Preparation of multifunctional and multireactive polypeptides via methionine alkylation

Article abstract of DOI:10.1021/bm300807b

We report the development of a new "click"-type reaction for polypeptide modification based on the chemoselective alkylation of thioether groups in methionine residues. The controlled synthesis of methionine polymers and their alkylation by a broad range of functional reagents to yield stable sulfonium derivatives are described. These "methionine click" functionalizations are compatible with deprotection of other functional groups, use an inexpensive, natural amino acid that is readily polymerized and requires no protecting groups, and allow the introduction of a diverse range of functionality and reactive groups onto polypeptides.

Full text of DOI:10.1021/bm300807b

Communication
pubs.acs.org/Biomac
Preparation of Multifunctional and Multireactive Polypeptides via
Methionine Alkylation
Jessica R. Kramer† and Timothy J. Deming*,†,‡
‡
†Department of Chemistry and Biochemistry and Department of Bioengineering, University of California, Los Angeles, California
90095, United States
S
* Supporting Information
ABSTRACT: We report the development of a new “click”-
type reaction for polypeptide modification based on the
chemoselective alkylation of thioether groups in methionine
residues. The controlled synthesis of methionine polymers and
their alkylation by a broad range of functional reagents to yield
stable sulfonium derivatives are described. These “methionine
click” functionalizations are compatible with deprotection of
other functional groups, use an inexpensive, natural amino acid
that is readily polymerized and requires no protecting groups,
and allow the introduction of a diverse range of functionality and reactive groups onto polypeptides.
he postpolymerization modification of polymers to
introduce a diverse range of functional groups has
protecting groups, and allow the introduction of a diverse
range of functionality and reactive groups onto polypeptides.
Because of its role in biology, the alkylation of thioether
groups in methionine has been studied for some time, with
pioneering studies being done by Toennies in the 1940s.20,21
The majority of work in this area has employed simple
alkylating agents, such as iodomethane to give the naturally
occurring, dietary supplement S-methyl-methionine,22,23 as well
as iodoacetic acid to form soluble derivatives and probe the
active sites of proteins.24−26 There are some reports on the use
of other activated alkyl halides to alkylate methionine, including
those containing alkene and alkyne groups, yet no reactions
were performed on the resulting sulfonium derivatives.27−31
Katchalski was the first to alkylate poly(L-methionine),
poly(Met), preparing both methyl and carboxymethyl
sulfonium derivatives from the corresponding alkyl bromides
and poly(Met) in neat formic acid.32 These polysulfoniums
were found to be stable and water-soluble and were studied for
their conformational and polyelectrolyte behavior. Despite the
simplicity of this reaction, which gives quantitative alkylation
with no side products, there have been few further studies on
these materials.33,34 More significantly, there have been no
reports on the use of any other functional reagents for
alkylation of poly(Met). This lack of activity may be due to
traditional difficulties in synthesis of poly(Met),35,36 as the
monomer, L-methionine N-carboxyanhydride (Met NCA), is
difficult to purify by standard methods, and the living
homopolymerization of Met NCA has not been demonstrated.
Realizing that the alkylation of methionine residues has
potential to be much broader in scope, we sought to develop
T
received much recent attention.1,2 This approach, where
different molecules are conjugated to reactive side-chain groups
in a polymer, is advantageous because it avoids the need to
prepare many different functional monomers and individually
optimize their polymerization conditions. Because of the need
for highly efficient side-chain conjugations and compatibility of
reactive groups with polymerization, “click”-type reactions are
typically employed.1−3 We are interested in the preparation of
side-chain-functionalized polypeptides because these can mimic
the diversity of post-translationally modified proteins found in
biology and may be useful for medical applications.4 Reactive
precursor polypeptides have been prepared that contain side-
chain alkyne,5−8 alkene,9−13 azide,14 or thiol15 groups for
modification using thiol/ene, thiol/yne, or azide/alkyne click
chemistry. However, these methods require the use of
expensive unnatural amino acids or prior introduction of
unnatural functional groups or protecting groups onto
polypeptide side chains, adding substantial cost, and often use
linkages (i.e., esters)5−14 that are unstable to deprotection
conditions necessary to create copolypeptides containing other
functional residues (e.g., lysine, glutamic acid). Here we report
the development of a new “click”-type reaction for polypeptides
that utilizes the unique reactivity of the thioether group found
in the natural amino acid methionine. Because it is not
protonated at low pH methionine is the most reactive
nucleophile present in peptides and proteins under acidic
conditions.16−19 We have found that methionine can undergo
chemoselective, broad scope, highly efficient alkylation
reactions in homo- and copolypeptides yielding stable
sulfonium derivatives. These “methionine click” functionaliza-
tions are compatible with deprotection of other functional
groups, use an inexpensive, natural amino acid that is readily
polymerized and requires no side-chain modification or
Received: May 23, 2012
Revised: May 25, 2012
Published: May 25, 2012
© 2012 American Chemical Society
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the synthesis of well-defined poly(Met) and subsequently
expand its alkylation chemistry as a means to create
polypeptides containing diverse and reactive functionality.
Our lab recently reported a method for straightforward
preparation of Met NCA in high yield and high purity,37 which
enables the further development of methionine polymers. We
also showed that statistical copolymers of equimolar Met NCA
with Nε-CBZ-L-lysine N-carboxyanhydride (Z-Lys NCA) could
be prepared with controlled chain lengths and narrow chain
length distributions using (PMe3)4Co initiator in THF. We
now report that homopolymerizations of Met NCA (eq 1) also
modification (i.e., alkylation) of the free thiol groups
challenging.15 For initial studies, poly(Met) was reacted with
a variety of alkylating reagents in DMF, deionized water, or 0.2
M aqueous formic acid. We observed, similar to previously
reported reactions on methionine amino acid,27−31 that only
activated alkyl bromides and iodides were able to react
efficiently with poly(Met) under mild conditions (Figure 2).
go to completion within a few hours at room temperature, yet
molecular weight analysis of these chains was prohibited by
aggregation of poly(Met).38 To determine chain lengths, we
polymerized Met NCA at different monomer-to-(PMe3)4Co
initiator ratios, and after complete monomer consumption,
active chains were end-capped with isocyanate terminated PEG
(Mn = 2000 Da).39 Compositional analysis of purified, end-
1
capped polymers by H NMR gave average poly(Met) chain
lengths that increased linearly with stoichiometry (Figure 1).
Figure 2. Alkylation of poly(Met). Reagents and conditions: R-X in
DMF, H2O, or 0.2 M aqueous formic acid, 20 °C. Yield is total isolated
yield of completely functionalized polypeptide. (a) Product was
dialyzed against 0.1 M aqueous NaCl to give X = Cl. (b) X = Br. (c) X
= I.
The significant exception in this comparison was allyl bromide/
iodide, which has been reported to alkylate methionine amino
acid,42 yet was found by us to react only sluggishly with
poly(Met) (vide infra). In addition to the previously described
products 3 and 4,32 these direct alkylation reactions were used
to prepare a number of new sulfonium derivatives of
poly(Met). Various haloacetyl derivatives reacted readily and
quantitatively to generate polysulfoniums bearing amide (5),
ester (6), and active ester (7) functionalities, where the latter
may be useful for further derivatization with nucleophiles such
as primary amines. Propargylic and benzylic/pseudobenzylic
halides also reacted efficiently with poly(Met) and allow the
introduction of a variety of useful functional groups into
polypeptides. This method for introduction of alkyne
functionality (8) is straightforward and is more economical
than other routes to install this click reactive group onto
polypeptides.5−8 The facile formation of pyridine-containing
sulfoniums (9, 10) allows incorporation of this basic
functionality that is otherwise difficult to introduce in
polypeptides. Phenyl-boronic-acid-containing polypeptides
(11) have also been of interest for their sugar-binding
abilities43,44 and now can be readily prepared with high degrees
of incorporation in a single step.
◆
Figure 1. (a) Molecular weight (Mn, ) as a function of monomer to
initiator ratio ([M]/[I]) for poly(Met) prepared by polymerization of
Met NCA using (PMe3)4Co in THF at 20 °C. (b) GPC
chromatogram (normalized LS intensity versus elution time in
arbitrary units (a.u.)) of glycopolypeptide 15, Mw/Mn = 1.14.
Although chain length distributions of these poly(Met) samples
could not be obtained, GPC analysis of an alkylated poly(Met)
(vide infra) was found to possess a narrow polydispersity index
(Mw/Mn) of 1.14, indicating that the parent poly(Met)s are also
well-defined (Figure 1). Poly(Met) was prepared in high yield
with precisely controlled chain lengths up to over 400 residues
long and could also be prepared as statistical and diblock
copolymers with other amino acids. (See the Supporting
Information (SI).) Overall, these data show that Met NCA,
similar to other NCAs,40,41 is able to undergo living
polymerization when initiated with (PMe3)4Co.
Although α-helical poly(Met) has low solubility in many
solvents, DCM and TFA being notable exceptions, it is soluble
enough in a variety of different media to allow facile alkylation.
This property provides a significant advantage over poly(L-
cysteine), which forms insoluble β-sheets that prohibit
preparation of long chains as well as make high yield
In contrast with the results above, unactivated alkyl halides,
especially those without adjacent multiple bonds, reacted either
sluggishly or not at all with poly(Met) under similar conditions.
This result is not surprising because in all previous work on
methionine alkylation using amino acids, peptides, or proteins
there are no reports of sulfonium formation using unactivated
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reagents.16−19,27−31,42 To expand further the scope of poly-
(Met) alkylation, we explored different means to increase the
reactivity of unactivated alkyl halides. Silver tetrafluoroborate is
known to promote thioether alkylation in small molecules,45,46
and we found that the addition of this reagent to alkylations in
acetonitrile promoted the complete reaction of poly(Met) with
unactivated alkyl halides (e.g., haloethyl compounds) (Figure
3). This strategy allowed the introduction of an even wider
Figure 4. Alkylation of poly(Met) using alkyl triflates. Reagents and
conditions: R-OTf, DCM/MeCN, 20 °C. Yield is total isolated yield of
completely functionalized polypeptide. (a) X = OTf. (b) Product was
dialyzed against 0.1 M aqueous NaCl to give X = Cl.
Figure 3. Alkylation of poly(Met) using AgBF4. Reagents and
conditions: R-X, AgBF4, MeCN, 50 °C. Yield is total isolated yield
of completely functionalized polypeptide. (a) Product was dialyzed
against 0.1 M aqueous NaCl to give X = Cl. (b) Product was dialyzed
against aqueous HCl at pH 2 with 0.1 M NaCl to give the polyketone,
14a, and X = Cl. (c) X = BF4.
Alkyl triflate modification of poly(Met) was also found to be a
facile method to introduce the azide functional group (17),
which gave polypeptides bearing the click reactive counterpart
to the alkyne groups introduced above. Other functional groups
that required silver salts for introduction onto poly(Met) could
also be introduced by the use of the corresponding alkyl
triflates. Therefore, ether (18) and glycoside (19) function-
alities were added to poly(Met) via the corresponding alkyl
triflates. Removal of the acetyl protecting groups from the
sugars in polypeptide 19 gave a water-soluble glycopolypeptide
with no signs of any degradation. (See the SI.) In general, all of
the above poly(Met) alkylations were found to cause no
polypeptide chain cleavage and gave polysulfoniums that were
stable in a variety of media, at different pH (2 to 10), at
elevated temperature (80 °C), and after storage for more than 3
months. (See the SI.)
The results above confirm that alkylation of poly(Met) is a
highly efficient process for preparation of functionalized
polypeptides. Many of the polysulfoniums reported here are
the first examples of methionine derivatives bearing these
reactive or functional groups, and, compared with other
methods, these reactions are also very cost-effective and
straightforward ways to introduce these groups onto poly-
peptides. We have shown that alkylation of poly(Met) is broad
in scope, is high yielding, occurs readily, can use equimolar
reagents, has a single reaction trajectory, gives stable products,
and purification of products is facile: characteristics that define
“click”-type reactions.3 For methionine alkylation to be
considered a “click” reaction, it must also be chemoselective.3
In peptides, polypeptides, and proteins, there can be many
nucleophilic functional groups that react with alkylating
reagents.42 Of these, the cysteine thiol group is most widely
utilized in protein and peptide alkylation reactions,53 as it
typically gives single products, in contrast with lysine amines
and histidine imidazoles, which give multiple alkylation
products. It is noteworthy that in water the thiol, amine, and
imidazole functional groups are highly reactive with alkylating
agents at near neutral pH and above, but their reactivity
diminishes greatly with decreasing pH.42,54 This trend is due to
protonation of all these nucleophilic amino acids at low pH,
variety of reactive and functional groups onto poly(Met), giving
additional new reactive and functional polypeptides. In
addition, use of silver salts now facilitated the facile
incorporation of alkene functionality (12) onto polypeptides,
which is useful for a variety of further modifications including
thiol/ene click reactions.9,10 Ethylene glycol derivatives (13)
were added to impart the water solubilizing and passivating
properties of PEG.47,48 Reactive ketone groups, useful for
conjugation in aqueous environments,49 were introduced in a
single step using the 1,3-dioxolane derivative (14) that
deprotects to give the water-soluble polyketone during acidic
workup. Glycopolypeptides, of interest as mimics of natural
glycoproteins,50,51 often require many synthetic steps, especially
for preparation of longer chains with high sugar content. Using
iodoethyl glycosides, high-molecular-weight and well-defined,
fully glycosylated polypeptides (15) were readily prepared in
excellent yield by poly(Met) alkylation.
Because the use of silver salts may not be desirable in some
applications, we also explored functionalization of poly(Met)
with alternative reactive reagents. Alkyl triflates are known to be
powerful alkylating agents,52 and, although these have not been
used to alkylate methionine in amino acids, peptides, or
proteins, we found that these react efficiently and cleanly with
poly(Met). Functional alkyl triflates were prepared in a
straightforward manner from a variety of hydroxyalkyl
compounds. These reagents reacted efficiently with poly(Met)
in organic solvents under mild conditions to give the fully
alkylated polymers (Figure 4). Because of the significant
difference in reactivity between alkyl triflate and bromide, this
method also allowed incorporation of alkyl bromide function-
ality onto poly(Met) (16). This electrophilic functionality can
be readily modified by subsequent reaction with different
nucleophiles, such as amines or thiols. We reacted poly-
sulfonium 16 with aminomethane sulfonic acid, which gave
quantitative incorporation of sulfonate functionality (16a) that
may be useful in mimicking sulfonated biopolymers (eq 2).
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which greatly decreases their reactivity. Because methionine is
more resistant to protonation, it is consequently the only highly
reactive nucleophilic amino acid at pH <3.16−19,27−31 To
highlight this chemoselectivity and show its utility for selective
alkylation of methionine in polypeptides, we prepared a
statistical copolymer of methionine and lysine and studied its
alkylation (Scheme 1). We chose lysine as a competing
groups are stable and useful for further polypeptide
modifications.
In summary, the alkylation of methionine residues in
polypeptides has all the features of a “click” reaction,2 and
consequently is an attractive means for preparation of
functionalized and side-chain reactive polypeptides. Aside
from the examples given here, these methods should also be
applicable to a variety of other alkylating reagents and
thioether-containing residues, such as S-alkyl cysteines. In
comparison with other methods for installation of functional
and click reactive groups onto polypeptides, the starting
material methionine is substantially less expensive than the
side-chain-modified or unnatural amino acids typically
employed, and poly(Met) is readily prepared with controlled
and high molecular weights, which makes these “methionine
click” reactions attractive for large-scale use. Facile incorpo-
ration of other click-reactive functional groups (e.g., alkyne,
azide, or alkene) by methionine alkylation also allows for
further chemoselective modification of polypeptides. Such
“secondary click” strategies, as shown in Scheme 1, allow
methionine alkylation to also utilize the broad diversity of
reagents already developed and available for existing click
conjugation methods.1,2
Scheme 1. Schematic Showing Chemoselective Alkylation of
Methionine Residues in the Presence of Excess Amine
Groups, Followed by Clicking of PEG-N3 to the Alkylated
a
Methionine Residues
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and spectral data for all new
compounds, polymerization data, and Mn versus [M]/[I]
plots. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: demingt@seas.ucla.edu. Fax: (+1) 310-794-5956.
■
a
Initial copolymer is poly[(Nε-TFA-L-lysine)0.8-stat-(Met)0.2]206, where
Notes
0.8 and 0.2 are mole fractions of lysine and methionine residues,
respectively. Reagents and conditions: (a) K2CO3, MeOH, H2O
(99%); (b) propargyl bromide, 0.2 M formic acid (94%); (c) α-
methoxy-ω-azidoethyl-poly(ethylene glycol) (Mn = 1000 Da), CuSO4,
ascorbic acid, PMDETA, H2O (95%).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the IUPAC Transnational Call in
Polymer Chemistry and the NSF under award nos. MSN
1057970 and DMR 0907453.
nucleophile because it is the most abundant nucleophile found
in proteins, it is more widely used in synthetic polypeptides
compared with histidine or cysteine, and it is known to
compete with thiol and imidazole groups in protein
alkylations.42,53 As is also true for cysteine and histidine
polypeptides, the synthesis of poly(L-lysine) requires the use of
protecting groups, and methionine alkylation needs to be
compatible with and orthogonal to deprotection chemistry to
be useful as a “click” reaction. Here we found that the
methionine residues of our copolymer could be alkylated with
propargyl bromide either before or after lysine deprotection to
give the same final product. These results show that methionine
sulfonium groups are stable to deprotection reactions (see the
SI) and alternatively that methionine residues can be alkylated
chemoselectively at pH 2.4 in the presence of a four-fold excess
of free amine groups (Scheme 1). Confirming this selectivity, a
control reaction of propargyl bromide with pure poly(L-lysine)
under identical conditions gave no alkylation products. (See the
Supporting Information.) All of the alkyne groups in the
copolymer prepared above were then quantitatively conjugated
with azide-terminated PEG chains (Scheme 1), showing that
alkyne reactivity is not compromised and that the sulfonium
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