is lower than 5 kJ/mol (approximatively 2 k b T at room temperature). This means that a sensible population of inverted chairs, of the order of 10% is expected at equilibrium, in contrast with no experimental evidence of the occurrence of this conformer. The behavior of -d- Tal is even more pronounced, displaying an inverted chair free energy close to zero or, for the β anomer, even negative. This should have been the case of idose, whose inverted chair conformers have been experimentally detected. On the contrary, the puckering free energy of idose inverted chairs simulated using the 45a4 set of parameters results to be greater than 10 kJ/mol, therefore ruling out the possibility of observing idose inverted chairs in equilibrium simulations. 

The gromos 45a4 force field appears to be unable not only to compare quantitatively with experimental and theoretical results, but — even more importantly — to reproduce the qualitative behavior of any of the two series. Given the ubiquitous presence of galactose and mannose in relevant oligo and polysaccharides of biological origin, the inability of the force field to prevent appearance of inverted chairs at room temperature seems to be a severe drawback, at least for out-of-equilibrium simulations. 

While the free energy of different ring conformers is certainly an important physical quan- tity, one should not overlook the importance of the kinetics of the conformational transitions. One might reason that alternate conformers might not be seen during equilibrium simula- tions, if the inverse transition rate is much longer than the typical time interval spanned by a simulation. This pragmatic approach could be hazardous, given the fast pace of increase in simulations sizes and lengths, but nevertheless appealing. Kr¨ autler and coworkers 28 re- ported that in 200 ns long simulation runs of β -d- Glc, β -d- Gal, β -d- Man and β -d- talose, all sugars but glucose remained for more than 99.9% of the time in the chair conformation, while glucose was found in boat and twisted conformation for the 0.7% of the time (giving a rough estimate of the characteristic time of escape from the chair conformer basin of 10 ns). 

Although 200 ns is a time much longer than that of most simulations, one should keep in mind that conformational transitions are stochastic events, and a characteristic time of 10 ns might lead to a considerable amount of “unwanted” conformers in simulation runs much shorter than 200 ns, but with more than just one sugar molecule in solution. We tested a setting which we consider to be representative of a typical simulation of medium to large size, namely, of a 25 ns long run at constant temperature and pressure of 512 β -d- Glc and 

