvents for poly-L-
methionine, it is likely that blocky copolymers were not formed.
To check for chain-breaking side reactions during these
copolymerizations, experiments to verify molecular weight
control were performed. Polymerization of equimolar mixtures
of Met NCA and Z-Lys NCA at different M:I ratios gave
copolypeptides whose lengths (Mn) increased linearly with M:I
stoichiometry and that possessed narrow chain length distribu-
tions (Mw/Mn; Figure 3). These data show the first instance where
well-defined methionine containing copolypeptides have been
prepared, and open the possibility for using methionine building
blocks in new biopolymer materials.
purified by both methods, were separately polymerized at M:I
ratios of 50 to 1, and their resulting chain lengths were
compared. All monomers were completely consumed under
these conditions and were able to form polypeptides with low
polydispersities in high yields. However, it was seen that
chromatographed NCAs always gave shorter chains compared
to those purified by recrystallization. Larger chain lengths in
these polymerizations are indicative of initiator quenching at
the beginning of the polymerizations caused by electrophilic
impurities (e.g., HCl) in the NCA. All polymers had higher than
theoretical molecular weights, which is partially due to the
known incomplete efficiency of (PMe3)4Co initiation in THF.41
Although not all of the (PMe3)4Co preinitiator is able to generate
active chains, this system is able to produce low polydispersity
polypeptides of controlled chain lengths and has been used to
prepare numerous well-defined block copolypeptides.3,4 As such,
the absence of chain-breaking side reactions makes this a useful
system to probe low levels of impurities in NCAs that might
be masked by the abundance of side reactions that occur in
uncontrolled polymerizations. Here, it was seen that the amounts
of initiator quenching impurities were significantly lower in the
chromatographed NCAs compared to recrystallized counterparts.
Recrystallized NCAs of high purity could also be obtained by
additional recrystallization (i.e., 8 vs 2 times), yet this is a time-
consuming and tedious process that also lowers yields. From
this study, it can be seen that chromatographic purification of
NCAs has a distinct advantage in minimizing initiator deactiva-
tion and chain quenching during polymerization, which is
important for the synthesis of well-defined homo and block
copolypeptides using any initiating system.
Conclusion
In summary, we have established that silica gel column
chromatography is a useful general method for rapid and
straightforward purification of NCA monomers. 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
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 chromato-
graphed NCAs required no further purification and could be
used directly to form high molecular weight polypeptides. This
method is also practical in that it requires less time than
recrystallization and often gives NCAs in improved yields. This
procedure is especially useful for the purification and polym-
erization of monomers that cannot be recrystallized, or crystal-
lize only with difficulty. Synthetic polypeptides are being
developed and studied for many diverse uses, and this meth-
odology greatly simplifies the purification of NCA monomers,
which should increase the accessibility to and synthetic pos-
sibilities for these important materials.
To highlight the potential of chromatographic NCA purifica-
tion for the development of new biopolymer materials, we have
conducted polymerization studies on Met NCA. This monomer
is infrequently used because it is difficult to purify, yet poly-
L-methionine has interesting fiber forming properties.24 Homo
and copolymerizations of Met NCA, usually used as a non-
crystallizable syrup,42,43 have been previously reported,44,45 yet
controlled polymerization has never been demonstrated. Met
NCA purified by chromatography, obtained as a white solid,
was successfully polymerized using (PMe3)4Co in THF at M:I
ratios up to 200 to 1 with complete conversion to polymer.
However, molecular weight measurement of the poly-L-me-
thionine was obstructed by its insolubility in common organic
solvents, where its R-helical chains tend to aggregate to form
gels.46 To enable molecular weight measurement, Met NCA was
copolymerized with Z-Lys NCA to form statistical copolypep-
Acknowledgment. This work was supported by the NSF
IGERT: Materials Creation Training Program (MCTP), DGE-
0654431; the NSF under award No. DMR 0907453; and the
California NanoSystems Institute.
Supporting Information Available. Details of the prepara-
tion and purification of each NCA described and relevant
spectral data. This material is available free of charge via the
Internet at http://pubs.acs.org.

3672 Biomacromolecules, Vol. 11, No. 12, 2010
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