Project LIFE12 ENV/IT/352
"Naturalised dyes replacing commercial colorants for environmentally
friendly leather dyeing and water recycle" (BIONAD)
Naturalized Dyes Synthesis
Bionad Life 12
Serichim, Torviscosa (UD), 29.01.2015
Serichim, 29/01/2015
1
SERICHIM ACTIVITIES UP TO 12° MONTH
 RECIPES SCALING UP
 ANALYTICAL METHODS AND DYES RAW MATERIALS
CHARACTERIZATION
 PROCESS FLOW SHEET
 FULL SCALE PREPARATION OF SUGAR BLOCK
Serichim, 29/01/2015
2
RECIPES SCALING UP AND FULL SCALE
PREPARATION
 LAB SCALE PREPARATION OF ORANGE DYE
 LAB SCALE PREPARATION OF YELLOW DYE
 LAB SCALE PREPARATION OF RED DYE
FULL SCALE PREPARATION OF SUGAR BLOCK
Serichim, 29/01/2015
3
RECIPE SCALING UP
• REPRODUCTION OF THE PROCEDURE IN LAB SCALE
Preparation of sugar block
Preparation of chromophore block
Coupling building blocks and final naturalized dyes isolation
• FOCUSE ON PROCEDURE CRITICAL POINTS FOR SCALE UP:





reaction mixture rheological behavior,
complexity of work-up and isolation steps,
Starting material specify consumption
Batch’s number
Solvent recovering and controlled waste
Serichim, 29/01/2015
4
Preparation of sugar block
H3C
OH
H3C
O
OH
1° STEP
HO
CH3
O
HO
O
CH3
O
O
OH
O
HO
OH
MeO
H3C
H2O
O
O
O
OH
HO
O
O
H3C
OMe
OH
INT 1
2° STEP
H3C
H3C
O
H3C
HN
H3C
CH3
O
N
O
CH3
O
H3C
H3C
MeO
O
OH
INT 3
Serichim, 29/01/2015
O
OMe
CH3
O
TsO
O
CH3
O
O
O
O
3° STEP
H3C
H3C
O
O
O
MeO
O
OH
OMe
INT 2
5
Preparation of sugar block
Lactose protection, synthesis of INT1
MeO
OH
O
HO
OMe
3
1
O
OH
O
HO
OH
HO
+ 4 H3C
OMe
p-TsOH
H2O
OH
3
2 H3C
OH
OH
H3C
O
2
H3C
H3C
2 CH3COCH3
OH
O
+
DCM
+
HCl
O
CH3
O
H2O
O
O
3
OMe
CH3
O
HO
CH3
O
O
+4 CH3OH
H3C
CH3
O
MeO
O
+ 2 H2O
H3C
O
O
OMe
OH
O
1
OH
DMP
O
INT 1A
3
MeO
O
+ 5 H3C
+ 2 H2O
O
O
CH3
Open
Structure
H3C
H3C
OMe
OH
O
CH3
CH3
CHO
O
MeO
O
OMe
OH
HO
CH3
O
1
H3C
HO
2
O
H3C
DMP
INT 1A
O
OH
HO
O
reflux
CH3
H2O
O
H3C
H3C
OH
2
H3C
CH3
H3C
H3C
O
+ 1 CH3COCH3
+ 1 CH3OH
MeO
O
OH
OMe
INT 1B
Serichim, 29/01/2015
6
Preparation of sugar block
Lactose protection, synthesis of INT1
Lab-scale synthesis
Esp
N°
Lactose, g
Mol DMP/
Mol
Lactose
Mol
HCl/Mol
Lactose
Mol NaHCO3
/Mol Lactose
INT1, g
Yield %
S1
40,0
17,6
3,3
1,5
35,8
63%
S7
40,0
10,0
3,3
1,5
31,1
55%
15,0
1,4
0,76
Production INT1
Lactose,
Kg
Lab-scale
S1
01/2015
Serichim, 29/01/2015
4,0
Kg DMP/
Kg INT1,
Kg 4% HCl/
Kg INT1
Kg 10%
NaHCO3
/Kg INT1
5,7
8,6
4,2
4,9
3,9
2,2
INT1,
Kg
Yield %
3,48
64%
7
Preparation of sugar block
Production of INT1
Serichim, 29/01/2015
8
Preparation of sugar block
H3C
Production of INT1
O
H3C
CH3
O
HO
O
CH3
O
O
O
H3C
H3C
O
 Protected lactose, light-yellow solid, 3,48 Kg, Yield 64%
 Analysis TLC, NMR
MeO
O
OH
OMe
Protected Lactose, INT 1B
Serichim, 29/01/2015
9
Preparation of sugar block
Production of INT1
NMR 1H, INT1, batch 01/2015
Serichim, 29/01/2015
10
Preparation of sugar block
Tosylation of primary alcohol, synthesis of INT2
H3C
H3C
O
H3C
O
O
H3C
H3C
DCM, 0-20°C, ovn
MeO
O
O
O
H3C
OMe
OH
O
CH3
O
pTsCl/TEA, DMAP
O
CH3
O
TsO
CH3
O
O
H3C
CH3
O
2
HO
O
H3C
INT 1
O
MeO
O
OH
OMe
+ TEA HCl
+ pTsCl
+ TEA
INT 2
Esp
N°
T,
°C
t,
h
Reaction
time,
h
Kg 10% Citric
acid/
Kg INT2
Kg 10%
NaHCO3
/Kg INT2
Yield%
S2
4-23°C
3h
23h
2,3
6,1
100%
S32
19-26°C
1h
4h
1,5
1,5
100%
(0,5 mol eq)




Serichim, 29/01/2015
Esp 20-30 g INT1
Mol TsCl/Mol INT1= 1,4, add in 2 portions of 0,7 mol eq
Mol TEA/Mol INT1= 1,5
Mol DMAP/Mol INT1= 0,1
11
Preparation of sugar block
Tosylation of primary alcohol, synthesis of INT2
Decomposition of unreacted pTsCl
H 3C
O
H 3C
CH3
O
TsO
O
H3C
CH3
O
INT 2
O
H 3C
O
O
O
OMe
+
10% NaHCO3
Solvent, rt, ovn
O
S
pTsCl
O
CH3
O
OH
H 3C
CH3
O
TsO
MeO
H 3C
O
H3C
O
O
O
+
MeO
H3C
H3C
O
Cl
OH
pTsOH
OMe
INT 2
O
Esp
N°
Kg 10%
NaHCO3
/Kg INT2
Solvent
Kg
Solvent
/Kg INT2
Yield%
S2
4,2
EtOAc
3,8
100%
S32
1,5
DCM
2,2
100%
(1,2 mol eq.)
Serichim, 29/01/2015
12
Preparation of sugar block
Production of INT2
 Protected lactoseTs, yellow-orange gel , 4,4 Kg, Yield 100%
 Analysis TLC, NMR
H3C
H3C
O
S
O
O
H3C
CH3
O
O
O
CH3
O
O
O
O
MeO
H3C
H3C
O
OMe
OH
Protected lactose Ts, INT2
INT1,
Kg
02/2015
Serichim, 29/01/2015
3,4
Kg 10%
Citric
acid/
Kg INT2
Kg 10%
NaHCO3
/Kg INT2
1,5
1,7
Kg 10%
NaHCO3
/Kg INT2
Kg DCM
/Kg INT2
INT2,
Kg
Yield
%
3,3
4,4
100%
Decomp.
step
1,7
13
Preparation of sugar block
Production of INT2
NMR 1H, INT2, batch 02/2015, pTsCl residue 1.1% w/w
Serichim, 29/01/2015
14
Preparation of sugar block
Reaction SN2 with Piperazine, synthesis of INT3
H3C
H3C
O
H3C
CH3
O
2
TsO
NH
O
CH3
H3C
O
O
O
H3C
H3C
H3C
H3C
INT 2
+
MeO
O
OH
+
O
OMe
INT2,
g
Mol
Pip/Mol
INT2
Kg Pip/
Kg INT3
Kg ACN
/Kg INT3
Kg
H2O/Kg
INT3
INT3,
g
Yield %
S3
46,0
10
1,5
11
10
38,9
97%
S9
16,0
5
0,75
5,9
3
14,2
100%

O
S
O
INT 3
Esp
N°


H
N
CH3
O
O
O
ACN, 80°C, 6h
OMe
OH
O
O
O
MeO
O
CH3
CH3
O
N
NH
HN
O
-
Piperazine amount reduction from 10 to 5 mol/mol INT2 gives still quantitative yield;
INT3 NMR spectra show traces of solvent and INT1 as major impurities. No dimer was
detected;
End product is a jelly compound: crystallization from n-hexane, MTBE, DCM/n-hexane
were unsuccessful.
Serichim, 29/01/2015
15
N
H2
Preparation of sugar block
Production of INT3, IN PROGRESS
H3C
O
H3C
HN
CH3
O
N
O
CH3
O
H3C
H3C
O
O
O
 Protected lactose Pip, yellow-orange gel, 1,9 Kg
 Expected Yield 100%
 Analysis TLC, NMR
MeO
O
OH
OMe
Lactose protected Pip, INT3
03/2015
Serichim, 29/01/2015
INT2,
Kg
Kg
Pip/Kg
INT3
Kg
ACN/Kg
INT3
INT3,
teoric
Kg
Yield
%
2,2
0,64
7,8
1,9
100%
16
Lab scale preparation of ORANGE DYE
 Chromophore block Orange 30 preparation
 Building blocks coupling
Serichim, 29/01/2015
17
Preparation of block Orange 30 chromophore
O 2N
Cl
1° STEP
O 2N
Cl
Orange 30 De-acetilato
N
N
Cl
Cl
N
N
CN
CN
N
N
Disperse Orange 30
OAc
O 2N
Cl
OH
2° STEP
N
Cl
N
CN
N
Orange 30 Succinato
O
O
Serichim, 29/01/2015
18
CO 2H
Preparation of block Orange 30 chromophore
Hydrolysis of acetyl group, synthesis of Orange 30 De-acetilate
O 2N
O 2N
Cl
Sol aq. K2CO3, MeOH
N
Cl
N
Cl
N
0-20°C, ov
Cl
+
+
N
N
N
De-acetilate Orange 30
OH
OAc
Esp N°
Mol MeOH/
Mol O30
Mol H2O/
Mol O30
T,
°C
KHCO3
CN
CN
Disperse Orange 30
AcOK
Reaction
crude
HPLC,
Area %



Orange 30 de-acetylated,
Analysis HPLC, NMR
Dark-red solid
Yield
%
Orange
Acetilate
Purity
(HPLC,
Area %
@275 nm)
S10
90
100
3-20
-
40%
-
-
S10bis
165
100
3-20
Concentrated
mixture
1,8%
91%
90%
S13
166
100
rt
untreated
Mixture
1,2%
92%
96%
S16
112
18
rt
untreated
Mixture
1,6%
91%
97%
Serichim, 29/01/2015
disperse orange (cromatos) dye HPLC @275 purity is 89% ;
water content is 42% (KF and weight loss determined);
19
Preparation chromophore block Orange 30
Esterification dye, synthesis of Orange 30 Succinate
O 2N
O 2N
Cl
O
O
Cl
O
+
N
Cl
Et3N, DMAP, THF
0-20°C, ov
N
N
Cl
N
dihydrofuran-2,5-dione
CN
CN
N
N
De-acetilato Orange 30
Orange 30 Succinato
OH
O
Exp
Solvent
T,
°C
Reaction
time,
h
Work up
description
Note
Purity
(HPLC,
Area %
@275 nm)
Yield %
S11
THF
3-25
24
Solvent change THF
/EtOAc Extraction
EtOAc
Difficult
phase
separation
89,3%
100%
S14
DCM
25
4,5
DCM extraction
Difficult
phase
separation
89,2%
98%
S26
DCM
rt
3
Partial DCM
evaporation with
precipitation of solid
Easy filtration
86,9%
98%
Serichim, 29/01/2015
O
CO 2H
20
Preparation chromophore block Orange 30
O 2N
Esterification dye, synthesis of Orange 30 Succinate
Cl
N
Cl
 Orange 30 succinate,
 Analysis HPLC, NMR
 Dark-red solid
N
CN
N
Orange 30 succinato
O
O
CO 2H
HPLC analysis, Area % @275 nm
Sample
Rt min
Orange 30 Succinate RIF
Orange 30 De-acetilate (S13)
S14P1 (3h15, 32°C)
S14Fin
Serichim, 29/01/2015
Disperse
Orange 30 %
12.96
1.2
1.2
1.8
Orange 30 DeAcet. %
8.47
1.4
91.7
-
Orange 30 Succinate
%
9.3
91.5
91.2
89.2
IMP1%
8.9
6.5
-
21
Coupling building blocks to synthesize Orange dye
STEP1
O2 N
Cl
H3C
O2 N
O
H3C
N
Cl
HN
+
N
O
O
N
H3C
H3C
CH3
WSC
or
TBTU/TEA
O
O
N
Cl
N
DCM, under N2, ov
CN
MeO
O
OH
Latt-Prot Pip
Orange 30 Succinato
N
OMe
Orange 30 -Lattosio
protetto
O
CO2H
Serichim, 29/01/2015
O
O
CN
O
CH3
O
N
Cl
O
O
C
Pip_Latt_Prot
22
Coupling building blocks to synthesize Orange dye
STEP2
O2 N
Cl
O 2N
Ammide Orange 30
Cl
ORANGE
N
Cl
N
N
N
CN
O
Cl
HCl 37% in acetone
O
N
N
N
Acetone, 0-ta, ov
O
N
OH
CN
O
O
OH
O
H3C
O
H3C
N
CH3
O
N
O
O
CH3
+ 2 MeOH
+ 3 MeCOMe
HO
O
OH
OH
OH
O
O
O
H3C
H3C
Serichim, 29/01/2015
HO
O
O
MeO
O
OH
OMe
23
Coupling building blocks to synthesize Orange dye
Coupling reaction, synthesis of Amide Orange 30
Exp N°
Solvent
Coupling
reactive
Mol Coupling
reactive/
Mol O ester
Mol INT3/
Mol O ester
HPLC
area %
Amide
Orange
Procedure FI
THF
DMTMM
1,1
1,15
S12A-B
DCM
WSC HClTBTUTEA
1,1
1,15
61% 71%
S23
DCM
WSC HCl
1,20
1,20
68,4%
DCM:
 Solution of INT3 in DCM,
 No change solvent in work-up
Serichim, 29/01/2015
24
Coupling building blocks to synthesize Orange dye
Coupling reaction, synthesis of Amide Orange 30
H3C
Cl
CN
H3C
N
O 2N
N
N
O
O
O
H3C
O
Cl
O
O
CH3
O
MeO
N
OMe
Ammide Orange 30
N
O
OH
O
O
O
H3C
CH3
 Amide Orange 30,
 Analysis HPLC, NMR
 Foamy Dark-red solid
Serichim, 29/01/2015
25
Coupling building blocks to synthesize Orange dye
Deprotection reaction, synthesis of Orange dye
Serichim, 29/01/2015
Exp N°
HPLC
area %
Orange Dye
Yield%
Procedure FI
73%
90%
S15A-B
53%-60%
74%-86%
S29
64%
88,7%
26
Coupling building blocks to synthesize Orange dye
Deprotection reaction, synthesis of Orange dye
O 2N
Cl
ORANGE DYE
N
N
O
Cl
O
N
 Orange Dye,
 Analysis HPLC_MS, NMR
 Foamy Dark-red solid
N
O
N
OH
CN
HO
O
OH
O
HO
O
OH
OH
OH
HPLC analysis, Area % @275 nm
Sample
Orange
Dye
%
Amide
Orange 30
%
Nominal MS
Rt min
Orange 30
amide (S23)
ORANGE STD
S29 solido wet
S29 AM-AL
Serichim, 29/01/2015
1066
12.6
10.5
11.6-11.9
16.0
17.6
68.4
2.2
11.0
1.4
9.5
0.2
4.4
2.9
1.5
2.5
18.0
3.4
0.3
13.5
899
9.3
Orange
dye
+Me
%
913
9.7
Orange
deacetilated
10.3
11.1
408
14.2
72.8
64.3
0.4
10.7
10.2
0.2
3.0
1.7
0.3
6.1
8.0
1.2
1.0
1.2
38.9
27
Coupling building blocks to synthesize Orange dye
Deprotection reaction, synthesis of Orange dye
Cl
O 2N
ORANGE DYE
N
O
N
O
Cl
N
N
N
O
OH
CN
O
HO
OH
O
OH
Serichim, 29/01/2015
OH
O
HO
OH
28
Lab scale preparation of YELLOW DYE
 Preparation of chromophore block Yellow 42
 Coupling building blocks
Serichim, 29/01/2015
29
Preparation of chromophore block Yellow 42
NO 2
NH
NH
NH
S
O
NO 2
STEP 1
N
S
O
O
O
Yellow 42
CO 2Et
Yellow 42 ester
STEP 2
NO 2
NH
N
S
O
O
CO 2H
Yellow 42 acid
Serichim, 29/01/2015
30
Preparation of chromophore block Orange 30
Alkylation reaction, synthesis of Yellow 42 ester
NO 2
NH
NO 2
NH
K2CO3, KI
NH
S
O
ClCH2CO2Et,
O
acetone, 50°C
O
Yellow 42
+
N
S
O
KCl
+ KHCO3
CO 2Et
Yellow 42 estere
Exp N°
Disperse
Yellow 42,
g
Mol EtCl
acetate/ Mol
Y42
Mol
K2CO3/
Mol Y42
Yellow 42
ester,
g
Purity (HPLC,
Area %)
@275 nm
Yield
%
S17
7,6
1,2
1,4
8,46
99%
90%
 Treatment of Yellow 42 via DCM extraction has been necessary due to its initial
water content (24% KF). Water content after treatment: 0,15% (K-F).
 Work-up: filtration of salts, evaporation of acetone, residue solubilized in EtOAc
and washed with brine.
Serichim, 29/01/2015
31
Preparation chromophore block Yellow 42
Hydrolysis of ester, synthesis of Yellow 42 acid
NO 2
NO 2
NH
O
NH
1. Sol aq. KOH
N
S
O
CO 2Et
Yellow 42 estere
Exp N°
O
THF, 0-20°C
+
N
S
2. Sol aq. HCl
O
Yellow 42 acido
Solvent
S18
4,0
THF
3,6
97,7
95,9%
S19
4,2
DCM
-
34%
-
Yellow 42
acid, g
Purity
Rt min
Yellow 42 acid
STD, lot 574784
Yellow 42 ester S17
S18A (2h45, ta)
S18 solido
S18 AM
S18 AL
Serichim, 29/01/2015
Yellow 42
acido %
7.64
Yield %
(HPLC,
Area %)
@275 nm
HPLC analysis, Area % @275 nm
Yellow 42
estere %
13.91
+ EtOH
CO 2H
Yellow 42
ester,
g
Sample
KCl
1.4
2.4
0.07
2.8
1.95
61.5
95.3
 Yellow 42 acid,
 Analysis HPLC
 yellow solid
99.6
98.6
-
97.1
97.7
2.8
0.4
21.1
1.1
32
Coupling building blocks to synthesize Yellow dye
STEP 1
NO 2
NO 2
NH
NH
S
S
N
O
O
WSC HCl
Yellow 42 acido
H3C
H3C
CH3
O
O
DCM, under N2, ov
HO
CH3
O
O
OMe
CH3
O
O
O
O
O
H3C
O
MeO
O
OH
Latt-Prot Pip
Serichim, 29/01/2015
Yellow 42 -Lattosio
protetto
O
N
H3C
N
N
O
H3C
HN
O
C
CO 2H
+
N
O
O
CH3
CH3
O
MeO
O
O
OMe
H3C
CH3
33
Coupling building blocks to synthesize Yellow dye
STEP 2
NO 2
NO 2
NH
NH
S
N
S
O
O
O
O
C
H3C
CH3
O
O
N
O
N
N
HCl 37% in acetone
N
+ 2MeOH
+ 3MeCOMe
O
acetone, 0-ta, ov
YELLOW
N
OH
HO
O
OMe
O
O
CH3
CH3
MeO
O
H3C
O
OH
HO
O
OH
HO
O
Serichim, 29/01/2015
O
O
HO
HO
Yellow 42 -Lattosio
protetto
CH3
34
Coupling building blocks to synthesize Yellow dye
Coupling reaction, synthesis of Amide Yellow 42
Exp N°
Solvent
Coupling
reactive
Mol
Coupling
reactive/
Mol Y acid
Procedure
FI
THF
DMTMM
1,1
S20
DCM
WSC HCl
1,20
S3
1,20
72%
S22
DCM
WSC HCl
1,20
S9
1,20
72%
Serichim, 29/01/2015
INT3
Batch
number
Mol INT3/
Mol Y acid
HPLC
area %
Amide
Yellow
1,15
35
Coupling building blocks to synthesize Yellow dye
Coupling reaction, synthesis of Amide Yellow 42
MeO
OMe
O
H3C
H3C
OH
O
O
S
O
O
CH3
CH3
CH3
NH
O
CH3
O
O
O
 Amide Yellow 42,
 Analysis HPLC
 Foamy orange-yellow solid
O
NO 2
N
N
N
C
O
HPLC analysis, Area % @275 nm
Yellow 42
acid
%
IMP
%
Amide Yellow
42
%
Rt min
13.8
8.5
12.2-12.6
9.6-11.5
17.5
Yellow 42 acid
(S18Fin)
96.5
2.0
S22 ORG
0.4
1.0
72.4
13.6
9.2
S20 ORG
0.2
-
72.2
11
12.0
Sample
Serichim, 29/01/2015
36
Coupling building blocks to synthesize Yellow dye
Deprotection reaction, synthesis of Yellow dye
Exp N°
HPLC
area %
Yellow Dye
Yield%
Procedure FI
68,4%
91%
S21
73,6%
93%
NO 2
NH
S
O
N
O
O
N
 Yellow Dye,
 Analysis HPLC
 Orange-yellow solid
N
OH
HO
O
O
OH
HO
O
OH
HO
HO
Serichim, 29/01/2015
37
Coupling building blocks to synthesize Yellow dye
Deprotection reaction, synthesis of Yellow dye
Serichim, 29/01/2015
38
Lab scale preparation RED DYE
 Preparation chromophore block Red 202
 Coupling building blocks
Serichim, 29/01/2015
39
Preparation chromophore block RED 202
O 2N
O 2N
Cl
Cl
STEP 1
N
N
N
N
CN
CN
AcHN
AcHN
N
Disperse RED 202
RED
De-acetilato
OAc
N
202
OH
STEP 2
O2 N
Cl
N
N
CN
AcHN
N
RED 202 Succinato
O
Serichim, 29/01/2015
O
CO2H
40
Preparation chromophore block RED 202
Hydrolysis of acetyl group, synthesis of RED 202 De-acetilate
O 2N
O 2N
Cl
Cl
Sol aq. K2CO3, MeOH
N
N
ta, ov
N
AcHN
N
Disperse RED 202
RED
De-acetilato
OAc
Esp N°
AcOK
+ KHCO
3
CN
CN
AcHN
+
N
N
202
Purity
OH
Mol MeOH/
Mol R202
Mol H2O/ Mol
R202
Yield %
Procedure FI
90
100
89%
95%
S24
143
34
89%
89%
S16 (Orange)
112
18
91%
97%
HPLC,
Area %
@275 nm
disperse red(cromatos) dye HPLC @275 purity is 90% ;
water content is 57% (KF and weight loss determined);
Serichim, 29/01/2015
41
Preparation chromophore block RED 202
Hydrolysis of acetyl group, synthesis of RED 202 De-acetilate
O 2N
 RED 202 de-acetylated,
 Analysis HPLC, NMR
 Dark-red solid
Cl
N
N
CN
AcHN
N
OH
Work-up:
 crude mixture was diluted with water to obtain H2O:MeOH 5:1,
 Filtration of solid and drying in oven at 45°C
Serichim, 29/01/2015
42
Preparation chromophore block RED 202
Esterification dye, synthesis of Red 202 Succinate
O2 N
Cl
O2 N
O
O
O
Et3N, DMAP, DCM
+
N
N
Cl
dihydrofuran-2,5-dione
N
rt
N
CN
AcHN
CN
N
AcHN
RED 202 De-acetilato
N
RED 202 Succinato
OH
O
O
CO2H
Esp N°
S26
Deacetilate,
g
Mol
Anydride/
Mol Deacet.
Succinate,
g
Reaction
time,
h
Purity
HPLC,
4,0
1,2
4,85
1,5
Yield %
4,9
3
86,9%
98%
4,0
24
93%
67%
Area %
@275
nm
(Orange)
S25
Serichim, 29/01/2015
43
Preparation chromophore block RED 202
Esterification dye, synthesis of Red 202 Succinate
O2 N
Work-up:
 acidification of carboxilate and elimination TEA HCl with HCl,
 Washes to eliminate DMAP,
 Concentration solvent
Cl
N
N
CN
AcHN
N
O
 Red 202 succinate,
 Analysis HPLC
 Dark-red solid
O
CO2H
Serichim, 29/01/2015
44
Coupling building blocks to synthetize RED dye
STEP 1
CN
Cl
N
O 2N
O
Cl
N
RED 202
succinato
N
WSC HCl
+
H3C
O
O
C
O
O
H3C
CH3
O
O
CH3
CH3
OH
Latt-Prot Pip
N
O
O
O
MeO
O
OMe
O
N
CH3
O
O
H3C
O
RED 202 -Lattosio
protetto
O
N
AcHN
DCM, under N2, ov
H3C
HN
CN
N
O
NHAc
O 2N
N
CO 2 H
N
H3C
H3C
O
O
O
O
OMe
CH3
CH3
OH
MeO
Serichim, 29/01/2015
45
Coupling building blocks to synthetize RED dye
STEP 2
Cl
Cl
O 2N
O 2N
N
N
CN
CN
+ 2MeOH
+ 3MeCOMe
N
N
N
N
AcHN
AcHN
O
O
O
HCl 37% in acetone
O
RED 202 -Lattosio
protetto
RED 202
O acetone, 0-ta, ov
C
C
O
N
N
O
CH3
CH3
HO
O
O
O
H3C
H3C
O
N
N
O
O
CH3
HO
O
O
OH
CH3
O
HO
O
OMe
OH
HO
OH
OH
MeO
Serichim, 29/01/2015
46
Coupling building blocks to synthetize RED dye
Coupling reaction, synthesis of Amide RED 202
Esp N°
Solvent
Coupling
reactive
Mol Coupling
reactive/
Mol R ester
Mol INT3/
Mol R ester
Procedure FI
THF
DMTMM
1,1
1,15
S27
DCM
WSC HCl
1,2
1,2
73%
S30
DCM
WSC HCl
1,20
1,20
73%
DCM:
 Solution of INT3 in DCM,
 No change solvent in work-up
Serichim, 29/01/2015
HPLC
area %
Amide
RED
 Amide RED 202,
 Analysis HPLC
 Foamy Dark-red solid
47
Coupling building blocks to synthetize RED dye
Deprotection reaction, synthesis of RED dye
Esp N°
HPLC
area %
RED Dye
Procedure FI
34%
S28
30%
S28Pur
43%
S31
30%
Yield%
94%
95%
NC
O
O
C
N
N
O
N
Cl
N
N
HO
OH
NHAc
OH
O
HO
 RED Dye,
 Analysis HPLC_MS
 Dark-red solid
O
O 2N
O
OH
Serichim, 29/01/2015
HO
OH
48
Coupling building blocks to synthetize RED dye
Deprotection reaction, synthesis of RED dye
Sample
Rt min
RED 202
amide (S27)
RED STD
S28 solid
S28 AM-AL
S28 Pur1
solid
Serichim, 29/01/2015
RED
202
%
9.1
9.4
9.5
RED
deac
?
13.5
2.6
2.1
34.4
29.6
-
7.7
6.9
-
4.2
0.7
-
23.7
3.3
9.4
2.9
1.8
5.1
39.7
2.9
33.3
1.4
3.9
42.7
9.4
1.6
10.2
0.4
4.1
8.4
Amide
RED 202
%
12.6
10.2
11.211.6
14.7
16.5
73.2
1.8
11.5
1.6
10.5
1.1
2.4
4.7
1.6
1.6
-
1.1
5.1
7.1
0.9
0.7
2.4
7.0
-
RED
succi
nato
?
13.7
6.26.4
1.73
49
Coupling building blocks to synthetize RED dye
Deprotection reaction, synthesis of RED dye
Serichim, 29/01/2015
50
Comparison between Serichim and Firenze samples
Analysis UV
Dye
orange
12D
lotto
λ nm
FW
Fi
441
937,2
Serichim
446
442
937,2
937,2
Fi
420
856,3
Serichim
417
417
856,3
856,3
Fi
425
959,8
498
498
959,8
959,8
Fi (measured in
Serichim)
Yellow
4217
Fi (measured in
Serichim)
Red 4d
Fi (measured in
Serichim)
Serichim
Serichim, 29/01/2015
mg/ml
mol/l
ABS
ε
(l/mol/
cm)
16300
0,034
0,089
3,63E-05
9,48E-05
1,237
1,326
34098
13995
4200
0,0968
0,100
1,13E-04
1,17E-04
0,695
0,616
6148
5275
12900
0,072
0,044
7,50E-05
4,58E-05
1,438
1,511
19169
32960
51
Comparison between Serichim and Firenze samples
Analysis UV
Serichim, 29/01/2015
52