Supplementary Data. Development of Tumor-Targeted Near Infrared Probes for Fluorescence Guided Surgery

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1 Supplementary Data Development of Tumor-Targeted Near Infrared Probes for Fluorescence Guided Surgery Lindsay E. Kelderhouse, Venkatesh Chelvam, Charity Wayua, Sakkarapalayam Mahalingam, Scott Poh, Sumith A. Kularatne, Philip S. Low * Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana Department of Chemistry, Indian Institute of Technology Indore, Indore , Madhya Pradesh, India On Target Laboratories, LLC, 1281 Win Hentschel Blvd, West Lafayette, Indiana * Corresponding author: Philip S. Low, Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN Tel: (765) Fax: (765) plow@purdue.edu. 1

2 Experimental Procedures Materials Amino acids for peptide synthesis were purchased from Chem-Impex Int. (Chicago, IL). N-Hydroxybenzotriazole (HOBt) and benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate) (PyBOP) were obtained from Peptide Int. (Louisville, KY). Solid phase peptide synthesis (SPPS) was performed using a standard peptide synthesis apparatus (Chemglass, Vineland, NJ). Unless otherwise specified, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). All peptide linkers and ligand targeted peptide dye conjugates were purified by preparative reverse phase (RP)-HPLC (Waters, xterra C18 10 µm; 19 x 250 mm) and LC/MS analyses were obtained using a Waters micromass ZQ 4000 mass spectrometer coupled with a UV diode array detector. Synthesis, purification and characterization of folate NIR probes The synthesis of folate-dylight 680, folate-dylight 750 and folate-ir800cw is outlined in Schemes 1 and 2. Under argon atmosphere, one mg of DyLight 680 maleimide, Dylight 750 maleimide (Thermo Scientific) or IR800CW NHS ester (Licor) was dissolved in anhydrous DMSO (100 µl) containing 50 equivalents of anhydrous diisopropylethylamine. A two-fold molar excess of a folate peptide linker 7 (EC-119) (folate-γ-asp-arg-asp-asp-cys-sh) or folate-eda (linker 8), synthesized as described previously 28-31, was dissolved in anhydrous DMSO (100 µl) and was added to the above mixture. Product formation was confirmed by LC-MS and the crude folate NIR probes were then purified by RP-HPLC with a gradient mobile phase of A= 10mM ammonium acetate (ph 7.0) and B= methanol; λ = 285 nm; solvent gradient 0% B to 30% B in 30 2

3 min. The pure folate NIR probes 1, 2 and 3, were analyzed by LC-MS (ESI) mass spectrometry (Figures 1-3) and were freeze dried and stored at 4 C until use for in vitro or in vivo studies. Synthesis, purification and characterization of PSMA targeted NIR probes Synthesis of DUPA-DyLight 680, DUPA-Alexa Fluor 647 and DUPA-IR800CW is outlined in Scheme 3. Under argon atmosphere, one mg of DyLight 680 or Alexa Fluor 647 maleimide (Thermo Scientific) or 800CW maleimide (Licor) was dissolved in anhydrous DMSO (100 µl) containing 50 equivalents of anhydrous diisopropylethylamine. A two-fold molar excess of a DUPA peptide linker 9, synthesized as described previously 23-24, dissolved in anhydrous DMSO (100 µl), was added to the above mixture and stirred at room temperature. The formations of products were confirmed by LC-MS. The crude DUPA NIR probes were then purified by preparative RP-HPLC with a gradient mobile phase of A= 10mM ammonium acetate (ph 7.0) and B= methanol; λ = 285 nm; solvent gradient 0% B to 80% B in 30 min. The pure DUPA NIR probes 4, 5 or 6, were analyzed by LC-MS (ESI) mass spectrometry (Figures 4-6) and were freeze dried and stored at 4 C until use for in vitro or in vivo studies. Tissue penetration of NIR dyes. Fresh porcine flesh was purchased from the Purdue Butcher Block and cut into slabs of 1cm thickness. One milliliter solutions of each dye (100 nm) was dispensed into 1.5 ml Eppendorf tube and placed under the porcine flesh. Images were taken using a Kodak Image Station operated with Kodak molecular imaging software (version 4.5; Carestream Molecular Imaging) as shown in Figure 7. Settings: [white light images: illumination 3

4 source = white light transillumination, acquisition time = sec, f-stop = 11, focal plane = 5, FOV = 160, binning = none]. Optical image settings for Alexa Fluor 647 and DyLight 680 conjugates: [illumination source = multiwavelength (625 nm), acquisition time = 4 min, f-stop = 2.80, focal plane = 5, FOV = 160, binning = 4 pixels]. Optical image settings for DyLight 750 and IR800CW conjugates: [illumination source = multiwavelength (755 nm), acquisition time = 4 min, f-stop = 2.80, focal plane = 5, FOV = 160, binning = 4 pixels]. The same tubes were also imaged using a Caliper Ivis Lumina II Imaging Station with Living Image 4.0 software as shown in Figure 8. Settings for imaging Alexa Fluor 647 and DyLight 680 conjugates: [lamp level: high; excitation: 645; emission: Cy5.5; epi illumination; binning: (M) 4; FOV = 7.5; f-stop=4; acquisition time=1s.] Settings for imaging DyLight 750 and IR800CW conjugates: [lamp level: high; excitation: 745; emission: ICG; epi illumination; binning: (M) 4, FOV=12.5; f-stop=4; acquisition time=1s]. 4

5 SCHEME 1. Synthesis of folate-dylight 680 and folate-dylight 750 from EC-119 linker. (a) DyLight 680 maleimide or Dylight 750 maleimide/dipea (50 equivalents)/ DMSO/ RT/ overnight/argon. 5

6 SCHEME 2. Synthesis of folate-ir800cw from folate-eda linker and IR800CW NHS ester. (a) IR800CW NHS ester/dmso/dipea (50 equivalents)/rt/overnight/argon. 6

7 SCHEME 3. Synthesis of DUPA-DyLight 680, DUPA-Alexa Fluor 647 and DUPA-IR800CW. (a) Alexa Flour 647 maleimide, DyLight 680 maleimide or IR800CW maleimide/ DMSO/ DIPEA (50 equivalents)/rt/overnight/argon. 7

8 Figure 1. LC/MS of folate-dylight 680 8

9 Figure 2. LC/MS of folate-dylight 750 9

10 Figure 3. LC/MS of folate-ir800cw 10

11 Figure 4. LC/MS of DUPA-DyLight

12 Dye structure not disclosed by manufacturer. Figure 5. LC/MS of DUPA-Alexa Fluor

13 Figure 6. LC/MS of DUPA-IR800CW 13

14 Figure 7: Imaging of NIR dyes through 1 cm of fresh porcine flesh using a Kodak Image Station. One ml of various fluorescent dyes (100nM) was placed in an Eppendorf tube beneath 1cm of fresh porcine flesh and imaged using a Kodak Image Station at the appropriate excitation wavelength. A: Alexa Fluor 647 (λ ex =625nm), B: DyLight 680 (λ ex =625nm), C: DyLight 750 (λ ex =755nm) and D: IR800CW (λ ex =755nm). 14

15 Figure 8: Imaging of NIR dyes through 1 cm of fresh porcine flesh using a Caliper Ivis Lumina II Imaging Station. One ml of various fluorescent dyes (100nM) was placed in an Eppendorf tube beneath 1cm of fresh porcine flesh and at the appropriate excitation wavelength. A: Alexa Fluor 647 (λ ex =645nm), B: DyLight 680 (λ ex =645nm), C: DyLight 750 (λ ex =745nm) and D: IR800CW (λ ex =745nm). 15