2942 Zhang et al.
Asian J. Chem.
in mass spectra and characterization of the factors affecting
the reaction. We illustrate the fragmentation pathways ofAPRs
with combination of computational calculations and it is impor-
tant for understanding the process of glycosylation between
sugars and peptides, even protein in complicated systems.
functional formulated with a mixture of Hartree-Fock exchange
energy and Becke's three-parameter 1988 gradient-corrected
exchange energy, as well as Lee-Yang-Parr (LYP) correlation
energy15. Atomic orbitals were described by a Gaussian-type
split valence shell 6-31++G(d,p) basis set, including polari-
zation and diffuse functions for all atoms16. Low-lying structures
of the molecular ions were obtained through Monte Carlo
conformational searches with a semi-empirical method (AM1)
using Spartan software ("SPARTAN, '04 Essential V2.0.0;
Wavefunction, Inc.: Irvine, CA, 2004") followed by geometry
optimizations at UB3LYP/6-31G(d) and UB3LYP/6-31++G
(d,p) levels.Additional DFT geometry optimizations for other
plausible low-lying structures, which likely were missed in
the conformational searches, also were performed. Harmonic
vibrational frequencies of all optimized structures were calcu-
lated to confirm the structures were at local minima (all real
frequencies). Relative enthalpies at 0 K (∆H°) were calculated
from the electronic energies and zero-point vibrational energies
(ZPVE) obtained within the harmonic approximation.All DFT
calculations were performed using the Gaussian 03 software
package.
EXPERIMENTAL
Sugars and six amino acids: arginine (Arg), asparagines
(Asn), glutamine (Gln), histamine (His), lysine (Lys) and
tryptophan (Trp), were purchased from Sigma (St. Louis, MO,
USA). Water was purified by Milli-Q distilled (Millipore,
Bedford, MA, USA). Unless otherwise indicated, all other
reagents and solvents were of analytical grade and were
purchased from Sigma-Aldrich (St. Louis, MO, USA). The Fmoc-
protected amino acids and the Wang resin were purchased from
Advanced ChemTech., Inc. (Louisville, KY, USA).
Sample preparation: Fmoc-protected amino acid and
pre-washed Wang resin mixed with N,N-dimethyl 4-amino-
pyridine (DMAP) was dissolved in DMF and reacted for 2 h
under nitrogen bubbled at room temperature, then washed
DMF (3 times), dichloromethane DCM (3 times) and dried
under nitrogen atmosphere. Acetylation of amino acids 1 mL
acetylation reagent (acetic acid:methanol = 1 : 4) (v/v) was
added to a mixture of Fmoc-protected amino acid with Wang
resin (20 mg) and 100 mL of 50 mM ammonium bicarbonate
and stirred for 3 h at room temperature. Fresh 20 % piperidine
in DMF (v/v) was added to theWang resin with Fmoc-protected
amino acid attached at room temperature and swirled for 20
min under nitrogen. The resin was filtered, washed with DMF,
DCM and dried under nitrogen gas. Wang resin with acetylated
amino acid was cleaved by trifluoroacetic acid (TFA) and filtered.
Acetylated amino acid solution was precipitated in tert-butyl
methyl ether, purified by HPLC, freeze-dried for experiments.
Maillard reaction model: The reaction model was set as
following: 0.1 M of amino acids and acetylated amino acids
were dissolved in 1 M d-glucose solution and got the ratio of
1:10 by molecular weight. Freezed-dry by SC250DDA Speedvac
Plus (Thermo Electron Corporation, Waltham, MA) the
samples to get the whiter power mixture and dry-heating the
samples in sealed vials for 1 h at 50, 70 and 90 °C, respectively
and cooling down the samples in -20 °C immediately after
reaction finished. All samples were dissolved with CH3CN:
H2O:FA (40/60/0.5:v/v/v) just before submitting to MS.
Mass spectrometer:All mass spectrometric experiments
were conducted using a quadrupole ion trap mass spectrometer,
LTQ Deca XP Plus (Finnigan LTQ, Thermo Finnigan, San
Jose, CA, USA) equipped with a home-made nanospray ion
source. The physical parameters of the interface, i.e. the
distance of the needle from the hole in the spray shield (1.2-
1.5 cm), the high voltage (2.8 - 3kV) added on the stainless
steel unit and the temperature of the heated capillary (180 °C),
were optimized at the flow rate of 25 mL/h. Electrospray
voltage was typically kept between 2.8-3.0 kV and the inlet
capillary was maintained at 180 °C. To obtain the spectra of
MS2, the normalized collision energy was varied with all the
other ion tuning conditions fixed.
RESULTS AND DISCUSSION
Amino acids, time and sugar effects onAmadori rearran-
gement products: In our model system, six selected amino
acids and their corresponding N-terminal acetylated forms
were reacted with three reducing sugars (D-glucose, β-lactose
and maltose) at 50, 70 and 90 °C, respectively. In MS spectra,
the main additional masses increased related to intermediates
(180 Da; binding with glucose) and toAmadori rearrangement
products (162 Da; condensation by loss one molecular of water),
compared with mass to charge ratio (m/z) of the original amino
acid in MS spectra (Table-1).Amadori rearrangement products
were formed easily for six amino acids and acetylated lysine,
it means that reducing sugars preferentially reacted with N-
terminal amino group on main chain (α-NH2) and of ε-NH2
on side chain of lysine, but not amino or imine groups on side
chain of other five amino acids at studied temperature (≤
90 °C). This demonstrated that reactivity of ε-NH2 group of
lysine is as high as α-NH2 of amino acids with reducing sugars
at relative low temperature in Maillard reaction. The results
also displayed that extended reaction time increased the yield
of Amadori rearrangement products. For example, it took 1 h
to obtain the yield of Amadori rearrangement products
(glucosylated lysine) about 50 % at 90 °C, but more than 3 h
at 70 °C and almost 20 h at 50 °C (data not show). All three
reducing sugars (D-glucose, β-lactose and maltose) could react
with amino acids noticeably at studied conditions. Among the
three reducing sugars, glucose was the most reactive with
amino compounds at relatively low temperature (50 °C). The
reaction rate of sugars followed the order: glucose > lactose >
maltose.
Fragmentation of Amadori rearrangement products
in MS2: The fragmentation pattern ofAmadori rearrangement
products in reducing sugar-amino acid model system was
systematically studied by ESI-MS2 and the MS2 spectra of
Amadori rearrangement products from D-glucose showed the
characteristic fragment ions of [M-18 + H]+, [M-36 + H]+,
Computational methods: Electronic energies were calcu-
lated in the framework of DFT using the unrestricted (U) hybrid
Zhang
