Novel Synthesis of N-Glycosyl Amino Acids Using T3P: Propylphosphonic Acid Cyclic Anhydride as Coupling Reagent

Article abstract of DOI:10.1007/s10989-017-9614-4

In this paper is presented a novel and simple synthetic pathway for obtaining new protected and unprotected N-glucosyl amino acids from 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl amine and Fmoc-l-amino acids. Three methodologies were evaluated, using the coupling reagents: N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate, diisopropylcarbodiimide and propylphosphonic acid cyclic anhydride. The obtained products using propylphosphonic acid cyclic anhydride showed less undesired species, easy purification and higher yields than the other two methodologies. Deprotection strategies widely used in solid phase peptide synthesis were applied to develop the synthetic pathway reported and achieve the final products. The protected and unprotected N-glucosyl amino acids were purified using solid phase extraction chromatography and characterized by high performance liquid Chromatography and nuclear magnetic resonance spectroscopy. Different amino acids (Fmoc-l-Asp(OtBu)OH, Fmoc-l-Phe(OH) and Fmoc-l-Lys(Boc)-OH) have been employed to demonstrate the simple and reproducible coupling methodology using propylphosphonic acid cyclic anhydride. The results showed that new protected and unprotected N-glucosyl amino acids can be obtained with high purity and the methodology could be used with any Fmoc-amino acid. The methodology developed could be considered as a synthetic tool for obtaining building blocks for glycopeptide synthesis and potential drugs candidates based on glycoconjugates.

Full text of DOI:10.1007/s10989-017-9614-4

Int J Pept Res Ther
DOI 10.1007/s10989-017-9614-4
Novel Synthesis of N-Glycosyl Amino Acids Using T3P®:
Propylphosphonic Acid Cyclic Anhydride as Coupling Reagent
Sandra Milena Pinzón Martín1 · Ricardo Fierro Medina1 ·
Carlos Arturo Iregui Castro2 · Zuly Jenny Rivera Monroy1 ·
Javier Eduardo García Castañeda3
Accepted: 10 July 2017
© Springer Science+Business Media, LLC 2017
Abstract In this paper is presented a novel and simple
synthetic pathway for obtaining new protected and unpro-
tected N-glucosyl amino acids from 2,3,4,6-tetra-O-acetyl-
β-d-glucopyranosyl amine and Fmoc-l-amino acids. Three
methodologies were evaluated, using the coupling reagents:
N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium tetra-
fluoroborate, diisopropylcarbodiimide and propylphospho-
nic acid cyclic anhydride. The obtained products using pro-
pylphosphonic acid cyclic anhydride showed less undesired
species, easy purification and higher yields than the other
two methodologies. Deprotection strategies widely used in
solid phase peptide synthesis were applied to develop the
synthetic pathway reported and achieve the final products.
The protected and unprotected N-glucosyl amino acids
were purified using solid phase extraction chromatogra-
phy and characterized by high performance liquid Chro-
matography and nuclear magnetic resonance spectroscopy.
Different amino acids (Fmoc-l-Asp(OtBu)OH, Fmoc-l-
Phe(OH) and Fmoc-l-Lys(Boc)-OH) have been employed
to demonstrate the simple and reproducible coupling meth-
odology using propylphosphonic acid cyclic anhydride.
The results showed that new protected and unprotected
N-glucosyl amino acids can be obtained with high purity
and the methodology could be used with any Fmoc-amino
acid. The methodology developed could be considered as
a synthetic tool for obtaining building blocks for glyco-
peptide synthesis and potential drugs candidates based on
glycoconjugates.
Keywords Glycoconjugate · N-glucosyl amino acids ·
Glycosidic bond · Propylphosphonic acid cyclic anhydride
Introduction
Carbohydrates, one of the most abundant biomolecules
in nature, are essential for the sustenance and survival of
living things (Varki 1993). These molecules participate
in a spectrum of recognition events and binding between
surface molecules of pathogens and target cells (Varki
1993). The carbohydrates are involved in cell–cell (can-
cer), cell–bacteria (Micobacterium tuberculosis, Escheri‑
chia coli, Elicobacter pilory, Vibrio colherae, Bordetelle
pertusiis, Pasteurella multocida, etc) cell–virus (influenza
virus, herpes virus, human immunodeficiency virus, ebola
virus, etc.), cell-parasite (e.g. plasmodium falciparum,
Leishmania, etc.), and cell–fungi (Candida albicans, Cryp‑
tococcus neoformans, etc.) interactions (Osanya et al. 2011;
Karlsson 1999; Levitz et al. 2015; Tripathi et al. 2015; Kato
and Ishiwa 2015). Carbohydrate and amino acids constitute
two important classes of building blocks used by nature
to build its vast repertory of molecules (Varki 1993). The
sugar amino acids have great potential as multifunctional
building blocks in the development of large varieties of
designer molecules. The demand for finding new molecules
for discovering new drugs is increasing (Chakraborty et al.
2005).
* Javier Eduardo García Castañeda
jaegarciaca@unal.edu.co
1
Chemistry Department, Universidad Nacional de Colombia,
Bogotá Carrera 45 No 26-85, Building 451, office 409,
Bogotá 11321, Colombia
2
Veterinary and Animal Science Faculty, Universidad
Nacional de Colombia, Bogotá Carrera 45 No 26-85,
Building 510, office 504, Bogotá 11321, Colombia
3
Pharmacy Department, Universidad Nacional de Colombia,
Bogotá Carrera 45 No 26-85, Building 450, office 203,
Bogotá 11321, Colombia
Vol.:(0123456789)
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Int J Pept Res Ther
Successful establishment of infection by bacterial path-
ogens requires adhesion to host cells, colonization of tis-
sues, and in certain cases cellular invasion (Pizarro-Cerda
and Cossart 2006). Conventional therapies involve the use
of medicines based on carbohydrates and peptides that pro-
duce bacterial death through different mechanisms. Anti-
adhesion therapy is an approach focused on preventing the
attachment of the bacteria to host tissues, without affecting
the bacteria’s life (Sharon and Ofek 2001). The adhesion
of many pathogenic organisms is mediated by lectins that
bind to complementary carbohydrates on the surface of the
host tissues (Sharon and Ofek 2001). However, the use of
monosaccharides or carbohydrates as possible therapeutic
agents show many limitations: requirement of high doses to
obtain an effective concentration at the site of action, low
residency time, susceptibility to glycosidases, and adverse
reactions. Another limitation is that obtaining oligosaccha-
rides implies complex synthetic pathways that affect yields.
The development of medicine based on carbohydrates is
focused on designing and obtaining small molecules that
are easy to synthesize, with high yields and low cost.
are not required. T3P® can be consider as a green, relatively
non-toxic, and non-allergic/non-sensitizing reagent. This is
an efficient coupling reagent as well as a water scavenger. It
activates the amino acid, forming active ester with the for-
mation of easily removable, non-hazardous, water-miscible
propanephosphonic acid as a by-product (Waghmare et al.
2014). The products can be isolated with high yields caus-
ing a very limited level of racemization without the use
of any additive (Basavaprabhu et al. 2013). The protected
and unprotected N-glucosyl amino acids were purified by
a methdology based on RP-SPE cromatography developed
in our laboratory. In the present study, six new N-glucosyl
amino acids were obtained from d-glucose and three amino
acids with different side chain structure, which gives them
a different nature in physiological conditions, The products
were obtained with high purity and were fully character-
ized by NMR.
Experimental Methods
General Procedures
In this respect, the amino acid-carbohydrate glycoconju-
gates can be an interesting approach to develop therapeu-
tic agents that can inhibit bacterial adhesion. The glycosyl
amino acids are the smallest glycoconjugates and are the
basic building blocks for complex glycoconjugates. The
synthesis of N-glucosyl amino acids has been focused on
obtaining protected derivatives to be used in solid-phase
synthesis of glycopeptides (Yao et al. 2010; Van Amei-
jde et al. 2002; Paolini et al. 2007). However, unprotected
N-glucosyl amino acids could be considered as a source of
new candidates for developing drugs, which has inspired
scientists to explore new methodologies for obtaining
unprotected N-glucosyl amino acids (Monsigny et al.
1998). The synthesis of N-glucosyl amino acids involves
the formation of the N-glycosydic bond between an amino-
glucopyranose and the carboxyl group of the amino acid.
The glycosidation reaction requires special conditions and
the use of toxic reagents that could be difficult to handle.
The coupling of amino acids and carbohydrates has been
done using many coupling reagents, such as uronium salts
(Paolini et al. 2007; Bejugam and Flitsch 2004), carbodi-
imides (Kornhauser and Keglević 1969; Wang et al. 2010),
and other methods (Yao et al. 2010; Van Ameijde et al.
2002; Valentekovic and Keglević 1976; Real-Fernández
et al. 2010; Arsequell and Valencia 1997; Kröger et al.
2004). In the present article, we show a new method for
coupling amino acids and carbohydrates using the pro-
pylphosphonic anhydride (T3P®). This reagent is used in
the amide bond formation, and it is very easy to use and
combines excellent reaction selectivity and high product
purity with good yields. It is an efficient coupling reagent;
in the reactions in which it participates, hazardous additives
Monitoring of Reactions of Compounds 4, 4b, 4c, 5, 5a, 5b,
5c, 6, 6a, 6b, and 6c
The reactions were monitored by RP-HPLC chromatogra-
phy using an Agilent Technologies 1260 Infinity Chromato-
graph. Method 1: Monolithic column Chromolith high-res-
olution Rp-18e (50×4.6 mm), solvent A: (Water and TFA
0.05%), solvent B: (ACN and TFA 0,05%), gradient elution
15/75/75/15%B in 1/11.5/13.5/17.5 min., Flow: 2 mL/min,
wavelength 302 and 210 nm, injection 10 μL (0.1 mg/mL
N-glucosyl amino acid). Method 2: Eclipse XDB-C18 col-
umn (3.5 µm; 4.6×150 mm): solvent A: (water and TFA
0.05%), solvent B: (ACN and TFA 0.05%), gradient elu-
tion 15/100/100/15%B in 2/47/54/64 min. Flow: 1 mL/min,
wavelength 302 and 210 nm, injection 10 μL (0.1 mg/mL
N-glucosyl amino acid).
Purification of Compounds 4, 4c, 5, 5c, 6 and 6c
RP-SPE columns were activated in accordance with the
supplier’s recommendations. The crude product was puri-
fied eluting an increasing gradient of ACN. The fractions
obtained were analyzed by means of RP-HPLC using
method 1.
NMR. The spectra were recorded in CDCl3 and D2O
solutions on an Avance 400 MHz spectrometer.
2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl amine (3)
was prepared according to standard methods (Mukhopad-
hyay et al. 2005; Fritz and Schenk 1959; Soli et al. 1999).
Obtaining to amine GlcAc4-NH2 was accomplished via the
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Int J Pept Res Ther
corresponding glucosyl azide GlcAc4-N3 (2) as reported
by Soli et al. 1999 from 1,2,3,4,6-Penta-O-acetyl-α-d-
glucopyranose (1).
After analyzing the methodologies, the methodology
using T3P® as coupling reagent was chosen for obtaining
N-glucosyl amino acids, compounds 5 and 6.
Fmoc‑l‑Phe‑O‑(2,3,4,6‑tetra‑O‑acetyl‑β‑D‑N‑glucopyran
osyl) (4) (C38H40N2O12, MW: 716.22 g/mol) Compound
4 was obtained as a white solid (195.5 mg, yield 75%).
1H-NMR (400 MHz, CDCl3) δ (ppm): 1.93 (s, 3H, 1 ×
COCH3), 2.04 (d, J=9.4 Hz, 6H, 2 × COCH3), 2.11 (s, 3H,
1 × COCH3), 3.12 (s, 2H, Phe β-CH2), 3.84 (d, J=7.9 Hz,
1H, H5), 4.13 (d, J=11.6 Hz, 1H, H-6), 4.22 (t, J=6.9 Hz,
2H, H-Fmoc), 4.31 (dd, J=12.8, 3.5 Hz, 1H, H-Fmoc), 4.41
(dd, J=29.5, 7.4 Hz, 2H, H-Fmoc and Phe α-CH), 4.87 (t,
J=9.5 Hz, 1H, H-2), 5.07 (d, J=7.5 Hz, 1H, H-4), 5.26 (t,
J=9.2 Hz, 1H, H-1), 5.32 (t, J=9.5 Hz, 1H, H-3), 6.80 (d,
J=7.4 Hz, 1H, NH), 7.18 (d, J=5.0 Hz, 1H, Phe-Ar), 7.31
(dt, J=10.5, 6.0 Hz, 5H, Phe-Ar), 7.43 (t, J=7.4 Hz, 2H,
Fmoc-Ar), 7.59–7.50 (m, 2H, Fmoc-Ar), 7.79 (d, J=7.5 Hz,
2H, Fmoc-Ar). 13C-NMR (100 MHz, CDCl3) δ (ppm): 20.6
(CH3), 20.7 (CH3), 37.3 (CH2), 47.1 (CH), 56.1 (CH), 61.7
(CH2), 67.3 (CH2), 68.1 (CH), 70.5 (CH), 72.6 (CH), 73.6
(CH), 78.2 (CH), 120.0 (CH), 125.0 (CH), 125.1 (CH),
127.1 (CH), 127.2 (CH), 127.8 (CH), 128.8 (CH), 129.5
(CH), 141.3 (C), 143.6 (C), 169.5 (C=O), 169.9 (C=O),
170.4 (C=O), 170.6 (C=O), 171.0 (C=O), 171.4 (C=O).
General Procedures for the Coupling Reaction
The N-glucosyl amino acids 4, 5, and 6 were obtained by
reaction between 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl
amine and Fmoc-l-amino acids, Fmoc-Phe-OH, Fmoc-
l-Asp(OtBu)-OH, or Fmoc-l-Lys(Boc)-OH, respectively.
The reactions were performed using three different meth-
odologies in order to evaluate the coupling reagents and
choose the best one to continue with the reactions. As a
model Fmoc-Phe-OH was used, and three methodologies
were compared (Fig. 2, step iv).
Method (a), (Fig. 2, step iv‑a)
Fmoc-Phe-OH (1 mmol) was mixed with TBTU (1 mmol),
HOBt (1 mmol) and DIPEA (3 mmol) using DMF as sol-
vent, and then 3 (1 mmol) was added. The mixture was
stirred at RT for 16 h. The progress of the reaction was
checked by RP-HPLC (Fig. 2a). Then the mixture was dis-
solved in CH2Cl2 and was washed successively with water
and saturated NaHCO3. The organic layer was dried with
Mg2SO4, filtered and concentrated to dryness. Compound 4
was purified via RP-SPE chromatography.
Fmoc‑l‑Asp‑(OtBu)‑O‑(2,3,4,6‑tetra‑O‑acetyl‑β‑d‑N‑gluco
pyranosyl) (5) (C37H44N2O14, MW: 740.28 g/mol) Com-
pound 5 was obtained as a white solid (405.5 mg, yield
1
57%). H-NMR (400 MHz, CDCl3) δ (ppm): 1.46 (s, 9H,
Method (b), (Fig. 2, step iv‑b)
3 × CH3—OtBu), 2.10 (m, 12H, 4 × COCH3), 2.59 (d,
J=16.8 Hz, 1H, CH2), 3.09 (d, J=16.5 Hz, 1H, CH2, Asp
β-CH2), 3.85 (d, J=9.9 Hz, 1H, CH, H-5), 4.10 (m, 1H,
CH2, H-6), 4.32 (m, 2H, CH2, H-6 and Fmoc-CH), 4.52
(m, 2H, Fmoc-CH2 and Asp α-CH), 4.99 (t, J=9.4 Hz, 1H,
H-2), 5.10 (t, J=9.7 Hz, 1H, H-4), 5.22 (t, J=9.2 Hz, 1H,
H-1), 5.36 (t, J=9.5 Hz, 1H, H-3), 7.35–7.28 (m, 2H, H-Ar),
7.44 (t, J=7.4 Hz, 3H, H-Ar, NH), 7.68 (t, J=7.7 Hz, 2H,
H-Ar), 7.80 (d, J=7.5 Hz, 2H, H-Ar). 13C-NMR (100 MHz,
CDCl3) δ (ppm): 20.5 (CH3), 20.6 (CH3), 20.7 (CH3), 20.8
(CH3), 28.0 (CH3), 36.5 (CH2), 47.0 (CH), 51.3 (CH), 61.6
(CH2), 67.7 (CH2), 68.1 (CH), 70.5 (CH), 72.5 (CH), 73.6
(CH), 78.4 (CH), 78.4 (CH), 82.0 (C), 120.1 (CH), 125.2
(CH), 127.2 (CH), 127.8 (CH), 141.3 (C), 143.6 (C), 156.2
(C=O), 169.7 (C=O), 169.9 (C=O), 170.8 (C=O), 171.2
(C=O), 171.4 (C=O), 171.5 (C=O).
Fmoc-Phe-OH (1 mmol) was mixed with 3 (1 mmol) and
DIC (1 mmol) using CH2Cl2 as solvent. The mixture was
stirred at RT for 16 h. The progress of the reaction was
checked via RP-HPLC (Fig. 2b). Then the mixture was dis-
solved in CH2Cl2 and was washed successively with water
and saturated NaHCO3. The organic layer was dried with
Mg2SO4, filtered, and concentrated to dryness. Compound
4 was purified via RP-SPE chromatography.
Method (c), (Fig. 2, step iv‑c)
Fmoc-Phe-OH (0.65 mmol) was mixed with 3 (1.2 mmol),
DIPEA (2 mmol) and EtOAc (10 mL) in a cold bath, and
then T3P® 50% EtOAc (1.5 mmol) was added. The mixture
was stirred at RT for 16 h. The progress of the reaction was
checked via RP-HPLC, and the disappearance of amino
acid was observed (Fig. 2b). Then the mixture was dis-
solved in CH2Cl2 and was washed successively with water
and saturated NaHCO3. The organic layer was dried with
Mg2SO4, filtered, and concentrated to dryness. Compound
4 was purified via RP-SPE chromatography.
Fmoc‑l‑Lys‑(Boc)‑O‑(2,3,4,6‑tetra‑O‑acetyl‑β‑d‑N‑gluco
pyranosyl) (6) (C40H51N3O14, MW: 797.34 g/mol) Com-
pound 6 was obtained as a white solid (389.2 mg, yield
1
92%). H-NMR (400 MHz, CDCl3) δ (ppm): 1.27 (s, 4H,
2 × Lys-CH2), 1.45 (s, 9H, CH3, 3 × Boc), 1.99 (s, 3H,
CH3, 1 × COCH3), 2.07 (m, 9H, 3 × COCH3), 3.12 (s, 2H,
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Int J Pept Res Ther
Fig. 1 Design of N-glucosyl-amino acids. a unprotected target molecules, b protected building blocks
Fig. 2 Comparative analysis
via RP-HPLC of 2,3,4,6-tetra-
O-acetyl-β-d-glucopyranosyl
amine and Fmoc-Phe-OH
coupling reaction for obtaining
4 (step iv), using three coupling
reagents, TBTU (a), DIC (b)
and T3P® (c). Reaction time
16 h
CH2, Lys-CH2), 3.83 (d, J=8.2 Hz, 1H, H-5), 4.12 (m, 3H,
CH2, H-6; Lys α-CH), 4.25 (m, 3H, CH2, H-6; Fmoc-CH),
4.40 (m, 2H, Fmoc-CH2), 4.94 (m, 1H, H-2), 5.08 (m, 1H,
H-4), 5.24 (m, 2H, H-1 and H-3), 6.99 (d, J=9.0 Hz, 1H,
NH), 7.34 (t, J=7.4 Hz, 2H, Fmoc-Ar), 7.42 (t, J=7.4 Hz,
2H, Fmoc-Ar), 7.62 (d, J=7.2 Hz, 2H, Fmoc-Ar), 7.78 (d,
J=7.5 Hz, 2H, Fmoc-Ar). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 20.5 (CH3), 22.3 (CH2), 28.4 (CH3), 29.6 (CH2),
30.9 (CH2), 34.1 (CH2), 47.1 (CH), 55.1 (CH), 61.9 (CH2),
67.2 (CH2), 68.1 (CH), 70.5 (CH), 73.7 (CH), 78.2 (CH),
90.2 (CH), 120.0 (CH), 125.1 (CH), 127.1 (CH), 127.8
(CH), 141.3 (C), 143.7 (C), 156.2 (C=O), 169.5 (C=O),
169.9 (C=O), 170.1 (C=O), 170.6 (C=O), 170.9 (C=O),
172.3 (C=O).
Methodology for Deprotection of Compounds 4, 5 and 6
To carry out the removal of side chain protective groups
OtBu and Boc (Fig. 3, step v), compounds 5 and 6 were
dissolved in a mixture of TFA/DCM (55% v/v) (Blondelle
and Houghten 1993), and the mixture was stirred at room
temperature for 2 h. Then the TFA was removed and prod-
ucts 5a and 6a were obtained. The removal of the Fmoc
group (Fig. 3, step vi) was then carried out, dissolving 4,
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Int J Pept Res Ther
Fig. 3 Synthetic pathway for obtaining unprotected N-glucosyl
amino acids. The OtBu and Boc groups were removed by treat-
ment with TFA/DCM (step v) obtaining 5a and 6a. Then 4, 5a, and
6a were treated with 4-methyl piperidine and the Fmoc group was
removed (step vi) for obtaining 4b, 5b and 6b, then acetyl groups
were removed by treatment with hydrazine hydroxide leads to 4c, 5c
and 6c (step vii), which correspond to unprotected target molecules
(Fig. 1a)
5a and 6a in 4-methylpiperidine/DMF (25% v/v) (Vergel
et al. 2014). The mixture was stirred at RT for 3 h and the
progress of the reactions was checked via RP-HPLC. Then
solvent was removed under reduced pressure and 4b, 5b,
and 6b were obtained.
l‑Asp‑O‑β‑1‑N‑(d‑glucopyranosyl) (5c) (C10H18N2O8, MW:
294.11 g/mol)
Compound 5c was obtained as a white solid (5.6 mg,
1
yield 20%). H-NMR (400 MHz, CDCl3) δ (ppm): 2.79
Finally, for obtaining 4c, 5c, and 6c (target molecules,
Fig. 1a), deacetylation of 4b, 5b, and 6b was carried out
with hydrazine hydroxide, 20%, (Fig. 3, step vii) (Khan
et al. 1996), the mixture was stirred at RT for 6 h, and the
progress of the reactions was checked via RP-HPLC. Then
acetone was added and solvent was removed under reduced
pressure. The compounds were pre-purified via RP-SPE
chromatography, and the products were recrystallized in
EtOH.
(dd, J = 17.7 and 8.3 Hz, 1H, Asp β-CH2), 2.91 (dd,
J = 17.7 and 4.6 Hz, 1H, Asp β-CH2), 3.33 (t, J = 8.9 Hz,
2H, H-5), 3.49–3.40 (m, 2H, H-2), 3.62 (dd, J = 12.4,
5.2 Hz, 1H, CH2, H-6), 3.78 (d, J = 13.9 Hz, 1H, CH2,
H-6), 4.25 (dd, J = 8.2, 4.7 Hz, 1H, Asp α-CH), 4.93 (d,
J = 9.1 Hz, 1H, 1-H),. 13C-NMR (100 MHz, CDCl3) δ
(ppm): 35.7 (CH2), 50.2 (CH), 60.4 (CH2), 69.1 (CH),
71.7 (CH), 76.3 (CH), 77.6 (CH), 79.3 (CH), 170.1
(C=O), 174.3 (C=O).
l‑Phe‑O‑β‑1‑N‑(d‑glucopyranosyl) (4c) (C15H22N2O6, MW:
326.15 g/mol)
l‑Lys‑O‑β‑1‑N‑(d‑glucopyranosyl) (6c) (C12H25N3O6, MW:
307.17 g/mol)
Compound 4c was obtained as a white solid (6.0 mg, yield
1
32%). H-NMR (400 MHz, CDCl3) δ (ppm): 3.06 (m 1H,
Compound 6c was obtained as a white solid (42.9 mg, yield
1
Phe β-CH2), 3.20 (m 1H, Phe β-CH2), 3.33 (m, 2H, 1 ×
H-2), 3.44 (m, 1H, H-5), 3.66 (m, 1H, CH2, H-6), 3.82
(m, 1H, CH2, H-6), 4.16 (m, 1H, Phe α-CH), 4.91 (d,
J=9.2 Hz, 1H, H-1), 7.24 (d, J=7.8 Hz, 3H, H-Ar Phe),
7.31 (m, 4H, H-Ar Phe).13C-NMR (100 MHz, CDCl3) δ
(ppm): 36.9 (CH2), 54.5 (CH), 60.5 (CH2), 69.1 (CH), 71.7
(CH), 76.4 (CH), 77.5 (CH), 79.1 (CH), 127.9 (CH), 129.0
(CH), 129.6 (CH), 171.3 (C=O).
73%). H-NMR (400 MHz, CDCl3) δ (ppm): 1.42 (m, 3H,
Lys-CH2), 1.63 (m, 3H, Lys-CH2), 1.89 (m, 3H, Lys-CH2),
2.92 (m, 2H, Lys β-CH), 3.34 (m 2H, H-2), 3.52–3.40 (m,
2H, H-5), 3.61 (m, 1H, H.6), 3.79 (d, J=14.3 Hz, 1H, H-6),
3.99 (t, J=6.4 Hz, 1H, Lys α-CH), 4.94 (d, J=9.2 Hz, 1H,
1-H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 21.09 (CH2),
26.3 (CH2), 30.1 (CH2), 39.0 (CH2), 53.2 (CH), 60.5 (CH2),
71.8 (CH), 76.4 (CH), 77.8 (CH), 79.4 (CH), 170.9 (C=O).
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Int J Pept Res Ther
Table 1 Coupling of
GlcAc4-NH2 to phenylalanine to
obtain product 4
Results and Discussion
Coupling reagent
Yield (%)
TBTU/HOBt
DIC
39
42
75
New unprotected N-glucosyl amino acids were designed
(Fig. 1a). Please note that the selected sugar motif is glu-
cose and the amino acid motif is phenylalanine, aspartic
residue, or lysine, whose lateral chain is non-polar aro-
matic, acidic, and basic, respectively. For all the designed
molecules, the 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl
amine is attached to the amino acid by the α-carboxylic
acid group. For obtaining these molecules, it is neces-
sary to use orthogonal protection groups. Thus, the acetyl
group was selected for the glucose residue and amino
acids were used with the Fmoc/tBu system of protection
(Fig. 1b).
T3P®/DIPEA
Reagents:
Fmoc-l-Phe-
OH (1 mmol), GlcAc4-NH2
(1 mmol), coupling reagent
(1 mmol), DIPEA (3 mmol),
16 h, RT
After obtaining protected N-glucosyl amino acids 4, 5,
and 6 using T3P® reagent, the RP-HPLC analysis was done
using two chromatographic methods: (1) using a mono-
lithic column Chromolith high resolution RP -18e (new
method) and (2) using a packed column XDB—C18 (con-
ventional method). A reduction of retention time, required
solvent volume, and analysis time required (run time)
was observed. These data show the benefits that a mono-
lithic column can give us in the analysis of hydrophobic
molecules, reducing several aspects such as analysis time
(60%) and consumption of solvents (50%). The purifica-
tion method via RP-SPE was simple, fast, and require less
solvent volumes, additionally the purified products were
obtained with higher yields and similar purity than conven-
tional Flash chromatography. The analysis monitoring via
RP-HPLC (using a monolithic column), and some steps of
the synthetic pathway fulfill some of the principles of green
chemistry.
The coupling of Fmoc amino acids and glucopyrano-
sylamine GlcAc4-NH2 was performed using different cou-
pling reagents, specifically (i) TBTU, (ii) DIC, and (iii)
T3P®, as will be discussed below (Fig. 2, step iv). Then
T3P® was chosen as coupling reagent for obtaining 4, 5
and 6. In the last step, the protective groups of N-glucosyl
amino acids were removed by treatment with TFA, 4-meth-
ylpiperidine and hydrazine hydroxide (Fig. 3).
Three different methodologies were analyzed and evalu-
ated for obtaining N-glucolsyl amino acids, using three
coupling reagents N,N,N′,N′-Tetramethyl-O-(benzotriazol-
1-yl)uronium tetrafluoroborate (TBTU) (Fig. 2a), diisopro-
pylcarbodiimide (DIC) (Fig. 2b), and propylphosphoninc
anhydride (T3P®) (Fig. 2c). The reactions were performed
using Fmoc-Phe-OH as starting reagent and monitored
using RP-HPLC, and the results are shown in Fig. 3.
In general, the synthesis involves less hazardous chemi-
cal synthesis and catalysts in the majority of the reactions.
It also was designed for energy efficiency. All reactions
were done under room temperature and atmospheric pres-
sure conditions. Characterization and purification methods
by reverse phase were done considering the use of safer
solvents and auxiliaries, which reduces the consumption
of organic reagents that can affect human health and the
environment.
The RP-HPLC analysis (C18 packed column) of com-
pound 4 formation using three different coupling rea-
gents is shown in Fig. 2. The chromatographic profile of
the reaction performed using TBTU (Fig. 2a) shows the
formation of several species that affect the purification
of 4. The incomplete reaction (at 16 h) can also be seen.
The profile for the coupling reaction using DIC (Fig. 2b)
shows the formation of two species with a higher reten-
tion time, which can produce difficulties in the purification
process, besides which the formation of N-acylurea pre-
cipitate, which causes difficulties for its physical separation
and consequently product purification. The profile for the
reaction using T3P® shows the disappearance of the amino
acid peak and the formation of only one species (Fig. 2c).
Table 1 shows obtained yields for Fmoc-l-Phe-OH cou-
pling to GlcAc4-NH2 using TBTU/HOBt, DIC or T3P®/
DIPEA generating 4. It was observed that T3P® reagent led
to higher yields. The reaction using T3P® by-products are
water soluble products, which reduces the time of purifica-
tion. Furthermore, DIC is a toxic reagent, and HOBt has
been classified as an explosive compound (Wehrstedt et al.
2005), while T3P® is non-toxic and non-allergenic and can
be classified as a green reagent (Waghmare et al. 2014).
Our results showed that it was possible obtain the
intermediates 4, 5 and 6 with high purity and good yields
(50–95%). The results suggesting that the coupling reac-
tion between Fmoc-amino acids and GlcAc4-NH2 using
T3P is affected by the side chain amino acid nature, specifi-
cally the reaction with Fmoc-Phe-OH proceeds with 75%
of yield, while using Fmoc-Leu-OH the yield was lower
(56%) (data no shown). The pathway described here can
be applied to the synthesis of protected N-glucosyl amino
acids, which are the building blocks for N-glycopeptide
synthesis.
It was also possible to obtain 4c, 5c, and 6c with high
purity. It was shown that deprotection reactions are the
critical point for obtaining unprotected N-glucosyl amino
acids. The pathway implies sequential deprotection steps
1 3

Int J Pept Res Ther
34828 “Diseño y síntesis química de péptidos y glicoconjugados
y evaluación de la actividad antibacteriana contra Escherichia coli
ATCC 25922.”
(Fig. 3). First, the side chain protective groups were
removed by treatment of N-glucosyl amino acids 5 and 6
with TFA/DCM solution. The progress of the reactions was
monitored via RP-HPLC; the retention time of 5a and 6a
was less than 5 and 6, respectively; this is due to the depro-
tection reaction, which yields fewer hydrophobic products.
When peaks for 5 or 6 disappeared, the reaction was con-
sidered finished. The Fmoc group removal was achieved
by treatment of 4, 5a, and 6a with 4-methylpiperidine in
DMF for obtaining 4b, 5b, and 6b, respectively. The reac-
tions were monitored by RP-HPLC; the peak of the starting
reagent disappeared, while the peak corresponding to the
by-product dibenzofulvene adduct was observed. The last
step involved the removal of acetyl groups by treatment of
4b, 5b, and 6b with Hydrazine hydroxide; this yields 4c,
5c, and 6c, respectively. Then the crude products were
pre-purified and recrystallized in EtOH. The yields and
purity grade suggest that deprotection reactions are affected
because of their amino acid side chain nature.
Compliance with Ethical Standards
Conflict of interest The authors declares that they have no conflict
of interest.
Ethical Approval This article does not contain any studies with ani-
mals performed by any of the authors.
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