General Considerations Involving Carbohydrates

Carbohydrates is a term that encompasses saccharides (mono-, di-, tri-, oligo-, poly-) and glycoconjugates, such as glycoproteins or glycolipids. Their structural diversity allows them to encode information for specific molecular recognition or protein folding. Indeed, glycosylation is one of the most common

post-translational modifications.^[102] Therefore, carbohydrates represent an important part of biology since small differences in the saccharide units can lead to important changes.

Figure 31 depicts how carbohydrates are essential in cell survival. Indeed, the cell surface is decorated with glycoconjugates that allow for the specific recognition with the receptors of neighbouring cells thus triggering communication events. On the other hand, monosaccharides are taken up by the cell using specific transporters and are assembled into oligosaccharides which are in turn converted into more complex glycoproteins or triglycerides.^[102] In addition, the monosaccharide metabolism leads to the production of adenosine triphosphate (ATP), which is the main energy source of the cell.^[103]

Figure 31. Schematic representation of cell surface glycoconjugates used for cell-cell communication and the uptake of monosaccharides used for carbohydrate biosynthesis. Image from reference.^[102]

Since the cell membrane is impermeable to polar molecules, the cellular uptake of monosaccharides is mediated by membrane-associated carrier

GLUT receptors use existing gradients in monosaccharide concentration between the external and internal sides of the cell membrane, while SGLT transporters mediate an energy-dependent transport against gradient concentration.^[104-105] To date, 14 different GLUT receptors have been identified with high degree of homology and, therefore, there is no selectivity for just one monosaccharide. For example, the most abundant GLUT, GLUT1, primarily transports glucose, but it can also carry galactose and mannose.^[104] GLUT proteins can be found in all human tissues, with varied distribution of the different subclasses, however they are overexpressed in cancer cell lines due to the higher demand in energy.^[104-108] The abundancy of GLUT transporters on cancer cell surfaces makes the use of glycosylation a powerful tool for cell-targeting.

Fluorescent dyes appended with monosaccharides, especially glucose and its derivatives, have been, therefore, used to study glucose uptake or for imaging purposes of cancer cells and tissues.^[109-113] For example, Park et al. reported the use of probes 72-73 to track the cellular uptake of glucose in different cancer cells (Figure 32).^[114] These cyanine dyes were labeled with α-glucose, 72, and β-glucose, 73. Both probes could enter different cell lines independently on the stereochemistry of the anomeric monosaccharide unit, with higher uptake in cancer versus healthy cell lines. In order to prove the involvement of the GLUT transporter in the cell entry of 72, inhibition experiments were conducted by adding free D-glucose in the cell media prior incubation of the probe. A concentration-dependent decrease in uptake could be observed, while, on the other hand, when L-glucose was used, the transport was not affected due to the fact that the L-isomer is not recognized by the GLUT protein.^[114]

Figure 32. Fluorescently labeled glucose probes 72-73. Cellular uptake in HeLa cells of 72 followed by a) confocal laser scanning microscopy and b) flow cytometry in the absence and in the presence of D- and L-glucose. Figure adapted from reference.^[114]

Further evidence of the involvement of GLUT transporters for the cellular uptake of monosaccharide-based molecules was reported by Lo and coworkers in 2013.^[105] In this case, the probe, an iridium phosphorescent complex, was appended with two different carbohydrates: β-glucose 74 and β-galactose 75, as shown in Figure 33. HeLa cells treated with probe 74 were found to have a higher amount of iridium compared to 75, suggesting the entry using two different

HO O

HO OH OH

O HO O

HO OH OH

O N

H

O N

H

O N

N

2

72

NH

O N

H

O N

N

2

73 b)

a)

when L-glucose was added to the cell media, suggesting that GLUT was responsible for the cell entry. Interestingly, probe 75 bearing the galactose unit was not affected by the presence of D- or L-glucose, pointing to the binding to a different transporter with higher affinity for galactose compared to glucose.

Figure 33. Phosphorescent iridium complexes containing β-glucose 74 and β-galactose 75 units. Cellular uptake of 74 (orange) and 75 (green) in HeLa cells in the presence of a) D-glucose and b) L-glucose. Figure adapted from reference.^[105]

GLUT transporters are operational when small molecules are concerned.

However, when dealing with larger structures, such as polymers, nanoparticles, micelles, there are other receptors that can promote cell entry, such as the family of lectins.[115-118]

Lectins are proteins that can bind carbohydrates reversibly, but with a high degree of specificity. They are involved in different cell processes, such as communication, adhesion or signalling and hundreds of lectins, differing in carbohydrate binding, structure and size, have been identified.^[119-120] The recognition of carbohydrates takes place exploiting non-covalent interactions

Ir N

N

X N

X N

N N

= R² O

HO OH OH

NO ₃

74 : R¹ = H; R² = OH R¹

75 : R¹ = OH; R² = H O

N N b)

a)

with the amino acid residues of the protein that are contained in the binding sites, such as H-bonding, ion-dipole, electrostatic and CH-π interactions or hydrophobic effects.^[121-123] Figure 34 shows, for example, the interaction between the calcium-rich domain of Pseudomonas aeroginosa Lectin A with a galactose unit.^[121]

Figure 34. Non covalent interactions between Pseudomonas aeroginosa Lectin A with galactose. Figure adapted from reference.^[121]

The binding to carbohydrates becomes stronger going from mono- to di-, tri- and tetrasaccharides. Moreover, lectins are multivalent and they tend to self-assemble in order to simultaneously bind several complementary glycoside units in a phenomenon also known as the “glycoside cluster effect”.^[124] For this reason, polymers carrying multiple carbohydrate functional groups, i.e.

glycopolymers, can engage in multivalent interactions with the lectins on the cell surface, thus enabling them to efficiently enter the cell.^[125]