<p> 1</p><p>2 Supplementary Figures</p><p>3</p><p>SH-PEG-Lactonolactone 55 4 50 45 A 40 35 5 30 25 20 15 10 6 5 %T 554000 3500 3000 2500 2000 1500 1000 500 50 7 45 SH-PEG-Coumarin 40 35 30 B 8 25 20 15 10 9 5 4000 3500 3000 2500 2000 1500 1000 500 10 wavelength (in nm)</p><p>11Fig S1: FT-IR spectra of thiolated PEG conjugated with (A) lactonolactone (B) 7-aminocoumarin 3- 12carboxylic acid. 13 14 15 16 17 18 19 20</p><p>21</p><p>22</p><p>23</p><p>24</p><p>25</p><p>26</p><p>27</p><p>28 Fig S2 NMR spectra of thiolated PEG conjugated to 7-aminocoumarin 3-carboxylic acid. 29</p><p>30</p><p>31 32</p><p>33 3.0</p><p>2.5 34 PEG conjugated Coumarine 2.0 Absorbance intensity 7-aminocoumarin 3-carboxylic acid 35 1.5</p><p>1.0 373nm</p><p>36 0.5 368nm</p><p>0.0 37 300 400 500 600 700 800 900 1000 1100 wavelength (in nm) 38</p><p>39</p><p>40Fig S3: UV-Vis analysis curve showing hypsochromic shift in λmax value for 7-aminocoumarin 3- 41carboxylic acid and its PEG conjugated derivative 42 43 44 45 46 47 48</p><p>49</p><p>50</p><p>51</p><p>52</p><p>53</p><p>54 FigS4: FT-IR spectra of bare gold nanoparticles 55</p><p>56</p><p>57</p><p>58</p><p>59</p><p>60</p><p>61 1.0 62 522nm Bare Gold Nanoparticles 0.8 63 Bioconjugated Gold nanoparticles 0.6 64 Absorbance 0.4 563nm</p><p>65 0.2</p><p>66 0.0 67 400 500 600 700 800 68 wavelength (in nm) 69 70 71 72 Fig S5: UV-Vis analysis data showing bathochromic shift in λmax value of gold nanoparticles up on 73 conjugation 74</p><p>75</p><p>76</p><p>77</p><p>78 A B 79</p><p>80</p><p>81</p><p>82</p><p>83</p><p>84 Fig S6: QELS Data (A) and TEM Micrograph (B) of conjugated gold nanoparticles</p><p>85</p><p>86</p><p>87</p><p>88</p><p>89</p><p>90</p><p>91 CH2OH CH2OH 92 OH O O OH OH</p><p>CH2OH CH2OH O 93 OH OH O O O O OH OH 94 OH OH</p><p>95 OH OH</p><p>96 Scheme S1: Synthesis of lactonolactone from lactose 97 98</p><p>99</p><p>100</p><p>101</p><p>CHO COOH HOOC 102 Aniline/Ethanol 70-80oC + CH2 HOOC 103 CHO O O</p><p>Conc H2SO4/ConcHNO3 104 0oC</p><p>105</p><p>COOH 106</p><p>O O 107 O2N</p><p>SnCl2/HCl RT 108</p><p>COOH 109</p><p>O O 110 H2N 111 112 Scheme S2: Synthesis of 7-aminocoumarin 3-carboxylic acid 113 114. 115</p><p>116</p><p>117</p><p>118</p><p>119 120</p><p>121</p><p>122 HO O O OMe 123 n Thioacetate N /RT 124 2</p><p>HO O 125 O SAc n</p><p>126 Tosylchloride/Et3N 60oC/RT 127 tsO O O SAc n 128 NaN3/RT</p><p>129 N N O O SAc n 130 LiAlH4 o 131 0 C Derivative 3. iv H2N O O SH 132 n</p><p>133 Scheme S3: - Synthesis of SH-PEG-NH2 from monomethoxy PEG 5000 134</p><p>135</p><p>136</p><p>137</p><p>138</p><p>139</p><p>140</p><p>141</p><p>142 143</p><p>144</p><p>145</p><p>HO O 146 O OMe n Tosylchloride 147 DMAP+Et3N 60oC</p><p> tsO O 148 O OMe Derivative 4. i n DMSO 149 Na2HP4</p><p>OHC O O OMe Derivative 4. ii 150 n</p><p>151 Thiacetate N2/RT</p><p>O 152 OHC O SAc n</p><p>NaOMe/MeOH 153 HCl/RT O OHC O SH 154</p><p>155</p><p>156Scheme S4: Synthesis of SH-PEG-CHO from monomethoxy PEG 2000 157</p><p>158</p><p>159</p><p>160</p><p>161</p><p>162</p><p>163</p><p>164</p><p>165 166</p><p>167</p><p>168 CH2OH CH2OH O OH HS O O 169 OH O OH O NH2 O n 170</p><p>OH 171 OH CH2OH CH2OH O 172 HO OH O OH HC OH HS O O 173 O N n H</p><p>174 OH OH 175</p><p>176 Scheme S5:- Coupling of lactonolactone to SH-PEG-NH2 to form Adduct 1 177</p><p>178</p><p>179</p><p>180</p><p>181 COOH</p><p>182 HS O CHO O O O n H N 183 2 1. Et N 3 184 COOH 2. NaBH CN 3 185 H HS O C O O 186 O NH n 187</p><p>188 Scheme S6:- Coupling of Coumarin derivative to SH-PEG-CHO to form Adduct 2 189 190</p><p>191 Suppelmentary Methods</p><p>1922.2 Synthesis of Targeting Moiety: Lactonolactone</p><p>193</p><p>194Lactonolactone was synthesised by oxidizing the sugar moiety Lactose17. Briefly, lactose (2g;) was</p><p>195dissolved in minimum amount of hot water followed by its addition to an iodine solution in</p><p>196methanol(3g/40mL) at 40˚C. The reaction mixture was stirred for 2 hours. Following this, a concentrated</p><p>197solution of potassium hydroxide in methanol was added drop-wise to the reaction mixture until the colour</p><p>198of iodine disappeared. The solution was then cooled externally in an ice bath which led to the</p><p>199precipitation of a crystalline product. The product was filtered, repeatedly washed with cold methanol and</p><p>200recrystallized using water/methanol system.</p><p>201</p><p>202The potassium salt of lactobionic acid thus formed was converted to its free acid form by passing it</p><p>203through a column of Acidic amberlite resin. The acidic elute was concentrated and evaporated several</p><p>204times with methanol to get the final lactone as a highly viscous colourless oil. The scheme for the</p><p>205synthesis of lactonolactone from lactose is shown in Scheme S1. IR spectra: - 3371, 2898, 1736, 1647,</p><p>2061421, 1226, 1139, 1077, 1035, 891, 787</p><p>207</p><p>2082.2.3 Synthesis of Fluorescent moiety: 7-aminocoumarin 3-carboxylic acid</p><p>209</p><p>210The scheme for the synthesis of fluorescent 7-aminocoumarin 3-carboxylic acid is shown in Scheme S2.</p><p>211Synthesis of the fluorescent moiety was performed in three consecutive steps, the first one involving the</p><p>212synthesis of 3-carboxycoumarin from Salicyldehyde and Malonic acid as per the procedure discussed by</p><p>213Besson et al18, followed by its controlled nitration at cold temperatures to give 7-nitrocoumarin 3- 214carboxylic acid. The later was reduced with SnCl2/HCl mixture to yield the final product as a bright</p><p>215yellow solid. </p><p>216</p><p>217Data for 7-aminocoumarin 3-carboxylic acid: 1H N.M.R: - 8.93(d, COOH), 8.52(d, ArH), 7.65 (d, ArH),</p><p>-1 2184.18 (NH2), 3.86 (CH=C) IR spectra (ᶹ/CM ): - 3428, 3369-3400 (d, primary amine), 3069, 2855, 1741</p><p>219(C=O for lactone), 1705 (C=O for carboxylic acid), 1610 () N-H), 1096 (C-O), 1001, 947, 845.</p><p>220</p><p>2212.2.4 Synthesis of SH-PEG-NH2 from monomethoxy PEG 5000</p><p>222</p><p>223The synthesis of hetero bi-functional PEG derivative from monomethoxy PEG 5000 involves a sequence</p><p>224of steps as illustrated in Scheme S3. The synthetic procedures have been referred from a few</p><p>225publications19, 20 and were performed with slight modifications. </p><p>226</p><p>227Derivative 3.i: - Monomethoxy PEG 5000 (1.0Eq) was dissolved in minimum amount of DMF followed</p><p>228by the addition of Potassium thioacetate (10.0 Eq) under inert atmosphere. The reaction mixture was</p><p>229allowed to stir under nitrogen atmosphere for about 24 hours. The progress of the reaction was monitored</p><p>230through TLC (methanol/CH2Cl2 system). After the completion of the reaction, the crude reaction mixture</p><p>231was treated with CH2Cl2 and excess thioacetate was washed by adding equal portions of saturated solution</p><p>232of NH4Cl and brine. The aqueous and the organic layer were then separated followed by 4-5 times</p><p>233extraction of the aqueous layer with CH2Cl2. Finally, the organic layer aliquots were combined, evaporated</p><p>234and purified over alumina (methanol/CH2Cl2 system) to get the final crude product as yellow oil with foul</p><p>235smell. The yellow oil when triturated with Diethyl ether gave pale yellow solid as the final product. 1H</p><p>-1 236N.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 ) – 3433</p><p>237(br, m), 2888 (br, vs), 1680, 1467, 1343 (asymmetric S=O stretch), 1281, 1250, 1112 (C-O stretch), 963,</p><p>238842</p><p>239 240Derivative 3.ii - Thioacetate derivative of PEG was tosylated using the procedure as follows: - AcS-PEG-</p><p>241OH (1.0eq) was dissolved in minimum amount of toluene followed by addition of base triethylamine</p><p>242(3.0eq) and p-tosylchloride (1.5eq). The reaction mixture was stirred for about 5 hrs at a maintained</p><p>243temperature of 60˚C after which it was left on stirring for another 10 hrs at ambient temperature. Progress</p><p>244of the reaction was monitored through TLC (methanol/CH2Cl2 system). Following the completion of the</p><p>245reaction, the solvent was removed from the reaction mixture over Buchi rotary evaporator. The crude</p><p>246product was then dissolved in CH2Cl2, and treated with 0.25M aqueous HBr solution and brine which</p><p>247served as washing agents for excess triethylamine and tosylchloride, respectively. The aqueous and</p><p>248organic layers were separated. The aqueous layer was extracted 4-5 times with CH2Cl2. Finally, the organic</p><p>249layer aliquots were combined, evaporated and purified over alumina (Methanol/ CH2Cl2) to get the</p><p>250product as pale yellow coloured oil, which upon trituration with ether yielded pale yellow final product. .</p><p>1 251 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 (t,</p><p>-1 252CH2-Ots), 7.31 (d, ArH), 7.79 (d, ArH) IR Spectra (ᶹ/cm ): - 3400 (absorbed water), 2888, 1735, 1673,</p><p>2531466, 1344 (asymmetric S=O stretch), 1281, 1112, 1034(S-O stretch), 980, 842. </p><p>254</p><p>255Derivative 3.iii - Derivative 3.ii (1.0eq) was dissolved in minimum amount of DMF followed by the</p><p>256addition of sodium azide NaN3 (1.25eq) under inert atmosphere at ambient conditions. The reaction</p><p>257mixture was allowed to stir at the same conditions for 24hrs. The formation of product was confirmed</p><p>258through TLC (CH2Cl2/MeOH). The crude product was then repeatedly precipitated out from the reaction</p><p>259mixture by addition of dry ether. Purification by chromatography over alumina (EtOAc/MeOH) yielded</p><p>260the final product as pale yellow oil which upon trituration with ether gave pale yellow coloured solid. . 1H</p><p>261N.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</p><p>262(ᶹ/cm-1): - 2936, 2888, 2098 (azide stretch), 1644, 1466, 1346, 1281, 1110, 1034, 981, 843.</p><p>263</p><p>264Derivative 3.iv Under complete Argon atmosphere, a solution of LiAlH4 (5.0eq) in dry DMF was stirred</p><p>265for about half an hour at -10 to 0˚C (maintained in ice/Sodium chloride bath) in a dry round bottom flask, 266before a solution of Azido-PEG-Thioacetate (1.0eq) in DMF was added drop wise into it. Stirring at the</p><p>267maintained conditions was continued for another 4hrs. The progress of the reaction was analysed via</p><p>268Ellman’s test for thiol group and ninhydrin test for the amino group. After the completion of the reaction,</p><p>269double distilled water was cautiously added. Lithium hydroxide thus precipitated out was filtered over a</p><p>270pad of celite and washed repeatedly with ethanol. The filterate was concentrated, dissolved in minimum</p><p>271amount of CH2Cl2 and purified over alumina (EtOAc/MeOH system) to yield colourless oil. Trituration</p><p>272with dry ether yielded off white coloured Solid. 1.1–1.28 (m, SH), 1.97-2.1 (s, NH 2), 2.23–2.56 (m, CH2),</p><p>-1 2733.51–3.76 (m, CH2-CH2-O-CH2-CH2) IR spectra (ᶹ/cm ): - 3369 (strong, primary amine), 2918 (SH</p><p>274stretch), 2887, 1598 (N-H bend), 1465, 1346, 1282, 1112, 963</p><p>275</p><p>276</p><p>2772.2.5 Synthesis of SH-PEG-CHO from monomethoxy PEG 2000</p><p>278</p><p>279A versatile hetero-bifunctional polyethylene glycol (PEG) derivative containing active end-groups thiol</p><p>280and aldehyde was efficiently prepared from monomethoxy PEG as per the scheme in scheme S4. Though</p><p>281the synthetic procedures were referred from a few publications19, 20, 21, the synthesis was performed with</p><p>282considerable modifications. </p><p>283</p><p>284Derivative 4.i - Purified monomethoxy PEG (1.0eq) was dissolved in minimum amount of toluene </p><p>285followed by the addition of Et3N (3.0eq), DMAP (catalytic amount; 0.25eq) and p-tosylchloride (1.5eq). </p><p>286The reaction mixture was then heated in an oil bath maintained at 80˚C for 72 hrs. The progress of the </p><p>287reaction was analysed through TLC (CH2Cl2/MeOH system). After completion of the reaction, the </p><p>288reaction mixture was cooled to room temperature, the solvent was evaporated and the crude oil thus </p><p>289obtained was treated with CH2Cl2. Excessive reagents were removed through vigorous washings with </p><p>290saturated solution of NaHCO3 & brine and 0.25M aqueous HBr. Finally, the aqueous and organic layers </p><p>291were separated. The Aqueous layer was extracted extensively with CH2Cl2. The organic layer aliquots 292were combined, evaporated and purified over alumina (CH2Cl2/MeOH system) to obtain crude final </p><p>293product as colourless oil. Trituration with dry ether gave the final product as off white coloured solid. 1H </p><p>294N.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), </p><p>2957.33 (d, ArH), 7.77(d, ArH) Ir spectra (cm-1): - 3432, 2884, 1735, 1647, 1598, 1466, 1345 (asymmetric </p><p>296SO2 stretch), 1281, 1234, (symmetric SO2 stretch), 1112, 949, 842</p><p>297Derivative 4.ii - A solution of MeO-PEG-Ots (1.0eq) in 15mL DMSO was treated with Na2HPO4</p><p>298(20.0eq) and the mixture was stirred in an oil bath maintained at 100˚C for 20hrs. The progress of the</p><p>299reaction was monitored through TLC (CH2Cl2/MeOH system). After the completion of the reaction, the</p><p>300cooled reaction mixture was filtered and the filtrate was precipitated out several times with dry ether. The</p><p>301precipitate thus obtained was dissolved in minimum amount of water, dialysed and lyophilized to obtain</p><p>1 302the final product as an off-white coloured solid. H N.M.R- 3.32 (s, O-CH3), 3.56-3.71 (m, CH2-CH2-O-</p><p>-1 303CH2-CH2), 3.81(CH2-CH2-O-CH2-CHO), 4.17 (d CH2-CHO), 9.71(s, CHO) IR spectra (ᶹ/cm ): -3412</p><p>304(absorbed water), 2820-2835 (overtone for aldehyde) 2885, 1735 (C=O stretch), 1467, 1254, 1192, 1110,</p><p>305953, 847</p><p>306</p><p>307Derivative 4.iii- CHO-PEG-OMe was subjected to thioacetylation in the next step. To a solution of</p><p>308derivative 4.ii (1.0eq) in DMF, 1.5 equivalents of potassium thioacetate were added under inert</p><p>309atmosphere. The reaction was allowed to stir continuously at room temperature and nitrogen/argon</p><p>310atmosphere for 24 hrs. The progress of the reaction was monitored through TLC (methanol/CH2Cl2</p><p>311system). The work up of the reaction mixture was done following the same steps as done for derivative 3.i</p><p>1 312which gave the final thioacetated product as a pale yellow solid. H N.M.R- 2.16 (s, CH3), 3. 43(s,</p><p>313AcS-CH2), 3.51-3.68 (m, CH2-CH2-O-CH2-CH2), 3.81(CH2-CH2-O-CH2-CHO), 4.17 (d CH2-CHO),</p><p>3149.71(s, CHO) IR spectra (ᶹ/cm-1): -3412 (absorbed water), 2820-2835 (overtone for aldehyde) 2885, 1735,</p><p>3151687, 1467, 1350, 1254, 1192, 1110, 1034 (S-O strech), 953, 847</p><p>316 317</p><p>318Derivative 4.iv - To a solution of Derivative 4.iii (1.0eq) in degassed methanol, 5 equivalents of NaOMe</p><p>319in MeOH was added. The mixture was allowed to stir overnight at room temperature. Then, the mixture</p><p>320was acidified to pH 1–2 using 0.1N HCl. Solvent from the reaction mixture was evaporated over Buchi</p><p>321rotary evaporator to give the crude product. Purification by silica gel chromatography (CH 2Cl2/MEOH</p><p>322system) gave the bifunctional derivative as colourless oil, which upon trituration with Dry ether gave off-</p><p>1 323white coloured solid. H spectra 1.1–1.28 (m, SH), 2.23–2.56 (m, CH2), 3.51–3.76 (m, CH2-CH2-O-CH2-</p><p>324CH2), 3.81(CH2-CH2-O-CH2-CHO), 4.17 (d CH2-CHO), 9.71(s, CHO) IR spectra: - 3420 (absorbed</p><p>325water), 2918 (SH stretch) 2820-2835 (overtone for aldehyde), 2884, 2110, 1735, 1466, 1351, 1253, 1099,</p><p>3261022, 953, 845</p><p>327 </p><p>328</p><p>329NOTE: The coupling reactions of the thiolated PEG with Gold nanoparticles, target specific and</p><p>330fluorescent moieties were done within 2-3days of their synthesis as the thiol linkages lack stability in</p><p>331oxidizing environment and have strong tendency to form disulfides. </p><p>332</p><p>333</p>