Definitions of kosmotropes
Ionic kosmotropes and chaotropes
Non-ionic kosmotropes and chaotropes
Trimethylamine N-oxide (TMAO)
Definitions of kosmotropes
The terms 'kosmotrope' (order-maker) and 'chaotrope'
(disorder-maker) originally denoted solutes that stabilized,
or destabilized respectively, proteins and membranes; thus chaotropes unfold proteins, destabilize hydrophobic aggregates and increase the solubility of hydrophobes whereas kosmotropes stabilize proteins and hydrophobic aggregates in solution and reduce the solubility of hydrophobes. A recent review makes a strong case for reverting to these meanings , however most present literature uses these terms to refer to the apparently correlating
property of increasing, or decreasing respectively, the structuring
of water (a review of structure-making and structure-breaking ions has been presented ). Recently, the terms kosmotropes
and chaotropes have been quantified in terms of hydrophobicity and hydrophilicity . Although useful, the terminology may sometimes be
misleading as such properties may vary dependent on the circumstances,
method of determination or the solvation shell(s) investigated.
For example a solute may not always act in the same way at
different concentrations or in the presence of macromolecules
or gels . Also
some solutes with less well-defined properties (for example, urea) are sometimes classified as kosmotropes 
and sometimes as chaotropes .
An alternative term used for kosmotrope is 'compensatory solute'
as they have been found to compensate for the deleterious
effects of high salt, and/or high chaotrope contents , (which destroy the natural hydrogen
bonded network of water) in osmotically stressed cells, but
again behavior as a kosmotrope in one system does not mean
that a solute may behave as a 'compensatory solute' in
another or even that it will stabilize the structuring of
water in a third. Both the extent and strength of hydrogen
bonding may be changed independently by the solute but
either of these may be, and has been, used as measures of
order making. It is, however, the effects on the extent of
quality hydrogen bonding that is of overriding importance
as true kosmotropes shift the local equilibrium
dense water (for example, ES) more dense water (for example, CS)
to the left and chaotropes shift it to the right.
The ordering effects of kosmotropes may be confused by their
diffusional rotation, which creates more extensive disorganized
junction zones of greater disorder with the surrounding bulk
water than less hydrated chaotropes. It seems clear that most
kosmotropes do not cause a large scale net structuring in
Temperature and pressure both have effects on the kosmotropic/chaotropic status with the effects disappearing at high temperatures, particularly at high concentrations . For example, at very high pressures (0.6 GPa) Na+ ions change from being weak kosmotropes into weak chaotropes as their links to water molecules are preferably broken . [Back to Top ]
Ionic kosmotropes and chaotropes
Ionic kosmotropes a should be treated differently from non-ionic kosmotropes,
due mainly to the directed and polarized arrangements of the
surrounding water molecules. They are best not described as 'structure-makers' or structure-breakers' in terms of their effects on the properties of water outside their immediate solvation shells .
Generally, ionic behavior parallels
the Hofmeister series. Large singly
charged ions, with low charge density (for example, Ph4B- (tetraphenylborate anion), Ph4As+ (tetraphenylarsonium cation), SCN-,
Cl-, NO3-, NH4+,
Cs+, K+, (NH2)3C+ (guanidinium) and (CH3)4N+ (tetramethylammonium) ions; exhibiting weaker interactions
with water than water with itself and thus interfering little
in the hydrogen bonding of the surrounding water), are chaotropes
whereas small or multiply-charged ions, with high charge density,
are kosmotropes (for example, SO42-,
HPO42-, Mg2+, Ca2+,
Li+, Na+, H+, OH- and HPO42-, exhibiting stronger
interactions with water molecules than water with itself and
therefore capable of breaking water-water hydrogen bonds). b The Jones-Dole coefficient has also been used to classify ionic kosmotropes and chaotropes with negative B coefficients indicating chaotropes and positive B coefficients indicating kosmotropes. Kosmotropes remain hydrated near the water surface, while the chaotropes lose their hydration sheath
. The radii of singly charged chaotropic ions are greater than
1.06 Å for cations and greater than 1.78 Å for anions
. Thus the hydrogen
bonding between water molecules is more broken in the immediate
vicinity of ionic kosmotropes than ionic chaotropes
this conclusion, a Raman spectroscopic study of the hydrogen-bonded
structure of water around the halide ions F-, Cl-,
Br- and I- indicates that the total extent of aqueous hydrogen bonding
increases with increasing ionic size 
and an IR study in HDO:D2O showed slow hydrogen bond reorientation
around these halide ions getting slower with respect to increasing
It is not unreasonable that a solute may strengthen some of
the hydrogen bonds surrounding it (structure making; for example, kosmotropic cations will strengthen the hydrogen bonds donated
by the inner shell water molecules) whilst at the same time
breaking some other hydrogen bonds (structure breaker; for example, kosmotropic cations will weaken the hydrogen bonds accepted
by the inner shell water molecules) ,
so adding to the confusion in nomenclature. Kosmotropic ions such as Na+ reduce the average diffusion of water by slightly less than that expected if the hydrated water was not diffusionally active .
Much local organization around a solute is entropically compensated by reduced organization between the water molecules further away , such that K+ and Cl- (and Ar) have almost identical hydration entropies . Other factors
being equal, water molecules are held more strongly by molecules
with a net charge than by molecules with no net charge; as
shown by the difference between zwitterionic and cationic
amino acids .
Rather surprisingly, chaotropic ions such as K+ slightly increase the average diffusion of water at moderate to high concentrations (0.25 - 2 M) , showing that they have a net destructive effect on the structuring in water at these concentrations. Weakly hydrated ions (chaotropes, K+,
Rb+, Cs+, Br-, I-,
guanidinium+) may be 'pushed' on to weakly hydrated
surfaces by strong water-water interactions with the transition
from strong ionic hydration to weak ionic hydration occurring
where the strength of the ion-water hydration approximately
equals the strength of water-water interactions in bulk solution
(with Na+ being borderline on the strong side and
Cl- being borderline on the weak side) . Strongly hydrated surfaces, where the water molecules are prevented from forming ES-like expanded water clusters, favors strongly hydrated kosmotropic ions and effectively repels weakly hydrated chaotropic ions . Neutron diffraction studies on two important chaotropes (guanidinium
and thiocyanate ions) show their very poor hydration, supporting
the suggestion that they preferentially interact with the
protein rather than the water .
In contract to the kosmotropes, there is little significant
difference between the properties of ionic and nonionic chaotropes
due to the low charge density of the former.
Optimum stabilization of biological macromolecule
by salt requires a mixture of a kosmotropic anion with a chaotropic
cation. As ionic kosmotropes primarily achieve their increased
structuring solely within their hydration shell, they partition
into the more dense (CS)
water where they can obtain this hydration water more readily,
whereas the ionic chaotropes, by avoiding interference with
water's hydrogen-bonded network, tend to clathrate formation
within the less dense (ES)
environment. Thus there is agreement with the defining characteristic
of an ionic chaotrope in that it partitions selectively into
low-density water whereas a kosmotrope partitions selectively
into high-density water .
The stabilizing of structured low-density water (by ionic chaotropes) in turn stabilizes
the hydration shell around lower molecular mass hydrophobes, as seen in the promoted association of polyene antibiotics by ionic kosmotropes and the stabilization of their solution by ionic chaotropes . [Back to Top ]
Non-ionic kosmotropes and chaotropes
Non-ionic kosmotropes are very soluble well-hydrated molecules
with little tendency to aggregate, having no net charge and
strongly hydrogen bonding to water, that stabilize the structure
of macromolecules in solution. They are preferentially solubilized
within the bulk of the solution and excluded from the solvation
layers of macromolecular surfaces. Consequentially, they decrease
the water diffusion around the proteins and the exchange rate
of backbone amide protons .
This leads to the dehydration of such surfaces and ensures
that they are less flexible and therefore more thermally stable
but less enzymatically active. c Kosmotropes reduce the volume of water available to hydrate
the larger surface exposed by denatured proteins, so tending
to prevent the denaturation process (the 'excluded volume'
effect). These kosmotropes may be divided into two groups which
act somewhat differently: (1) polyhydroxy compounds fit in
well with the hydrogen bonding arrangements, but stabilize
any cluster 'flickering' between a continuum of hydrogen bond rearrangements,
as their hydroxyl groups are similarly separated to water-water
separations (see sugar hydration),
and (2) zwitterions where the balance between hydrophilicity, f hydrophobicity, f anionic and cationic characteristics ensures
good solubility but only weak net interactions to surrounding
water. Many, if not all, such kosmotropes are also useful as cryoprotectants (see below).
Chaotropes break down the hydrogen-bonded network of water,
so allowing macromolecules more structural freedom and encouraging
protein extension and denaturation. Kosmotropes are stabilizing
solutes which increase the order of water (such as polyhydric
alcohols , trehalose,
trimethylamine N-oxide, glycine betaine, ectoine,
proline and various other zwitterions) whereas chaotropes
create weaker hydrogen bonding, decreasing the order of water,
increasing its surface tension (but see anomaly)
and destabilizing macromolecular structures (such as guanidinium
chloride and urea at high concentrations).
Glucose clearly acts as a kosmotrope, enhancing both hydrogen bonding and hydrophobic
interactions . Rather unexpectedly, whereas L-amino acids and D-glucose
prefer a less dense (ES)
environment, D-amino acids and L-glucose prefer more dense
a consequence of which may be that condensed polymers of the
former (rather than their optical isomers) may have formed
in primordial clays so setting the trend for the molecular
evolution . Although
the arrangement of oxygen atoms within water clusters may
be symmetrical, the energetic preference for particular hydrogen-bonding
arrangements ensures the necessary chirality.
a Trimethylamine N-oxide
c Ectoine; R varying
e Glycine betaine
Generally the kosmotropic/chaotropic nature
of a solute is determined from the physical bulk properties
of water, often at necessarily high concentration. The change
in the degree of structuring may be found, for example, using NMR ()
or vibrational spectroscopy. Protein-stabilizing
solutes (kosmotropes) increase the extent of hydrogen bonding
(reducing the proton and 17O spin-lattice
relaxation times) whereas the NMR chemical shift may increase
(showing weaker bonding for example, the zwitterionic kosmotrope, trimethylamine N-oxide) or decrease (showing stronger bonding for example, the polyhydroxy kosmotrope, trehalose). Trehalose
shows both a reduction in chemical shift and relaxation time,
as to a lesser extent does the protein stabilizer (NH4)2SO4,
whereas NaCl only shows a reduction in chemical shift and
the protein destabilizer KSCN shows an increase in relaxation
time and a reduction in chemical shift (note these NMR parameters
are both time and structurally averaged values, where the
weighting of the averaging is unclear) . Vibrational spectroscopy may make
use of the near-IR wavelength near 5200 cm-1 (v2 + v3 combination), which shifts towards
longer wavelength (smaller wavenumber) when hydrogen bonds
are stronger .
It should be noted however that ranking of kosmotropic/chaotropic
character by different measures shows inconsistencies. [Back to Top ]
Urea (H2NCONH2) is of particular interest because of its interactions with proteins. It can only strongly bind one water molecule using two hydrogen bonds, donating from a nitrogen atom and accepting onto the urea oxygen atom  but will also hydrogen bond more weakly to about 4 - 7 moles further water molecules per molecule of urea . Otherwise it can hydrogen bond to itself (see (b) opposite) or water with close to equal ease, and hydrogen bonds to water slightly weaker than water to itself but seems to increase the water-water interactions .
Different studies describe urea as either a poor chaotrope or poor kosmotrope over much of its concentration range [1171, 1233]. Its conformation is not well-defined in water  and it can fit into a tetrahedral water network showing little preference between ES-like and CS-like water clusters , perhaps behaving rather like a water dimer , although this is not shown in a molecular dynamics study . Urea with small clusters of water molecules have been modeled (see the almost planar cluster left)  . Other work has
shown that urea weakens both hydrogen bonding and hydrophobic
interactions . The idiosyncratic
behavior of urea may well be due to its concentration-dependent
nose-to-tail oligomerization (where the carbonyl group of one urea molecule accepts two hydrogen bonds to the two amines of the next urea molecule); cyclic hydrogen-bonding dimers and oligomers
behaving differently from monomers  and the system becoming more organized with increasing urea concentration  .Thus, when urea molecules are less than optimally hydrated, urea hydrogen bonds
to itself (see opposite) and any protein present (significantly involving the peptide
in the absence of sufficient water. It so becomes more hydrophobic
and hence more able to interact with further sites on the
protein, leading to localized dehydration-led denaturation (i.e. urea is preferred over water for binding to the protein backbone so leading to protein denaturation [1711, 2324]). By replacing and removing the water from the protein surface, urea acts in an opposite manner, in denaturing proteins, to increased pressure .
Both urea and guanidinium (see below) increase the radius of gyration of the intrinsically disordered protein ACTR (activator for thyroid hormone and retinoid receptors) in proportion to their concentrations .
Guanidinium  is a planar ion that may form weak hydrogen bonds
around its edge  but may establish strongly-held hydrogen-bonded
ion pairs to protein carboxylate groups, like commonly found
quaternary structural arginine-carboxylate 'salt' links. Also,
guanidinium possesses rather hydrophobic surfaces that may
interact with similar protein surfaces to enable protein denaturation
. Likewise, they have a preference for water/air surfaces . The hydrophobic surfaces also allow the formation of contact guanidinium-guanidinium ion pairing  and the similar contact guanidinium-arginine ion pairing  with the repulsion of the like, if somewhat dispersed, positive charges being compensated by the increased entropy and enthalpy of the released water molecules from the surfaces on forming the stacked pair. The action of guanidinium is highly dependent upon the concentration and counteranion with which it was paired. As an example, The salting out efficacy at low salt concentrations is as follows :
SCN- > (Guanidinium+)2
SO42- > (Guanidinium+)2 CO32- > Guanidinium+NO3- > Guanidinium+Cl-
denaturants (urea and guanidinium) may cause protein swelling and destructuring by
sliding between hydrophobic sites and consequently dragging
in hydrogen-bound water to complete the denaturation. The hydration of the counterion is important to the action of guanidinium with chloride ions, being only weakly hydrated, allowing the water molecules to easily rearrange around the surface and allowing the denaturation process outlined above whereas sulfate ions hold on to their water molecules very strongly preventing the water rearrangement and hence preventing the guanidinium-protein interactions and consequent denaturation .
One of the most important kosmotropes is the
non-reducing sugar α,α-trehalose, found in some bacteria, fungi, plants, and invertebrates where it accumulates in response to thermal stress (structure given above and described in ).
It should perhaps be noted that trehalose has a much more
static structure than the reducing sugars, due to its lack
of mutarotation, or the other common non-reducing disaccharide,
sucrose, due to its lack of a furan ring; its two glucose units being joined by a very stable glycosidic linkage. Both inelastic neutron scattering 
and 17O-NMR show that a rotational restriction
exists in trehalose-bound water, indicating that trehalose
is definitely a net water-structurer. These stronger interactions d (between the trehalose and water compared to water-water interactions)
increase the bioprotective effectiveness of trehalose 
by reducing the (re-)activity of the water molecules, creating a rigid shell around biomolecules and so reducing thermal stress . This effect is increased by the introduction of greater static hydration with trehalose-trehalose interactions . Trehalose
interferes with the tetrahedral network of water, structuring
out to at least the third solvation layer, e in such a way as
to reduce the amount of freezable water ; terahertz spectroscopy indicating 27% more hydration water (~190 mols/mol) than lactose and almost four times the hydration water than glucose .
This is consistent with an increase in the local structuring,
due in part to maximizing the number of singly hydrogen-bonded
water molecules (to trehalose) without any (trehalose) intramolecular
hydrogen bonds or oriented water molecules held by two (trehalose)
hydrogen bonds. There is also reduced tetrahedrality amongst
the associated water molecules .
Neutron diffraction studies indicate that the hydrogen bonds to water are somewhat shorter (trehalose-O-H···OH2 ~0.15 nm and trehalose-O···HOH ~0.15 nm) than those between water molecules but with an average of ~0.9 (trehalose-O···HOH) and ~0.15 (trehalose-O-H···OH2) hydrogen bonds per hydroxyl group respectively . At lower water contents, essentially all the water is associated
with single hydrogen bonds to trehalose or first shell water
with consequential reduction in the ability to form four hydrogen
bonds per water molecule but retaining an expanded structure.
Trehalose is particularly effective at stabilizing macromolecules
as it has a large hydrated volume (2.5 times that of sucrose,
indicating that there is less water for the same volume; that is, low-density water is present ). Thus, α,α-trehalose reduces bulk water dynamics at low concentrations (0.1 M), with each affecting ~550 water molecules . Such reduced bulk water dynamics equates to lowered water activity, so stabilizing biomolecules in the solution. This lack of direct effect (i.e. no trehalose-protein interactions) has also been shown by differential scanning calorimetry (DSC) and viscometry measurements on the myoglobin-trehalose-water system glass transition and protein denaturation temperatures .
Trehalose can also protect membranes (and whole organisms ) in anhydrobiosis by hydrogen
bonding directly to the phosphate groups in the phospholipids
at low water content, so spreading their head groups and reducing
the membrane's tendency to undergo phase transitions and thus
leak during rehydration .
Thus trehalose exhibits different mechanisms of action with proteins from those with membranes . As further protective action trehalose crystals, formed
at low water content, can lose (and regain) up to two molecules
of water per molecule, without changing their crystal structure
or volume [327, 1486]. The glycosidic bond linking the two glucose monomers in α,α-trehalose far more stable than the bond in sucrose, that has the labile β-D-fructofuranoside residue.
α,α-Trehalose produced by drought-tolerant rhizobacteria protects host plants against drought. These microorganisms produce trehalose under drying conditions to facilitate both their own survival and that of the plant being colonized .
α,α-Trehalose is also a natural cryoprotectant, suppressing ice formation in living organisms or tissue and resulting in a higher post-thaw survival rate. Other polyols also cryoprotect; such as ethylene glycol (poisonous but used as automobile antifreeze), propylene glycol ( used to reduce ice crystals in ice cream) and glycerol  (used to store biological and pharmaceutical products at low temperatures, see right). Other kosmotropes, such as trimethylamine N-oxide and proline are also cryoprotectants. Although cryoprotectants (not unexpectedly) affect the bulk water structure , the resultant structures vary with their structure and concentration. Cryoprotection, therefore, appears to be an interfacial phenomenon like kosmotopism.
Ectoine ((S)-2-methyl-3,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a kosmotrope and a widespread osmolyte, found in halophilic microorganisms. g It is a strongly hydrated zwitterion . As with TMAO, ectoine gives increasing static relative permittivity with increasing concentration, possibly protecting biomolecules against excess ions whilst delivering their osmotic protection against excessive extracellular salt 
Trimethylamine N-oxide (TMAO)
Trimethylamine N-oxide (TMAO) . is found in saltwater fish, sharks and crustaceans where they use it to counteract the high pressure at depth. As the concentration of TMAO increases, proteins become more compact and their radius of gyration decrease .. TMAO also balances the destabilization effects of urea that is also present. This allows organisms which contain both TMAO and urea to maintain their protein structure whilst adjusting their osmotic pressure. It exemplifies the difficulty in describing molecules as structure makers or breakers as molecules that can form very strong links to water may weaken water-water links further away; whether experimental evidence finds structure-making or -breaking depends on which of these molecules of water are examined. For example, infrared spectroscopy  and concentration-dependent terahertz/far-infrared (THz/FIR) and Raman spectroscopic techniques  both shows that
TMAO forms strong hydrogen bonds with water. Vibrational sum frequency spectroscopy shows that the methyl groups point away from hydrophobic surfaces indicating a more hydrophilic nature of methyl groups attached to this strongly electron-withdrawing quaternary nitrogen atom and helping to explain TMAO's depletion at such interfaces compared to the bulk solution . However, fluorescence studies using model hydrophobic compounds indicate that TMAO reduces hydrophobic interactions in aqueous solutions . The effect of pressure on TMAO solutions has been described .
TMAO's protein stabilizing effects seems to reverse the denaturing effect of urea  (betaine also does this ) . However, this counteracting role of TMAO in proteins under denaturing condition of urea is in contrast to its reinforcement of urea denaturation on hydrophobic macromolecules . [Back]
[Back to Top ]
a Perhaps ionic kosmotropes
should be called antichaotropes to distinguish them from non-ionic
The water dodecahedra
shown only as connected O-atoms of water, surrounding
the central ion..
b Opposite is shown a cartoon
illustrating the main difference between ionic chaotropes
and kosmotropes. Water molecules surrounding ionic chaotropes
tend towards a convex dodecahedral (clathrate) arrangement,
whereas those surrounding ionic kosmotropes tend towards
a puckered arrangement with a number of water molecules
lying close to the ion (see
elsewhere for more details), causing bent and broken
hydrogen bonding further out. The dodecahedral water cluster
may be thought of as constantly fluctuating (over a period
of a few ps or so, see ),
interconverting between convex and puckered forms with
the preferred clustering governed by the chaotropic-kosmotropic
balance. The strength of the ion-water links explains why the ionic kosmotropes remain hydrated near the interface, while the ionic chaotropes lose their hydration sheath . [Back]
c A few adventitious
and deleterious enzymatic and non-enzymatic reactions may
occur during lengthy dehydration, which may be reduced by
the presence of specific amphiphilic molecules that concentrate
at the interfaces .
d This 'stronger'
character of the system is sometimes referred to as 'less fragile'
e This description has been disputed by recent neutron diffraction experiments combined with empirical potential structure refinement simulations, that found trehalose solvation induces more minor modifications of the water structure. .
f Hydrophilicity and
hydrophobicity have been quantified using a 1-propanol cosolvent probing methodology . [Back]
g Osmolytes are molecules that help balance the osmotic pressure between the intracellular and extracellular compartments and so help maintain cell volume. Osmolytes can also prevent the denaturation of native proteins and promote the renaturation of unfolded proteins . [Back]