ABSTRACT Theenzymatic orchemical insertion ofanoligosaccharide sequence insome proteic molecules canbestow a [610272]

ABSTRACT Theenzymatic orchemical insertion ofanoligosaccharide sequence insome proteic molecules canbestow a
protein with resistance against proteolytic enzymes .Glycosyltransferases areenzymes that transfer aglycosyl moiety
towards ahydroxylated substrate (serine, threonine or,more scarcely, tyrosine) .The chemical bonding involves the
functionalization ofanisothiocyanate residue belonging tooaglucidic sequence, followed byitscoupling toamino groups
from thelateral chain oflysine, orperhaps arginine .Because tyrosine isaubiquitous amino acid invirtually allproteins, one
canimagine abonding sequence ofamonosaccharide oranoligosaccharide which isextremely economical andsimple .In
thepresent work, theresults concerning thecoupling ofafunctionalized maltose with anaromatic amine, followed byits
diazonium salt coupling with tyrosine, are presented .After separation and purification, theobtained product was
investigated through mass spectrometry .
ESI
–
IT
–
MS CHARACTERIZATION OF AMINO ACIDS
FUNCTIONALIZED WITH OLIGOSACCHARIDE DERIVATIVES
I. Șișu1, L. Bozin1, M.C. Pascariu1,2,3, N. Dincă4, A. Georgescu1, A. Dragomirescu1, E. Șișu1*
1“Victor Babeș” University of Medicine and Pharmacy of Timișoara, Faculty of Medicine, 2 Eftimie Murgu Sq., RO -300041, Timișoara
2National Institute of Research and Development for Electrochemistry and Condensed Matter, 144 Dr. A. P. Podeanu, RO -300569, Timi șoara
3“Vasile Goldiș” Western University of Arad, Faculty of Pharmacy, 86 Liviu Rebreanu, RO -310045, Arad
4“Aurel Vlaicu” University of Arad, Faculty of Food Engineering, Tourism and Environmental Protection, 2 Elena Drăgoi, RO -310330, Arad
* Correspondence: [anonimizat]
EXPERIMENTAL Maltose is linked to 4,4′-
diaminodiphenylmethane (DADFM) by reductive
amination, andtheobtained amino sugar isconverted to
itscorresponding diazonium saltwhich, during thenext
step, iscoupled with tyrosine according tothereaction
sequence illustrated inFig.1.
Mass Spectrometry Mass spectrometry was performed
onaHigh Capacity IonTrap (HC-IT)mass spectrometer
from Bruker (Bruker Daltonik, Bremen, Germany ).The
sample was infused into theMSinstrument byon-line
syringe pump electrospray .Nitrogen with a2Lmin-1flow
rate and atemperature of250°Cwas employed for
desolvation andasanebulizer gas(at7p.s.i.).
Figure 1
RESULTS AND DISCUSSION Thefunctionalized tyrosine was subjected tomass spectrometry analysis .Theinvestigation
ofthecouplingcompound’s structure was conducted inboth positive andnegative ionmodes .Inpositive ionmode, the
[M+H+]+=717.0and [M+Na+]+=739.0pseudomolecular ions were revealed, which were further isolated andsubjected to
fragmentation ;theobtained MS2spectra areshown inFigs.2Aand 2B.Innegative ionmode, the[M-H+]-=715.0
pseudomolecular ionwas isolated and subjected tofragmentation, giving theMS2spectrum shown inFig.2C.The
acronyms used forMS2fragments’ assignment donotobey theDommon and Costello convention, except forthering
fragmentation, andareillustrated inFig.2D.
704.2699.4680.3667.8
627.3583.2393.3
238.1225.1+MS2(739.0
024681012Intens .
100 200 300 400 500 600 700 m/zY3/Z1
A*3,5Y4
M+Na/2H2OM+Na/N2/CO2
M/2H2O
X1,5
M/H2O
249.1
256.2281.2
315.0329.2356.3
511.3482.9
469.3411.10.1385.8401.4
379.218. -MS2(553.0), 22.2-22.5min #(575-579)
051015Intens.
150 200 250 300 350 400 450 500 550 m/z
X1,4B
704.2699.4680.3667.8
627.3583.2393.3
238.1225.1+MS2(739.0
024681012Intens .
100 200 300 400 500 600 700 m/zY3/Z1
A*3,5Y4
M+Na/2H2OM+Na/N2/CO2
M/2H2O
X1,5
M/H2O
249.1
256.2281.2
315.0329.2356.3
511.3482.9
469.3411.10.1385.8401.4
379.218. -MS2(553.0), 22.2-22.5min #(575-579)
051015Intens.
150 200 250 300 350 400 450 500 550 m/z
X1,4
704.2699.4680.3667.8
627.3583.2393.3
238.1225.1+MS2(739.0
024681012Intens .
100 200 300 400 500 600 700 m/zY3/Z1
A*3,5Y4
M+Na/2H2OM+Na/N2/CO2
M/2H2O
X1,5
M/H2O
249.1
256.2281.2
315.0329.2356.3
511.3482.9
469.3411.10.1385.8401.4
379.218. -MS2(553.0), 22.2-22.5min #(575-579)
051015Intens.
150 200 250 300 350 400 450 500 550 m/z
X1,4B
679.4624.2653.8
582.8
486.1476.4
462.3
432.2 283.1255.1
553.0-MS2(715.0)
01234Intens .
100 200 300 400 500 600 700 m/zZ3
Z3/N2 X*2Y5/Y1C5
Y5B5
M/2H2OM/H2O/CO2
X1,5B4/H2OC
679.4624.2653.8
582.8
486.1476.4
462.3
432.2 283.1255.1
553.0-MS2(715.0)
01234Intens .
100 200 300 400 500 600 700 m/zZ3
Z3/N2 X*2Y5/Y1C5
Y5B5
M/2H2OM/H2O/CO2
X1,5B4/H2OC
624.2653.8
582.8
486.1476.4
462.3
432.2 283.1255.1
553.0-MS2(715.0)
01234Intens .
100 200 300 400 500 600 700 m/zZ3
Z3/N2 X*2Y5/Y1C5
Y5B5
M/2H2OM/H2O/CO2
X1,5B4/H2OC
O
OH
OHOHOH
OHOH
OOHOH
NH N CH
2NOH
NH
2CH COOHCH
2
B1
C1B2C2B3
C3
B4
C4Z5Y4Y3Y2
Z4Z3
Z2
B5
C5
B6Y1
Y2Z1A0,2
A*3,5X*2X1,5
X*3X1,4
O
OH
OHOHOH
OHOH
OOHOH
NH N CH
2NOH
NH
2CH COOHCH
2O
OH
OHOHOH
OHOH
OOHOH
NH N CH
2NOH
NH
2CH COOHCH
2
B1
C1B2C2B3
C3
B4
C4Z5Y4Y3Y2
Z4Z3
Z2
B5
C5
B6Y1
Y2Z1A0,2
A*3,5X*2X1,5
X*3X1,4
163.6627.1 673.0
699.1+MS2(717.0)
0.10.20.30.40.50.6Intens .
100 200 300 400 500 600 700 m/z717.0
249.1
256.2281.2
315.0329.2356.3
511.3482.9
469.3411.10.1385.8401.4
379.218. -MS2(553.0), 22.2-22.5min #(575-579)
051015Intens.
150 200 250 300 350 400 450 500 550 m/z436.8M/H2OM/CO2X1,4
X*3/N2B1
121.2A0,2
Y5/CO2511.2
555.2Y5A
163.6627.1 673.0
699.1+MS2(717.0)
0.10.20.30.40.50.6Intens .
100 200 300 400 500 600 700 m/z717.0
249.1
256.2281.2
315.0329.2356.3
511.3482.9
469.3411.10.1385.8401.4
379.218. -MS2(553.0), 22.2-22.5min #(575-579)
051015Intens.
150 200 250 300 350 400 450 500 550 m/z436.8M/H2OM/CO2X1,4
X*3/N2B1
121.2A0,2
Y5/CO2511.2
555.2Y5
163.6627.1 673.0
699.1+MS2(717.0)
0.10.20.30.40.50.6Intens .
100 200 300 400 500 600 700 m/z717.0
249.1
256.2281.2
315.0329.2356.3
511.3482.9
469.3411.10.1385.8401.4
379.218. -MS2(553.0), 22.2-22.5min #(575-579)
051015Intens.
150 200 250 300 350 400 450 500 550 m/z436.8M/H2OM/CO2X1,4
X*3/N2B1
121.2A0,2
Y5/CO2511.2
555.2Y5A
CONCLUSIONS 1.The coupling ofanartificial amino sugar with aaromatic amino acid was achieved forthefirsttime.
2.The experimental conditions which allow thecoupling reaction, without affecting thestructure oftheamino acid, were
established .3.The mass spectrometry conditions, intheESIionization variant, were determined, andtheobtained product
wascompletely characterized through CID-MS2experiments .Figure 2Nano (+)-ESI HC-IT-MSanalysis oftheamino acid functionalized bycoupling with anamino sugar :A)MS2
spectrum of[M+H]+=717.0pseudomolecular ion;B)MS2spectrum of[M+Na]+=739.0sodiated pseudomolecular ion;C)
MS2spectrum of[M-H]-=715.0pseudomolecular ion;D)Fragmentation scheme with clarification ofacronyms used for
spectra A-C.Experimental conditions :eluent MeOH :H2O=9:1;concentration 20pmol/L;acquisition time 3min;injection
speed oftheanalyte solution :250L/15min;isolation window :2a.m.u.
Acknowledgements .This work wassupported bytheRomanian National Authority forScientific Research (CNCS -UEFISCDI) through project PN-II-PCCA -2011 -142.