Multimodal switching of conformation and solubility in homocysteine derived polypeptides

Article abstract of DOI:10.1021/ja500372u

We report the design and synthesis of poly(S-alkyl-l-homocysteine)s, which were found to be a new class of readily prepared, multiresponsive polymers that possess the unprecedented ability to respond in different ways to different stimuli, either through

Full text of DOI:10.1021/ja500372u

Communication
pubs.acs.org/JACS
Multimodal Switching of Conformation and Solubility in
Homocysteine Derived Polypeptides
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 design and synthesis of
poly(S-alkyl-L-homocysteine)s, which were found to be a
new class of readily prepared, multiresponsive polymers
that possess the unprecedented ability to respond in
different ways to different stimuli, either through a change
in chain conformation or in water solubility. The
responsive properties of these materials are also effected
under mild conditions and are completely reversible for all
pathways. The key components of these polymers are the
incorporation of water solubilizing alkyl functional groups
that are integrated with precisely positioned, multi-
Figure 1. Schematic drawing showing multimodal environmentally
responsive behavior of poly(homocysteine) derivatives, which are
represented as coils and α-helices that are hydrophobic (red) or
hydrophilic (blue). Red = reduction; Ox = oxidation; Alk = alkylation;
responsive thioether linkages. This promising system
allows multimodal switching of polypeptide properties to
obtain desirable features, such as coupled responses to
multiple external inputs.
DeAlk = dealkylation; T = temperature; red arrows = reaction above
40 °C; blue arrows = reaction below 35 °C.
timuli responsive polymers are promising materials that
the responsive component typically undergoes a transition
Saspire to mimic responsive and adaptive biological systems,
between hydrophobic and hydrophilic states or a change in
chain conformation.4 While most of these materials respond
such as proteins and protein complexes that can react to
changes in pH, oxidation, and other stimuli.1 Polypeptides offer
only to a single type of stimulus, Hammond’s lab has reported
advantages as protein mimics since they adopt ordered chain
conformations (e.g., α-helices) that can also respond to stimuli
remarkable copolypeptides that are able to respond to both
temperature and pH.10 While these materials are sensitive to
and influence properties. Responsive polypeptides have
practical applications in devices and therapeutic delivery, such
as thermoresponsive hydrogel formation for cell scaffold
formation in vivo,2 or oxidation responsive nanocarrier
disruption for triggered drug release.3 However, synthetic
polypeptides are often only able to respond to a single stimulus
and sometimes only in an irreversible manner.4 The develop-
ment of polypeptides that can reversibly respond under mild
conditions to multiple stimuli, and in a distinct, predictable
manner for each stimulus, is an important step toward
realization of more sophisticated and useful materials.1 For
example, a polypeptide that undergoes a chemically induced
conformational change that in turn switches on thermores-
ponsive behavior would be valuable for creation of triggered-
responsive systems. Addressing this challenge, we have
developed polypeptides capable of responding to multiple
external stimuli (chemical, redox, temperature) by fully
reversible changes in their chain conformation or aqueous
solubility (Figure 1). This unique system allows multimodal
switching of polypeptide properties to obtain desirable features,
such as coupled responses to multiple external inputs.
two different stimuli, the polypeptide chains have only a single
response to both, a change in solubility. In effort to develop
polypeptides that are able to function as true multiresponsive
materials, i.e., able to react differently to different stimuli, we
have explored the chemistry and properties of polypeptides
containing side-chain thioether functional groups. We pre-
viously prepared thioether containing glycopolypeptides based
on L-cysteine residues, which were found to undergo α-helix to
coil transitions upon oxidation, while remaining water soluble.6
One limitation of this system was the irreversibility of this
transition, since the thioether groups had to be fully oxidized to
sulfones. Analogous α-helical glycopolypeptides based on L-
homocysteine residues behaved differently and remained α-
helical as sulfones.6
In related studies on oxidation of α-helical poly(L-
methionine), we observed that the fully oxidized poly(L-
methionine sulfone) also remains α-helical,11 similar to the
glycosylated poly(L-homocysteine)s.6 However, limited oxida-
tion to give poly(L-methionine sulfoxide) yielded a highly
water-soluble disordered coil that can be reduced back to the
Water-soluble polypeptides are known that are responsive to
Received: January 13, 2014
Published: April 2, 2014
temperature,5 oxidation,6 pH,7 light,8 or sugar binding,9 where
© 2014 American Chemical Society
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parent polymer under mild conditions.11 In a separate study, we
found that poly(L-methionine) can also be alkylated in high
yield to give water-soluble disordered coils in a fully reversible
process.12 While poly(L-methionine) readily undergoes these
redox and chemically induced reversible α-helix to coil
transitions, the poor water solubility of poly(L-methionine)
limits its utility as a stimuli responsive polypeptide. Here, we
sought to utilize the favorable molecular features found in
methionine to design and prepare analogous S-alkyl-L-
homocysteine based polypeptides, where water solubilizing
alkyl functional groups are connected to the multiresponsive
thioether linkages. The goal was to create water-soluble,
multistimuli responsive polypeptides capable of responding
reversibly and distinctly to temperature, alkylation, and
oxidation (Figure 1).
We began by re-examining the glycopolypeptide, poly(α-gal-
CH), 5 (Scheme 1, see Scheme S1), which shares the same core
⧫
Figure 2. (A) Molecular weight (Mn, ) and polydispersity index
H
■
(Mw/Mn, ) of poly(α-gal-C ), 5, as functions of monomer to
initiator ratio ([M]/[I]) using (PMe3)4Co initiator in THF at 20 °C;
(B−D) CD spectra, 0.1 mg/mL in DI water, 20 °C, molar ellipticity is
reported in deg·cm2·dmol−1. (B) Reversible oxidization of 5 (average
degree of polymerization =145); (C) reversible alkylation of 5; (D)
irreversible alkylation of 5. * = alkylated or oxidized polypeptide after
reduction using thioglycolic acid for 5o or 2-mercaptopyridine for 5Bn
and 5Me.
Scheme 1. Conformational Switching of Poly(homocysteine)
Derivatives
a
α-helical signatures and instead was indicative of a disordered
coil conformation (Figure 2B).15 This conformational switch
was completely reversible, since reduction of 5o with
thioglycolic acid regenerated unmodified, α-helical 5 (Figure
2B). Alkylation of 5 gave similar results, yet its chain
conformation could be switched either reversibly or perma-
nently, depending on the choice of alkylating agent. Alkylation
of 5 with a benzylic halide yielded water-soluble polysulfonium
5Bn, which adopted a disordered coil conformation (Figure
2C). This reaction was reversed to regenerate 5 by quantitative
dealkylation using 2-mercaptopyridine, as described previously
for poly(L-methionine) derivatives.12 Alkylation of 5 with
methyl iodide also yielded a conformationally disordered,
water-soluble polysulfonium, 5Me, yet this is a one-way switch
since this alkylation was irreversible (Figure 2D).12 5 and its
derivatives all remained water soluble at elevated temperature
(80 °C).
a
(a) H2O2, AcOH, H2O; (b) 4-bromomethyl phenyl acetic acid or
methyl iodide, H2O; dialysis with NaCl; (c) thioglycolic acid, H2O;
(d) 2-mercaptopyridine, H2O. Pathway d occurs for 5Bn and 8Bn, but
not for 5Me and 8Me.
These initial results showed that the combination of
optimally positioned reactive thioether groups and water
solubilizing sugar functionality found in 5 gave a soluble
polypeptide capable of reversibly switching between α-helix and
coil conformations in response to different stimuli. To test the
generality of this design and to add a thermoresponsive
element, we prepared new S-alkyl-L-homocysteine based
polypeptides containing water solubilizing ethylene glycol
(EG) repeats. Since it is known that attachment of short EG
segments onto polymer, and polypeptide, side-chains can
impart thermoresponsive behavior in water,5,10 we prepared
alkylated poly(L-homocysteine) derivatives containing either 2
or 4 EG repeats (poly(EG2-CH), 8a, and poly(EG4-CH), 8b,
respectively) (Scheme 2). Similar to α-gal-CH NCA, EG2-CH
NCA and EG4-CH NCA, obtained in high purity after
chromatography,16 were found to polymerize efficiently using
(PMe3)4Co initiator in THF at room temperature (see Tables
S2 and S3).14 Variation of NCA to initiator ratios gave
polypeptides whose lengths increased linearly with stoichiom-
etry and which possessed narrow chain length distributions
(Mw/Mn) (Figure 3A, see Figure S1). While polypeptide 8b
structure as poly(methionine) yet is water soluble. Like other
glycosylated α-amino acid-N-carboxyanhydride (NCA) mono-
mers we have reported,6,13 α-gal-CH NCA was found to
polymerize efficiently using (PMe3)4Co initiator in THF at
room temperature (see Table S1).14 Variation of α-gal-CH
NCA to initiator ratios gave glycopolypeptides whose lengths
increased linearly with stoichiometry and which possessed
narrow chain length distributions (Mw/Mn) (Figure 2A). After
removal of protecting groups, water-soluble, α-helical 5 was
obtained with reproducible and precisely controlled chain
lengths up to over 300 residues long.
To study the ability of 5 to respond to stimuli, samples were
subjected to either chemical alkylation12 or mild oxidation11
(Scheme 1). Unmodified 5 gave a circular dichroism (CD)
spectrum with characteristic minima at 208 and 222 nm,
indicating a greater than 95% α-helical conformation in DI
water at 20 °C (Figure 2B).15 Upon oxidation with 1% H2O2 to
the corresponding sulfoxide, 5o, the polypeptide remained
water soluble yet its CD spectrum showed complete loss of the
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a
Scheme 2. Synthesis of Poly(EGx-CH)
a
(a) 2M NaOH, H2O (95-98% yield); (b) Cl2CHOMe, DCM, 50 °C
(64-69% yield); (c) (PMe3)4Co, THF (90-99% yield).
Figure 4. (A) Influence of temperature on light transmittance (500
nm) through a sample of aqueous poly(EG4-CH)150, 8b. Solid red line
= heating; dashed blue line = cooling; 1 °C min−1. (B) Reversible
change in optical transmittance of aqueous 8b when temperature was
alternated between 30 °C (high transmittance) and 45 °C (low
transmittance); 5 min per each heating/cooling cycle. (C) Cloud point
temperatures of 8b measured in different Hofmeister salts (Na+
counterion) at concentrations up to 1.0 M. All polypeptides were
prepared at 3 mg/mL.
⧫
Figure 3. (A) Molecular weight (Mn, ) and polydispersity index
H
■
(Mw/Mn, ) of poly(EG4-C ), 8b, as functions of monomer to
sample (Figure 4B). The chain conformations of 8b were found
to remain highly α-helical when samples were heated well
above the cloud points, indicating that a change in chain
conformation was not responsible for the observed thermor-
esponsive behavior (see Figures S6−S8). The more hydrophilic
derivatives 8o and 8Bn were not thermoresponsive and
remained soluble in water or PBS buffer at elevated
temperature (80 °C) (see Figure S9). The observed cloud
point temperatures for 8b were higher (∼50 °C) at lower
molecular weights but were found to plateau at ∼40 °C for
chains greater than 50 residues long (see Figure S10). In PBS
buffer, the cloud point temperature of 8b decreased from 40 to
37 °C due to slight salting out of the polymer (see Figure S11).
To study this effect in more detail, we examined solutions of 8b
in the presence of different Hofmeister anions, since anions are
known to affect thermoresponsive properties of polymers more
than cations (Figure 4C).18 The effects of different salt
concentrations on the cloud point temperatures of polymer
8b followed trends similar to those seen with other
thermoresponsive polymers and allow some tuning of the
transition temperature.18,19 Similar to other thermoresponsive
polypeptides,5,10 it also is likely that the cloud point
temperature could be varied by copolymerization of EG4-CH
NCA with EGx-CH NCAs containing different EG repeat
lengths. Overall, polypeptide 8b was found to possess reversible
thermoresponsive behavior in water by undergoing a transition
between hydrophobic and hydrophilic states, with no change in
chain conformation.
initiator ratio ([M]/[I]) using (PMe3)4Co in THF at 20 °C; (B−D)
CD spectra, 0.1 mg/mL in DI water, 20 °C, molar ellipticity is
reported in deg·cm2·dmol−1. (B) reversible oxidization of poly(EG4-
CH)150, 8b; (C) reversible alkylation of 8b; (D) irreversible alkylation
of 8b. * = alkylated or oxidized polypeptide after reduction using
thioglycolic acid for 8o or 2-mercaptopyridine for 8Bn and 8Me.
was very soluble in water at all chain lengths tested, polypeptide
8a was only fully water soluble when <25 residues long and
hence not a good candidate as a thermoresponsive polymer.
When oxidized or alkylated, 8a was found to be water soluble
and show reversible changes in chain conformation (see Figures
S2−S4), yet these derivitatives were found to possess no
thermoresponsive properties in water (see Figure S5). With
excellent water solubility similar to glycopolypeptide 5, 8b was
found to adopt a >95% α-helical conformation in DI water at
20 °C (Figure 3B).15 Oxidation of 8b to give 8o or alkylation
with a benzylic halide to give 8Bn were also both found to
reversibly switch the chain conformations from α-helices to
coils without affecting solubility (Figure 3B,C). Irreversible
alkylation of 8b with methyl iodide also yielded a conforma-
tionally disordered, water-soluble polysulfonium, 8Me (Figure
3D). These results show that the water solubilizing EG repeats
in 8b are viable substitutes for the sugar residues in 5, as both
provide water solubility and confer similar responsiveness to
oxidation and alkylation. Based on comparison to other EG
containing polypeptides,5 8b was also anticipated to display
thermoresponsive behavior in water.
Poly(S-alkyl-L-homocysteine)s, as exemplified by polypeptide
8b, are a new class of readily prepared, multiresponsive
polymers that possess the unprecedented ability to respond
differently to different stimuli, either through a change in
conformation or in water solubility. Their responsive properties
are also effected under mild conditions and are fully reversible
for all pathways. The completely decoupled multiresponsive
Upon heating aqueous samples of 8b, sharp transitions from
clear solutions to opaque suspensions were observed, indicative
of the presence of a lower critical solution temperature for this
polypeptide (Figure 4A).17 These transitions were completely
reversible and could be repeated multiple times with no
observable persistent precipitation or other changes to the
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(16) Kramer, J. R.; Deming, T. J. Biomacromolecules 2010, 11, 3668.
(17) Laloyaux, X.; Fautre, E.; Blin, T.; Purohit, V.; Leprince, J.;
Jouenne, T.; Jonas, A. M.; Glinel, K. Adv. Mater. 2010, 22, 5024.
(18) Zhang, Y.; Furyk, S.; Bergbreiter, D. E.; Cremer, P. S. J. Am.
Chem. Soc. 2005, 127, 14505.
nature of these polypeptides makes them particularly attractive
as components in molecular devices or nanoscale assemblies
capable of sequential, or triggered, responses to different
stimuli, akin to switches capable of performing Boolean-like
operations.20 For example, water-soluble polypeptide 8Bn will
switch to a water insoluble state only when presented with a
dealkylation trigger AND an elevated temperature trigger. Such
triggers may also be provided biologically, and studies to use
these polymers under biological conditions are underway.11,12
Overall, the potential for tunability of poly(S-alkyl-L-
homocysteine)s, combined with their exceptional multires-
ponsive properties, makes them promising candidates for a
broad range of stimuli responsive material challenges.
(19) Deyerle, B. A.; Zhang, Y. Langmuir 2011, 27, 9203.
(20) Szaciłowski, K. Chem. Rev. 2008, 108, 3481.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and spectral data for all new
compounds, as well as additional polymerization data, Mn vs
[M]/[I] plots, temperature response studies, and CD spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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 MSN 1057970.
■
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