Quantitative analysis of selective glycosylation of saccharides with aromatic amines

Article abstract of DOI:10.1016/j.carres.2020.108171

Glycosylation, a part of the Maillard reaction, occurs non-enzymatically in food and biological processes. The selectivity of N-glycosylation was analyzed based on the reactivity of monosaccharides with aromatic amines, including aromatic amino acids, and the degree of molecular interaction (MI) measured using liquid chromatography. Furthermore, the chemical structures of reaction products were determined using X-ray crystallography and/or NMR. The possible reaction products were estimated in silico using the optimized energy values of different conformations. The MI energy values of amino groups and saccharides were calculated using in silico analysis using a model phase. Saccharides having larger MI values easily produced stable crystals of N-glycosides. The reaction rate of glucose (an energy saccharide) was slow, and it easily produced the Amadori compounds. The study of the reactivity of aromatic amines with saccharides, the measurement of the retention of monosaccharides on amino phase in chromatography, and the synthesis of N-glycosides for the determination of their structures will provide useful information about selective glycosylation for the modification of drug candidates to improve their water solubility.

Full text of DOI:10.1016/j.carres.2020.108171

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Quantitative analysis of selective glycosylation of saccharides with aromatic amines.
Toshihiko Hanai, Keiko Shimada, Nobuhiro Koyama, Yukio Nohara
PII:
S0008-6215(20)30542-5
DOI:
https://doi.org/10.1016/j.carres.2020.108171
CAR 108171
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To appear in: Carbohydrate Research
Received Date: 26 March 2020
Revised Date: 1 October 2020
Accepted Date: 2 October 2020
Please cite this article as: T. Hanai, K. Shimada, N. Koyama, Y. Nohara, Quantitative analysis
of selective glycosylation of saccharides with aromatic amines., Carbohydrate Research, https://
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Quantitative analysis of selective glycosylation of saccharides with aromatic amines.
Toshihiko Hanaia,b* Keiko Shimadab, Nobuhiro Koyamab, Yukio Noharab,c
a Health Research Foundation, Research Institute for Production development 4F, Shimogamo-
morimoto-cho, Sakyo-ku, Kyoto, 606-0806 Japan
b School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku, Tokyo, 108-8641
Japan
c Faculty of Pharmacy, Iryo Sosei University, Chuodai, Iwaki, Fukushima, 970-8551, Japan
Corresponding author: Toshihiko Hanai, hanai104@kf7.so-net.ne.jp Health Research Foundation,
Research Institute for Production development 4F, Shimogamo-morimoto-cho, Sakyo-ku, Kyoto,
606-0806 Japan
Abstract
Glycosylation, a part of the Maillard reaction, occurs non-enzymatically in food and biological
processes. The selectivity of N-glycosylation was analyzed based on the reactivity of
monosaccharides with aromatic amines, including aromatic amino acids, and the degree of molecular
interaction (MI) measured using liquid chromatography. Furthermore, the chemical structures of
reaction products were determined using X-ray crystallography and/or NMR. The possible reaction
products were estimated in silico using the optimized energy values of different conformations. The
MI energy values of amino groups and saccharides were calculated using in silico analysis using a
model phase. Saccharides having larger MI values easily produced stable crystals of N-glycosides.
The reaction rate of glucose (an energy saccharide) was slow, and it easily produced the Amadori
compounds. The study of the reactivity of aromatic amines with saccharides, the measurement of the
retention of monosaccharides on amino phase in chromatography, and the synthesis of N-glycosides
for the determination of their structures will provide useful information about selective glycosylation
for the modification of drug candidates to improve their water solubility.
Keywords
Glycosylation, Selective reaction, Molecular interaction, X-ray crystallography, NMR, in silico
1. Introduction
Saccharides are integral parts of biologically important compounds, such as glycoproteins,
glycosaminoglycans, proteoglycans, and cyclodextrins. They are structural elements in the cell walls
of bacteria and plants, and in the exoskeleton of arthropods. Glycolipids are another group of
conjugated oligosaccharides, that occur in tissues, in which the aglycon part is a long-chain base
coupled with fatty acids [1-3]. The non-enzymatic glycosylation occurs mainly on the ε-amino
groups of lysine residues and/or the α-amino group of the N-terminal amino acid [4,5].
Glycosylation has a profound effect on the structure of proteins, changing their functional and
biological properties. Glycosylation may alter drug their binding capacities and influence the
distribution and intensity of their pharmacological effects [6-10]. Moreover, an elevation in the
levels of glycosylated human serum albumin decreases its drug binding affinity dramatically [11, 12].
1

The reaction of glucose with amino acids and peptides has become a very important experimental
model in the food flavor and pharmaceutical industries for better understanding the mechanism of
food flavor and drug stability. Because of the amino acid and sugar functional groups present in their
structures, most of the reaction components formed during the initial stages of Maillard reaction as
well as the substrates are relatively polar [13]. Synthesis methods for glucosides and Amadori
compounds have been described [14-16]. A variety of glycosides were synthesized since Schiff
obtained such compounds by heating aniline or p-toluidine with D-glucose [17], and the molecular
properties were reported such as melting point and refractive index [18-44]. Several N-glycosides of
p-aminobenzoic acid and its ethylester were synthetically prepared for the purpose of
microbiological assay to investigate the biochemical meanings of N-glycoside [32]. Antitumor
effects of p-aminobenzoic acid N-xyloside Na was applied to eleven patients with a variety of solid
tumors were treated and positive results were observed [38-40], and activities of drug-metabolizing
enzymes and glutathione-related enzymes was reported [41]. β-C-Mannoside (4-(4-aminophenyl)
benzoic acid) was described as an inhibitor of E-selection [42]. Synthesis and evaluation of D-gluco-
pyranocyclopropyl amines as potential glycosidase inhibitors were reported [45]. Glycoproteins are
playing a central role in many important biological processes and disease developments; therefore,
new strategy for glycopeptide, neoglycopeptide, and glycoprotein synthesis was described [46].
On other hand, the saccharides were separated in a system using ion-exchange resins and borate
buffer [47-50] or aqueous ethanol [51-53]. The high-speed separation was achieved using a silica
gel-based ion-exchanger (Corasil AX, Vydac HC-SCX using an eluent composed of acetone, ethanol,
and water mixture), and amino columns using aqueous acetonitrile as the eluent. The retention time
difference depends on molecular interaction strength between a saccharide and a model phase. The
propylamine-bonded silica gels have been the most commonly used gels in liquid chromatography
because they can be easily handled, but the saccharides are not quantitatively recovered from the
packing material compared to that obtained by ion-exchangers, which is probably due to the
formation of N-glycosides or their Amadori rearrangement products.
The study of glycosylation mechanisms at the molecular level is important for understanding the
factors that control the alterations in glycosylation which occur during normal and abnormal
physiological states. Analyzing selective glycosylation of aliphatic amines was not easy due to lack
of chromophores. Initial theoretical reactivity of aliphatic amine reactivity indicated that the
reactivity was about the same as that of aromatic amino acids. Further, aromatic amines were
seemed to be reactive and the reaction products were easily crystalized. Therefore, the selective
glycosylation of aromatic amines with several monosaccharides was quantitatively analyzed by
measuring reaction rate and retention time difference using amino-phase liquid chromatography. The
stereo structures of reaction products were confirmed by synthesizing N-glycosides and determining
their structures by X-ray crystallography and/or NMR. The conformations were further analyzed
using in silico analysis.
2 Experimental
Previously, the reactivity of p-toluidine was performed in mixture of equal volume of 100 mM
p-toluidine and saccharide in aqueous solution at a temperature 55 . The reaction rate was measured
by reversed-phase liquid chromatography using a CapcellPak18 and 55% aqueous methanol, where
the decrease in the amount of p-toluidine was quantitatively measured at intervals. The rate of
disappearance of p-toluidine was plotted as a function of reaction time. These plots were linear when
2

drawn as a pseudo-first-order reaction. The rate constant was calculated from the slope of the line.
The Slope was related to the amount of p-toluidine and the reaction time was used as the reactivity.
Two peaks observed by ultraviolet absorption corresponded to p-toluidine and a reaction product.
The reactivity of biological amine α-amino group was further studied using phenylalanine, tyrosine
and tryptophan methylesters. Hydrochloride forms of phenylalanine, tyrosine, and tryptophan
methylesters, and free form tyrosine methylester were obtained from Sigma Chem. Co. Methanol
solutions of hydrochloride form amino acid methylesters were passed through small columns packed
with hydroxyl form of anion exchange resin, Dowel 1X8, and converted to free form before the
reaction. The reaction was performed in a mixture of equal volumes of 200 mM amino acid
methylester methanol solution and 200 mM saccharides aqueous solution at a temperature 55 . The
latter peak was further identified as glycoside by post column reaction detection using taurocyamine
[54], where free saccharide and glycoside peaks were detected. The reaction detection was selective
and sensitive for only reducing saccharides. The eluent was aqueous 40% methanol containing 0.5 M
ammonium acetate for the measurement of reaction rate of phenylalanine and tryptophan methylester,
and that used for tyrosine methylester was aqueous 30% methanol containing 0.5M ammonium
acetate. Measured reaction rates are summarized in Table 1.
Selective molecular interactions between saccharide and amino group were studied using
capacity ratios measured by liquid chromatography using an amino column in 80% aqueous
acetonitrile [55]. A model amino-phase was constructed from a hexenyl-bonded phase [56]. Initial
structures of saccharides were used for the molecular interaction (MI) analysis.
MI energy value (kcal mol-1) of the hydrogen bonding (MIHB), electrostatic (MIES), and van der
Waals (MIVW) can be calculated using the following equations [57].
MIHB = HB (molecule A) + HB (molecule B) – HB (molecule A and molecule B complex),
MIES = ES (molecule A) + ES (molecule B) – ES (molecule A and molecule B complex),
MIVW = VW (molecule A) + VW (molecule B) – VW (molecule A and molecule B complex).
Calculated MIHB and MIES values are summarized in Table 2 with log k values of saccharides. The
relative MIHB, MIES, and MIVW values indicate the contribution level.
Synthesis of p-toluidine N-glycosides of aromatic amines, p-toluidine, p-aminobenzoic acid, 4-
aminocinnamic acid, 3-amino-2-phenylpropionic acid, and 4-methoxyaniline (anisidine) with
arabinose, 2-deoxy-D-glucose, 2-deoxy-D-ribose, fucose, galactose, glucose, mannose, rhamnose,
ribose, xylose, and allose were achieved by following procedures. Equal mole of saccharide was
solved in water, and aromatic amine was solved in organic solvent. The mixtures were kept in dark at
ambient temperature until crystal was found. The crystal was filtrated, washed, and vacuum dried.
For example, synthesis of p-toluidine-2-deoxy-glucoside II: N-4-methylphenyl-β-D-
deoxyglucopyranosylamine1.0 gram of 2-deoxyglucose (6.0 mmol) in 5.0 mL of water, and 0.65 g of
p-toluidine (6.0 mmol) in 2.0 mL methanol solution were mixed and stored at ambient in dark place
for 24 hrs. Reaction mixture was solved in methanol. The crystal was filtrated and washed using
chilled ether. The crystal was recrystallized from hot acetonitrile, filtrated, and dried under vacuum.
Another example, synthesis of p-amino-cinnamic acid-mannoside XV: N-4-(3’-carboxyl-1’,2’-ene-
pheny)-β-D-mannopyranosylamine 1.8 gram of mannose was solved in 2.5 mL water, and 1.37 gram
of p-amino-cinnamic acid was solved in 21 ml of 80% ethanol at 55 . These solutions were mixed
at 55 and kept over-night at ambient. The crystal was solved in 40 mL of 80% ethanol at 55 ,
then recrystallized at ambient. The synthesis schemes of p-toluidine-2-dexoyglucoside (II) and p-
3

aminocinnamic acid mannoside (XV) are shown in Figures 1. Synthesis procedures of individual N-
glycosides are summarized in SP1.
The X-ray crystallographic analysis of p-toluidine N-glycoside crystals was performed using
Rigaku AFC5R diffractometer with graphite monochromatic Cu Ka radiation and a 12KW rotating
anode generator. The analysis was not completed for several crystals due to evaporation of crystals
during the measurement. Proton and carbon NMR spectra measured using Varian XL400 are
summarized in SP2 with their measured melting point, their elemental analysis results, and their
infrared spectra.
In silico analysis of molecular interactions and glycoside conformations was performed by
MM2 and MOPAC PM5 of CAChe program from Fujitsu. Model amino phase was constructed
based on a monomeric hexyldimethylsilicon ligand. The silicone trioxides were homogeneously
distributed and locked as a solid phase silica gel. The number of hexyldimethylsilicon molecules
depends on the necessary surface area to trap analytes. A part model phase is shown in Figure 2
where glucose is retained.
2. Results and discussions
2.1. Glycosylation rate analysis
The glycosylation of p-toluidine with several monosaccharides (arabinose, deoxyribose, fructose,
galactose, glucose, mannose, ribose, and xylose) was similar to that of aniline [58]. The reaction
rates were related to the HB energy values of monosaccharides calculated using MM program (r =
0.955, n = 8), and the correlation was superior than that between the amount of aldehyde form and
reaction rate (r = 0.731, n = 5) [59]. The correlation coefficients of reaction rate with the relative
amount of carboxyl group [60] and the reactivities of hemoglobin and saccharides [61] were 0.627 (n
= 7) and 0.405 (n = 6), respectively. Further study was performed with additional data of aromatic
amino acids. The reactivity of saccharides with glycine was order of galactose>mannose>glucose.
The aldopentaoses reacted more rapidly than aldohexoses [62]. However, mannose was the most
reactive with p-toluidine and the N-glycoside was easily crystalized. It may be the relative
hydrophobicity of mannose.
The reaction rates of phenylalanine, tyrosine, and tryptophan methylesters with mannose were
related to HB energy values of monosaccharides. The correlation coefficient between the HB values
and reaction rates of aromatic amines together including p-toluidine, benzylamine, aniline, and
dopamine was 0.945 (n = 7). The reaction rates of phenylalanine, tyrosine, and tryptophan
methylesters were 29, 16, and 6% of that of p-toluidine. The reaction rates were normalized relative
to those of mannose and phenylalanine, tyrosine, or tryptophan methylesters (Table 1). The HB
energy values of phenylalanine, tyrosine ad tryptophan methylesters were 0.306, -0.433, and -2.297
kcal mol-1, respectively. The correlation between the relative reaction rates of phenylalanine
methylester and HB energy values of saccharides was 0.764 (n = 8). The correlation was better than
other correlations such as ES energy values, amount of aldehyde, amount of carbonyl, and reactivity
with hemoglobin: 0.615 (n = 8), 0.516 (n = 5), 0.307 (n = 8) and 0.169 (n = 7), respectively. These
results indicated that the reactivity of phenylalanine methylester with saccharides could be predicted
by in silico analysis.
The reactivity of tyrosine methylester with saccharides was also investigated, and the reaction
rates and calculated HB energy values were correlated (r = 0.328, n = 8). The correlation coefficients
4

with ES energy values of saccharide, amount of aldehyde, amount of carbonyl and reactivity with
hemoglobin were 0.208 (n = 8), 0.201 (n = 5), 0.111 (n = 7) and 0.538 (n = 6), respectively.
The reactivity of tryptophan methylester with saccharides was very poor. The correlation
coefficients between reaction rate ad HB energy of saccharides, ES energy of saccharides, amount of
aldehyde, amount of carbonyl and activity with hemoglobin were 0.093 (n = 8), 0.189 (n =8), 0.522
(n = 5), 0.634 (n = 7), and 0.122 (n = 6), respectively. Tryptophan methylester was less soluble and
less reactive. The poor solubility of amino acid methylesters in water made it difficult to measure the
reaction rate. Accurate measurements should be performed in a closed system, otherwise evaporation
of methanol will contribute to the margin of error. However, these results indicated that N-
glycosylation occurred by direct condensation between an amino group and a hydroxyl group of
saccharides, because the reactivity was no correlated with amount of aldehyde or carboxyl group of
saccharides. The reactivity was also different from that of hemoglobin.
Saccharides originally in furanose form were converted to pyranose form as glycosides
according to the structures of N-glycosides determined by X-ray crystallography. Therefore, the
energy values calculated from pyranose form may not reflect the actual energy values. The
calculated HB values were well correlated for p-toluidine N-glycosides. Crystallization of these
amino acid N-glycosides was difficult to determine their stereo structures. The poor reactivity may
be related to their atomic partial charge (apc) of nitrogen atoms. Apc of phenylalanine, tylosine, and
tryptophan methyl-esters nitrogen atoms are -0.544, -0.595, and -0.599 au, respectively. These values
are similar to those of aliphatic amines. Apc of alanine and lysine ε-nitrogen atom are -0.576 and -
0.578 au, respectively. Furthermore, apc of methyl-, ethyl-, propyl-, and butyl-amine are -0.557, -
0.593, -0.656, and -0.655 au, respectively. On the other hand, apc of aniline, toluidine, anisidine, p-
amino benzoic acid, p-amino cinnamic acid, p-aminohydrocinnamic acid nitrogen atoms are -0.392, -
0.393, -0.393, -0.364, -0.365, and -0.389 au, respectively. Therefore, aromatic amines were selected
for the initial study.
2.2. Quantitative analysis of molecular interactions
Overall, the calculated MI energy values demonstrated a linear relation with the log k values
obtained by liquid chromatography using an amino phase [56]. MIVW is a major contributor in
reversed-phase liquid chromatography. MIHB is a major contributor in normal-phase liquid
chromatography, and MIES is a major contributor in ion-exchange liquid chromatography. Such MI
energy values highlight the specificity of the retention mechanisms in different types of
chromatography. However, in silico analysis of chromatography of saccharides using the amino
phase did not provide a simple answer. Neither MIHB nor MIES demonstrated a predominant MI;
and the correlation coefficients between log k and MIHB, and log k and MIES, were 0.015 and 0.082
(n = 7), respectively. The linear relation between MIHB + MIES was dependent on their log k values
(r = 0.556, n = 7). The correlation between log k and MI (HB + ES – 0.3VW) was 0.834 (n = 7).
Glucose, whose retention time was long, showed small MI values. The correlation improved without
glucose (r = 0.930, n = 6). The small MI value of glucose may reflect its poor compatibility with
glycosylation. Indeed, other saccharides produced their crystal p-toluidine N-glycosides easily,
whose structures were identified using X-ray crystallography and/or NMR. However, glucose and
aromatic amine mixtures easily formed Amadori compounds having a brownish coloration. Their
purification was difficult to obtain the crystal, and the glycosylation reaction rates were slow. This
5

can be explained by the chromatographic behavior and reactivity of 2-deoxysaccharides, which are
easily converted to N-glycosides in vitro [60].
2.3. Determination of chemical structure of N-glycosides
Furthermore, synthesis of pure N-glycoside crystals and the determination of stereo structures of
several glycosides by X-ray crystallography and/or NMR confirmed the selective synthesis of N-
glycosides. The reaction products of p-toluidine and several monosaccharides were p-toluidine N-
glycosides. The properties of N-glycosides including known melting points are summarized in SP1.
The stereo structures of some N-glycosides were already reported without NMR spectra; therefore,
these glycosides are included in SP1. The above results indicated that glycosylation occurs via a
condensation reaction between the amino group of aromatic amines and C1 hydroxyl group of
saccharides under these mild reaction conditions.
As shown in Figure 3, X-ray crystallography results clearly defined the stereo structures of N-
glycosides. The stereo structures of p-anisidine N-mannoside, p-toluidine N-galactoside, p-toluidine
N-L-arabinoside, p-toluidine N-glucoside, p-toluidine N-xyloside, p-toluidine N-deoxyglucoside were
βC1 form, whereas those of p-toluidine N-deoxyriboside, and p-toluidine N-riboside were α1C form.
NMR spectra also determined the structures of N-glycoside reaction products. The determined
structures are summarized in SP2.
In addition, the possible conformations of glycosides (αC1, α1C, βC1, β1C) were constructed
and these stereo structures were optimized using MM2 calculations. The structures used for the
calculation were pyranose form based on the results determined by X-ray crystallography. The FS
energy values supported the synthesized structures and the feasibility of synthesis. As summarized in
Table 3, the energy values of βC1 conformation of free saccharides were the lowest and produced
βC1 conformation of N-glycosides. Conformation of N-glycosides followed the free saccharides of
lowest energy values.
Furthermore, N-glycosides of D- and L-arabinose with mannose were synthesized, but synthesis
with 3-O-methylglucose failed. From this result, C3 hydroxyl group of saccharides is seemed to
affect the glycosylation. C3 hydroxyl group may form intramolecular hydrogen bonding with amino
group hydrogen. Therefore, if C3 hydroxyl group of N-glycosides is modified, the N-glycosides
should be stable. Axial C2 hydroxyl group of saccharides was stable and did not produce Amadori
rearrangement compounds, but easily produced the N-glycosides. This observation was supported by
attempted synthesis of Amadori compounds instead of N-glycosides by longer reaction time and
boiling the reaction mixtures. The reaction mixtures were analyzed by TLC, melting point, and IR.
Mannose and galactose did not produce Amadori compounds even after using reactive anisidine
instead of toluidine. Only glucose produced Amadori compound; however, the determination of
structure of Amadori compound was difficult. The conformations of phenylalanine methyl ester and
saccharides were analyzed by in silico analysis. The atomic partial charge (not given) calculated by
PM5 calculation indicated that C1 carbon of mannose and hydrogen of the amino group of
phenylalanine methyl ester were the most reactive. Therefore, it can be concluded that the reaction
occurred between C1 carbon of mannose and amino group.
Conclusion
6

Naturally occurring selective glycosylation was studied based on the reactivity of aromatic
amines including aromatic amino acids with several saccharides, liquid chromatographic retention of
saccharides on amino phase, and synthesis of N-glycosides of aromatic amines with various
saccharides. Faster the reaction rate, easier the crystallization of N-glycoside. N-Mannoside was
stable and easily crystalized. However, synthesis of N-glucoside was difficult, and the related
Amadori compounds were produced. On the other hand, saccharides were retained on the amino
phase with a combination of hydrogen bonding, electrostatic interaction, and van der Waals force.
Finally, the conformation of N-glycosides was determined by X-ray crystallography and NMR. The
conformation was either βC1 or α1C. The structure seemed to be controlled by C3 hydroxyl group.
The reaction selectivity and conformation were determined by in silico analysis. The feasibility of N-
glycoside synthesis, the calculated MI values by in silico chromatography, and the reactivity
analyses provided useful information about the selective glycosylation for modification of drug
candidates to improve their water solubility. These results confirmed the feasibility of N-glycoside
synthesis, selectivity of saccharides, and poor recovery from amino columns. In addition, they
provide a saccharide selection guide for development of medicines.
Declaration of Competing Interest
There are no conflicts of interest to declare.
Acknowledgements
Toshihiko Hanai (Ph.D.) is grateful to Professor, Dr. Kenichi Sato (Kanagawa University) and
Professor, Dr. Hiroshi Ueno (Nara women’s University) for their helpful suggestions.
Appendix A. Supplementary material
SP1 Synthesis methods of N-glycosides and their properties
SP2 Estimated structures of N-glycosides
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Table 1. Relative reaction rate of glycosylation, properties of saccharides, and glycosides
Table 2. Properties of saccharides and molecular interaction energy values with an amino phase
Table 3. Optimized energy values of p-toluidine N-glycosides
Figure captions
Figure 1 Reaction scheme of p-toluidine-2-deoxy-glucoside synthesis
10

Figure 2. Retention of glucose on a model hexylaminodimethylsilicon-bonded polysiloxane-phase
Small and large white bolls: hydrogen and carbon. Black balls: oxygen. Gray ball: Nitrogen.
Figure 3. Stereo structures of p-toluidine N-glycosides determined using X-ray crystallography.
SP1 Synthesis methods of N-glycosides and their properties
SP2 Estimated structures of N-glycosides
11

Table 3. Optimized energy values of p-toluidine N-glycosides
_________________________________________________________
Saccharides
αC1
α1C
βC1
β1C
-------------------------------------------------------------------------------------
α-L-Arabinopyranose
α-2-D-Deoxyribopyranose
β-2-D-Deoxyglucopyranose
β-D-Fucopyranose
11.9250 8.2309 9.6639 8.4839
0.9894 0.5398 3.2943 3.2915
3.6938 7.5065 2.9575 7.1324
10.3127 13.8608 9.9766 13.0550
10.2214 12.1731 10.0292 13.0785
9.8716 15.2204 9.3474 17.1718
10.2657 12.0745 8.2585 12.0633
9.8295 5.3960 9.6449 6.3754
9.3081 8.0930 6.2374 8.5509
13.6047 10.7181 16.0525 10.7131
β-D-Galactopyranose
β-D-Glucopyranose
β-D-Mannopyranose
α-D-Ribopyranose
β-D-Xylopyranose
α-L-Rhamnopyranose
(Anisidine-β-D-mannopyranose 12.5519 16.9066 12.2855 14.0853)
Bold indicates structures confirmed by X-ray crystallography; Unit: kcal mol-1

Table 1. Relative reaction rate of glycosylation and properties of saccharides and glycosides
___________________________________________________________________________
Sacccharide
pT1
PhAl2 Tyr3 Try4 HB5 Ald6
Car7
Hem8
------------------------------------------------------------------------------------------------------------------------------------------------------------------
α-2-D-Deoxyribopyrase 1.8
1.14 1.02 0.94 -5.48
-
-
-
-
α−L-Arabinopyranose
β-D-Mannopyranose
α-D-Ribopyranose
β-D-Xylopyranose
β-D-Galactopyranose
β-D-Glucopyranose
β-D-Fructopyranose
1.2
1.0
0.9
0.7
0.5
0.2
0.0
1.19 1.05 0.30 -9.31 0.28
0.03
1.00 1.00 1.00 -10.18 0.064 0.005 3.2
0.52 1.01 1.32 -9.44 0.05 10.0
0.35 0.48 1.84 -10.21 0.17
0.33 0.88 1.37 -12.14 0.082 0.02
-
0.02
2.9
2.8
0.28 0.70 1.36 -11.56 0.024 0.002 0.6
0.31 0.90 0.19 -13.59 0.7 4.5
-
1: Relative reaction rate of p-toluidine [55], 2: Relative reaction rate of phenylalanine
methylester, 3: Relative reaction rate of tyrosine methyl-ester, 4: Relative reaction ratio of
tryptophan methylester, 5: Hydrogen bonding energy calculated using molecular mechanics,
6: Aldehyde (%) from ref. [58],7: Carbonyl (%) from ref. [59], 8: Reactivity with hemoglobin
from ref. [60].

Table 2 Properties of saccharides and molecular interaction energy with an amino phase
________________________________________________________________________
Saccharides
hb
es
vw
MIHB
MIES
MIVW
k
β-D-Arabinofuranose
β-D-Fructofuranose
-5.778 5.827 2.318
-9.716 10.464 2.798
40.203 -2.545 142.615 2.07
32.456 2.042 154.428 2.61
31.268 3.068 147.301 3.79
31.156 -0.687 139.503 4.02
31.842 1.653 141.568 3.35
26.230 1.701 144.368 1.78
29.388 3.250 148.269 2.21
β-D-Galactopyranose -12.135 8.502 7.273
β-D-Glucopyranose
β-D-Mannopyranose
β-D-Ribofuranose
β-D-Xylofuranose
-11.562 8.332 6.473
-10.182 8.853 6.168
-6.291 6.155 1.966
-7.084 5.534 1.949
hb, es, vw: hydrogen bonding, electrostatic, van der Waals energy values; MIHB, MIES,
MIVW: molecular interaction energy values of hydrogen bonding, electrostatic, and van der
Waals force, respectively (kcal mol-1); k: measured retention ratio [53].

Highlights
ò The selective glycosylation of aromatic amines including aromatic amino acids with several
monosaccharides was quantitatively analyzed by measuring reaction rate and molecular
interaction strength using liquid chromatography, and synthesis of N-glycosides.
ò The determination of stereo structures was achieved by X-ray crystallography and/or NMR;
furthermore, in silico analysis.
ò These results provide information about selective glycosylation of drug candidates to
improve their water solubility and affinity with glycoproteins.

Declaration of competing interest
There are no conflicts of interest to declare.
Toshihiko Hanai, Ph.D.

SP1 Synthesis methods of N-glycosides and their properties
p-toluidine-arabinoside: N-4-methylphenyl-β- -arabinopyranosylamine I
D
4.0 gram of D- or (L-)arabinoside was solved in 7.0 mL of water, and 3.2 of gram p-toluidine in 5.0 mL of ethanol
solution were mixed and stored at ambient in dark place for 24 hrs. The crystal was filtrated and washed using
chilled ethanol. The crystal was recrystallized from acetonitrile-ethanol mixture, filtrated and dried under vacuum.
mp 125(125 [31]) , C12H17O4N, IR υmax 3300, 1620, 1515 cm-1, 1H NMR (DMSO) δ = 7.5 Hz, 4.30 (t, 1H, J
= 8.0 Hz, H-1), 3.49 (ddd, 1H, J = 5.0, 8.0 Hz, H-2), 3.43 (dd, 1H, J = 3.5, 8.5 Hz, H-3), 3.68 (bs, 1H, H-4),
3.45 (dd, 1H, J = 2.2, 11.5 Hz, H-5ax). 3.60 (dd, 1H, J = 3.2, 12.0 Hz, H-5eq), 6.57 (d, 1H, J = 8.3 Hz, H-2’,
6’), 6.90 (d, 1H, J = 8.3 Hz, H-3’, 5’), 2.15 (s, 3H, H-7’), 4.75 (d, 1H, J = 4.8 Hz, OH-2), 4.82 (d, 1H, J = 5.0
Hz, OH-3), 4.50 (d, 1H, J = 4.0 Hz, OH-4), 5.95 (d, 1H, J = 8.0 Hz, NH), 13C NMR, 85.6 (C-1), 70.3 (C-2),
73.4 (C-3), 67.9 (C-4), 65.5 (C-5), 144.7 (C-1’), 113.4 (C-2’, 6’), 129.3 (C-3’, 5’), 125.4 (C-4’), 20.2 (C-7’).
p-toluidine-2-deoxy-glucoside: N-4-methylphenyl-β-D-deoxyglucopyranosylamine II
1.0 gram of 2-deoxyglucose (6.0 mmol) in 5.0 mL of water, and 0.65 g of p-toluidine (6.0 mmol) in 2.0 mL
methanol solution were mixed and stored at ambient in dark place for 24 hrs. Reaction mixture was solved in
methanol. The crystal was filtrated and washed using chilled ether. The crystal was recrystallized from hot
acetonitrile, filtrated and dried under vacuum.
mp 188 , C13H19O4N, IR υmax 3350, 1620, 1530 cm-1, 1H NMR (DMSO) δ = 9.8 Hz, 4.63 (ddd, 1H, J = 1.5,
9.0, 11.0 Hz, H-1), 1.52 (ddd, 1H, J = 1.0, 11.5, 24.0 Hz, H-2ax), 1.98 (ddd, 1H, J = 1.5, 5.0, 12.0 Hz, H-2eq),
3.48 (dddd, 1H, J = 4.5, 9.0 Hz, H-3), 3.01 (dt, 1H, J = 8.5, 8.5 Hz, H-4). 3.12 (ddd, 1H, J = 2.3, 5.0, 9.0 Hz,
H-5), 3.44 (ddd, 1H, J = 1.0, 6.0, 13.0 Hz, H-6ax), 3.63 (ddd, 1H, J = 2.2, 5.5, 11.5 Hz, H-6eq). 6.56 (d, 1H, J
= 8.2 Hz, H-2’, 6’), 6.88 (d, 1H, J = 8.0 Hz, H-3’, 5’), 2.14 (s, 3H, H-7’), 4.85 (d, 1H, J = 4.5 Hz, OH-3), 4.81
(d, 1H, J = 4.5 Hz, OH-4), 4.34 (dd, 1H, J = 5.5, 6.5 Hz, OH-6), 6.20 (d, 1H, J = 9.0 Hz, NH); 13C NMR, 80.5
(C-1), 39.4 (C-2), 71.7 (C-3), 71.8 (C-4), 77.5 (C-5), 61.2 (C-6), 144.4(C-1’), 113.4 (C-2’, 6’), 129.2 (C-3’,
5’), 125.3 (C-4’), 20.2 (C-7’).
p-toluidine-2-deoxyriboside: N-4-methylphenyl-β-D-2-deoxyribopyranosylamine III
1.0 gram of 2-deoxyribose (7.5 mmol) was solved in 5.0 mL water, and 0.80 gram p-toluidine (7.4 mmol) was
solved in 4.0 mL methanol. The mixture was kept in dark for 24 hours. The crystal was filtrated and washed using
water and methanol. After dried with calcium-chloride, the crystal was recrystallized from hot acetonitrile.
mp 157-158 (167.5 [17]) , C12H17O3N, IR (KBr) υmax 3350 (OH), 1620 (CO), 1535 (NH) cm-1, MS (FAB)
m/z 224 [M+H]+, 1H NMR (DMSO) δ = 3.3 Hz, 4.59 (ddd, 1H, J = 3.0, 9.0, 11.0 Hz, H-1), 1.71 (ddd, 1H, J =
2.0, 5.0, 12.0 Hz, H-2ax), 1.81 (ddd, 1H, J = 2.2, 10.0, 21.0 Hz, H-2eq), 3.69 (dddd, 1H, J = 2.0, 5.0, 10.0 Hz.
H-3), 3.51(bdd, 1H, J = 1.5. 3.0 Hz, H-4). 3.65 (dd, 1H, J = 3.5, 12.3 Hz, H-5eq), 3.40 (dd, 1H, J = 12.0, 1.5
Hz, H-5ax), 6.56 (d, 1H, J = 8.3 Hz, H-2’,6’). 6.89 (t, 1H, J = 8.0, 8.0 Hz, H-3’, 5’), 2.14 (s, 3H, H-7’), 4.73 (d,
1H, J = 5.5 Hz, OH-3), 4.39 (d, 1H, J = 4 Hz, OH-4), 6.19 (d, 1H, J = 9.1 Hz, NH); 13C NMR, 80.4 (C-1), 34.8
(C-2), 63.1 (C-3), 66.9 (C-4), 65.7 (C-5), 144.2 (C-1’), 125.4 (C-2’), 129.2 (C-3’), 113.5 (C-4’), 129.2 (C-5’),
125.4 (C-6’), 20.2 (C-7’).
p-toluidine-fucoside: N-4-methylphenyl-β-D-fucopyranosylamine IV
1.0 gram of fucose (6.0 mmol) in 4.0 mL of water, and 0.71 g of p-toluidine (7.0 mmol) in 2.0 mL methanol
solution were mixed and stored at ambient in dark place for 120 hrs. The crystal was filtrated and washed
using chilled acetone. The crystal was recrystallized from hot acetonitrile, filtrated and dried under vacuum.
mp 153 (dec), C13H19O5N, IR υmax 3420, 1630, 1540 cm-1, 1H NMR (DMSO) δ = 8.0 Hz, 4.26 (t, 1H, J = 8.0
Hz, H-1), 3.38 (-, 1H, J = - Hz, H-2), 3.37 (-, 1H, J = - Hz, H-3), 3.46 (bdd, 1H, J = 2.0, 4.5 Hz, H-4), 3.60 (dd,
1H, J = 1.4, 6.5 Hz, H-5). 1.03 (d, 1H, J = 6.3 Hz, H-6), 6.89 (d, 1H, J = 8.0 Hz, H-3’, 5’), 6.57 (d, 1H, J = 8.0
Hz, H-4’, 6’), 2.15 (s, 3H, H-7’), 4.61 (d, 1H, J = 4.7 Hz, OH-2), 4.67 (d, 1H, J = 5.0 Hz, OH-3), 4.36 (d, 1H,
J = 4.5 Hz, OH-4), 5.88 (d, 1H, J = 8.0 Hz, NH); 13C NMR, 85.6 (C-1), 70.0 (C-2), 74.4 (C-3), 71.4 (C-4),
70.2 (C-5), 16.9 (C-6), 145.1 (C-1’), 129.2 (C-2’, 6’), 113.2 (C-3’, 7’), 125.2 (C-4’), 20.2 (C-7’).
1

p-toluidine-galactoside: N-4-methylphenyl-β-D-galactopyranosylamine V
1.8 gram of galactose (0.01 mol) in 4.0 mL of water, and 1.07 g of p-toluidine (0.01 mol) in 2.0 mL methanol
solution were mixed and stored at ambient in dark place for 48 hrs. The crystal was filtrated and washed using
chilled ether. The crystal was recrystallized from hot acetonitrile, filtrated and dried under vacuum.
mp 163.5(162.5[31]) , C13H19O5N, IR υmax 3350, 1620, 1530 cm-1, 1H NMR (DMSO) δ = 7.5 Hz, 4.16 (dd,
1H, J = 7.5, 8.5 Hz, H-1), 3.40 (dt, 1H, J = 4.5, 8.5, 8.5 Hz, H-2), 3.31 (td, 1H, J = 2.3, 3.0, 9.5 Hz, H-3), 3.67
(bt, 1H, J = 3.0, 3.0 Hz, H-4), 3.38 (bt, 1H, H-5). 3.40 (ddd, 1H, J = 1.6, 5.5, 11.5 Hz, H-6ax), 3.46 (ddd, 1H, J
= 1.5, 6.0, 11.5 Hz, H-6eq), 6.46 (d, 1H, J = 8.2 Hz, H-2’, 6’). 6.69 (d, 1H, J = 8.2 Hz, H-3’, 5’), 1.93 (s, 3H,
H-7’), 4.64 (d, 1H, J = 5.0 Hz, OH-2), 4.54 (d, 1H, J = 5.5 Hz, OH-3), 4.19 (d, 1H, J = 3.5 Hz, OH-4), 4.53
(dd, 1H, J = 5.5, 6.5 Hz, OH-6), 5.74 (d, 1H, J = 7.5 Hz, NH); 13C NMR, 96.8 (C-1), 81.2 (C-2), 85.2 (C-3),
79.4 (C-4), 86.2 (C-5), 71.6 (C-6), 155.6 (C-1’), 123.7 (C-2’, 6’), 139.3 (C-3’, 5’), 136.0 (C-4’), 29.8 (C-7’).
p-toluidine-glucoside: N-4-methylphenyl-β-D-glucopyranosylamine VI
1.8 gram of glucose (0.01 mol) in 4 mL of water, and 1.07 g of p-toluidine (0.01 mol) in 2 mL methanol
solution were mixed and stored at ambient in dark place for 24 hrs. The crystal was filtrated and washed using
dimethylformamide and methanol mixed solution. The crystal was recrystallized from hot acetonitrile,
filtrated and dried under vacuum.
mp 114 (114.5 [19], 116[31], 135.5[33]) , C13H19O5N, IR υmax 3300, 1625, 1530 cm-1, 1H NMR (DMSO) δ =
8.0 Hz, 4.29 (t, 1H, J = 8.0, 8.2 Hz, H-1), 3.03 (ddd, 1H, J = 5.0, 10.0 Hz, H-2), 3.13 (ddd, 1H, J = 5.0, 8.5 Hz,
H-3), 3.24 (ddd, 1H, J = 4.0, 8.5 Hz, H-4), 3.18 (dddd, 1H, J = 2.0, 5.0, 9.0 Hz, H-5). 3.64 (dddd, 1H, J = 2.0,
5.0, 11.5 Hz, H-6eq), 3.42 (td, 1H, J = 6.0, 11.5 Hz, H-6ax), 4.88 (d, 1H, J = 5.0 Hz, OH-2). 4.83 (d, 1H, J =
5.0 Hz, OH-3), 4.95 (d, 1H, J = 5.0 Hz, OH-4), 4.41 (bt, 1H, J = 5.0 Hz, OH-6), 6.90 (d, 1H, J = 8.3 Hz, H-3’,
5’), 6.58 (d, 1H, J = 8.5 Hz, H-2’, 6’), 2.15 (s, 3H, H-7’), 5.96 (d, 1H, J = 7.0 Hz, NH); 13C NMR, 85.6 (C-1),
73.2 (C-2), 70.4 (C-3), 77.8 (C-4), 77.3 (C-5), 61.1 (C-6), 144.5 (C-1’), 113.4 (C-2’, 6’), 129.3 (C-3’, 5’),
125.4 (C-4’), 20.2 (C-7’).
p-toluidine-mannoside: N-4-methylphenyl-β-D-mannopyranosylamine VII
1.8 gram of mannose (10 mmol) in 4.0 mL of water, and 1.07 g of p-toluidine (0.01 mol) in 2.0 mL methanol
solution were mixed and stored at ambient in dark place for 24 hrs. The crystal was filtrated and washed using
chilled ether. The crystal was recrystallized from hot acetonitrile, filtrated and dried under vacuum.
mp 176.5 (183.5 [19], 175[31]) , C13H19O5N, IR υmax 3350, 1620, 1525 cm-1, 1H NMR (DMSO) δ = 6.0 Hz,
4.70 (d, 1H, J = 9.0 Hz, H-1), 3.70 (bdd, 1H, J = 2.2, 5.0 Hz, H-2), 3.37 (dddd, 1H, J = 2.5, 5.5, 8.5 Hz, H-3),
3.35 (ddd, 1H, J = 4.5, 8.5, 9.0 Hz, H-4), 3.12 (bdddd, 1H, J = 2.0, 6.0, 9.0 Hz, H-5). 3.42 (ddd, 1H, J = 6.0,
11.5 Hz, H-6ax), 3.64 (dddd, 1H, J = 2.0, 5.0, 11.5 Hz, H-6eq), 6.63 (d, 1H, J = 8.2 Hz, H-2’, 6’). 6.89 (d, 1H,
J = 8.2 Hz, H-3’, 5’), 5.51 (s, 3H, H-7’), 4.78 (d, 1H, J = 5.0 Hz, OH-2), 4.71 (d, 1H, J = 5.0 Hz, OH-3), 4.71
(d, 1H, J = 4.5 Hz, OH-4), 5.52 (d, 1H, J = 10.0 Hz, NH); 13C NMR, 81.7 (C-1), 71.3 (C-2), 74.6 (C-3), 67.2
(C-4), 77.8 (C-5), 61.4 (C-6), 143.9 (C-1’), 113.8 (C-2’), 129.3 (C-3’), 125.7 (C-4’), 129.2 (C-5’), 113.5 (C-
6’), 20.2 (C-7’).
p-toluidine-rhamnoside: N-4-methylphenyl-α-L-rhamnopyranosylamine VIII
1.64 gram of rhamnose (10 mmol) in 2.5 mL of water, and 1.07 g of p-toluidine (10 mmol) in 2.0 mL
methanol solution were mixed and stored at ambient in dark place for 120 hrs. The crystal was filtrated and
washed using chilled acetone. The crystal was recrystallized from acetone, filtrated and dried under vacuum.
mp 153 (Dec)(151[31]) , C13H19O4N, IR υmax 3400, 1620, 1520 cm-1, 1H NMR (DMSO) δ = 1.0 Hz, 4.67
(bs, 1H, J = 1.0 Hz, H-1), 3.68 (bd, 1H, J = 8.0 Hz, H-2), 3.33 (ddd, 1H, J = 2.0, 3.0, 9.0 Hz, H-3), 3.12 (dt,
1H, J = 2.5, 9.0 Hz. H-4), 3.18 (dq, 1H, J = 6.0, 9.0 Hz, H-5). 1.08 (d, 1H, J = 6.0 Hz, H-6), 4.81 (d, 1H, J =
5.0 Hz, OH-2), 4.67 (d, 1H, J = 5.0 Hz, OH-3). 4.73 (d, 1H, J = 5.0 Hz, OH-4), 6.89 (d, 1H, J = 8.3 Hz, H-3’,
5’), 6.55 (d, 1H, J = 8.2 Hz, H-4’, 6’), 2.13 (s, 3H, H-1’), 5.44 (d, 1H, J = 10 Hz, NH); 13C NMR, 81.3 (C-1),
2

74.2 (C-2), 71.2 (C-3), 72.1 (C-4), 72.4 (C-5), 18.0 (C-6), 143.8 (C-1’), 113.8 (C-2’, 6’), 129.2 (C-3’, 5’),
125.6 (C-4’), 20.1 (C-7’).
p-toluidine-riboside: N-4-methylphenyl-β-D-ribopyranosylamine IX
0.50 gram of glucose (3.3 mmol) and 0.40g of p-toluidine (37 mmol) in 0.3 mL water solution were heat on
water-bath for 5 min. Solve crystal in 2.0 mL methanol and recrystallized. 540 mg crystal was solved in 5.4
mL non-aqueous ethanol, then added unhydrous sodium sulfate and dried. The crystal was filtrated and
washed using chilled ether. The crystal was recrystallized from hot acetonitrile, filtrated and dried under
vacuum.
mp 124 (129.5 [17]) , C12H17O4N, IR υmax 3350, 1620, 1520 cm-1, MS (FAB) m/z 240 [M+H]+, 1H NMR
(DMSO) δ = 4.6 Hz, 4.74 (dd, 1H, J = 2.4, 9.4 Hz, H-1), 3.63 (bd, 1H, J = 4.2 Hz, H-2), 3.72 (bs, 1H, H-3),
3.59 (bd, 1H, J = 11 Hz. H-4), 3.35 (bdd, 1H, J = 2.0. 5.0 Hz, H-5ax). 3.59 (bd, 1H, J = 11 Hz, H-5eq), 6.90 (d,
1H, J = 8.5 Hz, H-3’ 5’), 6.61 (d, 1H, J = 8.2 Hz, H-2’,6’). 2.15 (s, 3H, H-7’), 4.88 (d, 1H, J = 5.6 Hz, OH-2),
5.08 (d, 1H, J = 5.0 Hz, OH-3), 4.80 (d, 1H, J = 7.5 Hz, OH-4), 5.79 (d, 1H, J = 9.5 Hz, NH); 13C NMR, 81.7
(C-1), 70.1 (C-2), 70.2 (C-3), 68.0 (C-4), 63.1 (C-5), 144.0 (C-1’), 113.6 (C-2’, 6’), 129.3 (C-3’, 5’), 125.8
(C-4’), 20.2 (C-7’).
p-toluidine-xyloside: N-4-methylphenyl-β-D-xylopyranosylamine X
1.5 gram of xylose (10 mmol) and 1.07 g of p-toluidine (10 mmol) in 12 mL unhydrous ethanol solution were
mixed and added unhydrous sodiumsulfate, then stored at ambient in dark place for 48 hrs. The crystal was
filtrated and washed using benzene. The crystal was recrystallized from hot acetonitrile, filtrated and dried
under vacuum.
mp 116.5 (124.5 [17], 123[31], 124.5[36]) , C12H17O4N, IR υmax 3370, 1620, 1530 cm-1, 1H NMR (DMSO)
δ = 8.0 Hz, 4.19 (dt, 1H, J = 9.0, 1.0 Hz, H-1), 3.05 (ddd, 1H, J = 4.5, 8.0, 8.5 Hz, H-2), 3.32 (dt, 1H, J = 3.0,
8.5, 8.5 Hz, H-3), 3.32 (tdd, 1H, J = 5.0, 8.5, 9.5 Hz, H-4), 3.05 (dd, 1H, J = 9.5, 11.0 Hz, H-5ax). 3.51 (dd,
1H, J = 5.0, 11.0 Hz, H-5eq), 6.44 (d, 1H, J = 8.0 Hz, H-2’, 6’), 6.70 (d, 1H, J = 8.0 Hz, H-3’, 5’). 1.93 (s, 3H,
H-7’), 4.77 (d, 1H, J = 4.5 Hz, OH-2), 4.88 (bd, 1H, J = 3.0 Hz, OH-3), 4.82 (d, 1H, J = 5.0 Hz, OH-4), 5.75
(d, 1H, J = 8.0 Hz, NH); 13C NMR, 96.8 (C-1), 83.8 (C-2), 88.8 (C-3), 80.7 (C-4), 76.6 (C-5), 155.4 (C-1’),
123.6 (C-2’, 6’), 136.2 (C-3’, 5’), 123.7 (C-4’), 29.8 (C-7’).
p-anisidine-mannoside: N-4-methylphenyl-β-D-mannopyranosylsylamine XI
3.6 gram of mannose was solved in 3.3 mL water, and 2.46 gram of anisidine was solved in 1.5 mL 80%
ethanol at 50 . The crystal was solved in 42.5 mL of 70% ethanol, then, recrystallized at ambient.
mp 164 , C13H19O6N, IR υmax 3330, 1520, 1240 cm-1, 1H NMR (DMSO) δ = 7.1 Hz, 4.69 (H-1), 3.68 (H-2),
3.41 (H-3), 3.41 (H-4), 3.32 (H-5). 3.38 (H-6), 6.65 (H-4’, 6’). 6.69 (H-3’, 7’), 3.09 (H-1’), 4.75 (OH-2), 3.61
(OH-3), 3.59 (OH-4), 3.67 (NH); 13C NMR, 82.1 (C-1), 77.8 (C-2), 67.2 (C-3), 74.5 (C-4), 71.3 (C-5), 61.3
(C-6), 152 (C-5’), 115 (C-3’,7’), 114 (C-4’,6’), 140 (C-2’), 55.3 (C-1’).
p-anisidine-xyloside: N-4-methylphenyl-β-D-xylopyranosylsylamine XII
3.0 gram of xylose was solved in 2.5 mL water at 50 . 2.46 gram of anisidine was solved in 2.0 mL 80%
ethanol. The mixture of these solutions was kept in dark at ambient. The crystal was solved in 1.5 mL 80%
ethanol, then recrystallized at ambient.
mp 114(119.5[36]) , C12H17O5N, IR υmax 3400, 1520, 1250 cm-1, 1H NMR (DMSO) δ = 8.0 Hz, 4.21(H-1),
3.16 (H-2), 3.16 (H-3), 3.27 (H-4), 3.16 (H-5ax). 3.68 (H-5eq), 6.59 (H-4’, 6’). 6.69 (H-3’, 7’), 4.28 (OH-2),
4.98 (OH-3), 4.32 (OH-4), 5.76 (NH); 13C NMR, 68.7 (C-1), 73.0 (C-2), 77.6 (C-3), 69.9 (C-4), 66.2 (C-5),
152 (C-5’), 114 (C-3’,7’), 114 (C-4’,6’), 141 (C-2’), 55 (C-1’).
p-aminobenzoic acid-mannoside: N-4-carboxyphenyl-β-D-mannopyranosylsylamine XIII
3

3.6 gram of D-mannose was solved in 2.0 mL water, and 2.74 gram of p-aminobenzoic acid was solved in 21
mL acetone at 55 . These solutions were mixed at 55 then solution was kept overnight in dark at ambient.
The crystal was solved in 34 mL of 70% ethanol at 55 , then recrystallized at ambient.
mp 174.5 , C13H17O7N, IR υmax 3300, 1660, 1610 cm-1, 1H NMR (DMSO) δ = 9.0 Hz, 4.76 (H-1), 3.72 (H-2),
3.42 (H-3), 3.42 (H-4), 3.16 (H-5). 3.26 (H-6), 7.67 (H-3’, 7’). 6.77 (H-4’, 6’), 4.83 (OH-2), 3.65 (OH-3), 3.62
(OH-4), 4.36 (OH-6), 12.1 (OH-C1’), 6.50 (NH); 13C NMR, 80.9 (C-1), 78.0 (C-2), 66.7 (C-3), 74.3 (C-4),
70.9 (C-5), 61.2 (C-6), 167 (C-5’), 113 (C-3’, 7’), 113 (C-4’, 6’), 151 (C-2’), 119 (C-1’).
p-aminobenzoic acid-xyloside: N-4-carboxyphenyl-β-D-xylopyranosylsylamine XIV
3.0 gram of D-xyloside was solved in 2.0 mL water, and 2.74 of gram p-amino benzoic acid was solved in 21
mL acetone. The mixture was kept over-night in dark at ambient. The crystal was recrystallized from 70%
ethanol.
mp 146.5 , C12H15O6N, IR υmax 3300, 1660, 1610 cm-1, 1H NMR (DMSO) δ = 8.4 Hz, 4.37 (H-1), 3.17 (H-2),
3.17 (H-3), 3.28 (-4), 3.17 (H-5ax). 3.63 (H-5eq), 6.68 (H-4’, 6’). 7.67 (H-3’, 7’), 4.92 (OH-2), 5.03 (OH-3),
4.97 (OH-4), 12.1 (H-1’), 6.94 (NH); 13C NMR, 84.7 (C-1), 72.8 (C-2), 77.6 (C-3), 69.8 (C-4), 66.5 (C-5), 167
(C-5’), 112 (C-4’, 6’), 131 (C-3’, 7’), 151 (C-2’), 119 (C-1’).
p-amino-cinnamic acid-mannoside: N-4-(3’-carboxyl-1’,2’-ene-pheny)-β-D-mannopyranosylamine XV
1.8 gram of mannose was solved in 2.5 mL water, and 1.37 gram of p-amino-cinnamic acid was solved in 21
ml of 80% ethanol at 55 . These solutions were mixed at 55 and kept over-night at ambient. The crystal
was solved in 40 mL of 80% ethanol at 55 , then recrystallized at ambient.
mp 149 , C15H19O7N, IR υmax 3400, 1680, 1600 cm-1, 1H NMR (DMSO) δ = 8.0 Hz, 4.76 (H-1), 3.74 (H-2),
3.42 (H-3), 3.42 (H-4), 3.15 (H-5). 3.26 (H-6), 7.40 (H-5’, 9’), 6.76 (H-6’, 8’), 7.43 (H-3’), 6.19 (H-2’), 4.82
(OH-2), 3.65 (OH-3), 3.62 (OH-4), 4.36 (H-6), 12.0 (OH-1’), 6.34 (NH); 13C NMR, 81.0 (C-1), 78.0 (C-2),
67.0 (C-3), 74.3 (C-4), 70.9 (C-5), 61.2 (C-6), 168 (C-7’), 114 (C-6’, 8’), 130 (C-5’, 9’), 149 (C-4’), 145 (C-
3’), 123 (C-2’), 113 (C-1’).
p-amino-cinnamic acid-xyloside: N-4-(3’-carboxyl-1’,2’-ene-pheny)-β-D-xylopyranosylamine XVI
3.0 gram of xylose was solved in 2.0 mL water, and 3.26 gram of p-amino-cinnamic acid was solved in 40 mL
acetone at 55 . These solutions were mixed at 55 and kept over-night at ambient. The crystal was solved
in 140 mL of 80% ethanol at 70 , then recrystallized at ambient.
mp 147-150 , C15H19O7N, IR υmax 3400, 1680, 1620 cm-1, 1H NMR (DMSO) δ = 8.0 Hz, 4.36 (H-1), 3.16
(H-2), 3.16 (H-3), 3.29 (-4), 3.16 (H-5ax). 3.26 (H-5eq), 7.40 (H-5’, 9’). 6.60 (H-6’, 8’), 7.43 (H-3’), 6.18 (H-
2’), 4.91 (OH-2), 5.02 (OH-3), 4.97 (OH-4), 12.0 (OH-1’), 6.67 (NH); 13C NMR, 84.9(C-1), 72.8 (C-2), 77.6
(C-3), 69.8 (C-4), 66.4 (C-5), 168 (C-7’), 130 (C-5’, 9’), 113 (C-6’, 8’), 149 (C-4’), 145 (C-3’), 123 (C-2’),
114 (C-1’).
p-amino-hydrocinnamic acid-mannoside: N-4-(3’-carboxylpheny)-β-D-mannopyranosylamine XVII
0.54 gram of mannose was solved in 0.7 mL water at 60 , 0.50 gram of p-amino-hydrocinnamic acid was
solved in 2.0 mL acetone at 60 . The mixture was kept in dark at ambient. The recrystallization was
performed from 1.5 mL 80% ethanol.
mp 149 , C15H19O7N, IR υmax 3400, 1720, 1625 cm-1, 1H NMR (DMSO) δ = 9.0 Hz, 4.69 (H-1), 3.68 (H-2),
3.49 (H-3), 3.49 (H-4), 3.12 (H-5). 3.30 (H-6), 6.92 (H-5’, 9’). 6.92 (H-6’, 8’), 2.65 (H-3’), 2.42 (H-2’), 4.78
(OH-2), 3.63 (OH-3), 3.60 (OH-4), 4.32 (OH-6), 4.57 (NH); 13C NMR, 81.5 (C-1), 77.8 (C-2), 67.1 (C-3),
74.5 (C-4), 71.2 (C-5), 61.2 (C-6), 174 (C-7’), 129 (C-5’, 9’), 114 (C-6’, 8’), 144 (C-4’), 35.9 (C-3’), 29.7 (C-
2’), 130 (C-1’).
4-(4-aminophenyl)-butyric acid-mannoside: N-4-(4’-carboxylpheny)-β-D-mannopyranosylamine XVIII
4

1.8 gram of mannose was solved in 0.9 mL water at 70 , 1.79 gram 4-(4-aminophenyl)-butyric acid was
solved in 5.0 mL 80% ethanol at 70 . The mixture was kept in dark at ambient. The recrystallization was
performed from 6.0 mL of 80% ethanol.
mp 115.5 , C16H23O7N, IR υmax 3400, 1720, 1520 cm-1, 1H NMR (DMSO) δ = 8.5 Hz, 4.68 (H-1), 3.68 (H-2),
3.40 (H-3), 3.40 (H-4), 3.15 (H-5). 3.29 (H-6), 6.88 (-6’, 10’). 6.63 (H-7’, 9’), 2.41 (H-4’), 2.15 (H-3’), 1.70
(H-2’), 4.78 (OH-2), 3.63 (dOH-3), 3.60 (OH-4), 4.30 (OH-6), 5.57 (NH); 13C NMR, 81.5 (C-1), 77.8 (C-2),
67.1 (C-3), 74.5 (C-4), 71.2 (C-5), 61.2 (C-6), 174 (C-8’), 129 (C-6’, 10’), 114 (C-7’, 9’), 144 (C-5’), 33.6 (C-
4’), 33.0 (C-3’), 26.7 (C-2’), 130 (C-1’).
p-toluidine-alloside: N-4-ethylphenyl-b-D-allopyranosylamine IXX
721 mg of B-D-(+)-allose was solved in 0.8 mL water, and 429 mg p-toluidine was solved in 2.2 mL 80%
ethanol at 50 . The mixture was kept in dark at ambient. The crystal was recrystallized from 6.4 mL of
99.5% ethanol.
mp 134-135 , C13H19O5N, IR υmax 3300, 1620, 1520 cm-1, 1H NMR (DMSO), 4.32, 4.25 Hz (H-1), 5.30, 4.96
Hz (NH), 4.78, 4.61, 4.5 (H-2, 3, 4, 5, 6) The product was seemed to be a mixture of C1β and 1Cα.
5