Work Presentation

Prachi Gupta (16136)

Thesis Supervisor
Dr. Aasheesh Srivastava
Synthesis of Tyramine-conjugated polyaspartamide

O O
NH3Cl O
N N y
x H
+ N 1. DIPEA, DMF, 75 oC O H
n HN O
2. API, 65 oC NH
O

OH
N

HO N

Reagent MW Eq. mmol Density Amount

g/mol g/ml
Polysuccinimide 97 per 1.00 10.30 - 1g
unit
Diisopropylethylamine 129.25 0.80 8.27 0.742 1.44 ml
Tyraminehydrochloride 173.64 0.25 2.57 - 447 mg
3-(aminopropyl)imidazole 125.18 2.80 28.84 1.062 3.40 ml
 Thermo-response of the polymer
 Polymer solution shows turbidity at and above a certain temperature known as

the ‘cloud point’

 Stimuli which favour globular form of the polymer are expected to decrease the

cloud point

T ≥ 32 oC

Fig.1 Thermo-response of the polymer in 1M PB at pH 6.2 (10 mg/ml)

 Effect of polymer concentration on cloud point

Polymer concentration Cloud point

2 mg/ml 54
5 mg/ml 34
10 mg/ml 32
15 mg/ml 30
20 mg/ml 26

 Cloud point decreases as the polymer concentration increases

 Propensity of the polymer to remain in globular form is enhanced

 Effect of polymer concentration on cloud point
100 2 mg/ml
5 mg/ml
90 10 mg/ml
15 mg/ml
80 20 mg/ml

70
% T at 600 nm

60

50

40

30

20

10

0
10 20 30 40 50 60 70 80

Temperature (oC)
 Effect of buffer concentration on cloud point
5 mg/ml polymer concentration

100 1 M PB
500 mM PB
250 mM PB
50 mM PB
80 50 mM PB + 100ul NaCl
50 mM PB + 500ul NaCl
% T at 600 nm

10 mM PB

60

40

20

0
10 20 30 40 50 60 70 80 90

Temperature (oC)
 Suspension in water
 Polymer forms a suspension in water
Fig.2 Mobility
distribution of 5
mg/ml polymer
solution in water

Size = 186.6 nm

Zeta potential = - 45.70 mV

Poly dispersity Index = 0.36

Fig.3 Intensity distribution of 5 mg/ml

polymer solution in water
 Horse Radish Peroxidase catalyzed gelation

HO

N
N N
N N
N
HRP/H2O2 HO O
N OH
N OH
N N

OH
OH

Fig. 4 HRP catalyzes ether and phenolic type bond formation in presence of H 2O2
 Effect of buffer on gelation

SN Buffer Other conditions Observations  1 mg/ml HRP

1 10 mM PB HCl, RT Transparent, yellow gel  1 wt % H2O2
2 100 mM PB No HCl/HCl, RT Turbid, white gel  7 wt% polymer
3 500 mM PB No HCl/HCl, RT Turbid, white gel  2 wt% PEG
4 1 M PB No HCl/HCl, RT Turbid, white gel  2 wt % PASA

5 1 M PB HCl, 0 oC Turbid, white gel

6 100 mM Citrate No HCl (solubility issue) Coagulated polymer,

buffer white
7 10x PBS HCl (solubility issue) Coagulated polymer,
white
8 1 M PB Polyethylene glycol Turbid, white gel

9 1 M PB Poly(aspartamide) Turbid, white gel

Buffer pH = 6.2
Effect of polymer concentration on gelation
(a) (b) (c)

1 % w/v 2 to 5 % w/v 6 % w/v

Fig. 5 Vials (a) and (b) did not result into gelation even after 36 hours. Vial (c)
showed gelation in ~2 min.

Conditions:
 0.5 mmol H2O2 (1 wt % aq. solution) per mmol of tyramine
 1.5 mg HRP (1 mg/ml) per mmol of tyramine
 Room temperature
 1 M PB with pH 6.2