Efficient double glycoconjugation to naturalize high molecular weight disperse dyes

Article abstract of DOI:10.1016/j.carres.2011.10.036

Commercially available Disperse Orange 29 (1a) and Disperse Red 1 (2a) were elaborated to glycoconjugated species, following a new version of a previously-described 'naturalisation' procedure. Glutamic acid was chosen to achieve a double glycoconjugation,

Full text of DOI:10.1016/j.carres.2011.10.036

Carbohydrate Research 356 (2012) 104–109
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Efficient double glycoconjugation to naturalize high molecular weight
disperse dyes q
a
a
b,
b
Roberto Bianchini a, , Massimo Rolla , Jalal Isaad , Giorgio Catelani , Lorenzo Guazzelli ,
⇑
⇑
Massimo Corsi a, Marco Bonanni a
a Dipartimento di Chimica ‘Ugo Schiff’, Università di Firenze, via della Lastruccia 13, Sesto Fiorentino 50019 (FI), Italy
b Dipartimento Scienze Farmaceutiche, Università di Pisa, via Bonanno 33, Pisa 56126, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 August 2011
Received in revised form 21 October 2011
Accepted 23 October 2011
Available online 29 October 2011
Commercially available Disperse Orange 29 (1a) and Disperse Red 1 (2a) were elaborated to glycoconju-
gated species, following a new version of a previously-described ‘naturalisation’ procedure. Glutamic acid
was chosen to achieve a double glycoconjugation, which is essential to give to the original disperse dye a
water solubility suitable for reaching optimal dyeing conditions. UV–vis plot of the ‘naturalised’ species
showed negligible differences when compared to those of the commercial dyes.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Disperse azodyes
Glycoconjugation
Glutamic acid
Lactose
The naturalisation of dyes1 (i.e., the conjugation of dyes to sac-
charide species) has proven to be a valid method to prepare novel
dyeing agents. The main characteristics of these dyes are not only
their solubility in water, but also their ability to dye the majority of
synthetic and natural (wool, cotton) textiles.2 Moreover this prop-
erty is accompanied by the environmentally sound characteristic:
the overall process of dyeing does not require surfactants, mor-
dants, dispersing agents or other additives, generally required to
yield a good permeation of the dye into the fabrics.3
A limitation of the solubilising power of the saccharidic unit
when conjugated with the dye, is that the molecular weight of
the two portions must remain similar.4 We have observed that
large molecular sized dyes do not reach an acceptable hydrosolu-
bility when glycoconjugated with a single lactose unit, despite
the presence of a variety of linkers such as esters, ethers, amides,
alkylamino derivatives and mixed ethereal-esters, or amido-es-
ters.4 To extend the field of application of this new type of dyeing
agent, we studied a double glycoconjugation with lactose, bonded
through a difunctional linker to the chromophore. This should en-
hance the solubilising power of the saccharide according to the
molecular weight of the dye.
As a first example of this approach we recently prepared malon-
ic acid derivatives.1c,d Herein, we highlight that glutamic acid is a
particularly convenient functionality to link chromophoric moie-
ties to saccharides in the form of water soluble dyes. The procedure
allows the preparation of new materials able to dye and to reduce
the environmental impact of the waste produced by dyeing.5
CO2R1
CO2R1
i)
1a
2a
DO-29
DR-1
1b R1 = Et
1c R1 = H
2b R1 = Et
2c R1 = H
ii)
ii)
iii)
iii)
O
O
CO2R1
CO2R1
HN
HN
DO-29
DR-1
CO2R1
iv)
CO2R1
iv)
1d R1 = Et
1e R1 = H
2d R1 = Et
2e R1 = H
q
Part of this work has been presented in preliminary form at XVI European
Carbohydrate Symposium, Sorrento (I), 3–7 July 2011, abstract PO054.
Scheme 1. Preparation of glutamic acid derivatives (1e) and (2e). Reagents and
conditions: (i) (1b): KOH, 18-crown-6, BrCH2CO2Et, THF, 0 °C, rt, 8 h, 91%; (2b): NaH,
BrCH2CO2Et, THF, 0 °C–rt, 8 h, 72%; (ii) KOH, THF, H2O, rt, 98% (1c) 84% (2c); (iii) (1d)
(1) ClCOEt, TEA, THF; (2) diethyl glutamateꢀHCl, TEA, 90%; (2d): (1) SOCl2, TEA; DCM;
(2) diethyl glutamateꢀHCl, TEA, 90%; (iv) KOH, THF, H2O, rt, 98% (1e) 96% (2e).
⇑
Corresponding authors. Tel.: +39 0554573486; fax: +39 0554573531 (R.B.); tel.:
+39 0502219700; fax: +39 0502219660 (G.C.).
E-mail addresses: roberto.bianchini@unifi.it (R. Bianchini), giocate@farm.unipi.it,
giorgio.catelani@farm.unipi.it (G. Catelani).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.10.036

R. Bianchini et al. / Carbohydrate Research 356 (2012) 104–109
105
The synthetic path to obtain multiple glycoconjugated dyes
(Scheme 1) was developed for two commercially available prod-
ucts: Disperse Orange 29 and Disperse Red 1 (Fig. 1).
O
O
O
CH(OMe)2
O
O CH(OMe)2
HO
O
H2N
O
i)
O
Ethyl 2-bromoacetate was used in the presence of a base of suit-
able strength to set up a useful tag for glutamic acid coupling. Sub-
sequent hydrolysis of the ester groups of 1b and 2b led to
carboxylic acid derivatives 1c and 2c in good yields. The acid
groups were activated and coupled with glutamic acid, as the cor-
responding diethyl ester. In the case of 1c, ethyl chloroformate6
was used to assemble a mixed ethyl-acetyl carbonate, and com-
pound 1d was obtained in good yields. For 2c, better results were
obtained with thionyl chloride,7 and compound 2d was achieved
in excellent yield. Both glutamic diester derivatives 1d and 2d
were hydrolysed to dicarboxylic acids 1e and 2e completing the
elaboration of dyes 1a and 2a (Scheme 1).
The naturalising lactose derivative 4 was prepared through a
straightforward elaboration8 of lactose-triacetonide 3, synthesised
from lactose according to a well established protocol.9 Toluensulfo-
nyl chloride was employed to regioselectively introduce a good
leaving group on the primary hydroxyl group of 3. The subsequent
SN2 displacement with sodium azide and the final hydrogenolysis
afforded 60-amino-lactose-triacetonide 4 in 80% yield over three
steps (Scheme 2).8
The coupling of the elaborated dye and saccharide moieties was
carried out using as the acid activating reagent 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM),
prepared in situ by reaction of 2-chloro-4,6-dimethoxy-1,3,5-tri-
azine with N-methyl morpholine.10 Compounds 1e and 2e were
then converted to the corresponding bis-60-amino-lactose-triaceto-
nide glutamic derivatives 1f and 2f in acceptable to excellent yields
thus confirming the validity of the reaction strategy (Scheme 3).
Final removal of the acetal groups using TFA1 afforded the tar-
get compounds 1g and 2g (Scheme 4).
O
O
O
O
O
O
O
O
OH
OH
4
3
Scheme 2. Synthesis of 6’-aminotriacetonlactose dimethyl acetal (4). Reagents and
conditions: (i) (1) pyridine, MeCN, pTsCl, 0 °C–rt, ovn, 92%; (2) NaN3, DMSO, 90 °C,
20 h, 91%; (3) H2, Pd/C, MeOH, rt, ovn, 96%.
O
O
HO
NH
O
O
O
CH(OMe)2
O
O
O
O
O
1f , Dye = DO-29
2f , Dye = DR-1
1e
2e
i)
HN
Dye
O
O
O
CH(OMe)2
O
NH
O
O
O
O
OH
Scheme 3. Synthesis of bis-lactose glutamic amide derivatives (1f) and (2f).
Reagents and conditions: (i) DMTMM, THF, 0 °C to room temperature, overnight,
91% (1f):60% (2f).
60 (230–400 mesh) according to Still et al.11 1H and 13C NMR spec-
tra were recorded at 200 MHz, on Varian spectrometers in deuter-
ated solvents and are reported in parts per million (ppm) with
solvent resonance used as internal reference. When not specified
spectra are referred to mixtures of enantiomers, or diastereoiso-
mers. Mass spectra were taken from ThermoFisher LCQ-Fleet ion
trap instrument and spectra were registered with ESI +c technique.
Elemental analysis was carried out on a Perkin Elmer 240 C Ele-
mental Analyzer. UV–vis spectra were recorded on a Cary-4000
Varian spectrophotometer. The industrial dye 4-[2-methoxy-4-
(40-nitrophenylazo)phenylazo]-phenol 1a (Colour Index name:
Orange 29) and N-ethyl-N-(2-hydroxyethyl)-4-(4-nitropheny-
lazo)aniline 2a (Colour Index name: Disperse Red 1) are commer-
cially available (Sigma–Aldrich) and used as such.
These new naturalised dyes have similar chromophoric proper-
ties compared with the non-glycoconjugated species 1a and 2a
(Fig. 1). The intensities are comparable and for the kmax absorption
the shifts are 2 or 3 nm. The maximum difference observed is be-
tween the emax of dye 2a and its double glycoconjugated version
2g which was found to be around 1% (Fig. 2).
In conclusion, the strategy here reported, which employs glu-
tamic acid as linker between the chromophore and the sugar fur-
nishes chemically stable final naturalized dyes, thanks to the
ethereal and amidic bonds. Currently, progress to extend this
methodology to other saccharide derivatives is under
investigation.
1. Experimental
1.2. 60-Deoxy-60amino-2,3:5,6:30,40-tri-O-(isopropylidene)lactose
1.1. General methods
dimethyl acetal (4)
Thin layer chromatography (TLC) was performed using com-
mercially prepared 60-mesh silica gel aluminium foils (Merck;
60 Å F254) and spots were visualized with: (a) UV light (254 and
366 nm), (b) an acidic solution of p-anisaldehyde in ethanol. Flash
column chromatography (FCC) was carried out on Merck Silica Gel
A solution of protected lactose 39 (2.00 g, 3.92 mmol) in pyri-
dine/acetonitrile 2:1 (15 mL) was added dropwise to a solution of
para-4-methylbenzene-1-sulfonyl
chloride
(TsCl)
(0.90 g,
4.70 mmol) in pyridine/acetonitrile 2:1 (3 mL).8 The reaction mix-
ture was stirred at room temperature overnight, then water
OMe
N
OH
N
N
N
N
O2N
N
O2N
N
OH
Disperse Orange 29
1a
Disperse Red 1
2a
Figure 1. Disperse dyes employed for naturalisation.

106
R. Bianchini et al. / Carbohydrate Research 356 (2012) 104–109
under reduced pressure. The residue was purified by precipitation
OH
O
HO
from ether at 0 °C to afford 4 (1.36 g, 96%) as a light yellow powder
having NMR data and physico-chemical properties identical with
those of an authentic sample.4
O
HO
O
OH
HO
O
HO
1.3. Ethyl 2-{4-[2-methoxy-4-(40-nitrophenylazo)phenylazo]
phenoxy}acetate (1b)
OH
O
1g, Dye = DO-29
2g , Dye = DR-1
1f
2f
NH
O
i)
HN
Dye
Disperse dye 1a (1.5 g, 3.97 mmol) was added to a solution of
KOH (1.12 g, 19.89 mmol) in THF (15 mL) and the resulting mixture
was stirred at room temperature for 1.5 h. Ethyl 2-bromoacetate
(1.32 mL, 11.91 mmol) and catalytic quantity of 18-crown-6 were
added and the resulting solution was stirred at room temperature
for 20 h. The reaction mixture was filtered, the filtrate was diluted
with water (40 mL) and extracted with CH3Cl (4 ꢁ 30 mL). The or-
ganic phase was dried over Na2SO4, filtered, and the filtrate was con-
centrated under reduced pressure. Purification by FCC eluting with
4:9 EtOAc–petroleum ether afforded 1b (1.67 g, 91%) as an orange
solid, Rf 0.75 (4:9 EtOAc–petroleum ether), mp 158–160 °C (chrom).
1H NMR (200 MHz, CDCl3): d 8.44 (m, 2H, ArH); 8.16–7.91 (m, 4H,
ArH), 7.88–7.76 (m, 3H, ArH), 7.01 (m, 2H. ArH), 4.95 (s, 2H, OCH2-
CO), 4.11 (q, 2H, J = 7.1 Hz, CH2CH3), 3.77 (s, 3H, PhOCH3), 1.33 (t,
3H, J = 7.1 Hz, CH2CH3). 13C NMR (50 MHz, CDCl3): d 169.74 (CO),
159.75, 157.02, 155.67, 153.97, 148.80, 147.57, 144.71, 125.37,
124.76, 123.55, 118.83, 117.68, 115.81, 114.78 (ArC), 66.12 (OCH2-
CO), 61.22 (CH2CH3), 53.88 (PhOCH3), 14.16 (CH2CH3). MS (ESI) m/
z = 464.28 [M+1]+. Anal. Calcd for C23H21N5O6 (463.15): C, 59.61;
H, 4.57; N, 15.11. Found: C, 59.55; H, 4.48; N, 15.21.
OH
OH
OH
HO
O
O
HN
HO
O
HO
OH
Scheme 4. Synthesis of naturalized dyes (1g) and (2g). Reagents and conditions: (i)
TFA, H2O, rt, ovn, 99% (1g):78% (2g).
(20 mL) and ether (20 mL) were added to the crude solution and
the aqueous phase was separated and extracted with Et2O
(2 ꢁ 20 mL). The organic phases were combined and dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue (obtained as a colourless oil) was purified by FCC (3:2
EtOAc–petroleum ether, Rf 0.66) to afford the 60-tosyl derivative
(2.4 g, 92%) as a colourless oil.
Sodium azide (0.29 g, 4.65 mmol) was added to a solution of
3 (2.1 g, 3.1 mmol) in dimethyl sulfoxide (20 mL) and the result-
ing mixture was heated at 90 °C for 20 h. The mixture was
cooled, filtered, and the filtrate was diluted with water (50 mL)
and extracted with CH3Cl (4 ꢁ 30 mL). The organic phases were
combined, dried over Na2SO4, and concentrated in vacuo. The
residue (obtained as a colourless oil) was purified by FCC eluting
with 2:1 EtOAc–petroleum ether to afford the 60-azido derivative
(1.5 g, 91%) as a colourless oil, Rf 0.54, 2:1 EtOAc–petroleum
ether).
1.4. 2-{4-[20-Methoxy-4-(p-nitrophenylazo)phenylazo]
phenoxy}acetic acid (1c)
KOH (0.54 g, 9.69 mmol) was added to a solution of 1b (1.5 g,
3.23 mmol) in 1:1 THF–H2O (16 mL) and the resulting mixture was
stirred at room temperature for 2 h. The TLC indicated the disap-
pearance of the starting product 1b (Rf 0.86, EtOAc) and the forma-
tion of one major spot (Rf 0.1). The solution was diluted with
water (25 mL) and extracted with CH3Cl (3 ꢁ 20 mL). The aqueous
solution was acidified with 1 N HCl to pH 2 and extracted with EtOAc
(30 mL). The organic solution was dried over Na2SO4 and filtered, the
A solution of the prepared azido derivative (1.5 g, 2.8 mmol) in
CH3OH (25 mL) was hydrogenated in presence of 10% palladium on
carbon (200 mg) for 20 h at room temperature. The solution was
filtered through a pad of CeliteÒ and the filtrate was concentrated
Figure 2. UV–vis spectra of (2a) (cyano line), compared with (2f) (red line) and (2g) (brown line). Molar absorptivities (e
) are expressed in the Mꢂ1 cmꢂ1 dimension, as usual.

R. Bianchini et al. / Carbohydrate Research 356 (2012) 104–109
107
solvent was removed under reduced pressure to afford 1c (1.37 g,
98%) as a red solid, mp 238–240 °C. 1H NMR (200 MHz, CDCl3): d
8.41 (m, 2H, ArH); 8.15–7.91 (m, 4H, ArH), 7.87–7.76 (m, 3H, ArH),
7.02 (m, 2H. ArH), 4.84 (s, 2H, OCH2CO), 3.76 (s, 3H, PhOCH3). 13C
NMR (50 MHz, CDCl3): d 162.74 (CO), 159.77, 157.08, 155.67,
153.97, 148.80, 147.57, 144.71, 125.37, 124.74, 123.56, 118.83,
117.67, 115.82, 114.78 (ArCH), 66.98 (OCH2CO), 53.88 (PhOCH3).
MS (ESI) m/z = 436.11 [M+1]+. Anal. Calcd for C21H17N5O6 (435.12):
C, 57.93; H, 3.94; N, 16.09. Found: C, 57.88; H, 3.87; N, 16.32.
Anal. Calcd for C26H24N6O9 (564.16): C, 55.32; H, 4.29; N, 14.89.
Found: C, 55.22; H, 4.18; N, 15.12.
1.7. 2-{4-[20-Methoxy-4-(p-nitrophenylazo)phenylazo]
phenoxy}-acetyl-10-glutamic-bis-{4-O-[6-O-amino-6-deoxy-3,4-
O-(isopropylidene)-b-
isopropylidene-aldehydo-
D
-galactopyranosyl]-2,3:5,6-di-O-
-glucose dimethyl acetal}amide (1f)
D
N-Methyl morpholine (NMO, 0.88 mL,7.98 mmol) was added to
a solution of 1e (1.5 g, 2.66 mmol) in THF (15 mL) and the mixture
was stirred at room temperature for 5 min. The resulting solution
was cooled to 0 °C, 2-chloro-4,6-dimethoxy-1,3,5-triazine
(1.34 g,7.98 mmol) was added and the mixture was stirred at room
temperature. After 2 h, TLC indicated the formation of the activated
intermediate (Rf = 0.77, CH2Cl2–CH3OH 10:0.5) and the disappear-
ance of the starting acid (Rf = 0.1). Amino lactose 4 (2.70 g,
5.32 mmol) was added and the reaction mixture was stirred at
room temperature for 24 h when TLC showed the formation of
one major spot at (Rf = 0.39, CH2Cl2–CH3OH 10:0.5). The reaction
mixture was evaporated to dryness under reduced pressure and
the residue was dissolved in CH3Cl (20 mL) and washed with 5%
HCl (20 mL) and water (3 ꢁ 20 mL). The organic solution was dried
over Na2SO4 and filtered, the filtrate was concentrated under re-
duced pressure and the residue was purified by FCC eluting with
CH2Cl2–CH3OH 10:0.4 (Rf = 0.35) to afford 1f (3.73 g, 91%) as a
red foam, mp 143–146 °C (chrom). 1H NMR (200 MHz, CDCl3): d
8.43–8.38 (m, 2H, ArH); 8.12–7.89 (m, 4H, ArH), 7.79–7.63 (m,
3H, ArH), 7.03–6.97 (m, 2H. ArH), 4.87–4.51 (m, 7H), 4.38–4.35
(m, 2H), 4.28–4.24 (m, 3H) 4.15–3.94 (m, 12H), 3.74–3.49 (m,
14H), 3.44–3.41 (s, 12H, 4 ꢁ OCH3), 2.30–2.11 (m, 4H, CHCH2,
CH2CO), 1.60–1.20 (various overlapping signals, 36H). 13C NMR
(50 MHz, CDCl3): d 174.87, 173.64 (2 ꢁ CO), 162.33 (CO), 159.77,
157.08, 155.67, 153.97, 148.80, 147.57, 144.71, 125.37, 124.74,
123.56, 118.83, 117.67, 115.82, 114.78 (ArCH), 110.44, 109.57,
108.12 [C(CH3)2], 67.48 (OCH2CO), 53.54 (PhOCH3), 51.77 (CH),
29.11, 28.81, 27.66, 26.84,2 6.44, 25.68, 25.25, 24.47. Glycidic sig-
nals were also at 106.1, 103.5, 79.0, 78.0, 76.3, 76.6, 74.2, 72.6, 71.3,
70.7, 64.7, 40.3. UV–vis kmax = 417 nm. MS (ESI) m/z = 1543.11
[M]+. Anal. Calcd for C72H102N8O29 (1543.62): C, 56.02; H, 6.66; N
7.26. Found: C, 56.47; H, 6.69; N, 7.50.
1.5. 2-{4-[20-Methoxy-4-(p-nitrophenylazo)phenylazo]
phenoxy}-acetyl-(diethyl ester)-10-glutamic amide (1d)
Triethylamine (1.64 mL, 11.73 mmol) was added to a solution of
1c (1.70 g, 3.90 mmol) in THF (15 mL) and the mixture stirred at
room temperature for 5 min. To the resulting solution, cooled to
0 °C, ethyl chloroformate (1.13 mL, 11.73 mmol) was added, and
the mixture was stirred at 0 °C for 30 min until TLC indicated the
formation of the activated intermediate (Rf 0.72, EtOAc–petroleum
ether) and the disappearance of the starting acid (Rf 0.1). Diethyl L-
glutamate hydrochloride (0.94 g, 3.91 mmol) was then added, fol-
lowed immediately by triethylamine (0.54 mL, 3.91 mmol). The
reaction mixture was stirred at room temperature overnight, then
was again cooled to 0 °C and subjected to another cycle of activa-
tion and coupling using half the quantities listed above. The reac-
tion mixture was slowly allowed to warm to room temperature
and stirred for additional 24 h. TLC showed the formation of one
major spot (Rf = 0.44, EtOAc–petroleum ether 3:1). The solvent
was evaporated under reduced pressure and the residue was puri-
fied by FCC on silica gel, eluting with EtOAc–petroleum ether 3:1
(Rf = 0.44) to afford 1d (2.18 g, 90%) as a red amorphous foam,
mp 185–187 °C (chrom). 1H NMR (200 MHz, CDCl3): d 8.41 (m,
2H, ArH); 8.15–7.91 (m, 4H, ArH), 7.87–7.76 (m, 3H, ArH), 7.02
(m, 2H. ArH), 4.84–4.54 (m, 3H, OCH2CO, CH), 4.14 (q, 2H,
J = 7.0 Hz, CH2CH3), 3.76 (s, 3H, PhOCH3), 2.29 (m, 4H,
CHCH2CH2CO), 1.32 (t, 6H, J = 7.0 Hz, 2 ꢁ CH2CH3). 13C NMR
(50 MHz, CDCl3):
d
171.54, 171.11, 162.74 (3 ꢁ CO), 159.77,
157.08, 155.67, 153.97, 148.80, 147.57, 144.71, 125.37, 124.74,
123.56, 118.83, 117.67, 115.82, 114.78 (ArCH), 67.48 (OCH2CO),
61.24 (2 ꢁ CH2CH3), 53.57 (PHOCH3), 51.24 (CH), 28.54, 27.11
(CHCH2CH2CO), 14.21 (2 ꢁ CH3). Ms (ESI) m/z = 621.39 [M+1]+.
Anal. Calcd for C30H32N6O9 (620.22): C, 58.06; H, 5.20; N, 13.54.
Found: C, 57.98; H, 5.14; N, 13.60.
1.8. 2-{4-[20-Methoxy-4-(p-nitrophenylazo)phenylazo]
phenoxy}-acetyl-10-glutamic-bis-(60-amino-lactose)amide (1g)
1.6. 2-{4-[20-Methoxy-4-(p-nitrophenylazo)phenylazo]
phenoxy}-acetyl-10-glutamic acid (1e)
A solution of 1f (2.0 g, 1.29 mmol) in 90% aqueous CF3COOH
(15 mL) was stirred at room temperature overnight. The TLC indi-
cated the disappearance of starting material (Rf = 0.33, CH2Cl2–
CH3OH 10:0.5) and the formation of compound 1g (Rf = 0.1). The
violet solution was evaporated to dryness and repeatedly coevap-
orated with toluene (5 ꢁ 25 mL) at reduced pressure to give the fi-
nal product 1g (1.59 g, 99%) as red powder, mp 201–204 °C. 1H
NMR (200 MHz, D2O): d 8.35–8.29 (m, 2H, ArH, both anomers);
8.18–7.89 (m, 4H, ArH, both anomers), 7.81–7.64 (m, 3H, ArH, both
anomers), 7.74–7.68 (m, 2H, ArH, both anomers), 5.08–4.57 (m, 7H
both anomers) 4.24–4.17 (m, 8H, both anomers) 3.91–3.81 (m,
12H, both anomers), 3.74 (s, 3H, PhOCH3 both anomers), 3.43–
3.40 (m, 4H, both anomers) 2.29–2.07 (m, 4H, CHCH2, CH2CO).
13C NMR (50 MHz, D2O): d 176.97ꢂ173.54 (2 ꢁ CO), 162.55 (CO),
159.77, 157.08, 155.67, 153.97, 148.80, 147.57, 144.71, 125.37,
124.74, 123.56, 118.83, 117.67, 115.82, 114.78 (ArCH), 67.48
(OCH2CO), 53.54 (PhOCH3), 51.77 (CH), 29.11–26.87 (CHCH2,
CH2CO) other signals for glycidic moiety : 103.7, 79.6, 78.4, 74.1,
71.9, 61.5, 106.6,74.4, 77.8, 73.5, 77.6, 64.5). MS (ESI) m/
z = 1210.31 [M]+ Anal. Calcd for C50H66N8O27 (1210.40): C, 49.59;
H, 5.49; N, 9.25. Found: C, 49.44; H, 5.41; N, 9.39.
KOH (0.54, 9.67 mmol) was added to a solution of the diester 1d
(2 g, 3.22 mmol) in H2O–THF 1:1 (20 mL) and the mixture was stir-
red at room temperature for 20 h. TLC showed the disappearance of
the starting material (EtOAc, Rf = 0.83) and formation of one major
spot at the baseline. The reaction mixture was evaporated to dry-
ness under reduced pressure and the residue dissolved in water
(20 mL). The resulting solution was cooled in an ice bath and the
pH adjusted to 2 by dropwise addition of 1 N HCl. The resulting
suspension was extracted with CH3Cl (20 mL) and the organic
phase was dried over Na2SO4, filtered, and evaporated under re-
duced pressure to afford 1e (1.78 g, 98%) as a black powder, mp
271–275 °C (dec). 1e is soluble in water at pH >7. 1H NMR
(200 MHz, CDCl3): d 8.40 (m, 2H, ArH); 8.15–7.91 (m, 4H, ArH),
7.88–7.78 (m, 3H, ArH), 7.01 (m, 2H. ArH), 4.87–4.51 (m, 3H, OCH2-
CO, CH), 3.74 (s, 3H, PhOCH3), 2.29–2.07 (m, 4H, CHCH2, CH2CO).
13C NMR (50 MHz, CDCl3): d 176.97, 174.54 (2 ꢁ CO), 162.55
(CO), 159.77, 157.08, 155.67, 153.97, 148.80, 147.57, 144.71,
125.37, 124.74, 123.56, 118.83, 117.67, 115.82, 114.78 (ArCH),
67.48 (OCH2CO), 53.54 (PhOCH3). MS (ESI) m/z = 565.21 [M+1]+.

108
R. Bianchini et al. / Carbohydrate Research 356 (2012) 104–109
1.9. N-Ethyl-N-(2-(acetylacetoxy)ethyl)-4-(40-nitrophenylazo)
aniline (2b)
120H), 4.44–4.38 (m, 1H, CH), 4.29–4.12 (m, 6H, 2 ꢁ CH2CH3, OCH2-
CO), 3.69–3.24 (m, 6H, 2 ꢁ NCH2CH2, NCH2CH3), 2.32–2.25 (m, 4H,
CHCH2, CH2CO), 1.24–1.14 (m, 9H, 2 ꢁ CH2CH3, NCH2CH3). 13C NMR
To a solution of disperse dye 2a (1.2 g, 3.8 mmol) in THF
(24 mL), NaH (60% disperse in mineral oil, 352 mg, 8.8 mmol)
was added at room temperature. After 8 h, ethyl 2-bromoacetate
(1.1 ml, 9.9 mmol) was added dropwise and the reaction mixture
was stirred overnight and then carefully quenched with 1:1 satu-
rated NH4Cl–H2O (50 mL) at 0 °C. The whole was extracted with
EtOAc (50 mL), the organic phase was separated, washed with
brine (50 mL), and dried over Na2SO4. The suspension was filtered
under reduced pressure and the filtrate was evaporated to dryness.
The residue was purified by FCC, eluting with 98:2 CH2Cl2–EtOAc
to afford 2b as a red amorphous solid (1.1 g, 72%), Rf 0.55 (98:2
CH2Cl2–EtOAc), mp 86–88 °C (chrom). 1H NMR (200 MHz, CDCl3):
d 8.32–8.30 (m, 2H, 30-H, 50-H), 7.92–7.87 (m, 4H, 20-H, 60-H, 3-H,
5-H), 6.79–6.76 (m, 2H, 2-H, 6-H), 4.22 (q, 2H, J = 7.2 Hz, CH2CH3),
4.11 (s, 2H, OCH2CO), 3.78 (t, 2H, J = 5.8 Hz, OCH2CH2N), 3.69 (t, 2H,
J = 5.8 Hz, OCH2CH2N) 3.57 (q, 2H, J = 7.2 Hz, NCH2CH3), 1.30–1.24
(m 6H, NCH2CH3, CH2CH3). 13C NMR (50 MHz, CDCl3): d 170.1
(CO), 156.8, 151.4, 147.3, 143.6, 126.2, 124.6, 122.5, 111.3 (ArC)
69.0 (OCH2CO), 68.7 (OCH2CH3N), 61.0 (NCH2CH3), 50.1, 46.0,
14.2 (CH2CH3), 12.2 (NCH2CH3). MS (ESI) m/z = 401.11 [M+1]+. Anal.
Calcd for C20H24N4O5 (400.20): C, 59.99; H, 6.04; N, 13.99. Found:
C, 59.87; H, 5.97; N, 14.08.
(50 MHz, CDCl3):
d
171.19 (2 ꢁ CONH), 168.87 (CO), 156.60,
151.10, 147.3, 143.7, 126.2, 124.6, 122.6, 111.3 (ArC) 66.47, (OCH2-
CO), 61.57 (CH2CH3), 61.47 (NCH2CH2), 52.14 (CH), 48.77, 45.67
(NCH2CH2, NCH2CH3), 28.47, 26.25 (CHCH2, CH2CO), 13.97
(CH2CH3), 12.2 (NCH2CH3). MS (ESI) m/z = 558.08 [M+1]+. Anal.
Calcd for C27H35N5O8 (557.21): C, 58.16; H, 6.33; N, 12.56. Found:
C, 58.04; H, 6.18; N, 12.69.
1.12. N-Ethyl-N-(2-(20-carboxyglutamic acid-10-amido)
ethyloxy)-4-(4-nitrophenyl-azo)aniline (2e)
KOH (0.56 g, 10 mmol) was added to a solution of the diester 2d
(1.4 g, 2.5 mmol) in H2O–THF 1:1 (30 mL) and the mixture was stir-
red at room temperature overnight. TLC showed the disappearance
of the starting material (Rf = 0.35, 2:1 EtOAc–petroleum ether) and
the formation of a major spot at the baseline. The reaction mixture
was evaporated to dryness under reduced pressure and the residue
dissolved in water (30 mL). The resulting solution was cooled in an
ice bath and the pH adjusted to 2 with dropwise addition of 1 N HCl.
The resulting suspension was filtered and the cake was washed with
H2O until pH neutral. Compound 2e was obtained, after drying in an
oven at 60 °C overnight, as a red amorphous solid (1.2 g, 96%), mp
186–190 (dec). 1H NMR (200 MHz, acetone-d6): d 8.32–7.92 (m,
4H, 20-H, 30H, 50H, 60H), 7.90–6.79 (m, 4H, 80H, 90H, 110H, 120H),
4.37 (m, 1H, CH), 4.29–4.13 (s, 2H, OCH2CO), 3.68–3.21 (m, 6H,
NCH2CH2, NCH2CH3, NCH2CH2), 2.31–2.26 (m, 4H, CHCH2, CH2CO),
1.24–1.14 (t, 3H, J = 7.1 Hz, CH2CH3). 13C NMR (50 MHz, acetone-
d6): d 172.15 (2CONH), 168.87 (CO), 156.65, 151.17, 147.35,
143.74, 126.24, 124.65, 122.61, 111.31 (ArC) 66.51, (OCH2CO),
61.44 (NCH2CH2), 52.55 (CH) 48.71, 45.62 (NCH2CH2, NCH2CH3),
28.46, 26.33, (CHCH2, CH2CO) 12.28 (NCH2CH3). MS (ESI) m/
z = 502.32 [M+1]+. Anal. Calcd for C23H27N5O8 (501.24): C, 55.09;
H, 4.53; N, 13.97. Found: C, 54.84; H, 5.27; N, 14.19.
1.10. N-Ethyl-N-(2-(20-hydrocarboxy)ethyloxy)-4-(4-nitrophe-
nylazo)aniline (2c)
KOH (0.55 g, 9.8 mmol) was added to a solution of 2b (1.96 g,
4.9 mmol) in 1:1 THF–H2O (16 mL) and the resulting mixture
was stirred at room temperature for 2 h. The TLC indicated the dis-
appearance of the starting product (EtOAc–petroleum ether 3:7,
Rf = 0.66) and the formation of a major spot (Rf = 0.1). The solution
was diluted with water (25 mL) and extracted with CH3Cl
(3 ꢁ 20 mL). The aqueous solution was acidified with 1 N HCl to
pH 2 and extracted with CH3Cl (30 mL). The organic phase was
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure to afford 2c (1.54 g, 84%) as a red solid, mp 159–162 °C. 2c is
soluble in water at pH >7. 1H NMR (200 MHz, acetone-d6): d 8.32–
7.92 (m, 4H, 20-H, 30H, 50H, 60H), 7.90–6.79 (m, 4H, 80H, 90H, 110H,
120H), 4.31 (s, 2H, OCH2CO), 3.71–3.26 (m, 6H, 3 ꢁ NCH2–), 1.14
(t, 3H, J = 7.1 Hz, CH2CH3). 13C NMR (50 MHz, acetone-d6): d
168.87 (CO), 156.60, 151.10, 147.3, 143.7, 126.2, 124.6, 122.6,
111.3 (ArC) 66.47, (OCH2CO), 61.5 (NCH2CH3), 48.7, 45.7 (NCH2CH2,
NCH2CH3), 12.2 (NCH2CH3). MS (ESI) m/z = 373.35 [M+1]+. Anal.
Calcd for C18H20N4O5 (372.21): C, 58.06; H, 5.41; N, 15.05. Found:
C, 58.01; H, 5.34; N, 15.12.
1.13. N-Ethyl-N-(2-(20-carboxyglutamic-bis-{4-O-[6-O-amino-6-
deoxy-3,4-O-(isopropylidene)-b-
D-galactopyranosyl]-2,3:5,6-di-
O-isopropylidene-aldehydo-
D
-glucose dimethyl acetal}-10-
amido)ethyloxy)-4-(4-nitrophenyl-azo)aniline (2f)
N-Methyl morpholine (0.26 mL, 2.4 mmol) was added to a solu-
tion of 2e (0.5 g, 1.0 mmol) in THF (5 mL)and the mixturewas stirred
at room temperature for 5 min. The resulting solution was cooled to
0 °C, 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.42 g, 2.4 mmol) was
added and the mixture was stirred at room temperature. After 2 h
the TLC indicated the formation of the activated intermediate
(Rf = 0.79, 10:0.4 CH2Cl2–CH3OH) and the disappearance of the start-
ing acid (Rf = 0.1). Amino lactose 4 (1.0 g, 2 mmol) was added and the
reaction mixture was slowly allowed to warm to room temperature
and stirred overnight. TLC showed the formation of one major spot
(Rf = 0.35, 10:0.4 CH2Cl2–CH3OH). The reaction mixture was evapo-
rated to dryness under reduced pressure. The residue was dissolved
in CH2Cl2 (20 mL) and washed with a solution of 5% aq HCl (20 mL)
and H2O (3 ꢁ 20 mL). The organic solution was dried over Na2SO4
and filtered. The filtrate was concentrated under reduced pressure
and the residue was purified by FCC, eluting with 95:5 CH2Cl2–
CH3OH to afford 2f (0.86 g, 60%) as red foam having Rf 0.4 (95:5
CH2Cl2–MeOH), mp 96–98 °C (chrom). d 8.34–8.30 (m, 2H, 30-H, 50-
H); 7.93–7.88 (m, 4H, 20-H, 60-H, 3-H, 5-H), 7.67 (d, 1H, J = 7.2 Hz,
CONHCH), 7.22–7.19 (m, 1H, CHCONHCH2), 6.81–6.78 (m, 2H, 2-H,
6-H), 6.69–6.66 (m, 1H, CH2CH2CONHCH2) 4.57–4,48 (m, 3H),
4.37–4.34 (m, 3H), 4.31–4.26 (m, 3H), 4.19–4.16 (m, 2H), 4.11–
4.09 (m, 1H), 4.08–3.97 (m, 8H), 3.95–3.87 (m, 4H), 3.83–3.69 (m,
7H), 3.63–3.37 (m, 17H), 3.25–3.18 (m, 1H), 3.14 (s, 1H), 3.07 (s,
1.11. N-Ethyl-N-(2-(20-carboxyglutamic(diethyl ester)-10-amido)
ethyloxy)-4-(4-nitrophenylazo)aniline (2d)
Thionyl chloride (0.24 mL, 3.3 mmol) was added to a solution of
2c (1.12 g, 3.0 mmol) in CH2Cl2 (12 mL) at 0 °C. The whole was stir-
red at 0 °C for 1.5 h and then at room temperature for 3 h. The dark
red solution was thereafter added dropwise to an ice precooled
solution of
L-glutamic acid diethyl ester hydrochloride (0.7 g,
3.0 mmol) and TEA (2.1 mL, 15.1 mmol) in CH2Cl2 (12 mL). Stirring
was continued for 2 h at 0 °C and then at room temperature over-
night. The mixture was diluted with CH2Cl2 (24 mL) and washed
with H2O (2 ꢁ 50 mL). The organic phase was separated, dried over
Na2SO4, and the resulting suspension filtered under reduced pres-
sure. The filtrate was collected and evaporated to dryness to afford
2d as a crude red solid (1.5 g, 90%) which was used without further
purification for the next step. 1H NMR (200 MHz, CDCl3): d 8.32–
7.92 (m, 4H, 20-H, 30H, 50H, 60H), 7.90–6.79 (m, 4H, 80H, 90H, 110H,

R. Bianchini et al. / Carbohydrate Research 356 (2012) 104–109
109
1H), 2.43–2.12 (m, 3H), 2.03–1.91 (m, 1H), 1.48–1.38 (m, 12H), 1.32
(s, 12H), 1.29–1.25 (m, 15H). 13C NMR (50 MHz, CDCl3): d 172.3 (CO),
171.4 (CO), 169.0 (CO), 156.7, 151.2, 147.4, 143.7, 126.2, 124.6,
122.6, 111.4, (ArCH), 110.4 (2C), 110.2 (2C), 108.4 (2C), 108.2,
107.2, 103.4, 103.3, 78.9 (2C), 77.8, 77.5 (2C), 77.0, 76.8, 76.7 (2C),
76.6 (2C), 74.0, 73.9 (2C), 73.8, 72.4, 71.8, 70.7, 69.2 (2C), 64.5,
57.9, 57.4, 56.2, 56.0, 51.9, 49.8, 46.0, 40.4, 31.6, 29.0, 28.1 (2C),
27.2, 27.1, 26.4, 26.3, 26.2 (2C), 25.6 (2C), 24.0 (2C), 12.2. MS (ESI)
m/z = 1480.41 [M]+. Anal. Calcd for C69H105N7O28 (1480.60): C,
55.97; H, 7.15; N, 8.54. Found: C, 55.22; H, 6.94; N, 8.61.
Anal. Calcd for C47H69N7O26 (1148.08): C, 49.17; H, 6.06; N, 8.54.
Found: C, 49.04; H, 5.87; N, 8.61.
Acknowledgements
We fully acknowledge Ente Cassa di Risparmio di Firenze for the
grants that allowed us to buy the Varian-4000 spectrophotometer
and the ThermoFisher LCQ-Fleet Esi-Mass apparatus.
References
1.14. N-Ethyl-N-(2-(20-carboxyglutamic bis[60amidoyl-lactose]-
10-amido)ethyloxy)-4-(4-nitrophenyl-azo)aniline (2g)
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A solution of 2f (0.7 g, 0.47 mmol) in 90% aqueous TFA (5 mL)
was stirred at room temperature overnight. The TLC showed the
disappearance of the starting material (Rf 0.4, 95:5 CH2Cl2–MeOH)
and the formation of a major spot with Rf 0.1. The violet solution
was repeatedly co-evaporated with toluene (5 ꢁ 25 mL) at reduced
pressure. The residue was treated with acetone (10 mL) at room
temperature overnight and the fine suspension was centrifuged
(1500 rpm) for 15 min. The supernatant liquid was removed and
the solid was dried in vacuo at room temperature to obtain 2g as
a red powder (0.42 g, 78%), mp 145–148 °C. 1H NMR (200 MHz,
DMSO-d6): d 8.32–7.92 (m, 4H, 20-H, 30-H, 50-H, 60-H); 7.90–6.79
(m, 4H, 80-H, 90-H, 110-H, 120-H), 5.08–4.57 (m, 4H), 4.33–4.13
(m, 3H, OCH2CO), 3.68–3.21 (m, 6H, NCH2CH2, NCH2CH3,
NCH2CH2), 2.31–2.26 (m, 4H, CHCH2, CH2CO), 1.24–1.14 (t, 3H,
J = 7.0 Hz, NCH2CH3). 13C NMR (50 MHz, DMSO-d6): d 172.33
(2 ꢁ CONH), 168.66 (CO), 156.48, 151.17, 147.35, 143.74, 126.24,
124.54, 122.57, 111.28, (ArCH), 66.51 (OCH2CO), 61.44 (NCH2CH2),
52.55 (CH), 48.71, 45.62 (NCH2CH2, NCH2CH3), 28.46, 26.33,
(CHCH2, CH2CO) 12.28 (NCH2CH3). MS (ESI) m/z = 1148.23 [M+1]+.
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