Synthesis of a Perylenediimideaminoacid with protecting groups
Georg von Langen Schule
Holzminden
Problem statement/ background:
Heparin
Fluorescent marking of Heparin (glycosaminoglycan, GAG) for better
detection in blood! Heparines are therapeutically used substances as
blood clotting inhibitors. Chemically they are polysaccharides
(glycosaminoglycanes), consisting of a variable number of aminosugars.
Aspired changes through the asymmetric Perylenediamide:
Strategy for solution of producing Alloc-Py-Boc
Performed production steps:
1. Checking reactivity and selectivity of the reaction of the Alloc- and Boc amino acid
onto the perylene, respectively.
2. Analogical checking synthesis instruction out of the doctorate of Fritz Thomas
Wetzel from Freising. Revision of 3 reaction channels:
- Collecting of information for the procedure (internet research)
- Creation of reaction equation and stoichiometric computation
- Determination of the properties of the educts (safety indication etc.):
-perylene
-Alloc protecting group
-Boc protecting group
-imidazole
-needed solvents
BOC‐Py‐BOC‐Syn
Boc-Py-Boc-Syn
m(BOC)+20% ÜS
Micro approach in testing tubes!
2.2 parts Boc
1.0 part Perylen
in imidazole 140 °C/ 1h
Processing:
extract with CH2Cl2/ 2M HCl in
silica gel 60 column
Perylen
Addition of the amino acid groups to the GAG-molecules.
Production of a perylenediimidaminoacid through insertion of protected amino acids.
Precursors:
target molecule:
NH
NH2 HCl
O
O
O
Alloc-1,2-diaminoethan hydrochlorid
O
O
O
H2N
O
N
O
. HCl
O
NH
O
n(Boc)
0,25
mmol
M(BOC_HCl)
167,3
g/mol
M(Perylen)
392,3
g/mol
n(Perylen) Imidazol (Lsm.)
0,12
1
mmol
g
Conclusions:
• detection of Boc-Py-Boc and
Alloc-Py-Boc was successful
through MS structure determination.
→I.) Synthesis of pure
Boc-Py-Boc was successful!
→ II.) Synthesis of pure
Alloc-Py-Alloc is not possible!
→III.) Synthesis of a Boc-Py-Boc
and Alloc-Py-Boc mixture was successful!
Alloc‐Py‐Boc‐Syn
procurement quantity
m(BOC)+20% ÜS
50
mg
Micro approach in testing tubes
2.2 parts Alloc
1.0 part Perylene
in Imidazol 140 °C/ 1h
Processing:
extract with CH2Cl2/ 2M HCl in
silica gel 60 column
N
O
m(Perylen)
48,85
mg
m(Alloc)+20%ÜS
54,00
m(BOC)
41,67
mg
n(Boc)
0,25
mmol
m(Alloc)
45,00
mg
n(Alloc)
0,25
mmol
m(Perylen)
97,70
mg
Alloc-Py-Alloc-Syn
O
O
O
O
m(BOC)
41,67
mg
50
mg
Alloc-Py-Boc-Syn
Micro approach in testing tubes!
1.2 parts Boc
1.2 parts Alloc
1.0 part perylene
in imidazole 140 °C/ 1h
Processing:
extract with CH2Cl2/ 2M HCl in
silica gel 60 column
O
O
O
University of Heidelberg
Prof. Dr. Krämer
M(BOC_HCl)
167,3
g/mol
M(Alloc_HCL)
180,67
g/mol
n(Perylen) Imidazol (Lsm.)
0,25
2
mmol
g
Alloc‐Py‐Alloc‐Syn
m(Alloc)+20% ÜS
m(Alloc)
41,67
mg
50
mg
m(Perylen)
45,24
mg
n(Alloc)
0,23
mmol
n(Perylen) Imidazol (Lsm.)
0,12
1
mmol
g
M(Perylen)
392,3
g/mol
M(Alloc_HCL)
180,67
g/mol
M(Perylen)
392,3
g/mol
-
Creation of synthesis instruction
Ordering chemicals and laboratory equipment
Performing reaction in compliance with safety- and hazard statements
Processing with silica gel
MS-Analytics and evaluation
Documentation of results
• Separation of both substances from III.) did not succeed completely
with the used silica gel 60 column.
→IV.) Full separation eventually possible with different eluent
and different column material.
Perylen-3,4,9,10-tetracarbonsäuredianhydrid
Glycine-tert-butylester-hydrochlorid
Reaction equations:
O
Imidazol
O
O
O
O
140°C/ 1h
O
2x
Analytics:
MeOH / CH2Cl2 1:20
1. Boc-Py-Boc
NH2 + O
O
O
O
Alloc
Py
Alloc
O
O
N
N
O
O
O
O
Boc
Py
Boc
no
reaction
C36H30N2O8
M=618,632 g/mol
LC:
Column:
Eluent:
Flow:
Temp:
Gradient:
UV:
MS:
Method:
Masses:
Dionex Ultimate 3000RS UHPLC
Phenomenex Kinetex C18, 2.1x100mm, 1.7µ
A= H2O + 0,1% formic acid, B= ACN + 0,09% formic acid
0,550ml/min
40°C
0 min A 100% B 0%, 22min A 5% B 95%, 27min A 5% B 95%
200-500 nm
Bruker micrOTOF II
ESI +
70-1600
Alloc
Py
Boc
2. Alloc-Py-Boc
O
O
O
NH
+
NH2 + O
O
O
O
O
N
O
O
NH
O
O
O
C36H29N3O8
N
NH2
M=631,6308 g/mol
O
O
N
N
O
O O
Alloc
Py
Alloc
Ansatz
Boc
Py
Boc
Ansatz
Boc
Py
Alloc
Ansatz
O
O
N
N
C36H30N2O8
O
O O
M = 631,63
M = 618,632
O
Boc after
Boc
Py 30 min Py
60 min
Boc 90 min Alloc
Ansatz
Ansatz
C36H29N3O8
O
O
140°C/ 1h
O
O
O
Imidazol
O
NH
O
O
O O
1.
2.
O
O
O
Imidazol
3. Alloc-Py-Alloc
140°C/ 1h
O
2x
O
NH
+ O
O
O
O
N
O
O
O
NH
O
HN
O
O
C36H28N4O8
N
NH2
O
M=644,6295 g/mol
Synthesis instruction Alloc-Perylen-Boc:
1. Add 195,4mg of perylene to 4g of imidazole molten at 110°C and stir until a suspension is formed. Then add 100mg (0.6mmol) G‐Boc and 108mg (0.6mmol) D‐Alloc , heat the suspension to 140°C. TLCs are taken for reaction observation (dichloromethane/methanol 20:1 used as eluent).
After 90 min the reaction is stopped, cooled to room temperature, the solid reaction product is collected in 40 ml of dichloromethane and the suspension is stirred in the ultrasonic bath and filtrated. Edulcorate the filtrate 2x with 40 ml of a solution of NaH2PO4 (10g in 120ml H2O) each in the separatory funnel. The product dissolves in the organic phase. Red slurry forms between the phase boundaries which can be flushed out with the organic phase. Edulcorate the organic phase 3x with 20ml of water each. The organic phase is dried through sodium sulphate without the slurry and the dichloromethane is removed with the rotary evaporator at 50°C without vacuum.
2. Dissolve the solid in dichloromethane and separate in a column filled with silica gel 60. Mobile phase (80:1 dichloromethane : methanol). The products appear as two red bands, whereas the Boc‐Py‐Boc substance is the highest and underneath it is the Boc‐Py‐Alloc substance on the TLC. In the particular elutions the solvent is removed with the rotary evaporator and the different products are dried in vacuum!
Synthesis instruction Boc-Perylene-Boc:
1. Initial weight: 50 mg (0,3mmol) glycerine‐tert.‐butylester hydrochloride (G‐Boc)
Initial weight: 45,24 mg (0,12 mmol) perylene added to 1 g imidazole molten at 110°C in a 25ml pear shaped flask. Stir the mixture until a uniform solution/suspension is formed. Add D‐Alloc at stirring and at given temperature and heat up to 140°C. TLCs for reaction observation after 30, 60 and 90 min (dichloromethane/methanol 20:1 used as eluent). Stop reaction after 90 min. Cool down to room temperature and add 40ml dichloromethane while stirring the suspension. Add NaH2PO4
(10g in 120ml H2O) and extract in separatory funnel. The product is dissolved in the organic phase and the imidazole in the water phase. Edulcorate the organic phase 3x with 20ml water each; dry through sodium sulphate. Filtration and following concentration of the organic phase with rotary evaporator (without vacuum at 45°C bath temperature).
2. Dissolve the solid in dichloromethane. Separate in column filled with silica gel 60. Cleaning off byproducts in the sample (dichloromethane/methanol 80:1 used as eluent). Qualitative control with TLC: products give off red bands.
Literature cited:
1. 2. 3. 4.
http://www.sigmaaldrich.com/safety‐center.html
http://www.chemiedidaktik.uni‐wuppertal.de/alte_seite_du/material/milch/milcheiweiss/aminosauren.pdf
Langhals, Heinz; Jona, Wolfgang: Synthese für substituierte Perylene. Universität München 1997
Langhals, Heinz; Jona, Wolfgang. The synthesis of perylenebisimide monocarboxylic acids. European Journal of Organic Chemistry (1998), (5), 847‐851.
5. Büttner, Jörn: Neue Synthesewege zu NIR‐Farbstoffen auf Basis der Quaterrylenbisimid. Universität München 2006
6. Wetzel, Fritz Thomas: Synthese von neuen Verbindungen auf Basis der Perylen‐3,4:9,10‐bis(dicarboximide) für Anwendungen im Flüssigkristall‐ Bereich und als optische Schalter. Universität München 2003
7. Onken, Kristian: Synthese und Charakterisierung neuartiger, ambipolarer organischer Halbleiter, basierend auf Perylencarboximid‐
Systemen kombiniert mit funktionalisierten Spirobifluoreneinheite. Universität Kassel 2007
8. Kampner, Michaela: Schulversuche mit Milch und Einsatzmöglichkeiten im Chemieunterricht. Universität Wuppertal 2005
9. Kuhn, Isabelle: Verbesserung: DC‐ Trennung von Aminosäuren. Phillipps‐ Universität Marburg 2006
10. Krämer, Prof. R.: Mix‐and‐measure fluorescence assays for the selective detection of glycosaminoglycans. Universität Heidelberg
11. Gesetze/ Schulordnung der Georg von Langen BBS
Involved persons: Willi Krieger, Vanessa Kaußen, Jessica Grieger, Maximilian Conradi, Thorsten Böddeker, Vanessa Harms, Max Gajfulin, Celina Peters, Alexandra Schuster, Teresa Weskamp, Rohit Patel, Jörn Lönneker, Dr. Carsten Wendelstorf