d- aldopyranoses, a system composed of the respective sugar ring in a cubic simulation box filled with 504 water molecule was set up. The SPC 96 model has been used to describe the water molecules employed to hydrate each of the pyranoses. The settle algorithm has been used to make water rigid $^{97}$, and all bond lengths in the sugar molecules have been constrained using the shake algorithm $^{98}$. An integration step of 0.2 fs has been used for every phase of the simulations. Before starting each run, a 100 ps long molecular dynamics simulation with no bias has been performed to equilibrate the different sugars in their $^{4}$C$_{1}$ conformer. The metadynamics part of the run consisted in a 4 ns long simulation, using Gaussian potential functions of height 0.5 kJ/mol and with 0.05 rad for both angular variables, to build up the biasing potential. Gaussian functions have been placed every 200 integration steps. The Nos´ e–Hoover 99,100 thermostat and the Parrinello–Rahman 101 barostat have been applied to simulate isothermal-isobaric conditions at 300 K and 1 atm using relaxation times of 0.1 and 1.0 ps, respectively. The simulation box edges have been kept orthogonal, and have been rescaled using an isotropic pressure coupling, which controlled the trace of the pressure tensor. Each metadynamics run has been then followed by a 4 ns long equilibrium molecular dy- namics run at the same thermodynamic conditions, using the set of Gaussians placed during the metadynamics to generate the time-independent biasing potential. At a difference with Ref. 50, where the Lagrangian metadynamics with truncated Gaussians has been employed, we make use of the standard direct metadynamics. During this run, the histogram ρ$_{bias}$ ( θ, φ ) has been collected by sampling configurations every 40 fs on a grid of 60 × 60 points. The combined metadynamics–umbrella sampling has been first applied to the calculation of the puckering free energy profile of β -d- Glc, employing the standard gromos 45a4 pa- rameter set $^{35}$. In Fig. 3 we present the free energy profile as a function of the Cremer–Pople angular variables θ and φ , showing isolines separated by 10 kJ/mol (upper panel). In order to facilitate the comprehension of the plot, the projection of the free energy profile onto the φ = 0 plane (lower panel) is also provided: the lower contour of the colored region in the projected profile allows to easily identify the minima along the θ coordinate (corresponding IV. PUCKERING FREE ENERGY OF GLUCOSE 12