CHARACTERIZATION BY SOFT IONIZATION MASS SPECTROMETRY [610263]

CHARACTERIZATION BY SOFT IONIZATION MASS SPECTROMETRY
METHODS OF MONO – AND OLIGOSACCHARIDES FUNCTIONALIZED AT THE
ANOMERIC CENTER

Loreta Andrea Bo žin1#, Mihai -Cosmin Pascariu1,2#, Anca Dragomirescu1,
Georgeta Simu1, Nicolae Dinc ă3, Eugen Ș ișu1*

1“Victor Babeș” University of Medicine and Pharmacy of Timișoara, Faculty of Medicine, 2
Eftimie Murgu Sq., RO -300041, Timișoara, Romania
2“Vasile Goldiș” Western University of Arad, Faculty of Pharmacy, 86 Liviu Rebreanu, RO –
310045, Arad, Romania
3“Aurel Vlaicu” U niversity of Arad, Faculty of Food Engineering, Tourism and
Environmental Protection, 2 Elena Drăgoi, RO -310330, Arad, Romania
e-mail: [anonimizat]
#equal contribution

Abstract

In order to obtain oligosaccharides functionalized with amine groups, al iphatic diamines and
polyamines were coupled with maltose by using reductive amination reactions . The
synthesized compounds were characterized through electrospray ionization mass spectrometry
(ESI-MS), while finer structural det ails were obtained by using MS2.

Introduction

In the hands of organic chemists, t he reductive amination reaction is a powerful tool for
building C -N bonds. Its importance is revealed by the fact that vol ume 59 of Organic
Reactions series (Ed. L.E. Overman et al. – John Wiley & Sons, 2002 ) is completely dedicated
to the reductive amination reaction , which uses boranes and their derivatives as reducing
agents (2100 reference citations). A multitude of substrates, including aliphatic, aromatic and
mixed aldehydes, but also aliphatic and aromatic ketones, can be reductively aminated with
primary or secondary amines belonging to the aliphatic or aromatic series, and even by using
the ammonium ion.
Dedicated literature abounds with sugar compounds modified with amines, especially in
relation with the processes of analytical detection of biological trace samples. The attachment
of aromatic amines (some possessing fluorescence) allows the detection of sugars by using
high performance liquid chromatography (HPLC) and capillary electrophoresis (CE)
techniques.
Keeping in mind our experience regarding the analysis and derivatization of dextrans and
maltodextrins [1 -3], we set out to synthesize some maltoses aminated with different aliphatic
amine components. In this paper , the obtained results r egarding the derivatization through
reductive amination of maltose, as well as the analysis through electrospray ionization mass
spectrometry of the obtained compounds, are presented.

Experimental

The reactions took place in round flasks made of glass, w hich were equipped with magnetic
stirrers, heating sources and straight condensers. The reagents ratios vary depending on the
amine, while the working temperature (45 -90 °C) depends on the solubility of the
participating reacting species. The progress of t he reaction was monitored through TLC ,

visualization of spots being made with 5% ethanolic ninhydrin. The isolated compound s were
purified by ion exchange chromatography, using ammonium hydroxide solutions of various
concentrations as mobile phases . The pr oduct s were finally brought in solid state through
lyophilization. Before being subjected to physical -chemical analysis, the products were
investigated regarding their purity through TLC and for identity by reactin g them with
ninhydrin, all compounds givin g positive results during the se tests.
The spectral characterization through HCIT -MS analysis of maltose derivatives , obtained
following the reductive amination reaction with different amine components , was done on a
High Capacity Ion Trap (HCIT) Ultra PTM instrument (Bruker Daltonik, Bremen, Germany) ,
belonging to the Institute of Chemistr y Timișoara of Romanian Academy and courtesy of dr.
Gh. Ilia. In all cases, the samples were injected in the spectrometer with the help of a
microsyringe, attached to a push -syringe, with a speed of 250 μL h-1. Nitrogen was used as a
nebulizer gas, with a flowrate of 5 L min-1 and at 7 p.s.i., and at a 300 °C desolvation
temperature. The instrument was programmed to operate in positive ion mode, at an ESI
potential of 3.0 kV.
For the analysis, the sample s were dissolved in MeOH/H 2O (1:1 v/v) to a concentrat ion of 5 -7
pmol μL-1 (stock solution s), which w ere diluted as needed.

Results and discussion

The reaction s that occur during the derivatization of maltose through reductive amination are
presented in Scheme 1 , while the reaction conditions, which depend on the amine being used ,
are presented in Table 1 .

Scheme 1. The reductive amination reactions of maltose with different amine components

Table 1. The reaction conditions for derivatization of maltose with various amin e components

No. Aliphatic amine
(mol) Substrate
(mol) Reducing agent
(mol) Acid
(mol) Time
(h) Control
MS Chemical
control
1 AcONH 4 maltose Na(CN)BH 3 AcOH 25 [M+H]+ Ny#
15·10-3 1.5·10-3 20·10-3 21·10-3 344.00 +
2 HMD maltose Na(CN)BH 3 AcOH 24 [M+H]+
92·10-3 9·10-3 18·10-3 50·10-3 443.00 +
3 DETA maltose Na(CN)BH 3 AcOH 120 [M+H]+
90·10-3 3·10-3 9·10-3 30·10-3 430.00 +
4 TETA maltose Na(CN)BH 3 AcOH 74 [M+H]+
210·10-3 9·10-3 20·10-3 120·10-3 473.00 +
#Ny – ninhydrin (ethanolic solution of ninhydrin)

In all cases, the [M+H]+ pseudomolecular ion ’s peak was revealed (see Table 1 ). This was
isolated and fragmented through CID, obtaining the MS2 spectrum (and also, whenever
possible, the MS3 and even the MS4 spectr um), which gave the minimal n umber of structural
data (characteristic fragmentations for the sugar and, respectively , the amine component). To
give an example, the MS2 and, respectiv ely, MS3 spectra of the product obtained after
amination of maltose with ammonium acetate are shown in Figs. 1 and 2. To facilitate the
assignments , the main fragmentations of the analyzed product are presented in Scheme 2 . The
MS3 spectrum of the P fragment (see Scheme 2 ), which is also the base peak, gives all
structural details that confirm the structure of the synthesized product.

100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0 100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0MS3
[M+H+]+ =MpMp-2H2O
Mp-2H2O-CH2OP
Mp-E-2CH2O
Mp-E-2CH2O-H2O Un
100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0 100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0MS3
100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0 100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0 100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0 100.1181.0
205.9224.0
278.0308.07. +MS2(344.0 )
0.00.51.01.52.02.53.04x10Intens .
100 150 200 250 300 350 m/z344.0MS3
[M+H+]+ =MpMp-2H2O
Mp-2H2O-CH2OP
Mp-E-2CH2O
Mp-E-2CH2O-H2O Un

Figure 1. The MS2 spectrum obtained after (+) -ESI-HCIT -MS analysis of the aminated
derivative of maltose, following ammonium acetate reductive amination

9.
05101520Intens .
60 80 100 120 140 160 180 m/z181.0163.1
145.0115.183.1
69.1+MS3(181.0 )
PP-H2O
P-2H2OP-2H2O-CH2OUn
Un9.
05101520Intens .
60 80 100 120 140 160 180 m/z181.0163.1
145.0115.183.1
69.1+MS3(181.0 )9.
05101520Intens .
60 80 100 120 140 160 180 m/z181.0163.1
145.0115.183.1
69.1+MS3(181.0 )9.
05101520Intens .
60 80 100 120 140 160 180 m/z181.0163.1
145.0115.183.1
69.1+MS3(181.0 )
PP-H2O
P-2H2OP-2H2O-CH2OUn
Un

Figure 2. The MS3 spectrum obtained after (+) -ESI-HCIT-MS analysis of the aminated
derivative of maltose, following ammonium acetate reductive amination

Scheme 2. Fragmentation possibilities f or the aminated derivative of maltose , obtained
through the reductive amination with ammonium acetate

Conclusions

The derivatization of the anomeric site through attachment of aliphatic amines is done in mild
conditions with satisfying yields and with procedures that were established by this research.
The determination of the mass spectrometry c onditions for ESI -HCIT -MS analysis of the
aminated derivatives of maltose gives clear, sharp and reproducible spectra. Owing to the
reactivity of the free amine groups, t he thus obtained aminated derivatives can be used in
many complexation reactions with hydrophilic agents.

Acknowledgements

This work was supported by the Romanian National Authority for Scientific Research
(CNCS -UEFISCDI) through project PN -II-PCCA -2011 -142. Part of the research was
performed at the Center of Genomic Medicine from the ‘Vi ctor Babe ș’ University o f
Medicine and Pharmacy of Timiș oara, POSCCE 185/48749, contract 677/09.04.2015 .

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