Carbohydrates fluorescent probes

Carbohydrates are not fluorescent. Therefore, one usually binds fluorophores covalently to small carbohydrates so that to carry out interaction or conformational studies between these carbohydrates and proteins. Also, fluorophores can be bound covalently to the proteins, and thus interaction between small carbohydrates and the protein is carried out by following variation of the fluorophores observables. See for examples (Monsigny et al. 1980, Khan et al. 1988, Yu and Pettigrew, 2003). 

Calcofluor Wliite (Fig. 3.31) is a fluorophore that binds to carbohydrate residues. Type of interaction depends on the secondary structure of the carbohydrate residues and fluorescence parameters of calcofluor are sensitive to this spatial secondary structure. 

Dissolved in water, fluorescence maximum of Calcofluor White is located between 435 and 438 nm (Fig. 3.32.b), while m an alc( l such as isobutanol or when bound to human serum albumin it fluoresces at 415 nm (Hg. 3.32a). In presence of ai- acid glycoprotein, fluorescence maximum of calcofluor shifts toward 439 nm when the fluorophore is at low concentrations and toward 448 lun when it is present at high concentrations (Fig. 3.32c). The sHft compared to water observed in presence of ai-acid glycoprotein is the result of calcofluor binding on the carbohydrates (40% in weight) of the protein. 

The role of the Tip residues in the catalytic site of qpimeiase has been investigAed by studying tbe energy transfer inhiUtion from Tip residues to NAD. 

EjxitaCion of Tip residues of ephnerase at 295 nm induces an emis m spectrum with two maxima at 330 and 435 nm. These peaks cunespmd to the emission of the Tip residues and of NAD respective. The at 435 nm is result of energy transfer from 

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During this period, Laurie also investigated the preparation of radiolabeled, iodo and bromo sugars via carbohydrate boranes (with J.-R. Neeser), fluorescent probe-sugar conjugates (with M. Yalpani), sugar ferrocene derivatives (with M. J. Adam), and extended his studies of the conformations of polycyclic sugar derivatives. 

Monovalent and multivalent fluorescent probes can be utilized to evaluate the influence of oligosaccharide clustering on recognition by cell-surface lectins. Fluorescence microscopy and flow cytometry are appropriate methods to visualize the corresponding receptor-carbohydrate interactions. 

Thermal intensity quenching can be performed also to study the dynamics of the carbohydrate residues of a i-acid glycoprotein. This protein contains 40% carbohydrate by weight and has up to 16 sialic acid residues (10-14% by weight). The fluorescent probe calcofiuor white binds to the carbohydrate residues of a i-acid glycoprotein and... 

The chiral unit is also important in the design of carbohydrate-selective boronic acid-based probes. " PET-based fluorescent probes containing the stereogenic centers were also developed to enhance selectivities towards sugars. Representative examples include the chiral sensors (R)-C-l, (iS)-C-l, (R)-C-2 and (iS )-C-2 and the achiral probes C-3a, C-3b and C-4 (Figure 4.4). The results of this study show that the chiral probes are more selective... 

Figure 4.4 Structure of chiral fluorescent probes for carbohydrates and their achiral analogs.

Several imino-alditols have been prepared from non-carbohydrate sources. 2,S-Dideoxy-2,S-imino-D-mannitol has been synthesized from a pyroglutamic acid derivative (see Vol. 27, p. 209, ref. 59 for the use of pyroglutamic acid in the synthesis of other imino-alditols), and 1-deoxy-L-allonojirimycin has been made from a protected L-serine derivative. A protected D-serine aldehyde provided access to a l,2,4-trideoxy-l,4-imino-D-eryrAro-pentitol derivative for incorporation into DNA, and reduction of the minor natural amino acid, 4-L-hydroxyproline, gave l,3,4-trideoxy-l,4-imino-D-eryrAro-pentitol which, after Af-acylation with a fluorescent probe, was incorporated into oligonucleotides. ... 

A long-wavelength probe 29 signaling carbohydrates in aqueous solutions by increasing of fluorescence was developed by Akkaya and Kukre on the basis of a symmetrical squaraine dye containing two phenylboronic acid functions [89]. The emission maximum of this probe is at 645 nm. A maximal response of about 25% was found for fructose. 

The design of fluorescent sensors is of major importance because of the high demand in analytical chemistry, clinical biochemistry, medicine, the environment, etc. Numerous chemical and biochemical analytes can be detected by fluorescence methods cations (H+, Li+, Na+, K+, Ca2+, Mg2+, Zn2+, Pb2+, Al3+, Cd2+, etc.), anions (halide ions, citrates, carboxylates, phosphates, ATP, etc.), neutral molecules (sugars, e.g. glucose, etc.) and gases (O2, CO2, NO, etc.). There is already a wide choice of fluorescent molecular sensors for particular applications and many of them are commercially available. However, there is still a need for sensors with improved selectivity and minimum perturbation of the microenvironment to be probed. Moreover, there is the potential for progress in the development of fluorescent sensors for biochemical analytes (amino acids, coenzymes, carbohydrates, nucleosides, nucleotides, etc.). 

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