Miriam’s comment on: Reversible Changes in Solution pH Resulting from Changes in Thermoresponsive Polymer Solubility

By Miriam I. Otero Rivera
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In this article by Bergbreiter’s group, present the effect of a LCST event on the solution pH of a polymer like poly(N isopropylacrylamide) better known as PNIPAM. They present different experiments were they change pendant groups on PNIPAM and all of these resulting polymer derivatives had lower critical solution temperature (LCST) properties. In the experiments they show how after heating the solution to its LCST the polymer experience a dehydration and the polymer phase separates from solution. In several experiments, the presence of a cationic or anionic pendant groups, that have low or high pH values respectively, its stabilization is dependent of its solvation, and after the LCST event, the species becomes neutral since it is more stable in the dehydrated state resulting from the LCST phenomenon. These experiments show that changes in the environment of the polymers results in great changes in solution acidity and basicity. Also they show that these process of hydration and dehydration is reversible by cooling the solution they obtain the polymer back in solution and the pH was revert to its initial value, this process of heating and cooling was reversible for up to 100 cycles.
Similarly, the article studied the effect of the concentration of the polymer, and the mole % loading of the pendant group (carboxylic acid) in changing the bulk solution pH as a function of temperature. First they saw that if the pH of the solution is significantly different from the pKa of the carboxylic acid (4.76) there is no change in pH. They also saw that at lower mole % loading of carboxylic acid the cloud point curve is most narrow and that at lower concentration of the polymer the clouding curves are broader. Also, studies with solutions containing added salts (LiCl or LiBr) show that the solution’s ionic strength does not significantly affect the ΔpH, although the clouding curve’s onset changes. Finally, they performed experiments with kosmotropic salts, which lower the LCST below room temperature due to the Hofmeister effect, as a result the addition of these salts to solutions lead to the same behavior of changing the pH of the solution, and also the process was reversible. This experiment was visualized using the pH-indicator phenolphtalein. In this experiment they also used visible spectroscopy to show how  reaching  the LCST temperature produce an inflection, before a drastic change in absorbance.  This article show how properties in the pendant group of a polymers change pH of solution as a result of changes in solubility because of changes in temperature, showing the thermoresponsiveness of polymers. This article was one of relevance to our laboratory because we work with LCST events on supramolecular G-quadruplexes. This article can be used as a reference to know the resulting effects on solubility and pH of an LCST event on the systems with which we work in our lab.


Rafa’s comment on: Thermal Switching of Thermoresponsive Polymer Aqueous Solutions

By Rafael A. Brito

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Thermal switches are of great importance to thermal management in a wide variety of applications. A common characteristic associated with thermal switching is thermal conductivity. After noticing the change in thermal conductivity across LCST transitions, Zhiting Tian started researching polymers for this purpose. Poly(N-isopropylacrylamide) (PNIPAM) is the most studied thermoresponsive polymer and has a workable LCST of 32° C. The LCST transition of PNIPAM changes the chain-like formation of the polymer into an aggregation that shows a drastic decrease of thermal conductivity. This sharp change is due to LCST transitions being second-order, which are characterized by being almost instantaneous when the corresponding temperature is reached. Thermal conductivity was measured by applying a powerful approach: the transient thermal grating technique. It is used by heating a solution as a function of position creating a grating of temperature. This grating allows the use of the one-dimensional heat equation, which can be solved to give a relation between the thermal conductivity and temperature. The thermal conductivity can then be calculated using 𝑘 = 𝜌𝑐𝑝α, where 𝜌 is the density,  is the specific heat capacity and α which is a function of temperature. After the setup was completed, solutions of varying concentrations were analyzed. For the solution with the highest concentration, the thermal switching ratio was measured to be 1.15 across the LCST transition. This shows a significant change between the two states of the polymer. The thermal conductivity of the PNIPAM aqueous solutions increases with temperature, the same as with water, until reaching the LCST. Then a drastic change is observed in the solution. The thermal switching ratio of PNIPAM aqueous solutions across the transition keeps increasing with increasing concentration, which is expected from the equation. To explain the thermal conductivity change due to the transition between the two modes of the polymer, the authors used the idea that the homogeneous phase of the solution separates into two phases that increases the thermal interface resistance resulting in a lower effective thermal conductivity.

As a summary, they reported the first direct measurement of thermal conductivity change in PNIPAM aqueous solutions across the LCST using a powerful approach, the laser-induced transient thermal grating technique. The results show an abrupt thermal conductivity drop across the transition temperature. The potential of using thermoresponsive polymer aqueous solutions of higher-order phase transitions for thermal switch applications has been demonstrated throughout this paper’s work.


Maxier’s comment on: Macromolecular Crowding Modifies the Impact of Specific Hofmeister Ions on the Coil–Globule Transition of PNIPAM

By Maxier Acosta Santiago

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While the recently discussed Cremer paper described efforts towards understanding the effect on the LCST of PNIPAM by salts of the Hofmeister’s series, Sakota’s article went somewhat deeper into the study of this phenomenon by taking in consideration molecular crowding. Sakota’s group decided to use a PEG polymer as a crowding agent. Crowding may affect the biding of anions from the Hofmeister’s series and the PNIPAM resulting in a change in the LCST. First, they studied the presence of PEG in a PNIPAM solution showing that the crowding agent reduces the LCST. Kosmotropic anions, that decrease the LCST, but chaotropes increase the LCST. For the Hofemeister series effect on LCST we can go back to Luis Prieto’s blog post and Cremer’s paper which explains this effect better.

When the article begins to look at the presence of PEG at different salt concentrations, they see a close correlation between the LCST and the Hofmeister series. Yet, for the chaotropes ClO4⁻ and SCN⁻, the presence of PEG lead to a larger increase in the LCST. From here they decide to apply different theories to explain the results. Within the examined theories they discuss around thermodynamics of the system. Their explanation evolves as follows, even if the organization via LCST of PNIPAM is not thermodynamically favorable, the overlapping excluded volumes of PEG and the PNIPAM particles increase the translational entropy of water molecules in solution, which makes the formation of the system possible.

Although this paper brings something new to the table to discuss (molecular crowding and LCST), I do have some concerns. When taking into consideration so many different factors like molecular crowding, salt, and the responsive system itself, we should look deeper into the behavior. To limit certain factors, they maintained certain constant concentrations throughout the paper. Yet, in the discussion, I feel they lacked additional experimental results or computational studies (using molecular and/or coarse-grained simulations) to support their theoretical thermodynamics analysis.

Minelise’s comment on: Synthesis and Direct Observation of Thermoresponsive DNA Copolymers

By Minelise E. Rivera

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In this paper, Li and Schroeder use single molecule techniques to have a direct observation on DNA-PNIPAM copolymers. First, they synthetized DNA-PNIPAM copolymers using a two-step strategy based on polymerase chain reaction (PCR) for generating linear DNA backbones containing dibenzocyclooctyne-dUTP, then grafted thermoresponsive side branches (PNIPAM) onto DNA backbones using copper-free click chemistry. Subsequent single molecule fluorescence spectroscopy studies unveiled more clearly the molecular heterogeneity association with the stretching and relaxing of the polymer above and below their LCST. Their results showed that intramolecular conformational dynamics of DNA-PNIPAM copolymers are affected by properties of the branches like molecular weight, density, leading to a change in transition temperatures. In other words, the single molecule technique provided a better understanding in a molecular perspective of chemically heterogeneous and stimuli-response polymers.

As I read this paper and looked for information to better understand it, I was amazed by the details with which they worked with throughout their study. I would have thought of working better with a bunch of them instead of just single molecules. It didn’t cross my mind that someone was going to, not only synthesized the molecule, but also study its characteristics. It was very interesting to learn about the methods that they used for characterization and synthesis. It got me wondering if those methods were the only ones that would work in this case and why. But, what I think that was very useful for me is that I got to understand better the importance of the LCST and the role that it played in their system. It reminded me of our project in which the SGQ self-assembles into the SHS and how it is to study it and understand its influence in the SHS as it was important for the copolymers with which it was worked with in the paper.

Diana’s comment on: Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

By Diana Silva Brenes

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Relating molecular structure to function is the first step and one of the greatest challenge to understand nature’s designs or to make novel “functional designs” of our own. This paper by the Chilkoti group begins with statistical analysis of some of the most relevant proteins displaying LCST and UCST behavior. By analyzing the peptide sequences, the authors identify as common motif for both behaviors a high glycine & proline content. Furthermore, for LCST abundance of aromatic residues seems to be a requirement whereas UCST peptides seem to be encoded by a pair of zwitterionic residues.
To test if these observations lead to LCST/UCST phenomena, over 80 model peptides were recombinantly synthesized and their thermoresponsive behavior was measured by UV absorbance while changing the temperature. Each peptide presented the predicted behavior, giving support to their observations. Furthermore, by comparing a few selected examples, they show how an increase in hydrophobicity leads to an increased UCST cloud point and how eliminating one of the residues from azwitterionic pair turns a UCST peptide to an LCST peptide.
The LCST and UCST behavior is, however, a complex phenomenon dependent on protein-protein versus protein-water interactions, which in turn are modulated by more factors aside from the sequence of the protein. The possible scenarios are limitless, and the authors give insight on the most significant: peptide length, concentration, and pH (charge state of protonable atoms).
The robustness of the behavior encoded in the rules they found can be seen by a hybrid peptide containing both an LCST portion and a UCST one. The resulting peptide displays both behaviors, albeit at different temperatures from the “parent” sequences.
Finally, the authors show that searching for the characteristics they determined as important for LCST/UCST behavior throughout the human proteome produces examples of proteins whose function could very well be related to a thermoresponsive behavior, highlighting the applicability of their observations to understand the phenomena that make life as we know it possible.

Quiroz, 2015. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

Valeria’s comment on: Reversible Regulation of Thermoresponsive Property of Dithiomaleimide-Containing Copolymers via Sequential Thiol Exchange Reactions

By Valeria Burgos Caldero

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The main purpose of this article was to synthesize multi-responsive polymers that could be reversibly modified to adjust their LCST. Indeed, these researchers were able to develop a system in which multiple thiol exchanges were made, and in turn, they could determine how the thiols affected the transition temperature of the polymer. They used a copolymer containing P(TEGA) and DMMA. By performing transmission measurements at various temperatures, they concluded that as the thiol changed, the transition temperature of polymer varied depending on the resulting hydrophobicity. More polar functionalities increased the transition temperature and less polar ones decreased it. They were able to demonstrate the reversibility of the modifications since they managed to return to their original functionality after various thiol exchanges. Finally, they implemented a fluorescence signal to monitor the reaction progress. They found that thioglucose quenches the polymer’s fluorescence while making the compound soluble throughout the range of temperatures. With these findings, a wide range of possibilities were opened, since now, if you want a polymer for a specific type of function where a specific temperature response is needed, it is easily accessible by adding the corresponding thiol to the polymer solution. The mechanism of turning off the fluorescence may give access to reversible systems in aqueous conditions.
In general, I found it much simpler to prepare for this article than for the first one I presented. I feel that by doing these exercises of presenting scientific articles I have been acquiring maturity in the analysis process since it was difficult for me to understand articles in the beginning. Something that I found missing in the article is that they never explained the experimental procedure on how they achieved reversibility after adding different thiols to the same sample. I liked that they used common thiols, some of which we use in our research and others that maybe we could apply. In general, the article relates a lot to the research I’m doing with Diana. It could be useful to try to see the stimuli-responsive variations in the compounds that we are synthesizing. Maybe because it is related to variations in functionalities with thiols, similar to my own research, I found it more enjoyable to prepare the discussion and understand the material in the article.

Tang, 2016. Reversible Regulation of Thermoresponsive Property of Dithiomaleimide-Containing Copolymers via Sequential Thiol Exchange Reactions

Luxene’s comment on: Selective Tuning of Elastin-like Polypeptide Properties via Methionine Oxidation

By Luxene Belfleur

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Figure 1. Kekule structure of and synthesis of ELP 2 and 3. Cartoon representation of ELPs solution, the LCST behavior and measurement of ELPs

In this article, the authors reported the LCST modulation of Elastin-like polymers (ELP) by the modification towards oxidation reactions of the methionine thioester group of the ELP 1 to form sulfoxide and sulfone ELPs derivatives 2 and 3 respectively (Figure 1). They isolated ELP 1 from plasmid DNA of E.coli bacteria and purified it by SDS-Page. In acidic media, using 30% hydrogen peroxide and 1% of acetic acid or formic acid in water, they obtained ELP derivatives 2 and 3 respectively and the molecular weight and structural elucidation of ELP 1 and its derivatives 2 and 3 had been examined and confirmed by mass spectrometry and NMR respectively.
They performed turbidity experiments to determine the cloud points of those three ELPs. As expected, both ELP derivatives 2 and 3 had a higher LCST behavior (55 °C and 43 °C for 2 and 3 respectively) compared to ELP 1 which is 25 °C (Figure 1). This is due to the increasing of water solubility of the modified ELPs (2 and 3), which required more energy to break the interaction between water and them (ELP 2 and 3) in order to evacuate the hydration shell around these ELPs and favored the collapse of the letters. Moreover, it was expected that ELP 3 would have a higher cloud point than the ELP 2 counterpart, however, this was not the case because the large dipole moment of ELP 3 favored intramolecular and intermolecular interactions between them and the protein respectively which promoted a decrease of the water solubility. They evaluated the influence of the I¯ and NO3¯ anions on the phase transition of the three ELPs and found that those anions had no significant effect on the LCST point of the ELP 1 while I¯ increased the LCST of ELP 2 and NO3¯ decreased the solubility of ELP 3 in agreement with the Hofmeister series effect.
They reported an interesting and straightforward work by turning the LCST behavior of ELPs towards synthetic modification. This work has inspired and motivated my team for designing and synthesizing sulfoxides and sulfones containing guanosine derivatives in order to both, stabilize the thioester containing guanosine derivatives compounds that we synthesize, and modulate the LCST properties of the SGQs that will be made from oxidizing 8ArG derivatives product.

Luis’ comment on: Guanidinium can both Cause and Prevent the Hydrophobic Collapse of Biomacromolecules

By Luis A. Prieto


Heyda, Jungwirth and Cremer collaborated in a study of guanidinium (Gnd+) salts and their effect in lower critical solution temperature (LCST). They studied molecular details of the cause of these transitions by IR-ATR and molecular dynamics simulation where they wanted to understand how Gnd+ salts interacted with the backbone of the Elastin-like peptides (ELP). In previous studies ELPs showed a change in LCST that followed the Hofmeister series in sodium salts but using Gnd+ salts proved to be different, especially Guanidinium thiocyanate (GndSCN) that at low concentrations the LCST decreases, but at high concentrations the LCST increases. They studied particular phenomenon using ATR-IR where they found GndSCN binds strongly with ELPs and resulted in an interesting behavior when the concentration of salt is increased. At low concentrations the polymer collapsed (salting-out) because of cross-linking of the peptide and at high concentrations resolubilization occurred (salting-in). Other salts followed typical behavior of salting-in (guanidinium chloride, weak binding) or salting-out (guanidinium sulfate, poor binding). Coarse-grain and all atom simulations corroborated this finding where they found particular detail of the interaction of the carbonyl groups of the peptide backbone with Gnd+, most likely through H-bonds.

The thermodynamics of this paper I found particularly interesting since it reminded me of everything that I have to re-learn.  An attractive experiment was that they used a melting point apparatus to measure the LCST, meaning the use of small amount of sample to gather fundamental information of the system which is also the case with ATR-IR. An elegant work and also inspirational since our lab works with responsive systems and we will definitively see if we can do the LCST measurement with a melting point instrument. I got to say that I particularly like the all atom simulations and Figure 5, where we can see in molecular detail the interactions of the salts with the peptide where thiocyanate and Gnd+ interact strongly with the hydrophobic parts (V, G) and hydrophilic part (peptide bond), respectively.

LAPC: Heyda, 2017. Guanidinium can both Cause and Prevent the Hydrophobic Collapse of Biomacromolecules


Our last three papers…

mAGi16-Top2_whiteBelow are the references to our last three papers. I will post a brief overview of each one soon, but in the meantime:

Structural studies of supramolecular G-quadruplexes formed from 8-aryl-2’-deoxyguanosine derivatives. García-Arriaga, M.; Hobley, G.; Rivera, J. M.,  J. Org. Chem.2016, 81, Advance Online Publication; DOI: 10.1021/acs.joc.6b01113. PMID: 27303787

  • The first 50 people can download a free reprint of the paper directly from the publisher by going to this link.
  • Abstract. Self-assembly is a powerful tool for the construction of complex nanostructures. Despite the advances in the field, the development of precise self-assembled structures remains a challenge. We have shown that in the presence of suitably sized cations like K+, 8-aryl-2′-deoxyguanosine (8ArG) derivatives self-assemble into sets of coaxially stacked planar tetramers, we term supramolecular G-quadruplexes (SGQs). Previously, we reported that when the 8-aryl group is a phenyl ring with a meta-carbonyl group, the resulting supramolecule is a hexadecamer, which is remarkably robust as illustrated by its isostructural assembly in both organic and aqueous environments. We report here a detailed three-dimensional structure of the SGQs formed by lipophilic, and hydrophilic, 8ArG derivatives with either 8-(meta-acetylphenyl), 8-(para-acetylphenyl), and 8-(meta-ethoxycarbonylphenyl) groups. The chirality and close contacts between the subunits impose different levels of steric and electrostatic constraints on opposite sides of the tetrads, which determine their preferred relative orientation. The balance between attractive non-covalent interactions juxtaposed with repulsive steric and electrostatic interactions explains the high cooperativity, fidelity and stability of these SGQs. These structural studies, together with titration experiments and molecular dynamics simulations provide insight on the mechanism of formation of these SGQs.

Organic Nanoflowers From a Wide Variety of Molecules Templated By A Hierarchical Supramolecular Scaffold. Negrón, L. M.; Diaz, T. L.; Ortiz-Quiles, E. O.; Dieppa, D.; Madera-Soto, B.; Rivera, J. M., Langmuir 2016, 32 (10), 2283–2290. DOI: 10.1021/acs.langmuir.5b03946; PMCID: PMC4896646

  • Abstract. Nanoflowers (NFs) are flowered-shaped particles with overall sizes or features in the nanoscale. Beyond their pleasing aesthetics, NFs have found a number of applications ranging from catalysis, to sensing, to drug delivery. Compared to inorganic based NFs, their organic and hybrid counterparts are relatively underdeveloped mostly because of the lack of a reliable and versatile method for their construction. We report here a method for constructing NFs from a wide variety of biologically relevant molecules (guests), ranging from small molecules, like doxorubicin, to biomacromolecules, like various proteins and plasmid DNA. The method relies on the encapsulation of the guests within a hierarchically structured particle made from supramolecular G-quadruplexes. The size and overall flexibility of the guests dictate the broad morphological features of the resulting NFs, specifically, small and rigid guests favor the formation of NFs with spiky petals, while large and/or flexible guests promote NFs with wide petals. The results from experiments using confocal fluorescence microscopy, and scanning electron microscopy provides the basis for the proposed mechanism for the NF formation.

Tuning Thermoresponsive Supramolecular G-Quadruplexes. José E. Betancourt & José M. Rivera, Langmuir 2015, 31 (7), 2095-2103. DOI:10.1021/la504446k; PMCID: PMC4863471 [Free PMC Article]

  • Abstract. Thermoresponsive systems are attractive due to their suitability for fundamental studies as well as their practical uses in a wide variety of applications. While much progress has been achieved using polymers, alternative strategies such as the use of well-defined nonpolymeric supramolecules are still underdeveloped. Here we report three 8-aryl-2′-deoxyguanosine derivatives (8ArGs) that self-assemble in aqueous media into precise thermoresponsive supramolecular G-quadruplexes (SGQs). We report the synthesis of such derivatives, studies of their isothermal self-assembly, and the thermally induced assembly to form higher-order meso-globular assemblies we term supramolecular hacky sacks (SHS). The lower critical solution temperature (LCST) that indicates the formation of the SHS was modulated by changing (a) intrinsic parameters (i.e., structure of the 8ArGs); (b) extrinsic parameters such as the salt used to promote the formation of the SGQ; and (c) supramolecular parameters such as the coassembly different 8ArGs to form heteromeric SGQs. Changes in the intrinsic parameters lead to LCST variations in the range of 28–59 °C. Modulating extrinsic parameters such as replacing KI with KSCN abolishes the thermoresponsive phenomenon whereas changing the cation from K+ to Na+or adjusting the pH (in the range of 6–8) has negligible effects on the LCST. Modulating supramolecular parameters results in transition temperatures that are intermediate between those obtained by the respective homomeric SGQs, although the specific proportions of the subunits are critical in determining the reversibility of the process. Given the extensive applications of thermoresponsive polymers, the nonpolymeric supramolecular counterparts presented here may represent an attractive alternative for fundamental studies and biorelevant applications.