1
Supplementary Figures
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3
4
5
6
%T
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8
9
10
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13
14
15
16
17
18
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20
55
50
45
40
35
30
25
20
15
10
5
A
554000
50
45
40
35
30
25
20
15
10
5
3500
4000
3500
SH-PEG-Lactonolactone
3000
2500
2000
1500
1000
500
SH-PEG-Coumarin
B
3000
2500
2000
1500
1000
500
wavelength (in nm)
Fig S1: FT-IR spectra of thiolated PEG conjugated with (A) lactonolactone (B) 7-aminocoumarin 3carboxylic acid.
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25
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30
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Fig S2 NMR spectra of thiolated PEG conjugated to 7-aminocoumarin 3-carboxylic acid.
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33
34
3.0
2.5
PEG conjugated Coumarine
2.0
35
7-aminocoumarin 3-carboxylic acid
Absorbance intensity
1.5
373nm
1.0
36
37
368nm
0.5
0.0
300
400
500
600
700
800
900
1000
1100
wavelength (in nm)
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40
41
42
43
44
45
46
47
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Fig S3: UV-Vis analysis curve showing hypsochromic shift in λmax value for 7-aminocoumarin 3carboxylic acid and its PEG conjugated derivative
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53
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56
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59
60
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FigS4: FT-IR spectra of bare gold nanoparticles
1.0
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Bare Gold Nanoparticles
522nm
0.8
Bioconjugated Gold nanoparticles
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0.6
64
65
66
67
68
69
70
71
72
73
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Absorbance
0.4
563nm
0.2
0.0
400
500
600
700
800
wavelength (in nm)
Fig S5: UV-Vis analysis data showing bathochromic shift in λmax value of gold nanoparticles up on
conjugation
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79
A
80
81
82
83
84
85
86
87
88
89
90
91
Fig S6: QELS Data (A) and TEM Micrograph (B) of conjugated gold nanoparticles
B
CH2OH
92
CH2OH
OH
O
OH
93
CH2OH
OH
OH
O
O
OH
OH
OH
96
97
98
O
OH
94
95
O
OH
CH2OH
O
O
OH
OH
Scheme S1: Synthesis of lactonolactone from lactose
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100
101
102
CHO
COOH
HOOC
+
103
CH2
Aniline/Ethanol
70-80oC
HOOC
O
CHO
104
O
Conc H2SO4/ConcHNO3
0oC
105
COOH
106
O2N
107
O
O
SnCl2/HCl
RT
108
COOH
109
110
111
112
113
114
115
116
117
118
119
H2N
O
Scheme S2: Synthesis of 7-aminocoumarin 3-carboxylic acid
.
O
120
121
122
HO
O
O
OMe
123
n
Thioacetate
N2/RT
124
HO
125
O
O
SAc
n
126
Tosylchloride/Et3N
60oC/RT
tsO
127
O
O
SAc
n
128
129
NaN3/RT
N
N
O
O
SAc
n
130
131
LiAlH4
0oC
H2N
Derivative 3. iv
O
O
132
133
134
135
136
137
138
139
140
141
142
SH
n
Scheme S3: - Synthesis of SH-PEG-NH2 from monomethoxy PEG 5000
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144
145
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HO
O
O
OMe
n
Tosylchloride
DMAP+Et3N
60oC
147
tsO
O
148
O
OMe
Derivative 4. i
OMe
Derivative 4. ii
n
DMSO
Na2HP4
149
OHC
O
O
150
n
151
Thiacetate N2/RT
O
152
OHC
O
SAc
n
NaOMe/MeOH
HCl/RT
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O
154
OHC
O
SH
155
156
157
158
159
160
161
162
163
164
165
Scheme S4: Synthesis of SH-PEG-CHO from monomethoxy PEG 2000
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167
168
169
CH2OH
HS
CH2OH
O
O
O
O
NH2
OH
O
n
170
OH
171
OH
CH2OH
CH2OH
O
172
173
OH
O
OH
HO
HS
HC
O
O
OH
O
OH
OH
O
N
H
n
174
OH
OH
175
176
177
Scheme S5:- Coupling of lactonolactone to SH-PEG-NH2 to form Adduct 1
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COOH
HS
O
CHO
O
n
183
H2N
O
O
1. Et3N
184
2. NaBH3CN
COOH
185
186
HS
H
C
O
NH
O
O
O
n
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188
189
Scheme S6:- Coupling of Coumarin derivative to SH-PEG-CHO to form Adduct 2
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191
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Suppelmentary Methods
2.2 Synthesis of Targeting Moiety: Lactonolactone
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Lactonolactone was synthesised by oxidizing the sugar moiety Lactose17. Briefly, lactose (2g;) was
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dissolved in minimum amount of hot water followed by its addition to an iodine solution in
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methanol(3g/40mL) at 40˚C. The reaction mixture was stirred for 2 hours. Following this, a concentrated
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solution of potassium hydroxide in methanol was added drop-wise to the reaction mixture until the colour
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of iodine disappeared. The solution was then cooled externally in an ice bath which led to the
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precipitation of a crystalline product. The product was filtered, repeatedly washed with cold methanol and
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recrystallized using water/methanol system.
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The potassium salt of lactobionic acid thus formed was converted to its free acid form by passing it
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through a column of Acidic amberlite resin. The acidic elute was concentrated and evaporated several
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times with methanol to get the final lactone as a highly viscous colourless oil. The scheme for the
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synthesis of lactonolactone from lactose is shown in Scheme S1. IR spectra: - 3371, 2898, 1736, 1647,
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1421, 1226, 1139, 1077, 1035, 891, 787
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2.2.3 Synthesis of Fluorescent moiety: 7-aminocoumarin 3-carboxylic acid
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The scheme for the synthesis of fluorescent 7-aminocoumarin 3-carboxylic acid is shown in Scheme S2.
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Synthesis of the fluorescent moiety was performed in three consecutive steps, the first one involving the
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synthesis of 3-carboxycoumarin from Salicyldehyde and Malonic acid as per the procedure discussed by
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Besson et al18, followed by its controlled nitration at cold temperatures to give 7-nitrocoumarin 3-
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carboxylic acid. The later was reduced with SnCl2/HCl mixture to yield the final product as a bright
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yellow solid.
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Data for 7-aminocoumarin 3-carboxylic acid: 1H N.M.R: - 8.93(d, COOH), 8.52(d, ArH), 7.65 (d, ArH),
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4.18 (NH2), 3.86 (CH=C) IR spectra (ᶹ/CM-1): - 3428, 3369-3400 (d, primary amine), 3069, 2855, 1741
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(C=O for lactone), 1705 (C=O for carboxylic acid), 1610 () N-H), 1096 (C-O), 1001, 947, 845.
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2.2.4 Synthesis of SH-PEG-NH2 from monomethoxy PEG 5000
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The synthesis of hetero bi-functional PEG derivative from monomethoxy PEG 5000 involves a sequence
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of steps as illustrated in Scheme S3. The synthetic procedures have been referred from a few
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publications19, 20 and were performed with slight modifications.
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Derivative 3.i: - Monomethoxy PEG 5000 (1.0Eq) was dissolved in minimum amount of DMF followed
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by the addition of Potassium thioacetate (10.0 Eq) under inert atmosphere. The reaction mixture was
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allowed to stir under nitrogen atmosphere for about 24 hours. The progress of the reaction was monitored
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through TLC (methanol/CH2Cl2 system). After the completion of the reaction, the crude reaction mixture
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was treated with CH2Cl2 and excess thioacetate was washed by adding equal portions of saturated solution
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of NH4Cl and brine. The aqueous and the organic layer were then separated followed by 4-5 times
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extraction of the aqueous layer with CH2Cl2. Finally, the organic layer aliquots were combined, evaporated
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and purified over alumina (methanol/CH2Cl2 system) to get the final crude product as yellow oil with foul
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smell. The yellow oil when triturated with Diethyl ether gave pale yellow solid as the final product. 1H
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N.M.R 2.15 (S, CH3), 3.14 (t, AcS-CH2), 3.60-3.68 (m, CH2-CH2-O-CH2-CH2), IR Spectra (ᶹ/cm-1) –
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3433 (br, m), 2888 (br, vs), 1680, 1467, 1343 (asymmetric S=O stretch), 1281, 1250, 1112 (C-O stretch),
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963, 842
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Derivative 3.ii - Thioacetate derivative of PEG was tosylated using the procedure as follows: - AcS-PEG-
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OH (1.0eq) was dissolved in minimum amount of toluene followed by addition of base triethylamine
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(3.0eq) and p-tosylchloride (1.5eq). The reaction mixture was stirred for about 5 hrs at a maintained
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temperature of 60˚C after which it was left on stirring for another 10 hrs at ambient temperature. Progress
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of the reaction was monitored through TLC (methanol/CH2Cl2 system). Following the completion of the
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reaction, the solvent was removed from the reaction mixture over Buchi rotary evaporator. The crude
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product was then dissolved in CH2Cl2, and treated with 0.25M aqueous HBr solution and brine which
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served as washing agents for excess triethylamine and tosylchloride, respectively. The aqueous and
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organic layers were separated. The aqueous layer was extracted 4-5 times with CH2Cl2. Finally, the organic
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layer aliquots were combined, evaporated and purified over alumina (Methanol/ CH2Cl2) to get the
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product as pale yellow coloured oil, which upon trituration with ether yielded pale yellow final product. .
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1
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(t, CH2-Ots), 7.31 (d, ArH), 7.79 (d, ArH) IR Spectra (ᶹ/cm-1): - 3400 (absorbed water), 2888, 1735, 1673,
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1466, 1344 (asymmetric S=O stretch), 1281, 1112, 1034(S-O stretch), 980, 842.
H N.M.R 2.15 (s, S- CH3), 2.42 (s, CH3) 3.12 (t, AcS-CH2), 3.50-3.72 (m, CH2-CH2-O-CH2-CH2), 4.12
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Derivative 3.iii - Derivative 3.ii (1.0eq) was dissolved in minimum amount of DMF followed by the
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addition of sodium azide NaN3 (1.25eq) under inert atmosphere at ambient conditions. The reaction
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mixture was allowed to stir at the same conditions for 24hrs. The formation of product was confirmed
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through TLC (CH2Cl2/MeOH). The crude product was then repeatedly precipitated out from the reaction
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mixture by addition of dry ether. Purification by chromatography over alumina (EtOAc/MeOH) yielded
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the final product as pale yellow oil which upon trituration with ether gave pale yellow coloured solid. . 1H
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N.M.R 2.15 (s, S-CH3), 3.14 (t, AcS-CH2), 3.32(t, CH2) 3.53-3.68 (m, CH2-CH2-O-CH2-CH2) IR spectra
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(ᶹ/cm-1): - 2936, 2888, 2098 (azide stretch), 1644, 1466, 1346, 1281, 1110, 1034, 981, 843.
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Derivative 3.iv Under complete Argon atmosphere, a solution of LiAlH4 (5.0eq) in dry DMF was stirred
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for about half an hour at -10 to 0˚C (maintained in ice/Sodium chloride bath) in a dry round bottom flask,
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before a solution of Azido-PEG-Thioacetate (1.0eq) in DMF was added drop wise into it. Stirring at the
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maintained conditions was continued for another 4hrs. The progress of the reaction was analysed via
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Ellman’s test for thiol group and ninhydrin test for the amino group. After the completion of the reaction,
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double distilled water was cautiously added. Lithium hydroxide thus precipitated out was filtered over a
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pad of celite and washed repeatedly with ethanol. The filterate was concentrated, dissolved in minimum
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amount of CH2Cl2 and purified over alumina (EtOAc/MeOH system) to yield colourless oil. Trituration
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with dry ether yielded off white coloured Solid. 1.1–1.28 (m, SH), 1.97-2.1 (s, NH2), 2.23–2.56 (m, CH2),
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3.51–3.76 (m, CH2-CH2-O-CH2-CH2) IR spectra (ᶹ/cm-1): - 3369 (strong, primary amine), 2918 (SH
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stretch), 2887, 1598 (N-H bend), 1465, 1346, 1282, 1112, 963
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2.2.5 Synthesis of SH-PEG-CHO from monomethoxy PEG 2000
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A versatile hetero-bifunctional polyethylene glycol (PEG) derivative containing active end-groups thiol
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and aldehyde was efficiently prepared from monomethoxy PEG as per the scheme in scheme S4. Though
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the synthetic procedures were referred from a few publications19, 20, 21, the synthesis was performed with
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considerable modifications.
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Derivative 4.i - Purified monomethoxy PEG (1.0eq) was dissolved in minimum amount of toluene
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followed by the addition of Et3N (3.0eq), DMAP (catalytic amount; 0.25eq) and p-tosylchloride (1.5eq).
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The reaction mixture was then heated in an oil bath maintained at 80˚C for 72 hrs. The progress of the
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reaction was analysed through TLC (CH2Cl2/MeOH system). After completion of the reaction, the
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reaction mixture was cooled to room temperature, the solvent was evaporated and the crude oil thus
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obtained was treated with CH2Cl2. Excessive reagents were removed through vigorous washings with
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saturated solution of NaHCO3 & brine and 0.25M aqueous HBr. Finally, the aqueous and organic layers
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were separated. The Aqueous layer was extracted extensively with CH2Cl2. The organic layer aliquots
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were combined, evaporated and purified over alumina (CH2Cl2/MeOH system) to obtain crude final
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product as colourless oil. Trituration with dry ether gave the final product as off white coloured solid. 1H
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N.M.R: - 2.39 (s, ArCH3), 3.32 (s, O-CH3), 3.53-3.66 (m, CH2-CH2-O-CH2-CH2), 4.13 (t, CH2-CH2-Ots),
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7.33 (d, ArH), 7.77(d, ArH) Ir spectra (cm-1): - 3432, 2884, 1735, 1647, 1598, 1466, 1345 (asymmetric
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SO2 stretch), 1281, 1234, (symmetric SO2 stretch), 1112, 949, 842
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Derivative 4.ii - A solution of MeO-PEG-Ots (1.0eq) in 15mL DMSO was treated with Na2HPO4
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(20.0eq) and the mixture was stirred in an oil bath maintained at 100˚C for 20hrs. The progress of the
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reaction was monitored through TLC (CH2Cl2/MeOH system). After the completion of the reaction, the
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cooled reaction mixture was filtered and the filtrate was precipitated out several times with dry ether. The
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precipitate thus obtained was dissolved in minimum amount of water, dialysed and lyophilized to obtain
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the final product as an off-white coloured solid.1H N.M.R- 3.32 (s, O-CH3), 3.56-3.71 (m, CH2-CH2-O-
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CH2-CH2), 3.81(CH2-CH2-O-CH2-CHO), 4.17 (d CH2-CHO), 9.71(s, CHO) IR spectra (ᶹ/cm-1): -3412
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(absorbed water), 2820-2835 (overtone for aldehyde) 2885, 1735 (C=O stretch), 1467, 1254, 1192, 1110,
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953, 847
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Derivative 4.iii- CHO-PEG-OMe was subjected to thioacetylation in the next step. To a solution of
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derivative 4.ii (1.0eq) in DMF, 1.5 equivalents of potassium thioacetate were added under inert
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atmosphere. The reaction was allowed to stir continuously at room temperature and nitrogen/argon
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atmosphere for 24 hrs. The progress of the reaction was monitored through TLC (methanol/CH2Cl2
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system). The work up of the reaction mixture was done following the same steps as done for derivative 3.i
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which gave the final thioacetated product as a pale yellow solid. 1H N.M.R- 2.16 (s, CH3), 3.
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AcS-CH2), 3.51-3.68 (m, CH2-CH2-O-CH2-CH2), 3.81(CH2-CH2-O-CH2-CHO), 4.17 (d CH2-CHO),
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9.71(s, CHO) IR spectra (ᶹ/cm-1): -3412 (absorbed water), 2820-2835 (overtone for aldehyde) 2885, 1735,
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1687, 1467, 1350, 1254, 1192, 1110, 1034 (S-O strech), 953, 847
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43(s,
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Derivative 4.iv - To a solution of Derivative 4.iii (1.0eq) in degassed methanol, 5 equivalents of NaOMe
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in MeOH was added. The mixture was allowed to stir overnight at room temperature. Then, the mixture
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was acidified to pH 1–2 using 0.1N HCl. Solvent from the reaction mixture was evaporated over Buchi
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rotary evaporator to give the crude product. Purification by silica gel chromatography (CH 2Cl2/MEOH
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system) gave the bifunctional derivative as colourless oil, which upon trituration with Dry ether gave off-
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white coloured solid. 1H spectra 1.1–1.28 (m, SH), 2.23–2.56 (m, CH2), 3.51–3.76 (m, CH2-CH2-O-CH2-
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CH2), 3.81(CH2-CH2-O-CH2-CHO), 4.17 (d CH2-CHO), 9.71(s, CHO) IR spectra: - 3420 (absorbed
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water), 2918 (SH stretch) 2820-2835 (overtone for aldehyde), 2884, 2110, 1735, 1466, 1351, 1253, 1099,
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1022, 953, 845
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NOTE: The coupling reactions of the thiolated PEG with Gold nanoparticles, target specific and
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fluorescent moieties were done within 2-3days of their synthesis as the thiol linkages lack stability in
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oxidizing environment and have strong tendency to form disulfides.
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