Glycopolypeptides with a redox-triggered helix-to-coil transition

Article abstract of DOI:10.1021/ja3007484

Conformation-switchable glycopolypeptides were prepared by the living polymerization of glycosylated l-cysteine-N-carboxyanhydride (glyco-C NCA) monomers. These new monomers were prepared in high yield by coupling of alkene-terminated C-linked glycosides of d-galactose or d-glucose to l-cysteine using thiol-ene "click" chemistry, followed by their conversion to the corresponding glyco-C NCAs. The resulting glycopolypeptides were found to be water-soluble and α-helical in solution. Aqueous oxidation of the side-chain thioether linkages in these polymers to sulfone groups resulted in disruption of the α-helical conformations without loss of water solubility. The ability to switch chain conformation and remain water-soluble is unprecedented in synthetic polymers, and allows new capabilities to control presentation of sugar functionality in subtly different contexts.

Full text of DOI:10.1021/ja3007484

Communication
pubs.acs.org/JACS
Glycopolypeptides with a Redox-Triggered Helix-to-Coil Transition
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
The ability of simple synthetic homopolypeptides, such as
ABSTRACT: Conformation-switchable glycopolypepti-
des were prepared by the living polymerization of
glycosylated L-cysteine-N-carboxyanhydride (glyco-C
NCA) monomers. These new monomers were prepared
in high yield by coupling of alkene-terminated C-linked
glycosides of D-galactose or D-glucose to L-cysteine using
thiol−ene “click” chemistry, followed by their conversion
to the corresponding glyco-C NCAs. The resulting
glycopolypeptides were found to be water-soluble and α-
helical in solution. Aqueous oxidation of the side-chain
thioether linkages in these polymers to sulfone groups
resulted in disruption of the α-helical conformations
without loss of water solubility. The ability to switch
chain conformation and remain water-soluble is unprece-
dented in synthetic polymers, and allows new capabilities
to control presentation of sugar functionality in subtly
different contexts.
poly-L-lysine or poly-L-glutamate, to undergo helix-to-coil
transitions in aqueous solution is well known.11 In these
polymers, the transition occurs upon protonation or deproto-
nation of the side-chain functional groups where the charged
polymers are disordered and the neutral forms adopt stable α-
helices. The resulting uncharged, α-helical polypeptides are
sparingly soluble in water and precipitate above micromolar
concentrations, essentially making these helix-to-coil transitions
a solubility switch.11 Numerous conformation switchable
synthetic polypeptides have been reported, including those
that respond to pH,12 light,13 or sugar binding,14 but none has
good water solubility in both the ordered and disordered
conformational states. Polypeptides that undergo helix-to-coil
transitions and remain soluble in organic solvents are known,15
but these are not amenable to biomimetic studies or
biotechnological applications. To obtain polypeptides that can
undergo a conformational change and remain water-soluble, we
prepared glycopolypeptides based on L-cysteine residues
(Schemes 1 and 2). The key design features in these polymers
he development of stimuli-responsive polymers has
a
Treceived much recent interest. These materials are Scheme 1. Synthesis of glyco-C NCA Monomers
promising since they aim to mimic adaptive biological systems,
trigger release of therapeutics, or amplify signals in biosensors.1
Polymers have been prepared that are responsive to light,2 pH,3
temperature,4 or oxidation,5 where the responsive component
typically undergoes a transition between hydrophobic and
hydrophilic states.6 Polypeptide and protein materials offer an
additional stimulus response mechanism since they adopt
ordered chain conformations (e.g., α-helices) that can be
disrupted under mild conditions. Thermal responsive elastin
a
Reagents and conditions: (a) CBz-Cl, THF:H2O 1:9, NaOH (98%
mimetics7 and pH responsive synthetic polypeptides8 have both
yield); (b) PPh3, THF:H2O 9:1 (98% yield); (c) 2a or 2b, 2,2-
dimethoxy-2-phenylacetophenone, DMF, 365 nm (94% yield); (d)
taken advantage of ordered/disordered conformation tran-
sitions to drive the formation or disruption of aqueous self-
assemblies such as vesicles and micelles. However, these
conformational changes act primarily as a solubility switch, and
thus do not replicate the more subtle structural changes found
in biological systems, such as in motor proteins9 and membrane
proteins involved in signal transduction,10 where bulk phase
separation does not occur. Addressing this challenge, we have
developed new glycopolypeptides that undergo α-helix-to-coil
transitions upon oxidation and possess unprecedented good
water solubility in both conformational states. A fine energetic
balance was achieved by careful placement of oxidizable
thioether groups combined with solubilizing and functional
sugar moieties to give polymers with switchable, soluble
conformations potentially useful for applications in sensing,
diagnostics, and biomimetics.
Cl2CHOMe, DCM, 50 °C (63−67% yield). 4a = α-gal-C NCA (R1 =
OAc; R2 = H). 4b = α-glc-C NCA (R1 = H; R2 = OAc).
are the incorporation of thioether linkages and stable 100%
glycosylation via use of C-linked glycosides. The polar
glycosides provide excellent nonionic, pH- and buffer-tolerant
water solubility, and add the potential for selective binding to
biological targets, such as sugar binding cell surface receptors.
The thioether groups provide an oxidation sensitive switch
from less-polar thioether to highly polar sulfone groups.
We had previously prepared stable glycopolypeptides based
on L-lysine residues, using amide linkages to attach different
Received: January 23, 2012
Published: February 23, 2012
© 2012 American Chemical Society
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Communication
Scheme 2. Polymerization of glyco-C NCAs and
a
Glycopolypeptide Deprotection
a
Reagents and conditions: (a) (PMe3)4Co, THF (98% yield); (b)
NH2NH2·H2O, (95% yield). For 4a and 5a: R1 = OAc; R2 = H. For 4b
and 5b: R1 = H; R2 = OAc. For 6a (poly(α-gal-C)): R3 = OH; R4 = H.
For 6b (poly(α-glc-C)): R3 = H; R4 = OH.
◆
Figure 1. (a) Molecular weight (Mn, ) and polydispersity index
■
(Mw/Mn, ) of poly(α-glc-C) as functions of monomer to initiator
ratio ([M]/[I]) using (PMe3)4Co in THF at 20 °C. (b) GPC
chromatograms (normalized LS intensity in arbitrary units (a.u.)
versus elution time) of glycopolypeptides after initial polymerization of
α-glc-C NCA to give a poly(α-glc-C)58 homoglycopolypeptide (A)
and after chain extension by polymerization of Nε-CBz-L-lysine-N-
carboxyanhydride to give a poly(α-glc-C)58-block-poly(CBz-Lys)290
diblock glycopolypeptide (B).
monosaccharides.16 These polymers displayed high α-helical
contents in aqueous media and only became partially
disordered when heated to ∼90 °C. L-Lysine is an inherently
helicogenic residue,11which combined with the ability of side-
chain amide groups to cooperatively H-bond favored formation
of stable α-helices in this polymer. To obtain glycopolypeptides
with lower helical stability, we chose to build off of L-cysteine,
since the presence of the β-heteroatom weakens formation of
α-helices.11 Synthetic glycopolypeptides using O- and S-linked
glycoside derivatives of L-serine and L-cysteine have been
prepared,17 yet this approach is not practical due to difficulties
both in monomer synthesis and polymerization, and these
materials also do not allow for conformational switching. In our
design, we used the method of Dondoni to attach the alkene-
terminated C-linked glycosides of D-galactose and D-glucose to
L-cysteine using thiol−ene “click” chemistry,18 followed by
conversion to the corresponding new α-amino acid N-
carboxyanhydride (glyco-C NCA) monomers (Scheme 1).
Although these monomers use non-native linkages, it has been
well demonstrated that C-linked glycopeptides can bind targets
with nearly equal affinity and conformation as native O-linked
analogues,19 and have been widely utilized when stable
glycoprotein mimetics are desired.20 Our approach allows
formation of the glycosylated amino acids in high yield, is
amenable to a variety of sugars, and imparts these residues with
many desirable features for polymer formation. The un-
branched, thioether-containing side chains are long and
hydrophobic and hence should favor α-helix formation in
water, the spacing of sugars away from the backbone should
relieve steric crowding and assist NCA polymerization, and the
incorporation of thioether groups allows for facile postpolyme-
rization stimulus response via oxidation.
The glyco-C NCA monomers were found to polymerize
efficiently using (PMe3)4Co initiator in THF at room
temperature (Scheme 2), yielding side-chain-protected poly-
mers in excellent yields (see Supporting Information (SI)).21
Variation of monomer to initiator ratios for each glyco-C NCA
gave glycopolypeptides whose lengths increased linearly with
stoichiometry, and which possessed narrow chain length
distributions (Mw/Mn). Data for the α-D-glucose-L-cysteine
monomer (α-glc-C NCA) are shown in Figure 1, and data for
the α-D-galactose-L-cysteine monomer (α-gal-C NCA) and
statistical copolymers are given in the SI. Soluble, 100%
glycosylated high-molecular-weight polypeptides were prepared
with reproducible and precisely controlled chain lengths up to
ca. 200 residues long, which is difficult to achieve using
postpolymerization glycosylation strategies.22 Chain extension
experiments using sequential monomer additions to see if
glyco-C NCAs can be incorporated into diblock copolymers
(Table 1, see SI) all proceeded in high conversion to yield
predictable compositions, and with no evidence of inactive
chains by GPC analysis (Figure 1). Overall, these data show
that the glyco-C NCAs were able to undergo living polymer-
ization when initiated with (PMe3) 4Co, similar to conventional
NCAs.21 Furthermore, both poly(α-glc-C) and poly(α-gal-C)
were found to have good water solubility after removal of
protecting groups (Scheme 2).
To investigate solution conformations, circular dichroism
(CD) spectra of the poly(glyco-C)s were measured in PBS
buffer (pH 7.4) after purification and removal of all residual
cobalt ions by extensive dialysis against deionized water. Both
poly(α-glc-C) and poly(α-gal-C) were found to be soluble in
both DI water and PBS buffer at 20 °C (>50 mM), and gave
CD spectra with characteristic minima at 208 and 222 nm
indicating predominantly α-helical conformations (Figure 2, see
SI).23 CD analysis of a thin film of poly(α-gal-C) cast from a
solution in DI water also showed that this polymer is
predominantly α-helical in the solid state (see SI). The
thioether groups in these polymers can be oxidized to either
sulfoxide or sulfone functionalities, both of which have
increased polarity. A similar change of polarity has also been
used by Hubbell who oxidized thioether groups in synthetic
copolymers to disrupt vesicular assemblies.5b Complete
oxidation of our poly(glyco-C)s using hydrogen peroxide/
acetic acid gave quantitative conversions to the sulfone
derivatives, poly(glyco-CO2)s, by NMR analysis. To confirm
these structures, we also prepared a fully characterized sulfone
monomer (α-gal-CO2 NCA), and polymerized it to obtain a
polymer that showed properties identical to oxidized poly(α-
gal-C) samples (see SI). We also found no polymer backbone
degradation after oxidation (see SI).
The oxidized poly(glyco-CO2)s also had good solubility in DI
water and PBS buffer at 20 °C (>50 mM), yet their CD spectra
showed loss of the α-helical minima at 208 and 222 nm.
Instead, the spectra were characteristic of random coil
conformations (Figure 2, see SI),11,23 and showed that
oxidation of the thioether groups to sulfones in poly(glyco-
C)s was able to destabilize the α-helices and cause a transition
to disordered conformations. Examination of the corresponding
sulfoxide, i.e. poly(α-gal-CO), prepared by milder oxidation of
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Table 1. Synthesis of Diblock Copolypeptides Using (PMe3)4Co in THF at 20 °C
b
c
first segment
diblock copolymer
a
a
d
e
first monomer
25 Lys NCA
second monomer
Mn
Mw/Mn
DP
Mn
131 880
88 330
Mw/Mn
DP
yield (%)
100 α-glc-C NCA
15 980
27 550
1.15
1.09
61
58
1.01
1.03
305
290
97
96
25 α-glc-C NCA
100 Lys NCA
a
First and second monomers added stepwise to the initiator; number indicates equivalents of monomer per (PMe3)4Co. Lys NCA = Nε-CBz-L-
b
lysine-N-carboxyanhydride. Molecular weight and polydispersity index after polymerization of the first monomer (as determined by GPC/LS).
c
d
Molecular weight and polydispersity index after polymerization of the second monomer (as determined by GPC/LS and 1H NMR). Total degree
e
of polymerization of diblock glycopolypeptide. Total isolated yield of diblock glycopolypeptide.
unprecedented switching from soluble α-helices to soluble
disordered conformations.
While other synthetic homopolypeptides undergo triggered
helix-to-coil transitions coupled with a change in water
solubility, the incorporation of sugars in our polymers imparts
good water solubility for both conformers. The solubility of
both conformations is advantageous since it allows for the
presentation of sugar functionalities from different polymer
conformations in solution, which may be used to tune their
interactions with biomolecules and biological surfaces in
different applications.24 Also, oxidative switching of the chain
conformations occurs in aqueous solution, making the helix-to-
coil transition amenable for use in devices in vitro, or in
aqueous self-assembled materials, such as in glycosylated
carriers for drug delivery. The oxidation of thioether groups
in proteins is known to occur naturally, and the consequent
perturbed folding can serve as a signal for the oxidized protein
to be degraded by the proteasome.25 In a similar manner, the
poly(glyco-C)s may also be able to sense oxidative environ-
ments in biological systems and respond to them by changing
conformation or morphology of self-assembled structures.
Figure 2. Circular dichroism spectra and corresponding structures of
parent and oxidized glycopolypeptides. (a) Conversion of poly(α-gal-
C) 6a to poly(α-gal-CO2) 7a. (b) Circular dichroism spectra of poly(α-
gal-C)108 Mn = 33 200 Da (solid line), and poly(α-gal-CO2
)108 Mn = 36
650 Da (dashed line). (c) Conversion of poly(α-gal-CH) 15a to
poly(α-gal-CHO2) 16a. d) Circular dichroism spectra of poly(α-gal-
CH)88 Mn = 28 280 Da (solid line) and poly(α-gal-CHO2
) Mn = 31
88
ASSOCIATED CONTENT
■
100 (dashed line). All samples analyzed at concentrations of 0.5 mg/
mL in PBS buffer at 20 °C. Molar ellipticity is reported in
deg·cm2·dmol−1. Conditions (i) = H2O2, H2O−AcOH (98% yield).
S
* Supporting Information
Experimental procedures and spectral data for all new
compounds; polymerization data, Mn vs [M]/[I] plots, X-ray
crystal structure of α-gal-CO2 NCA, and CD spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
poly(α-gal-C), revealed that this functionality with intermediate
polarity is not sufficient to destabilize the α-helical
conformation (see SI). The increased polarity of the sulfone
groups likely drives the helix-to-coil transition by interacting
strongly with solvent water molecules, which can disrupt
hydrophobic packing of the polypeptide side chains and
increase steric crowding around the backbone to destabilize
the α-helical conformation. This substantial change in chain
conformation from such small molecular changes in these
samples suggests that even subtle shifting in the balance of
forces that dictate chain conformation can have large effects.
This hypothesis was further validated by preparation and study
of the homologous sample poly(α-gal-CH), based on L-
homocysteine residues, where the thioether group is separated
from the peptide backbone by one additional methylene unit
(Figure 2, see SI). Poly(α-gal-CH) is similar to poly(α-gal-C) in
that both are water-soluble and α-helical; however, upon
AUTHOR INFORMATION
■
Corresponding Author
demingt@seas.ucla.edu
Notes
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 no. MSN
1057970.
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