Synthesis of glycosylamines: Identification and quantification of side products

Article abstract of DOI:10.1081/CAR-100104862

The synthesis of some glycosylamines (1-amino-1-deoxy-D-glucose, 1-amino-1-deoxy- D-galactose and 1-amino-1-deoxylactose) was carried out by treatment of the corresponding reducing sugars with ammonium hydrogencarbonate in concentrated ammonia. The reaction mixture was first analyzed by capillary electrophoresis with indirect absorbance detection and high performance anion-exchange chromatography with pulsed amperometric detection. Beside glycosylcarbamate, a known reaction by-product, fructose and lactulose were detected during the synthesis of 1-amino-1-deoxyglucose and 1-amino-1-deoxylactose, respectively. Quantification of glycosylamines was carried out by micellar electrokinetic chromatography with UV detection of their 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives; lactulosylamine was thus detected in the synthesis mixture of 1-amino-1-deoxylactose. The Fmoc-glycosylamines were easily purified from the other components of the crude synthesis mixtures.

Full text of DOI:10.1081/CAR-100104862

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SYNTHESIS OF GLYCOSYLAMINES: IDENTIFICATION
AND QUANTIFICATION OF SIDE PRODUCTS
a
a
b
b
b
Cristiana Campa , Ivan Donati , Amedeo Vetere , Amelia Gamini & Sergio Paoletti
a
F.B.C. S.r.l. , AREA Science Park, Padriciano 99, Trieste, I-34012, Italy
b
Department of Biochemistry, Biophysics and Macromolecular Chemistry , University of
Trieste , Via L. Giorgieri 1, Trieste, I-34127, Italy
Published online: 16 Aug 2006.
To cite this article: Cristiana Campa , Ivan Donati , Amedeo Vetere , Amelia Gamini & Sergio Paoletti (2001) SYNTHESIS
OF GLYCOSYLAMINES: IDENTIFICATION AND QUANTIFICATION OF SIDE PRODUCTS, Journal of Carbohydrate Chemistry,
20:3-4, 263-273, DOI: 10.1081/CAR-100104862
To link to this article: http://dx.doi.org/10.1081/CAR-100104862
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J. CARBOHYDRATE CHEMISTRY, 20(3&4), 263–273 (2001)
SYNTHESIS OF GLYCOSYLAMINES:
IDENTIFICATION AND QUANTIFICATION OF
SIDE PRODUCTS
Cristiana Campa,1,2,* Ivan Donati, Amedeo Vetere,2
1,2
2
2
Amelia Gamini, and Sergio Paoletti
1
F.B.C. S.r.l., AREA Science Park, Padriciano 99,
I-34012 Trieste, Italy
Department of Biochemistry, Biophysics and
2
Macromolecular Chemistry, University of Trieste, Via L. Giorgieri 1,
I-34127 Trieste, Italy
ABSTRACT
The synthesis of some glycosylamines (1-amino-1-deoxy-D-glucose, 1-amino-
1-deoxy- D-galactose and 1-amino-1-deoxylactose) was carried out by treat-
ment of the corresponding reducing sugars with ammonium hydrogencarbon-
ate in concentrated ammonia. The reaction mixture was first analyzed by
capillary electrophoresis with indirect absorbance detection and high perfor-
mance anion-exchange chromatography with pulsed amperometric detection.
Beside glycosylcarbamate, a known reaction by-product, fructose and lactu-
lose were detected during the synthesis of 1-amino-1-deoxyglucose and 1-
amino-1-deoxylactose, respectively. Quantification of glycosylamines was
carried out by micellar electrokinetic chromatography with UV detection of
their 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives; lactulosylamine was
thus detected in the synthesis mixture of 1-amino-1-deoxylactose. The Fmoc-
glycosylamines were easily purified from the other components of the crude
synthesis mixtures.
*Corresponding author. Fax: ꢀ39-040-6763691; E-mail: campa@bbcm.univ.trieste.it
2
63
Copyright © 2001 by Marcel Dekker, Inc.
www.dekker.com

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CAMPA ET AL.
INTRODUCTION
Glycosylamines are currently used for the preparation of glycopeptides, de-
tergents, N-hydroxysuccinimidyl esters and for the synthesis of glycopoly-
1–3
mers. There are two main drawbacks related to the preparation and use of gly-
cosylamines: their low stability, due to rapid hydrolysis in neutral or weakly
1–4
acidic solutions, and the formation of N-glycosylcarbamate and of diglycosy-
1
–3,5–8
lamine as secondary reaction products.
Besides the interest to identify and
quantify reaction side-products, it is important to provide an accurate evaluation
of product yields in order to guide studies for reaction optimization. Furthermore,
amine by-products might interfere when glycosylamines are used to synthesize
substances of specific biological relevance. Among the reported methods for gly-
1,4,5
cosylamines synthesis,
we investigated that performed with ammonium hy-
drogencarbonate in concentrated ammonia solution at 42°C, which is claimed to
1
give quantitative yields with negligible amounts of side-products. Capillary
electrophoresis/indirect UV detection (CE/indirect UV), anion-exchange chro-
matography/pulsed amperometric detection (HPAEC-PAD), micellar electroki-
netic chromatography (MEKC)-UV and ion spray mass spectrometry allowed for
the quantification of many components in the synthesis mixtures: the starting re-
ducing sugars and their isomerization products, glycosylcarbamates, glycosy-
lamines and amino side-products. A method for purification of glycosylamines is
also presented.
RESULTS AND DISCUSSION
Analysis of the Crude Synthesis Mixtures
9–11
CE/indirect UV
turned out to be an ideal tool to analyze the untreated
synthesis mixtures of glycosylamines. A rapid migration of the anionic analytes
1
2
was achieved by adding tetradecyltrimethylammonium bromide (TTAB) to the
separation buffer. The high pH (12.5) of the medium also prevented the hydrolysis
1
,4
of the formed glycosylamines. In Figure 1 the electropherograms of glucosy-
lamine (Glc-1-NH ) (A), galactosylamine (Gal-1-NH ) (B) and lactosylamine
2
2
1
3
(
Lac-1-NH ) (C) synthesis mixtures are shown. In each panel, peak 3 refers to the
starting sugar. In Figure 1A, peak 2 can be assigned to glucosylcarbamate.
2
1
,3,5
The
strong acidity of the carboxylic group induces the short migration time. Moreover,
the disappearance of peak 2 was observed on decreasing the sample solution pH
(from 10 to 4). At the same time an increase in the glucose signal occurred. Those
evidences can be accounted for by the low stability of carbamates under acidic con-
14
ditions. Similarly, peaks 2 in panels B and C were assigned to galactosylcarba-
mate and lactosylcarbamate, respectively. Due to the high alkalinity of the synthe-
sis medium, peaks 3ꢁ in Figure 1 were attributed to fructose (panel A) and lactulose
(
4-O-ꢂ- D-galactopyranosyl- D-fructofuranose) (panel B), resulting from isomer-
15–17
ization of glucose and lactose, respectively.
by comparison with standards.
Such assignments were confirmed

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SYNTHESIS OF GLYCOSYLAMINES
265
Figure 1. CE/ indirect UV of untreated freeze-dried synthesis mixtures (5 mg in 1 mL of 0.1 mM
NaOH) of: (A) Glc-1-NH : (1) 0.7 mM galacturonic acid (i.s.), (2) glucosylcarbamate, (3ꢁ) fructose,
3) glucose, (4) contribution from glucosylamine; (B) Gal-1-NH : (1) 0.7 mM galacturonic acid (i.s.),
2) galactosylcarbamate, (3) galactose, (4) contribution from galactosylamine; (C) Lac-1-NH : (1)
.7 mM galacturonic acid (i.s.), (2) lactosylcarbamate, (3ꢁ) lactulose, (3) lactose, (4) contribution
from lactosylamine.
2
(
(
2
2
0
No isomers of galactose in the Gal-1-NH mixture were found. Due to the
2
scarce ionization, glycosylamines contributed to the peak 4. Unfortunately, since
the signal did not re-attain the baseline, an accurate quantification of glycosy-
lamines was precluded. CE/indirect UV monitored formation of side-products.
Figure 2A shows the molar percentages of glucose, fructose and glucosylcarba-
mate as a function of the Glc-1-NH synthesis time.
2
Fructose was detected only after 12 hours. Analogous data for the crude Lac-
1
-NH mixture showed that lactulose could be observed after 4 hours (Figure 2B).
2
These results were confirmed analyzing the untreated synthesis mixtures by

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CAMPA ET AL.
Figure 2. Glycosylamine synthesis monitored by CE/indirect UV: (A) mol percentages of glucose,
fructose and glucosylcarbamate, [(mol(x)36h/mol(Glc) )ꢃ100], as a function of the Glc-1-NH syn-
thesis time; (B) mol percentages of lactose, lactulose and lactosylcarbamate, [(mol(x)36h
mol(Lac) )ꢃ100], as a function of the Lac-1-NH synthesis time (percentages of glycosylcarbamates
0
2
/
0
2
have been indirectly evaluated by the yield of the reactions, the identity of the products and the mass
balance). In the inserts the magnifications of the corresponding graphics are reported.
1
8–20
HPAEC-PAD
(Figure 3). Quantification of glycosylamines was again pre-
cluded, their retention time being close to the dead time. In crude Glc-1-NH man-
2
nose was also detected (about 6% of the amount of glucose), as expected on the ba-
1
6
sis of glucose isomerization occurring in alkaline conditions. The presence of
carbamate derivatives (not shown here) was detected by HPAEC-PAD using
2
1
highly alkaline eluents, as reported elsewhere.
In order to verify whether or not those isomers could form glycosylamines
during the syntheses of Glc-1-NH and Lac-1-NH , acidification of the crude Glc-
2
2
1
-NH , Gal-1-NH and Lac-1-NH solutions was carried out (Table 1). As ex-
2 2 2
1–4
pected, a net increment of glucose, galactose and lactose percentages was ob-
served, due to the hydrolysis of the corresponding glycosylamines. The amount of
fructose remained unchanged and only lactulose increased from 6.2% to 11.6%,
suggesting that lactulosylamine could be formed during the lactosylamine synthe-
sis.

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SYNTHESIS OF GLYCOSYLAMINES
267
Figure 3. HPAEC-PAD analysis of untreated freeze-dried synthesis mixtures of glycosylamines
dissolved in 0.1 mM NaOH: (A) Glc-1-NH
NH (3.0 mg/mL).
2
(4.0 mg/mL); (B) Gal-1-NH
2
(5.0 mg/mL); (C) Lac-1-
2
a, b
Table 1. Mol Percentages of Sugars in the Synthesis Mixtures of Glycosylamines at Different
pH
Glc-1-NH
2
Gal-1-NH
2
Lac-1-NH
2
c
d
e
f
g
pH
Glc (%)
Fru (%)
Gal (%)
Lac (%)
Lactulose (%)
1
2
8
6
4
7.1
7.2
20.2
47.0
5.4
5.2
4.9
5.0
6.5
6.8
23.9
54.6
7.0
7.3
19.9
56.0
6.2
6.5
10.3
11.6
a. injections of freeze-dried reaction mixtures (2.0 mg/mL) were performed 30 min after stable pH
readings were attained; b. quantification of each species was achieved using calibration curves of cor-
responding genuine standards (data consistent with literature9); c. [mol(Glc36h)/mol(Glc
mol(Fru36h)/mol(Glc )] ꢃ 100; e. [mol(Gal36h)/mol(Gal )] ꢃ 100; f. [mol(Lac36h)/mol(Lac
00; g. [mol(Lactulose36h)/mol(Lac )] ꢃ 100.
0
) ꢃ 100; d.
[
1
0
0
0
)] ꢃ
0

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CAMPA ET AL.
Quantification and Purification of Glycosylamines
Direct quantification of glycosylamines and of other amino compounds pre-
sent in the synthesis mixture was accomplished by a derivatization of the reaction
2
2
mixtures with N-fluorenyl-methoxycarbonyl (Fmoc) chloride. Figure 4 shows
2
3,24
the MEKC-UV electropherograms
of Glc-1-NH (A), Gal-1-NH (B) and
2 2
Lac-1-NH (C) reaction mixtures treated with Fmoc-chloride. For Glc-1-NH the
2
2
formation of detectable amounts of fructosylamine was excluded. In the electro-
pherogram of Fmoc-Lac-1-NH synthesis mixture (Figure 4C), the small signal
2
was ascribed to lactulosylamine, as confirmed by comparison with the mobility of
Fmoc-lactulosylamine ad hoc synthesized.
Although the detected amount of lactulosylamine was only about 8% with
respect to the main amino-sugar, its interference in the subsequent reactions of
lactosylamine cannot be excluded. No diglycosylamines were detected as con-
firmed also by the analysis of the Fmoc- treated mixture by ion-spray mass spec-
trometry (data not shown). The methods used allowed for identification of the
Figure 4. Electropherograms of crude glycosylamines derivatized with Fmoc; (A) Glc-1-NH
0.8mg/mL); (B) Gal-1-NH (1.26 mg/mL); (C) Lac-1-NH (1.0 mg/mL).
2
(
2
2

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SYNTHESIS OF GLYCOSYLAMINES
269
1
3
Figure 5.
C NMR spectra of: (A) peracetylated Glc-1-NH
2
from crude synthesis mixture purified
with flash chromatography; (B) peracetylated Glc-1-NH
ments (ppm): 20.57–20.71 (COCH ), 23.38 (COCH ), 61.60 (C-6), 68.09 (C-4), 70.58 (C-2), 72.65
C-5), 73.51 (C-3), 78.20 (C-1(ꢂ)), 169.53–171.06 (COCH ).
2
purified as Fmoc-derivative. Peak assign-
3
3
(
3
amino components contributing to the unresolved peak 4 in Figure 1C. As the re-
actions with Fmoc were quantitative, the yields of the synthesis of glycosy-
lamines could be calculated by measuring the amount of the Fmoc-glycosy-
2
5
lamines formed: the yields were equal to 75%, a value lower than that
1
reported. The obtained Fmoc-derivatives were readily separated from the reduc-
ing sugars on a C-18 cartridge, and the free glycosylamines were then recovered
2
2
by simple treatment with aqueous ammonia.
The success of the purification
1
3
procedure was confirmed by C NMR, as clearly shown by the coincidence of
the spectra reported in Figure 5: panel A is relative to peracetylated Glc-1-NH2
separated from the crude amination reaction and panel B refers to peracetylated
Glc-1-NH after Fmoc removal.
2

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CAMPA ET AL.
CONCLUSIONS
Reliable methods have been developed for the analysis of glycosylamines and
of side-products formed during their preparation. In the synthesis mixtures of glu-
cosylamine, the side-products glucosylcarbamate, fructose and mannose were iden-
tified and quantified; in the synthesis of lactosylamine, the side-products lactosyl-
carbamate, lactulose and the corresponding lactulosylamine were detected. In the
production galactosylamine, only galactosylcarbamate was found as a side-product.
EXPERIMENTAL
Chemicals
D-glucose, D-galactose, lactose, N-fluorenylmethoxycarbonyl chloride,
sodium dodecylsulfate and sodium tetraborate were from Sigma (St. Louis, MO,
USA). Tetradecyltrimethylammonium bromide and the 50% (w/w) NaOH solution
used for the eluent preparation in HPAEC-PAD were from Aldrich (St. Louis, MO,
USA). Reversed phase chromatography was performed on Sep-Pak® C18 car-
tridges (Waters, Milford, USA).
Preparation of Glycosylamines1
The reducing carbohydrate (0.2 M) and ammonium hydrogen carbonate (0.2
M), dissolved in aqueous 16 M ammonia, were left for 36 h at 42°C.
Reaction of Glycosylamines with Fmoc22
0.1 mmol of glycosylamine in saturated aqueous sodium bicarbonate (5 mL)
were added to Fmoc-Cl (0.4 mmol) in dioxane (5.0 mL). The mixture was stirred
overnight at room temperature. A C18 cartridge (Sep-Pak®, 10 g) conditioned in
water was first washed with water to remove salt and reducing carbohydrates, and
then with methanol. The glycosylamine-Fmoc derivative was recovered after con-
centration and freeze-drying. The reaction was quantitative (gravimetric test on
pure commercial Glc-1-NH and Gal-1-NH ). Fmoc was removed by stirring gly-
2
2
cosylamine-Fmoc (0.2 mmol) in 15% aqueous ammonia (20 mL) overnight. The
resulting mixture was filtered (0.45 ꢄm), concentrated and freeze-dried to obtain
the glycosylamine (96%). TLC (propanol: 1N ammonia: water, 6:2:1) showed the
presence of pure glycosylamine, and no UV-absorbing material.
Capillary Electrophoresis
The system was an Applied Biosystems HPCE Model 270A-HT with Tur-
bochrom Navigator (4.0) software. The fused silica column (72 cm (50 cm to de-
tector) ꢃ 50 ꢄm I.D.ꢃ 375 ꢄm O.D.) was from Supelco (St. Louis, MO, USA).

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SYNTHESIS OF GLYCOSYLAMINES
271
All runs were done at 30°C. Samples were loaded under vacuum at a pressure of
6.9 kPa (1.5 s). Before sample injection, a 2-min conditioning of the capillary
1
with the buffer followed a 2-min washing with 0.1 N NaOH (vacuum pressure 67.6
kPa). CE/indirect UV on glycosylamines solutions in 0.1 mM NaOH was per-
9
–12,26
formed using 6 mM sorbate at pH 12.5 ꢀ 0.5 mM TTAB
(10 kV; 256 nm at
the anode). Positive signals were obtained by reversing the output polarity of the
detector. Quantification of Fmoc-glycosylamines was achieved by MEKC using
20 mM tetraborate (pH 9.4) ꢀ 25 mM SDS (20 kV, 260 nm at the cathode).
HPAEC-PAD
The system (Dionex, Sunnyvale, CA, USA) consisted of an isocratic pump
(IP20), a pulsed amperometric detector (ED40) and PeakNet 5.1 software (10 ꢄL
loop). A CarboPac PA1 anion-exchange column (250 ꢃ 4 mm I.D.) plus guard col-
umn (50 ꢃ 4 mm I.D.) were used. The flow-through detection cell contained a gold
working electrode (1.0-mm diameter) and an Ag/AgCl reference electrode; the
counter electrode was the titanium cell body across the 25 ꢄm thin layer channel
from the working electrode. The waveform parameters were E ꢅꢀ0.05V,
det
t ꢅ440 ms (t ꢅ240 ms), E ꢅꢀ0.8V, t ꢅ180 ms, E ꢅ ꢆ0.2V, t ꢅ360 ms,
det
int
ox
ox
red
red
with 1 s response time. The eluent was 0.01 M NaOH ꢀ 2 mM Ba(CH COO)
3
2
flowing at 1.0 mL/min.
Mass Spectrometry
Mass spectra were recorded on an API-I PE SCIEX quadrupole mass spec-
trometer equipped with an articulated ion spray connected to a syringe pump for
sample injection. The solvents were acetone or 50% aqueous methanol containing
formic acid (0.1%). The injection flow rate was 0.1 mL/h; the ionspray voltage was
5
600 V.
Nuclear Magnetic Resonance
13
C NMR spectra were recorded on a Jeol EX400 (100.6 MHz) spectrometer.
All experiments were carried out in CDCl ; chemical shifts are reported in ppm
3
downfield from tetramethylsilane. The glycosylamines were peracetylated before
2
7
their NMR analysis, to achieve stable derivatives. The peracetylated glycosy-
lamines in the crude synthesis mixture were separated from the peracetylated re-
ducing sugars by flash chromatography (ethyl acetate).
ACKNOWLEDGMENT
This work was supported by a research grant of F.B.C. S.r.l. to the Depart-
ment of Biochemistry, Biophysics and Macromolecular Chemistry of the Univer-

ORDER
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CAMPA ET AL.
sity of Trieste and by Grant no. 9903263827 of Italian MURST (Progetti di Inter-
esse Nazionale). The authors thank Prof. Cataldi (Department of Chemistry, Uni-
versity of Basilicata) for the use of the HPAEC-PAD instrument.
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Received November 22, 2000
Accepted April 19, 2001

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