Full Papers
doi.org/10.1002/ejoc.202100672
Chemical synthesis of the O-glycosidic series: p-aminophenyl-ß-D-
PBS and incubated with fluorescent neoglycoproteins (fixed
concentrations) and compounds (range of concentrations) and
glucopyranoside(O-1) was synthesized as a white solid (97% yield)
from p-nitrophenyl-ß-D-glucopyranoside according to an already
published protocol.[23] Characterization data were identical with
those already described.
°
incubated 4 h at 4 C. Then, the plates were carefully rinsed with
PBS. 200 uL of fresh PBS was added for the fluorescence readout
(λex=485 nm, λem=530 nm). The amount of neoglycoproteins
stayed in interaction with cells was compared with the amount of
neoglycoprotein in absence of product.
p-aminophenyl-ß-D-galactopyranoside (O-2) was synthesized as a
white solid (91% yield) from p-nitrophenyl-ß-D-galactopyranoside
according to an already published protocol.[23] Characterization data
were identical with those already described.
Tyrosinase inhibition tests: Tyrosinase inhibitor activity was
measured by spectrophotometry based one the method presented
by Masamoto et al.[25] 10 μL of inhibitor solution at different
concentrations in DMSO were placed into 96-wells microplate
mixed with ammonium formate buffer (60 μL, 50 mmol/L, pH 6.4)
and 20 μL (0.8 mg/mL) of tyrosine was added. Just before the
absorbance read at 450 nm, 10 μL of mushroom tyrosinase (5000 U/
p-aminophenyl-ß-D-fucopyranoside(O-3) was synthesized as a white
solid (97% yield) from p-nitrophenyl-ß-D-fucoopyranoside accord-
ing to an already published protocol.[23] Characterization data were
identical with those already described.
°
mL) was added. The mixture was incubated at 27 C for 10 min. The
absorbance value was recovered every 20 sec during 10 min.
Arbutine were used as a positive control. Each measurement was
performed at least in triplicate. The IC50, corresponding to the
needed concentration to observe 50% loss of the enzyme activity,
were calculated with GraphPad.
Biological procedures
Protein expression and purification: Production and purification of
DtGlyE159Q was performed as described previously by Guillotin
et al.[19] Escherishia coli Rosetta (DE3) transformed with expression
plasmids were grown in LB medium supplemented with chloram-
°
phenicol (34 μg/mL) and kanamycin (30 μg/mL) at 37 C until
OD600 reached 0.6. Induction was then done by addition of 1 mM
Acknowledgements
°
IPTG and incubated overnight a 25 C. Cells were harvested, lyzed
by freeze-thaw cycles and sonication. Then, supernatant was
clarified by heat treatment for 15 min at 70 C before centrifugation.
Finally, supernatant was loaded on a Nickel column (HisPure,
Thermo Scientific) and purified by elution with lysis buffer
containing 500 mM imidazole. The concentration of the various
fractions collected was obtained by Bradford assay.
°
The authors thank the regions Centre Val de Loire and Bretagne
for their financial support.
Conflict of Interest
Lectin Array assays: Lectin Array assays were performed according
GlycoDiag’s protocol already described.[26] Briefly, GLYcoPROFILEs
were performed on LEctPROFILE® plates from GLYcoDiag (Orléans,
France). The interaction profiles of each compound were deter-
mined through an indirect method based on the inhibition by the
compound of the interaction between a specific couple lectin-
glycan (neoglycoproteins or glycoproteins). Briefly, a mix of neo-
glycoproteins or glycoproteins (fixed concentration) and the
corresponding compounds (range of concentrations) prepared in
PBS supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 is deposed
in each well (50 μL each) in triplicates and incubated two hours at
room temperature. After washing with PBS buffer, the conjugate
streptavidine-DTAF for fluorescence plate or extravidine-peroxydase
for absorbance palte is added (50 μL) and incubated 30 min more.
The plate was washed again with PBS. Finally, 100 μL of PBS was
added for the readout of fluorescent plate performed with a
fluorescence reader (λex=485 nm, λem=530 nm, Fluostar OPTIMA,
BMG LABTECH,France) For the absorbance plate, the plate was
washed with PBS buffer, and a solution of OPD (SIGMAFAST™ OPD
(o-Phenylenediamine dihydrochloride), 100 μl) for the detection of
the peroxidase activity. The plate was incubated 15 min protected
from light. The coloration was stopped by adding HCl (100 μl,
1 mM) and the readout performed with an absorbance reader. The
signal intensity is inversely correlated with the capacity of the
compound to be recognized by the lectin and expressed as
inhibition percentage with comparison with the corresponding
tracer alone (Neoglycoproteins or glycoproteins).
The authors declare no conflict of interest.
Keywords: Arbutin analogs · Carbohydrates · Enzyme catalysts ·
Thioglycoligase · Whitening agents
[1] a) S. Kunachak, P. Leelaudomlipi, S. Wongwaisayawan, Aesthetic Plast
Surg 2001, 25, 114–117; b) B. R. Nelson, D. J. Fader, M. Gillard, S. R.
Baker, T. M. Johnson, J. Am. Acad. Dermatol. 1995, 32, 623–626; c) K. B.
Penney, C. J. Smith, J. C. Allen, J. Invest. Dermatol. 1984, 82, 308–310;
d) Y. Kobayashi, H. Kayahara, K. Tadasa, T. Nakamura, H. Tanaka, Biosci.
Biotechnol. Biochem. 1995, 59, 1745–1746.
[2] a) M. Seiberg, C. Paine, E. Sharlow, P. Andrade-Gordon, M. Costanzo, M.
Eisinger, S. S. Shapiro, J. Invest. Dermatol. 2000, 115, 162–167; b) A.
Greatens, T. Hakozaki, A. Koshoffer, H. Epstein, S. Schwemberger, G.
Babcock, D. Bissett, H. Takiwaki, S. Arase, R. R. Wickett, R. E. Boissy, Exp.
Dermatol. 2005, 14, 498–508.
[3] a) A. Palumbo, M. d’Ischia, G. Misuraca, G. Prota, Biochim. Biophys. Acta
1991, 1073, 85–90; b) K. Jimbow, H. Obata, M. A. Pathak, T. B. Fitzpatrick,
J. Invest. Dermatol. 1974, 62, 436–449.
[4] a) W. Westerhof, T. J. Kooyers, J. Cosmet. Dermatol. 2005, 4, 55–59;
b) J. J. Nordlund, P. E. Grimes, J. P. Ortonne, J. Eur. Acad. Dermatol.
Venereol. 2006, 20, 781–787.
[5] a) C. Ramírez, K. Pham, M. F. E. Franco, M. Chwa, A. Limb, B. D.
Kuppermann, M. C. Kenney, Neurotoxicology 2013, 39, 102–108; b) G. H.
Kim, K. A. Cheong, A.-Y. Lee, Ann. Dermatol. 2017, 29, 715–721.
[6] G. N. Selezenev, D. M. Popov, N. G. Selezenev, Vopr. Biol., Med. Farm.
Khim. 2012, 18–21.
[7] Y.-J. Lim, E. H. Lee, T. H. Kang, S. K. Ha, M. S. Oh, S. M. Kim, T.-J. Yoon, C.
Kang, J.-H. Park, S. Y. Kim, Arch. Pharmacal Res. 2009, 32, 367–373.
[8] a) M. Seiberg, Pigm. Cell Res. 2001, 14, 236–242; b) D. Cerdan, G.
Redziniak, C. A. Bourgeois, M. Monsigny, C. Kieda, Exp. Cell Res. 1992,
203, 164–173; c) R. E. Boissy, Exp. Dermatol. 2003, 12 Suppl 2, 5–12.
[9] a) K. Sugimoto, T. Nishimura, K. Nomura, K. Sugimoto, T. Kuriki, Chem.
Pharm. Bull. 2003, 51, 798–801; b) A. Garcia-Jimenez, J. A. Teruel-Puche,
J. Berna, J. N. Rodriguez-Lopez, J. Tudela, F. Garcia-Canovas, PLoS One
NeoPROFILE@ Assays: NeoPROFILE@ Assays were performed
according GlycoDiag’s protocol already described.[27] Compounds
interactions with carbohydrate recognition receptors expressed at
the surface of NHEK cells were measured and achieved with
fluoresceinylated neoglycoproteins according to GLYcoDiag tech-
nology. (α-Galactose-BSA, ß-Glucose-BSA, α-Rhamnose-BSA). Briefly,
cells were first grown to confluence (80–90%) in 96-well plates.
Once the confluence reaches, cells were washed several times with
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