Supporting Information

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Supporting Information

1. Synthesis of FMOC-ethylenediamine-cyanocobalamin

Cyanocobalamin (1.00 g, 0.736 mmol) and 1,1’-carbonyldi-(1,2,4-triazole) (483 mg, 2.94 mmol) were added to a round bottom flask (100 mL). Anhydrous DMSO (15 mL) was added to the flask and stirred at room temperature for 2 h. Ethylenediamine (159 μL, 2.93 mmol) was then added and stirred for an additional 2.5 h. The solution was divided into 5 mL aliquots and diluted by 10x with 2:1 diethyl ether: DCM, centrifuged to a pellet and decanted.

The pellet was dissolved in DMF and 4 eq of FMOC-OSu (982 mg, 2.91 mmol) was added to a round bottom flask (100 mL). The solution was reacted for 3 h and then precipitated with diethyl ether before being centrifuged and decanted. A red product was observed.

The product was dissolved in 10 mL of MeOH and centrifuged and decanted again to remove unreacted FMOC-OSu. A light-pink pellet was observed. The supernatant was diluted to 30:70 MeOH:H2O and loaded onto purified by flash chromatography (reverse phase, C-18 Ultra, 30 g).

A binary gradient solvent system (solvent A: 0.1 v/v% trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used with an equilibrating ratio (A:B) of 1:0 (35 mL/ 5 min).

The solvent gradient was varied from a ratio of 1:0 to 60:40 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (807 mg, 65.8% yield)

2. Synthesis of FMOC-trans-1,4-diaminocyclohexane-cyanocobalamin

Cyanocobalamin (1.00 g, 0.736 mmol) and 1,1’-carbonyldi-(1,2,4-triazole) (483 mg, 2.94 mmol) were added to a round bottom flask (100 mL). Anhydrous DMSO (15 mL) was added to the flask and stirred at room temperature for 2 h. trans-1,4-Diaminocyclohexane (334 mg, 2.93 mmol) was then added and stirred for an additional 2.5 h. The solution was divided into 5 mL aliquots and diluted by 10x with 2:1 diethyl ether: DCM, centrifuged to a pellet and decanted.

The solvent gradient was varied from a ratio of 1:0 to 60:40 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (795 mg, 62.8 yield)

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3. Synthesis of FMOC-1,8-octanediamine -cyanocobalamin

Cyanocobalamin (1.00 g, 0.736 mmol) and 1,1’-carbonyldi-(1,2,4-triazole) (483 mg, 2.94 mmol) were added to a round bottom flask (100 mL). Anhydrous DMSO (15 mL) was added to the flask and stirred at room temperature for 2 h. 1,8-Octanediamine (421 mg, 2.92 mmol) was then added and stirred for an additional 2.5 h. The solution was divided into 5 mL aliquots and diluted by 10x with 2:1 diethyl ether: DCM, centrifuged to a pellet and decanted.

4. Synthesis of FMOC-p-phenylenediamine-cyanocobalamin

Cyanocobalamin (1.00 g, 0.736 mmol) and 1,1’-carbonyldi-(1,2,4-triazole) (483 mg, 2.94 mmol) were added to a round bottom flask (100 mL). Anhydrous DMSO (15 mL) was added to the flask and stirred at room temperature for 2 h. p-Phenylenediamine (316 mg, 2.93 mmol) was then added and stirred for an additional 2.5 h. The solution was divided into 5 mL aliquots and diluted by 10x with 2:1 diethyl ether: DCM, centrifuged to a pellet and decanted.

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5. Synthesis of FMOC-ethylenediamine-cobalamin-ethyl

FMOC-Eda-Cbl-CN (105 mg, 0.063 mmol) was dissolved in 10mL dry MeOH, then 5% w/v NH4Br was added with 100 eq. Zn powder. The reaction was allowed to react for 30 minutes and periodically checked for a dark-grey/black solution indicative of the super nucleophilic Co(I) species. Then, 4 eq of bromoethane was added and the reaction was shaken for 4 hours. The solution was centrifuged to pellet the Zn, and the supernatant was decanted. The supernatant was diluted to 50:50 MeOH:H2O. After dilution, the solution was centrifuged to pellet any

precipitated FMOC-Cbl-Ethyl. The supernatant was loaded onto the C18-Ultra 30g column after equilibration of the column. The pellet was dissolved with 10 mL DMF and diluted to 40 mL with H2O. The solution was centrifuged again and the supertant was loaded. This process was repeated three times.

A binary gradient solvent system (solvent A: 0.1 v/v% trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used with an equilibrating ratio (A:B) of 1:1 (35 mL/min, 5 min). The solvent gradient was varied from a ratio of 30:70 to 85:15 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (62.5 mg, 59.4% yield)

6. Synthesis of FMOC-trans-1,4-diaminocyclohexane-cobalamin-ethyl

FMOC-(trans)Dach-Cbl-CN (158 mg, 0.092 mmol) was dissolved in 10mL dry MeOH, then 5%

w/v NH4Br was added with 100 eq. Zn powder. The reaction was allowed to react for 30 minutes and periodically checked for a dark-grey/black solution indicative of the super

nucleophilic Co(I) species. Then, 4 eq of bromoethane was added and the reaction was shaken for 4 hours. The solution was centrifuged to pellet the Zn, and the supernatant was decanted. The supernatant was diluted to 50:50 MeOH:H2O. After dilution, the solution was centrifuged to pellet any precipitated FMOC-Cbl-Ethyl. The supernatant was loaded onto the C18-Ultra 30g column after equilibration of the column. The pellet was dissolved with 10 mL DMF and diluted to 40 mL with H2O. The solution was centrifuged again and the supernatant was loaded. This process was repeated three times. A binary gradient solvent system (solvent A: 0.1 v/v%

trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used with an

equilibrating ratio (A:B) of 1:1 (35 mL/min, 5 min). The solvent gradient was varied from a ratio of 30:70 to 85:15 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (66 mg, 41.8% yield)

7. Synthesis of FMOC-1,8-octanediamine-cobalamin-ethyl

FMOC-Oda-Cbl-CN (334 mg, 0.191 mmol) was dissolved in 10mL dry MeOH, then 5% w/v NH4Br was added with 100 eq. Zn powder. The reaction was allowed to react for 30 minutes and periodically checked for a dark-grey/black solution indicative of the super nucleophilic Co(I) species. Then, 4 eq of bromoethane was added and the reaction was shaken for 4 hours. The solution was centrifuged to pellet the Zn, and the supernatant was decanted. The supernatant was

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diluted to 50:50 MeOH:H2O. After dilution, the solution was centrifuged to pellet any

precipitated FMOC-Cbl-Ethyl. The supernatant was loaded onto the C18-Ultra 30g column after equilibration of the column. The pellet was dissolved with 10 mL DMF and diluted to 40 mL with H2O. The solution was centrifuged again and the supernatant was loaded. This process was repeated three times.

A binary gradient solvent system (solvent A: 0.1 v/v% trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used with an equilibrating ratio (A:B) of 1:1 (35 mL/min, 5 min). The solvent gradient was varied from a ratio of 30:70 to 85:15 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (163 mg, 48.7% yield)

8. Synthesis of FMOC-p-phenyldiamine-cobalamin-ethyl

FMOC-Pda-Cbl-CN (334 mg, 0.195 mmol) was dissolved in 10mL dry MeOH, then 5% w/v NH4Br was added with 100 eq. Zn powder. The reaction was allowed to react for 30 minutes and periodically checked for a dark-grey/black solution indicative of the super nucleophilic Co(I) species. Then, 4 eq of bromoethane was added and the reaction was shaken for 4 hours. The solution was centrifuged to pellet the Zn, and the supernatant was decanted. The supernatant was diluted to 50:50 MeOH:H2O. After dilution, the solution was centrifuged to pellet any

precipitated FMOC-Cbl-Ethyl. The supernatant was loaded onto the C18-Ultra 30g column after equilibration of the column. The pellet was dissolved with 10 mL DMF and diluted to 40 mL with H2O. The solution was centrifuged again and the supernatant was loaded. This process was repeated three times.

A binary gradient solvent system (solvent A: 0.1 v/v% trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used with an equilibrating ratio (A:B) of 1:1 (35 mL/min, 5 min). The solvent gradient was varied from a ratio of 30:70 to 85:15 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (267 mg, 80.8% yield)

9. Synthesis of ethylenediamine-cobalamin-ethyl

FMOC-Eda-Cbl-Ethyl (109 mg, 0.0653 mmol) was dissolved in 5 mL dry DMF, then 10 eq of piperidine was added and the reaction was stirred for 1.5 h. The solution was precipitated with ether and centrifuged. The pellet was dissolved in 2 mL MeOH and diluted to 10% MeOH with H2O. A C18-Ultra 30g column was conditioned with H2O (35 mL/min, 5 min) and solution was loaded onto column. A binary gradient solvent system (solvent A: 0.1 v/v% trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used. The solvent gradient was varied from a ratio of 0:100 to 50:50 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (94.4 mg, 99.7% yield)

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10. Synthesis of trans-1,4-diaminocyclohexane-cobalamin-ethyl

FMOC-(trans)Dach-Cbl-Ethyl (85 mg, 0.0494 mmol) was dissolved in 5 mL dry DMF, then 10 eq of piperidine was added and the reaction was stirred for 1.5 h. The solution was precipitated with ether and centrifuged. The pellet was dissolved in 2 mL MeOH and diluted to 10% MeOH with H2O. A C18-Ultra 30g column was conditioned with H2O (35 mL/min, 5 min) and solution was loaded onto the column. A binary gradient solvent system (solvent A: 0.1 v/v%

trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used. The solvent gradient was varied from a ratio of 0:100 to 50:50 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (40.3 mg, 54.4% yield)

11. Synthesis of 1,8-octanediamine-cobalamin-ethyl

FMOC-Oda-Cbl-Ethyl (163 mg, 0.930 mmol) was dissolved in 5 mL dry DMF, then 10 eq of piperidine was added and the reaction was stirred for 1.5 h. The solution was precipitated with ether and centrifuged. The pellet was dissolved in 2 mL MeOH and diluted to 10% MeOH with H2O. A C18-Ultra 30 g column was conditioned with H2O (35 mL/min, 5 min) and solution was loaded onto the column. A binary gradient solvent system (solvent A: 0.1 v/v% trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used. The solvent gradient was varied from a ratio of 0:100 to 50:50 A:B. MeOH was removed under reduced pressure, and the

fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (85 mg, 59.7 % yield)

12. Synthesis of p-phenylenediamine-cobalamin-ethyl

FMOC-Pda-Cbl-Ethyl (267 mg, 0.156 mmol) was dissolved in 5 mL dry DMF, then 10 eq of piperidine was added and the reaction was stirred for 1.5 h. The solution was precipitated with ether and centrifuged. The pellet was dissolved in 2 mL MeOH and diluted to 10% MeOH with H2O. A C18-Ultra 30g column was conditioned with H2O (35 mL/min, 5 min) and solution was loaded onto the column. A binary gradient solvent system (solvent A: 0.1 v/v% trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used. The solvent gradient was varied from a ratio of 0:100 to 50:50 A:B. MeOH was removed under reduced pressure, and the

fractions were frozen at -80ᵒC before lyophilization, yielding a red solid (119.9 mg, 51.4 % yield).

13. Synthesis of BODIPY-ethylenediamine-cobalamin-ethyl

BODIPY FL (0.9 eq, 3.58 mg, 0.0122 mmol) and 10 eq DIPEA were combined in 5 mL dry- DMF. Then, 0.9 eq of HATU (4.65 mmg, 0.0122 mmol) were added. After 5 minutes, 1 eq of Eda-Cbl-Ethyl (19.6 mg, 0.0136 mmol) was added and the solution was reacted for 1.5 h. The solution was precipitated with ether and the pellet was dissolved in 3 mL MeOH and diluted to

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10% MeOH with H2O. A C18-Ultra 12 g was conditioned with H2O (15 mL/min, 5 min) and the solution was loaded onto the column. A binary gradient solvent system (solvent A: 0.1 v/v%

trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used. The solvent gradient was varied from a ratio of 10:90 to 25:75 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80C before lyophilization which yielded a red solid (4.5 mg, 23.0% yield)

This procedure was repeated with trans-1,4-diaminocyclohexane-cobalamin-ethyl (19.6 mg, 0.0131 mmol) producing BODIPY-trans-1,4-diaminocyclohexane-cobalamin-ethyl in (5.0 mg, 20.7% yield)

14. Synthesis of TAMRA-ethylenediamine-cobalamin-ethyl

5-TAMRA (0.9 eq, 6.58 mg, 0.0153 mmol) and 10 eq. DIPEA were combined in 5 mL dry- DMF. Then, 0.9 eq of HATU (5.81 mg, 0.0153 mmol) were added. After 5 minutes, 1 eq of Eda- Cbl-Ethyl (24.5 mg, 0.0170 mmol) was added and the solution was reacted for 1.5 h. The

solution was precipitated with ether and the pellet was dissolved in 3 mL MeOH and diluted to 10% MeOH with H2O. A C18-Ultra 12 g was conditioned with H2O (15 mL/min, 5 min) and the solution was loaded onto the column. A binary gradient solvent system (solvent A: 0.1 v/v%

trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used. The solvent gradient was varied from a ratio of 10:90 to 25:75 A:B. MeOH was removed under reduced pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (4.2 mg, 13.3% yield )

This procedure was repeated with trans-1,4-diaminocyclohexane-cobalamin-ethyl (32.4 mg, 0.216 mmol) producing TAMRA-trans-1,4-diaminocyclohexane-cobalamin-ethyl (11.3 mg, 27.3% yield)

This procedure was repeated with 1,8-octanediamine-cobalamin-ethyl (24.2 mg, 0.016 mmol) producing TAMRA-1,8-octanediamine-cobalamin-ethyl (13.2 mg, 42.9% yield)

This procedure was repeated with p-phenylenediamine-cobalamin-ethyl (40.0 mg, 0.210 mmol) producing TAMRA-trans-1,4-diaminocyclohexane-cobalamin-ethyl (12.0 mg, 23.5% yield)

15. Synthesis of Cy3-ethylenediamine-cobalamin-ethyl

Cyanine-3 (0.9 eq, 8.7 mg, 0.0162 mmol) and 10 eq. DIPEA were combined in 5 mL dry-DMF.

Then, 0.9 eq of HATU (6.2 mg, 0.0163 mmol) were added. After 5 minutes, 1 eq of Eda-Cbl- Ethyl (26 mg, 0.018 mmol) was added and the solution was reacted for 1.5 h. The solution was precipitated with ether and the pellet was dissolved in 3 mL MeOH and diluted to 10% MeOH with H2O. A C18-Ultra 12 g column was conditioned with H2O (15 mL/min, 5 min) and the solution was loaded onto the column. A binary gradient solvent system (solvent A: 0.1 v/v%

trifluoroacetic acid (TFA)/H2O; solvent B: 0.1% v/v% TFA/MeOH) was used. The solvent gradient was varied from a ratio of 10:90 to 25:75 A:B. MeOH was removed under reduced

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pressure, and the fractions were frozen at -80ᵒC before lyophilization which yielded a red solid (9.1 mg, 23.7%)

This procedure was repeated with trans-1,4-diaminocyclohexane-cobalamin-ethyl (15 mg, 0.010 mmol) producing Cy3-trans-1,4-diaminocyclohexane-cobalamin-ethyl in (10 mg, 51.5% yield)

Characterization

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Figure S1. Chromatogram of Purified Cbl-Ethyl with overlayed ESI MS spectra. Monitored at 360 nm

Figure S2. Chromatogram of purified Acyl-Eda-Cbl-Ethyl with overlayed ESI MS spectra.

Monitored at 360 nm

min

2 4 6 8 10 12 14 16

mAU

0 10 20 30 40 50 60

13.223

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Figure S3. Chromatogram of purified BOD-Eda-Cbl-Ethyl with overlayed ESI MS spectra.

Monitored at 360 nm

Figure S4. Chromatogram of purified BOD-(trans)Dach-Cbl-Ethyl with overlayed ESI MS spectra. Monitored at 360 nm

min

2 4 6 8 10 12 14 16

mAU

0 10 20 30 40 50 60 70

11.348

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Figure S5. Chromatogram of purified TAMRA-Eda-Cbl-Ethyl with overlayed ESI MS spectra.

Monitored at 360 nm

Figure S6. Chromatogram of purified TAMRA-(trans)Dach-Cbl-Ethyl with overlayed ESI MS spectra. Monitored at 360 nm

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Figure S7. Chromatogram of purified TAMRA-Oda-Cbl-Ethyl with overlayed ESI MS spectra.

Monitored at 360 nm

Figure S8. Chromatogram of purified TAMRA-Pda-Ethyl with overlayed ESI MS spectra Monitored at 360 nm.

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Figure S9. Chromatogram of purified Cy3-Eda-Cbl-Ethyl with overlayed ESI MS spectra.

Monitored at 360 nm

Figure S10. Chromatogram of purified Cy3-(trans)Dach-Cbl-Ethyl with overlayed ESI MS spectra. Monitored at 360 nm

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Additional photolysis data:

Figure S11. Photoproduct formation of TAMRA-Eda-Cbl-Ethyl at 60 minutes of irradiation with 550 nm or 510 nm. No significant difference was found between photoproduct formation with 510 nm light and photoproduct formation of controls.