µMap photoproximity labeling enables small

molecule binding site mapping

Supporting Information

Sean W. Huth§1,2, James V. Oakley §1,2, Ciaran P. Seath1,2, Jacob B. Geri1,2, Aaron
D. Trowbridge1,2, Dann L. Parker, Jr3., Frances P. Rodriguez-Rivera3, Adam G.
Schwaid4, Carlo Ramil4, Keun Ah Ryu5, Cory H. White5, Olugbeminiyi O.
Fadeyi5, Rob C. Oslund5, David W. C. MacMillan*1,2

1
Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States
2
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
3
Discovery Chemistry, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States
4
Discovery Chemistry, Merck & Co., Inc., Cambridge, Massachusetts 02141, United States
5
Merck Exploratory Science Center, Merck & Co., Inc., Cambridge, Massachusetts 02141,
United States

§These authors contributed equally to this work.

*Corresponding authors. Email: [email protected].

S0
Table of Contents

Supporting Figures ................................................................................................................. 2

General Considerations .........................................................................................................18

General materials ..................................................................................................................18

General Procedures ...............................................................................................................19

Procedures for site–of–labelling proteomic experiments .......................................................22

Modified peptide MS/MS data ..............................................................................................28

Syntheses ..............................................................................................................................63

Spectroscopic data ................................................................................................................88

References........................................................................................................................... 119

S1
Supporting Figures

Fig. S1. Selective labelling of carbonic anhydrase

The labelling reaction was carried out according to the general procedure using bovine carbonic
anhydrase (Sigma–Aldrich, C2624) and sulfonamide–PEG3–Ir (1) in DPBS, and 4–
sulfamoylbenzoic acid (Sigma–Aldrich, C11804).

S2
Fig. S2. Selective labelling of BRD4.

The labelling reaction was carried out according to the general procedure using recombinant
(His)6-tagged BRD4 and (+)-JQ–1–PEG3–Ir (5) or (–)-JQ–1–PEG3–Ir. The Labeling ratio of
BRD4 in lane 1 to BRD4 in lane 2 is 2.4-fold greater.

S3
Fig. S3. BRD4 MS Open Search
The BRD4 proteomics data was searched with MSFragger via an Open workflow to identify
modification masses from 0-700 Da. Among the highest confidence modifications by PSMs was
the mass corresponding to the intact carbene insertion product of the diazirine-PEG3-biotin probe.

S4
 Representative Western blot (n = 3)

lane 1 2 3 1 2 3
Dz-biotin (100 µM) + + + + + +
Ir diol (0.8 µM) + — — + — —
Ir-dasatinib(0.8 µM) — + + — + +
dasatinib (20 µM) — — + — — +
BSA
BTK(1.0 µM)
(1.0 µM)
BSA (1.0 µM)
BTK (1.0 µM)

Streptavidin 800 Total Protein Stain

lane BTK:BSA (normalized intensity ratio)

1 0.38
2 4.7
3 0.93

Fig. S4. Selective labelling of BTK.

The labelling reaction was carried out according to the general procedure using BTK
(ThermoFisher, PR5442A), dasatinib–PEG3–Ir (4), and dasatinib (Sigma–Aldrich, SML2589).

S5
Fig. S5. Selective labelling of CDK2 / cyclin A complex

The labelling reaction was carried out according to the general procedure using recombinant CDK2
(Thermo Fisher, PV3267), AT7519–PEG3–Ir (9), and AT7519 (Cayman Chemical, 16231).

S6
 Representative Western blot (n = 3)

lane 1 2 3 1 2 3
Dz-biotin (100 µM) + + + + + +
Ir diol (0.8 µM) + — — + — —
Ir-lenalidomide (0.8 µM) — + + — + +
lenalidomide (20 µM) — — + — — +
CRBN (1.0 µM)

BSA (1.0 µM)

Streptavidin 800 Total Protein Stain

lane CRBN:BSA (normalized intensity ratio)

1 0.34
2 5.4
3 2.0

Fig. S6. Selective labelling of CRBN

The labelling reaction was carried out according to the general procedure using CRBN (abcam,
ab162449), lenalidomide–PEG3–Ir (6), and lenalidomide (astatech).

S7
 Representative Western blot (n = 3)

lane 1 2 1 2
Dz-biotin (100 µM) + + + +
Ir-rapamycin (0.4 µM) + + + +
rapamycin (20 µM) + — + —
mTOR (0.5 µM)
BSA (0.5 µM)
FKBP12 (1.0 µM)
Streptavidin 800 Total Protein Stain

Target:BSA (normalized intensity ratio);

lane mTOR:BSA/FKBP12:BSA

1 0.59/0.051
2 4.9/0.42

Fig. S7. Selective labelling of mTOR/FKBP12

The labelling reaction was carried out according to the general procedure using recombinant
mTOR (Invitrogen), FKBP12 (R&D systems), rapamycin–PEG3–Ir (7), and rapamycin.

S8
Fig. S8. Selective labelling of STAT3

The labelling reaction was carried out according to the general procedure using recombinant
STAT3 (Acro), MM-206-PEG3-Ir (10), and MM-206 (9).

S9
Fig. S9. Volcano Plot of Modified Peptides for Carbonic Anhydrase

S10
Fig. S10. Volcano Plot of Modified Peptides for BRD4

S11
Fig. S11. Volcano Plot of Modified Peptides for BTK

S12
Fig. S12. Volcano Plot of Modified Peptides for CDK2

S13
Fig. S13. Volcano Plot of Modified Peptides for CRBN

S14
Fig. S14. Volcano Plot of Modified Peptides for FKBP12

S15
Fig. S15. Volcano Plot of Modified Peptides for MTOR

S16
Fig. S16. Volcano Plot of Modified Peptides for STAT3

S17
General Considerations
Synthetic methods
Organic solvents were purified according to the method of Grubbs1. Water was purified using a
Millipore Milli–Q Integral Water Purification System. Organic solutions were concentrated under
reduced pressure on a Büchi rotary evaporator using a water bath. 1H NMR spectra were recorded
on a Bruker UltraShield Plus Avance III 500 MHz unless otherwise noted and are internally
referenced to residual solvent signals. Data for 1H NMR are reported as follows: chemical shift (δ
ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m = multiplet, dd =
doublet of doublets, dt = doublet of triplets…etc, br = broad), coupling constant (Hz) and
integration. Irradiation of samples was performed in either a PennOC Photoreactor for small–
molecule sensitization experiments (PennOC, Pennsburg, PA, Model M1) or a Biophotoreactor
(Efficiency Aggregators, Richland, Tx, Fisher, NC1558343 BPR200). Spectral analysis of light
sources was performed using a UPRtek MK350 handheld spectrometer. Chromatographic
purification was carried out using a Biotage Isolera Prime flash chromatography system with
SilaSep flash cartridges (60 mesh) and UV detection. 13C NMR spectra were recorded on a Bruker
UltraShield Plus Avance III 500 MHz (125 MHz) and data are reported relative to the solvent
employed. High resolution mass spectra and intact protein mass spectra were obtained from the
Princeton University Mass Spectral Facility and Princeton Proteomics & Mass Spectrometry Core.

General materials
All buffers and synthetic starting materials were used as received from commercial sources.
Ascorbic acid (BP321–500) and ethanol (BP2818100) were purchased from Fisher Scientific
(Pittsburgh, PA). Bovine serum albumin (BSA) (A7906), Eppendorf Protein LoBind tubes
(Z666505), iBright Prestained Protein ladder (LC5615) were purchased from Thermo Scientific
(Rockford, IL). TBST (IBB–581X) was purchased from Boston BioProducts (Ashland, MA). 5M
Sodium chloride (S24600–500.0) was purchased from Research Products International (Mt.
Prospect, S19 IL). 12% Criterion TGX precast gels (5671044) and 4x Laemmli sample buffer
(161–0747) were purchased from Bio–Rad (Hercules, CA). 20% SDS solution (351–066–721) was
purchased from Quality Biological (Gaithersburg, MD). Biotin–PEG3–diazirine (3), Ir-G1 alkyne2

S18
(12), Ir-G3 DBCO3 (14), Ir-G2 PEG3 CO2H (16), (+)-JQ-1-PEG3 Ir (4), (–)-JQ-1-PEG3 Ir, and
(His)6-tagged BRD44 (15), and MM-2065 (9) were prepared as previously reported.

N N CO2—
CO2—
F3C F3C
H F3C F
F Me
N O Me Me
Me
O N
HO N
N F
N F
O O Ir
O Ir F
F N
N
HN O N
H HN MeO
HO N
Me
O NH Me Me F
Me F F3C
S H F3C
CO2H
CO2H

Biotin-PEG3-diazirine (3) Ir-bpydiol (11) Ir-G1 alkyne (12)

CO2—
F3C F3C
F PF6 F

Me N Me N

N F N F
Ir Ir
F F
N O N
H
N N N N
HO2C N
H
F O O F
F3C F3C
CO2H
Ir-G2 acid (13)
Ir-G3 DBCO (14)

F3C
F PF6
F3C
F PF6
Me N
Me N
N F
Cl
Ir N
F F
N Ir
H H F
N N N N N
Me
* O H
3 HO2C N N
O O F O
N N F3C 3
O F
Me S N F3C
Me
(+)-JQ1-PEG3-Ir (4)
(–)-JQ1-PEG3-Ir (15) Ir-G2 PEG3-CO2H (16)

General Procedures
General Western blot protocol
Gel electrophoresis was performed using a Bio–Rad Criterion Vertical Electrophoresis Cell tank,
Bio–Rad PowerPac Basic Power Supply, and Criterion TGX tris–glycine polyacrylamide gel
cassettes (SDS/Tris). After electrophoresis, gels were transferred from precast cassettes to
nitrocellulose or PVDF membranes using an iBlot 2 gel transfer device (Thermo Fisher, IB21001,

S19
IB23001), and washed with water. The membranes were then immersed in REVERT total protein
stain (Li–Cor, 926–11011) for 5 minutes. Excess stain was decanted, membranes washed with
6.7:30:63.3 AcOH:MeOH:H2O and imaged using a Li–Cor Odyssey CLx scanner in the 700 nm
channel. The membranes were washed with water, then immersed in Odyssey Blocking Buffer
(Li–Cor, 927–50000) and incubated for 1 hour. The blocking solution was then decanted, and 35
mL of fresh blocking buffer containing 70 µL of Tween 20 was added. This mixture was rocked
for 5 minutes. Afterwards, 1.5 µL of IRDye 800CW streptavidin (Li–Cor, 926–32230) was added
and the mixture incubated for 1 hour. The blocking buffer was then decanted, and the membranes
were washed with 1X TBST (3 x 5 min) and water before imaging via Li–Cor Odyssey CLx
scanner in the 800 nm channel. Pixel densitometry was performed using Image Studio Lite V. 5.2
(Li–Cor). The streptavidin 800 channel pixel density was then divided by the total protein stain
700 channel pixel density to provide a normalized biotinylation signal for each protein band.

General procedure for in vitro labelling Selectivity analysis

To a 0.5 mL Eppendorf tube was added a stock of target protein, either as provided commercially
or in DPBS to a final concentration of 1.0 µM. Then, the appropriate photocatalyst–conjugate was
added (to a final concentration of 0.9 µM) from a concentrated DPBS stock solution. To off
competition control samples, excess inhibitor is added first (to a final concentration of 10 µM) and
allowed to incubate at 24 °C for 5 minutes before the addition of the appropriate photocatalyst
conjugate (to a final concentration of 0.9 µM). In free photocatalyst control samples, the Ir-
diolbppy (11) is added (0.9 molar equivalents). Biotin-PEG3 diazirine (3) is then added to all
samples (to a final concentration of 100 µM) from a 1 mM stock in DPBS. The samples are then
briefly vortexed and placed in a transparent, airtight vessel (see image below) and allowed to
incubate in the dark at 24 °C for 30 minutes under H2O–saturated N2 gas. The samples are then
irradiated with at for 10 minutes at 450 nm in the airtight vessel. Following irradiation, 4x Laemmli
sample buffer w/ ß–mercaptoethanol was added (to 1X) and the samples were then denatured on a
heating block at 95 °C for 10 minutes. The samples were then analyzed by Western blot according
to the Western Blot General Protocol described above using a 10% PAGE gel.

S20
All irradiations were performed on an M2 photoreactor (Acceled) outfitted with a 450 nm LED
plate (Acceled). Irradiation intensity was set to 100% (corresponding to an output of 2.2 W) and
fan speed was set to 5200 rpm.

S21
Procedures for site–of–labelling proteomic experiments

General procedure for in vitro binding site determination experiment (CA,

BRD4, CDK2, BTK, CRBN, FKBP12, mTOR, and STAT3)

Sample preparation and irradiation

To a 0.5 mL Eppendorf tube was added a stock of target protein, either as provided commercially
or in DPBS to a final concentration of 1.0 µM (at least 4 µg of target protein per replicate is
recommended). Then, the appropriate photocatalyst–conjugate was added (to a final concentration
of 0.9 µM) from a concentrated DPBS stock solution. To off competition control samples, excess
inhibitor was added first (to a final concentration of 10 µM) and allowed to incubate at 24 °C for
5 minutes before the addition of the appropriate photocatalyst conjugate (to a final concentration
of 0.9 µM). In free photocatalyst control samples, the Ir-diolbppy (11) is added (0.9 molar
equivalents). Biotin-PEG3 diazirine (3) is then added to all samples (to a final concentration of
500 µM) from a 2.5 mM stock in DPBS. The samples are then briefly vortexed and placed in a
transparent, airtight vessel and then allowed to incubate in the dark at 24 °C for 30 minutes under
H2O–saturated N2 gas. The samples are then irradiated with at for 10 minutes at 450 nm in the
airtight vessel.

Mass spec sample preparation

Following irradiation, samples were reduced via DTT (Millipore-Sigma, 3860) (added to final
concentration of 10 mM) for 30 minutes at room temperature, followed by cysteine alkylation with
IAA (Sigma-Aldrich, I6125) (15 mM final concentration) for 30 minutes at room temperature in
the dark. IAA was then quenched with DTT (20 mM final concentration) for 15 minutes at room
temperature. Following this, samples were cleaned up via an SP3 bead protocol. 6 Reaction
mixtures were diluted 1:1 with reconstitution solution, before adding 100 ug total of SP3 beads
and homogenizing the solution. The bead-containing solution was then diluted 1:1 with ethanol
and incubated on a Thermo-Mixer at 1000 RPM for 5 minutes at room temperature. Following
bead binding, beads were pelleted on a magnetic rack and supernatant was discarded. Beads were
washed 3 times with 180 uL 80% ethanol in water solution. Following washes, beads were

S22
resuspended in 50 uL of 50 mM ammonium bicarbonate (Sigma-Aldrich, 09830) containing 0.5
µg of MS-grade trypsin (Thermo, 90057) and digested on a Thermo-Mixer at 1000 RPM and 37°C
overnight. Following digestion, supernatant was quenched with 0.5 uL Optima-Grade formic acid
(Fisher, A117-50). Sample was passed through a 0.22-micron spin filter column (CoStar, 98231-
UT-1), before being transferred to an LCMS vial for proteomics analysis.

General procedure for in-cell binding site determination experiment (BRD4 in

Jurkat cells)

Protocol for in cellulo binding site mapping

240M Jurkat cells at 2 million/ml (grown in RPM1-1640) cells were removed from T75 flasks and
divided into 6 portions (T75 flasks, 20 mL each). 4, (+)-JQ-1-Ir (5 μM final concentration, from a
20 mM stock in DMSO) was added to 3 flasks (Targeted), and 15, (-)-JQ-1-Ir (5 μM final
concentration, from a 20 mM stock in DMSO) was added to the remaining 3 flasks (negative
control) and the samples were incubated at 37 °C for 3 hours. After incubation, the cells were
removed, collected, and resuspended in DPBS buffered saline (1 mL/plex) and transferred to 1.5
mL Eppendorf tubes. Diazirine-PEG3-desthiobiotin (23) was added to each tube (250 μM) and the
samples were incubated at 23 °C for an additional 30 minutes. The tubes were subsequently
irradiated in a Photoreacor M2 (Acceled) equipped with a 450 nm LED plate (Acceled) for 10
minutes at 100% intensity. The cells were then washed twice with cold DPBS (4 °C) and
resuspended in 1 mL of 1X RIPA with 1X protease inhibitor cocktail. The lysed cells were
incubated on ice for 15 minutes and then sonicated (35%, 3 x 10s with 30s rest). The lysate was
then centrifuged at 15x1000g for 20 mins at 4 °C and the supernatant collected. The concentration
of the cell lysate was measured by BCA assay.

In-cell labeling sample preparation for mass spectrometry

For label free whole cell proteomics samples for target enrichment, Jurkat lysates were loaded onto
Pierce Streptavidin beads (Thermo, 88816) (0.5 mg Lysate per replicate, 50 uL of beads) and
incubated at 4C overnight. The next day, samples were reduced via DTT (Millipore-Sigma, 3860)
S23
(added to final concentration of 10 mM) for 30 minutes at room temperature, followed by cysteine
alkylation with IAA (Sigma-Aldrich, I6125) (15 mM final concentration) for 30 minutes at room
temperature in the dark. IAA was then quenched with DTT (20 mM final concentration) for 15
minutes at room temperature. Samples were then washed 3X with 1% SDS in DPBS ,3X with 1
M NaCl in DPBS, and 3X with 10% EtOH in DPBS. Finally, samples are washed 3X with 50 mM
Ammonium Bicarbonate buffer and resuspended in Ammonium Bicarbonate buffer (50 mM)
(Sigma-Aldrich, 09830) containing 0.5 ug of MS-grade trypsin (Thermo, 90057) per replicate and
digested at 37C overnight. Sample was passed through a 0.22-micron spin filter column (CoStar,
98231-UT-1), before being transferred to an LCMS vial for proteomics analysis.

For the in-cellulo binding site experiments, Jurkat lysates were loaded onto Pierce streptavidin
beads overnight at 4C (2.1 mgs of lysate, 200 uL beads). The next day, samples were reduced via
DTT (Millipore-Sigma, 3860) (added to final concentration of 10 mM) for 30 minutes at room
temperature, followed by cysteine alkylation with IAA (Sigma-Aldrich, I6125) (15 mM final
concentration) for 30 minutes at room temperature in the dark. IAA was then quenched with DTT
(20 mM final concentration) for 15 minutes at room temperature. Samples were then washed 3X
with 1% SDS in DPBS ,3X with 1 M NaCl in DPBS, and 3X with 10% EtOH in DPBS. Samples
were then washed 2X with Optima grade water, before being resuspended in 100 uL of Streptactin
BXT elution buffer (IBA, 2-1042-025), and allowed to incubate with rotation overnight at 4C.
Following elution, the resulting supernatant was cleaned up via SP3 bead protocol. Reaction
mixtures were diluted 1:1 with reconstitution solution, before adding 100 ug total of SP3 beads
and homogenizing the solution. The bead-containing solution was then diluted 1:1 with ethanol
and incubated on a Thermo-Mixer at 1000 RPM for 5 minutes at room temperature. Following
bead binding, beads were pelleted on a magnetic rack and supernatant was discarded. Beads were
washed 3 times with 180 uL 80% ethanol in water solution. Following washes, beads were
resuspended in 50 uL of 50 mM ammonium bicarbonate (Sigma-Aldrich, 09830) containing 0.5
µg of MS-grade trypsin (Thermo, 90057) and digested on a Thermo-Mixer at 1000 RPM and 37°C
overnight. Following digestion, supernatant was quenched with 0.5 uL Optima-Grade formic acid
(Fisher, A117-50). Sample was passed through a 0.22-micron spin filter column (CoStar, 98231-
UT-1), before being transferred to an LCMS vial for proteomics analysis.

S24
General mass-spectrometry and analysis procedures

For all binding site experiments, samples were injected (~100 ng per injection) onto a trap cartridge
(C18 Pepmap, 5 µM particle size, 5 mm length, 300 µM internal diameter) and then separated on
an analytical column (C18 ReproSil AQ, 1.9 µM particle size, 100mm length, 75 µM internal
diameter) at 500 nL/min with a nanoElute LC system connected in line to a Bruker TimsTOF Pro
2. The column temperature was maintained at 40 °C. Peptides were eluted via a water/acetonitrile
gradient (buffer A = 0.1% formic acid/water, buffer B = 0.1% formic acid/acetonitrile; flow rate
0.5 uL/min; gradient: start at 2% B, then increase to 35% B over 60 min, increase to 95% B over
half a min, hold at 95% for 2.25 min.) Scans were performed in positive ion, PASEF mode over a
m/z range of 100-1700 with a cycle time of 0.5s. Ion mobility parameters were set to 0.85-1.3
V•s/cm2, with a ramp time of 100 ms, accu. time of 100 ms, 100% duty cycle, 9.43 Hz ramp rate,
and MS averaging set to 1. Precursor m/z window was 0.5, with an abundance cutoff of 2%.

For the label free DIA experiment, samples were injected (~100 ng per injection) onto a trap
cartridge (C18 Pepmap, 5 µM particle size, 5 mm length, 300 µM internal diameter) and then
separated on an analytical column (C18 ReproSil AQ, 1.9 µM particle size, 100mm length, 75 µM
internal diameter) at 500 nL/min with a nanoElute LC system connected in line to a Bruker
TimsTOF Pro 2. The column temperature was maintained at 40 °C. Peptides were eluted via a
water/acetonitrile gradient (buffer A = 0.1% formic acid/water, buffer B = 0.1% formic
acid/acetonitrile;flow rate 0.5 uL/min; gradient: start at 2% B, then increase to 35% B over 20 min,
increase to 95% B over half a min, hold at 95% for 2.25 min.) Scans were performed in positive
ion, dia-PASEF mode over a m/z range of 100-1700 with a ramp time of 100 ms, Accu. time of
100 ms, and a duty cycle of 100%, ramp rate of 9.43 Hz, MS averaging set to 1. Absolute thresholds
were set to 10 for MS peaks, and 5000 for mobility peaks.

FragPipe DDA analysis

Analysis was performed in FragPipe v19.1. (MSFragger 3.7, IonQuant-1.8.10, philosopher 4.8.0,
python 3.9.12)789 Spectra were searched via LFQ-MBR (IM-MS), with a precursor mass tolerance
of 20 ppm and fragment mass tolerance of 20 ppm. Cleavage was specified as stricttrypsin with 2
missed cleavages. Modifications selected included cysteine alkylation as a fixed modification, and
S25
N-terminal deamidation and methionine oxidation as a variable modification. Spectra were
searched against a database containing the corresponding proteins (e.g. Carbonic Anhydrase 2)
and common contaminant proteins. An open search was performed with a mass window of 0 to
700 Da on the BRD4 example to identify modification masses, whereby the intact carbene
insertion product proved to be the highest confidence identified modification mass (see figure
S3).10 Searches were performed for the intact carbene insertion product mass of +616.2542 on any
residue. Results consist of at least 2 independent replicates per condition (targeted vs. off-
compete). The in-vitro search results were parsed for modification sites with high intensities vs.
the off-compete replicates, as well as passing a P-value and PSM threshhold. A detailed,
standardized, identification analysis example is provided below. For the in-vivo data, a nearly
identical protocol is used, but the insertion product mass is set to +586.2978 Da, corresponding to
the desthiobiotin probe. Because a much smaller number of modified peptides are detected in vivo
(including none in the off-compete), said modified residues were individually reviewed for
confidence and shown to all be binding site proximal.

Data Analysis to identify binding site residues in vitro

Due to the catalytic nature of our labeling platform, there are many modified peptides detected in
the in-vitro experiments. Thus, it is important to utilize certain criteria such as intensity change to
determine which peptides are the highest confidence. Below is a detailed workflow for
identification of "high-confidence” modified residues in the in-vitro experiments.

Upon completion of FragPipe analysis, the “combined.modified.peptide” file is opened in Perseus

(v2.0.7.0). Intensity values are annotated as the “main” input and all other descriptors are placed
in the categorical section. Data is then transformed (Log base 2), and missing values are replaced
via normal distribution (0.3 width, 1.8 downshift). Data is annotated as either “targeted” or “off-
compete” sample. Normalization is performed via median subtraction. Following this process, a
volcano plot is generated utilizing a t-test for statistical significance. Resulting volcano plots were
plotted in GraphPad Prism 9. Only residues with high confidence (P value > 1.3) and those with
the highest enrichment are selected. Further discrimination is achieved via PSM count: if a sample
contains less than 2 average PSMs across replicates, it is discarded. Excel sheets are color coded
to denote which peptides meet the criteria and which are discarded. (Yellow – included, red –
S26
discarded due to PSMs being low). Applying this method across all 6 examples yields high-
confidence binding site residues. Figures S9-S15 above contain volcano plots for all examples.

Data Independent Analysis for BRD4 Jurkat proteomics via DIANN

Data is processed via DIANN 1.8.1. Parameters set as follows: trypsin/P digestion, 3 missed
cleavages, 3 max. variable modifications, N-term M excision, Ox(M), Ac(N-term) and C-
carbamidomethylation. Peptide length range was 7-30, precursor charge range 1-4, m/z range 300-
1800, and fragment ion range 200-1800. Mass accuracy and MS accuracy are both set to 10. The
following settings on the algorithm are checked: “Use isotopologues”, “MBR”, “No shared
spectra”, “Heuristic protein inference”. Precursor FDR was set to 1%. A spectral library was used
generated via DIANN from all known human proteins (In-Silico specral library). Resulting
matrix.pg file was opened in Perseus (v2.0.7.0). Intensities are inputted as “main”, the rest of the
descriptors are categorical. Data is then transformed (Log base 2). Data is annotated as either
“targeted” or “inactive control” sample. Normalization is performed via median subtraction.
Following this process, a volcano plot is generated utilizing a t-test for statistical significance.
Resulting volcano plots were plotted in GraphPad Prism 9 for final figures.

S27
Modified peptide MS/MS data

Modified peptides for binding site mapping of Carbonic Anhydrase

Residue H2
Missed cleavages: 0
Charge: 2
m/z [Da]: 815.36
RT [sec]: 823.7156
Peptide Prophet Probability: 0.9656

Residue Q134
Missed cleavages: 0
Charge: 3
m/z [Da]: 957.141
RT [sec]: 1331.1376
Peptide Prophet Probability: 0.9999

S28
S29
Modified peptides for binding site mapping of BRD4

Residues F79, A80

Missed cleavages: 0
Charge: 4
m/z [Da]: 758.3548
RT [sec]: 3010.9946
Peptide Prophet Probability: 1.0

Residues A80, W81

Missed cleavages: 0
Charge: 4
m/z [Da]: 758.3548
RT [sec]: 2988.5149
Peptide Prophet Probability: 0.9998

S30
Residues W81, P82
Missed cleavages: 0
Charge: 3
m/z [Da]: 1010.81
RT [sec]: 3148.5615
Peptide Prophet Probability: 1.0

S31
Residues H77, Q78
Missed cleavages: 0
Charge: 4
m/z [Da]: 758.359
RT [sec]: 3026.5215
Peptide Prophet Probability: 0.9975

S32
Residues Q84
Missed cleavages: 1
Charge: 4
m/z [Da]: 855.924
RT [sec]: 2917.3277
Peptide Prophet Probability: 0.9963

S33
S34
Modified peptides for binding site mapping of BTK

Residue K406
Missed cleavages: 1
Charge: 3
m/z [Da]: 823.43
RT [sec]: 3309.5057
Peptide Prophet Probability: 1.0

Residue E407
Missed cleavages: 0
Charge: 2
m/z [Da]: 875.938
RT [sec]: 2288.7469
Peptide Prophet Probability: 1.0

S35
Residue K417
Missed cleavages: 1
Charge: 3
m/z [Da]: 700.355
RT [sec]: 2341.7363
Peptide Prophet Probability: 1.0

S36
Residue H491
Missed cleavages: 1
Charge: 4
m/z [Da]: 584.531
RT [sec]: 2034.0514
Peptide Prophet Probability: 0.9999

S37
Residue Y551
Missed cleavages: 0
Charge: 2
m/z [Da]: 1089.992
RT [sec]: 2368.5013
Peptide Prophet Probability: 0.9998

S38
S39
Modified peptides for binding site mapping of CRBN

Residue D149
Missed cleavages: 0
Charge: 2
m/z [Da]: 961.957
RT [sec]: 2796.1322
Peptide Prophet Probability: 0.9999

Residue F150
Missed cleavages: 0
Charge: 3
m/z [Da]: 641.64
RT [sec]: 2734.5065
Peptide Prophet Probability: 1.0

S40
Residue G151
Missed cleavages: 1
Charge: 4
m/z [Da]: 881.67
RT [sec]: 2918.6311
Peptide Prophet Probability: 0.9719

S41
Residue I152
Missed cleavages: 0
Charge: 2
m/z [Da]: 961.957
RT [sec]: 2774.7212
Peptide Prophet Probability: 1.0

S42
Residue I154
Missed cleavages: 0
Charge: 3
m/z [Da]: 641.64
RT [sec]: 2671.4165
Peptide Prophet Probability: 0.9986

S43
Residue Y349
Missed cleavages: 0
Charge: 4
m/z [Da]: 1007.483
RT [sec]: 2854.4781
Peptide Prophet Probability: 1.0

S44
Residue P352
Missed cleavages: 0
Charge: 4
m/z [Da]: 1007.483
RT [sec]: 2934.6481
Peptide Prophet Probability: 1.0

S45
S46
Modified peptides for CDK2

Residue K9
Missed cleavages: 1
Charge: 3
m/z [Da]: 720.03
RT [sec]: 2199.0788
Peptide Prophet Probability: 1.0

Residue T137
Missed cleavages: 1
Charge: 3
m/z [Da]: 795.09
RT [sec]: 2713.2312
Peptide Prophet Probability: 1.0

S47
Residue P294
Missed cleavages: 1
Charge: 4
m/z [Da]: 690.863
RT [sec]: 2341.2518
Peptide Prophet Probability: 0.9985

S48
Modified peptides for binding site mapping of mTOR and FKBP12

FKBP12 B.

Residue G52
Missed cleavages: 1
Charge: 3
m/z [Da]: 612.984
RT [sec]: 2467.7319
Peptide Prophet Probability: 1.0

S49
Residue K53
Missed cleavages: 1
Charge: 3
m/z [Da]: 612.984
RT [sec]: 2459.7038
Peptide Prophet Probability: 1.0

S50
Residue R58
Missed cleavages: 1
Charge: 3
m/z [Da]: 925.778
RT [sec]: 2323.203
Peptide Prophet Probability: 1.0

S51
mTOR

Residue L2261
Missed cleavages: 0
Charge: 3
m/z [Da]: 541.956
RT [sec]: 2190.918
Peptide Prophet Probability: 1.0

S52
Residue N2262
Missed cleavages: 0
Charge: 3
m/z [Da]: 541.956
RT [sec]: 2191.4534
Peptide Prophet Probability: 0.9995

S53
Residue H2265
Missed cleavages: 0
Charge: 3
m/z [Da]: 541.956
RT [sec]: 2247.1214
Peptide Prophet Probability: 0.9853

S54
Residue H2277
Missed cleavages: 0
Charge: 3
m/z [Da]: 727.001
RT [sec]: 2321.0623
Peptide Prophet Probability: 1.0

S55
S56
Modified peptides for binding site mapping of STAT3

Residue Q198
Missed cleavages: 1
Charge: 3
m/z [Da]: 903.434
RT [sec]: 3547.3579
Peptide Prophet Probability: 1.0

Residue V921
Missed cleavages: 1
Charge: 3
m/z [Da]: 672.339
RT [sec]: 1823.3031
Peptide Prophet Probability: 1.0

S57
Modified peptides for binding site mapping of BRD4 in Jurkat cells

Residue V90
Missed cleavages: 1
Charge: 4
m/z [Da]: 848.435
RT [sec]: 3226.0172
Peptide Prophet Probability: 1.0

S58
Residue K91 (A)
Missed cleavages: 1
Charge: 4
m/z [Da]: 848.435
RT [sec]: 3194.0339
Peptide Prophet Probability: 0.9923

S59
Residue K91 (B)
Missed cleavages: 1
Charge: 4
m/z [Da]: 848.435
RT [sec]: 3196.7134
Peptide Prophet Probability: 0.9995

S60
Residue W81 (A)
Missed cleavages: 0
Charge: 3
m/z [Da]: 795.406
RT [sec]: 2847.057
Peptide Prophet Probability: 0.85

S61
Residue W81 (B)
Missed cleavages: 0
Charge: 3
m/z [Da]: 795.406
RT [sec]: 2749.7623
Peptide Prophet Probability: 0.8909

S62
Syntheses
Ir-diolbppy (11)
—OTf
CO2H CO2—

CF3 CF3
OH F F
Me Me Me

Me N HO N
N K2CO3 (2 equiv.) N
MeCN F F
+ Ir
Ir F
N MeCN F DCM/MeOH(3:1)
DCM/MeOH (3;1)
N
HO N 40 °C, 12 hhr
40 °C, 12 N
HO
Me Me F
F Me Me
CF3
CF3
17 CO2H
CO2H

S63
To a two–neck 50 ml round bottom flask equipped with a reflux condenser and stir bar was added
[Ir(dCO2HdFCF3ppy)2(MeCN)2]OTf (17) (500 mg, 0.487 mmol), 2,2’–([2,2’–bipyridine]–4,4’–
diyl)bis(propan–2–ol) (146 mg, 0.535 mmol), and potassium carbonate (269 mg, 1.95 mmol).
Subsequently, DCM (16.4 mL) and MeOH (5.5 mL) was added to produce a yellow heterogenous
solution. The solution was then heated to 40 °C and let to stir under positive nitrogen pressure for
12 hours. The solution was then let to cool to room temperature, concentrated, and purified
16 via
normal phase chromatography (10% to 50% MeOH/DCM) to afford (16) (100 mg, 19.4% yield)
as a bright yellow powder.

1
H NMR (500 MHz, d6–DMSO): δ 8.86 (s, 2H), 8.41 (s, 2H), 8.03 (d, J = 6.0 Hz, 2H), 7.82 (dd,
J = 5.8, 1.6 Hz, 2H), 7.54 (s, 2H), 6.79 (ddd, J = 12.1, 9.1, 2.5 Hz, 2H), 5.86 (dd, J = 8.3, 2.4 Hz,
2H), 1.63 (s, 3H), 1.62 (s, 3H)

13
C NMR (125 MHz, d6–DMSO): δ 170.1, 169.0 (d, J = 6.7 Hz), 166.3 (dd, J = 259.8, 12.8 Hz),
166.1, 164.0 (dd, J = 262.3, 13.1 Hz), 157.0, 156.3 (d, J = 6.9 Hz), 151.9, 151.3, 147.0 (d, J = 6.0
Hz), 127.8 (m), 126.6, 123.2 (q, J = 273.2), 122.7–122.2 (m), 122.4, 115.2 (d, J = 17.2 Hz), 100.5
(t, J = 27.2 Hz), 72.6, 31.1, 31.0

19
F NMR (376 MHz, d4–MeOD): δ –62.04 (6F, s), = –104.60 (2F, dt, J = 12.9, 8.9 Hz), –108.29
(2F, td, J = 12.6, 3.4 Hz)

HRMS (ESI–TOF) m/z calcd. for C42H30F10IrN4O6+ ([M + H]+) 1069.1629, found 1069.1659.

S64
Sulfonamide–PEG3–azide (17)

O O
O O S
EDCI, HOBt·H2O NH2
S H
NH2 Azide-PEG3-amine N
O
HO
DIPEA, DMF
DIEA, DMF O O
O
N3 17
O

To an 8 mL vial was added 0.5 mL of DMF, 4–sulfamoylbenzoic acid (20.0 mg, 0.10 mmol), and
DIPEA (53.5 μL, 0.30 mmol). Then, EDCI (30.4 mg, 0.16 mmol) and HOBt•H2O (21.5 mg, 0.16
mmol) were added and the mixture was stirred at room temperature for 20 minutes before the
addition of 2–(2–(2–(2–azidoethoxy)ethoxy)ethoxy)ethan–1–amine (27.1 mg, 0.12 mmol). This
mixture was then stirred at room temperature for 12 hours. The mixture was then concentrated and
purified via reverse phase column chromatography (H2O/MeCN, 0–70%) to give sulfonamide–
PEG3–azide (23) (28.8 mg, 82% yield) as an opaque viscous oil.

1
H NMR (500 MHz, d6–DMSO): δ 8.71 (t, 1H, J = 5.8 Hz), 7.99 (d, 2H, J = 8.6 Hz), 7.89 (d,
2H, J = 8.5 Hz), 7.38 (br, 2H), 3.62–3.56 (m, 2H), 3.56–3.52 (m, 10H), 3.44 (q, 2H, J = 5.8 Hz),
3.39–3.37 (m, 2H)

13
C NMR (125 MHz, d6–DMSO): δ 165.8, 147.0, 137.6, 128.3, 126.0, 70.3, 70.2, 70.1, 70.1, 69.7,
69.2, 50.4

HRMS (ESI–TOF) m/z calcd. for C15H24N5O6S+ ([M + H]+) 402.1447, found 402.1419.

S65
Sulfonamide–PEG3–Ir (1)

CO2—
H 2N O
CF3
S F
O Me Me

MeO N
H
N N
F
O Ir
O F
O
N
N N
O N
N O
1 Me F
Me CF3
CO2H

To an 8 ml vial was added sulfonamide–PEG3–azide (17) (5.0 mg, 0.012 mmol, 1 equiv.), Ir–G1
alkyne (12) (13.7 mg, 0.012 mmol, 1 equiv.), CuSO4 (62.0 mg, 0.025 mmol), Sodium ascorbate
(24.7 mg, 0.12 mmol), and 2.5 ml of 1:1 t–BuOH:H2O. The reaction was stirred overnight at 35
°
C in the dark. The resultant cloudy yellow mixture was then concentrated and purified via reverse
phase chromatography (0–25% MeCN/H2O) followed by reversed phase HPLC (0–90%
MeCN/H2O w/ 0.1% TFA) to yield sulfonamide–PEG3–Ir photocatalyst (1) as a yellow solid (1.1
mg, 6.0% yield)

1
H NMR (500 MHz, d4–MeOD): δ 9.04 (s, 1H), 8.90 (s, 1H), 8.54 (s, 2H), 8.35 (s, 2H), 8.10 (d,
J = 5.8 Hz, 1H), 8.08 (d, 1H, J = 5.8 Hz), 8.00 (s, 1H), 7.90 (s, 4H), 7.83 (dd, 1H, J = 6.1, 2.2 Hz),
7.81 (dd, 1H, J = 5.9, 2.0 Hz), 7.50 (s, 1H), 7.49 (s, 1H), 6.78 (ddt, 2H, J = 11.5, 9.1, 2.3 Hz), 5.86
(td, 2H, J = 5.6, 2.8 Hz), 4.52 (t, 2H, J = 5.1 Hz), 4.46 (s, 1H), 3.83 (t, 2H, J = 5.0 Hz), 3.77–3.44
(m, 12H), 3.22 (s, 3H), 1.70 (s, 3H), 1.69 (s, 3H), 1.67 (s, 3H), 1.66 (s, 3H)

13
C NMR (125 MHz, d4–MeOD): δ 170.8, 170.1, 168.9 (d, J = 7.4 Hz), 168.7, 166.2 (dd, J =
258.7, 11.4 Hz), 163.9 (dd, J = 262, 13.1 Hz), 163.3, 157.5, 157.4, 156.3 (m), 152.5, 152.3, 152.0,
147.7, 147.0 (m), 145.7, 138.9, 129.0, 127.9, 127.6, 127.4, 125.6, 124.4, 124.1, 123.9, 123.3 (q, J
= 273.7 Hz), 122.6–122.0 (m), 115.1 (m), 100.5 (t, J = 27.1 Hz), 78.8, 78.0, 71.4, 71.4, 71.3, 71.2,
70.3, 58.2, 51.3, 51.3, 41.0, 38.3, 27.6, 27.2

S66
19
F–NMR (376 MHz, MeOD): δ –62.04 (s, 3F), –62.07 (s, 3F) –104.84 (dt, 2F, J = 12.3, 8.2 Hz),
–108.39 (qd, 2F, J = 12.3, 4.1 Hz)

HRMS (ESI–TOF) m/z calcd. for C61H57F10IrN9O12S+ ([M + H]+) 1522.3317, found 1522.3359.

S67
Dasatinib–PEG3–azide (18)

OH
Cl Me O Cl Me
EDCI, HOBt·H2O
N O NH O N O NH
Azide-PEG3-acid
O O
N DIEA, DMFDMF
Triethylamine, N
S S
N N N N
O
NH N3 NH
N N 18
Me Me

To an 8 mL vial was added acid–PEG3–azide (30.4 mg, 0.12 mmol), 0.5 mL of DMF, and
triethylamine (71.2 μL, 0.51 mmol). Then, EDCI (35.3 mg, 0.18 mmol) and HOBt•H2O (25.0 mg,
0.18 mmol) were added and the mixture was let to stir at room temperature for 20 minutes before
the addition of dasatinib (50.0 mg, 0.102 mmol). This mixture was then stirred at room
temperature. After 12 hrs, the mixture was concentrated and purified normal phase column
chromatography (DCM/MeOH, 0–20%) to give dasatinib–PEG3–azide (24) (36 mg, 49% yield)
as an off–white solid.

1
H NMR (500 MHz, d6–DMSO): δ 11.47 (br, 1H), 9.88 (s, 1H), 8.22 (s, 1H), 7.41 (d, 1H, J =
5.8 Hz), 7.34–7.21 (m, 2H), 6.05 (s, 1H), 4.17 (t, 2H, 5.7 Hz), 3.65 (t, 2H, 6.2 Hz), 3.59 (t, 2H, J
= 4.9 Hz), 3.56–3.49 (m, 14H), 3.41–3.37 (m, 2H), 2.60 (t, 2H, J= 5.8 Hz), 2.56 (t, 2H, J = 6.2
Hz), 2.41 (s, 3H), 2.24 (s, 3H)

13
C NMR (125 MHz, d6–DMSO): δ 171.6, 165.6, 163.1, 162.8, 160.4, 157.4, 141.3, 139.3, 134.0,
132.9, 129.5, 128.7, 127.5, 126.1, 83.1, 70.3, 70.2, 70.2, 70.1, 69.7, 66.5, 61.9, 56.4, 52.8, 50.4,
44.0, 35.2, 26.0, 18.8

HRMS (ESI–TOF) m/z calcd. for C32H43ClN9O6S+ ([M + H]+) 717.2698, found 717.2661.

S68
Dasatanib–PEG3–Ir (4)

CO2—
CF3
F
Me Me

MeO N

N
Me F
Me
Ir
F
NH N N N
N
S NH N N O N N O
Cl O
Me F
N O N Me CF3
O O O
CO2H 4

To an 8 ml vial was added dasatinib–PEG3–azide (18) (8.6 mg, 0.012 mmol), Ir–alkyne (3) (13.7
mg, 0.012 mmol), CuSO4 (6.2 mg, 0.025 mmol), sodium ascorbate (24.7 mg, 0.12 mmol), and 2.5
ml of 1:1 t–BuOH:H2O. The reaction was let to stir over night at 35 °C in the dark. The resultant
cloudy yellow mixture was then concentrated and purified via reverse phase chromatography (0–
25% MeCN/H2O) followed by reversed phase HPLC (0–90% MeCN/H2O w/ 0.1% TFA) to yield
the photocatalyst as a yellow solid (0.8 mg, 3.7% yield)

1
H NMR (500 MHz, d4–MeOD): δ 9.04 (s, 1H), 8.90 (s, 1H), 8.36 (s, 2H), 8.13 (s, 1H), 8.09 (d,
J = 5.8 Hz , 1H), 8.07 (d, J = 5.8 Hz , 1H), 8.03 (s, 1H), 7.83 (dd, J = 5.8, 1.9 Hz, 1H), 7.81 (dd, J
= 5.8, 1.9 Hz, 1H), 7.49 (s, 1H), 7.48 (s, 1H), 7.33 (dd, 1H, J = 7.6, 2.0 Hz), 7.26–7.18 (m, 2H),
6.78 (ddt, 2H, J = 11.5, 9.1, 2.4 Hz), 6.03 (s, 1H), 5.85 (ddd, 2H, J = 8.4, 6.0, 2.4 Hz), 4.56 (t, 2H,
J = 5.0 Hz), 4.48 (s, 2H), 4.23 (t, 2H, J = 5.6 Hz), 3.93–3.81 (s, 2H), 3.77–3.61 (m, 6H), 3.62–3.43
(m, 10H), 3.21 (s, 3H), 2.77 (t, 2H, J = 5.5 Hz), 2.68 (m, 3H), 2.56 (2t, H, J = 6.1 Hz), 2.45 (s,
3H), 2.3 (s, 3H), 1.71 (s, 3H), 1.70 (s, 3H), 1.66 (s, 3H), 1.65 (s, 3H)

13
C NMR (125 MHz, d4–MeOD): δ 173.0, 170.7, 168.9 (d, J = 7.2 Hz), 167.4, 166.1 (dd, J =
257.8, 12 Hz), 164.3, 163.9 (dd, J = 260.6, 13.7 Hz), 163.3, 163.2, 162.8, 158.6, 157.5, 157.4,
156.3 (m), 152.4 (m), 152.2, 152.0, 147.0 (m), 145.6, 142.3, 140.3, 134.3, 134.2, 130.1, 129.5,
128.3, 127.9, 127.6, 127.4, 126.8, 125.7, 124.1, 123.9, 123.3 (q, J = 273.3 Hz), 122.5–121.9 (m),
115.1 (m), 100.5 (t, J = 27.2 Hz), 84.0, 78.8, 78.0, 73.8, 71.4, 71.4, 71.3, 70.3, 67.5, 64.4, 62.6,
58.2, 57.4, 53.8, 51.3, 51.3, 44.6, 35.9, 28.7, 27.7, 27.2, 25.6, 18.7

S69
19
F–NMR (376 MHz, MeOD): δ –61.97 (s, 3F), –62.03 (s, 3F), –104.76 (p, 2F, J = 8.5 Hz), –
108.31 (m, 2F)

HRMS (ESI–TOF) m/z calcd. for C77H75ClF10IrN14O12S+ ([M + H]+) 1837.4568, found
1837.4624.

S70
AT7519–PEG3–azide (19)

N3 O
O
N3 O oxayl chloride, DMF,
O Cl O O
DCM;
Cl
O then DIPEA, NH O N
then DIEA, O
HO2C NH
N
Cl H
O N
H NH
HN 19
N
Cl
O
N
N
H

Azido–PEG3–acid (98.8 mg, 0.40 mmol) and dimethyl formamide (5.8 mg, 0.080 mmol) were
dissolved in anhydrous DCM at room temperature in an oven–dried vial under N2. Oxalyl chloride
(101 mg, 0.80 mmol) was then added dropwise and the mixture stirred for one hour. The mixture
was then evaporated under high vacuum for 16 hours to remove all volatile products and suspended
in 4.0 mL DCM to afford a 0.1 M colorless stock solution which was used without further
purification. AT7519 (6.9 mg, 0.018 mmol) and DIPEA (13.9 mg, 0.108 mmol) were combined in
1 mL DCM, then 60 µL of the freshly prepared 0.1 M stock solution of 3–(2–(2–(2–
azidoethoxy)ethoxy)ethoxy)propanoyl chloride (0.020 mmol) was added. After 16 hours, the
mixture was subjected to chromatography (25 g SiO2, 1% MeOH/DCM to 20% MeOH/DCM, 25
mL/min) to afford the product (19) as a white solid (8.8 mg, 73%).

1
H NMR (500 MHz, d1–CDCl3): δ 9.78 (s, 1H), 8.42 (s, 1H), 7.29 (d, 1H, J = 2.0 Hz), 7.23 (dd,
1H, J = 9.3, 6.6 Hz), 7.20 (s, 1H), 6.86 (d, 1H, J = 8.1 Hz), 4.50 (m, 1H), 4.03 (dtd, 1H, J =
10.9, 7.2, 4.0 Hz), 3.83 (m, 1H), 3.71 (m, 2H), 3.59 (m, 10H), 3.32 (m, 2H), 3.07 (ddd, 1H, J =
14.2, 11.7, 2.8 Hz), 2.69 (ddd, 1H, J = 14.1, 11.8, 2.9 Hz), 2.56 (m, 2H), 1.95 (m, 2H), 1.42 (m,
2H)

13
C NMR (125 MHz, d1–CDCl3): δ 163.1, 161.2, 135.4, 133.1, 132.6, 131.1, 128.2, 122.6, 121.5,
70.7, 70.6, 70.5, 70.4, 70.0, 67.5, 50.6, 46.4, 44.6, 40.7, 33.6, 32.5, 31.7

S71
HRMS (ESI–TOF) m/z calcd. for C25H33Cl2N8O6+ ([M + H]+) 611.1985, found 611.1997

S72
AT7519–PEG3–Ir (9)
CO2—
CF3
F
Me Me

MeO N

N
F
Ir
F
N
N
Cl O N N O
Cl Me F
O N Me CF3
NH O N O O
O CO2H 9
N
H
HN N

To an 8 ml vial was added (19) (9.0 mg, 0.015 mmol), Ir–alkyne (3) (16.5 mg, 0.015 mmol),
CuSO4 (7.3 mg, 0.030 mmol), sodium ascorbate (29.5 mg, 0.147 mmol), and 2.9 ml of 1:1 t–
BuOH:H2O. The reaction was let to stir over night at 35 °C in the dark. The resultant bright yellow
mixture was then concentrated and purified via reverse phase chromatography (0–20%
MeCN/H2O), followed by normal phase chromatography (0–15% MeOH/DCM), and then finally
reverse phase HPLC (0–90% MeCN/H2O) to yield photocatalyst (9) as a bright yellow powder
(6.7 mg, 26 % yield)

1
H NMR (500 MHz, Methanol–d4) δ 9.06 (s, 1H), 8.91 (s, 1H), 8.36 (s, 2H), 8.34 (s, 1H), 8.09
(dd, 2H, J = 9.2, 5.8 Hz), 8.04 (s, 1H), 7.84 (dd, 1H, J = 5.8, 1.9 Hz), 7.81 (dd, 1H, J = 5.8, 2.0
Hz), 7.53–7.43 (m, 5H), 6.78 (dd, 2H, J = 12.6, 8.9 Hz), 5.85 (ddd, 2H, J = 7.9, 5.3, 2.3 Hz), 4.56
(t, 2H, J = 5.1 Hz), 4.49 (s, 2H), 4.47–4.40 (m, 2H), 4.09–4.02 (m, 1H), 3.99 (d, 1H, J = 13.9 Hz),
3.87 (t, 2H, J = 5.2 Hz), 3.71 (t, 2H, J = 6.3 Hz), 3.63–3.51 (m, 8H), 3.21 (s, 3H), 3.17 (t, 2H, J =
13.0 Hz), 2.79–2.71 (m, 1H), 2.72–2.64 (m, 1H), 2.63–2.54 (m, 1H), 1.98–1.92 (m, 1H), 1.92–
1.85 (m, 1H), 1.71 (s, 3H), 1.71 (s, 3H), 1.67 (s, 3H), 1.66 (s, 3H), 1.63–1.55 (m, 1H), 1.50–1.43
(m, 1H)

13
C NMR (126 MHz, Methanol–d4) δ 168.4, 166.0 (m), 165.4, 165.3 (m), 163.6 (dd, J = 257.3,
12.4 Hz), 161.5 (dd, J = 261.8, 13.1 Hz), 160.6, 160.5, 155.6, 155.2, 153.7, 150.6, 145.2 (m),

S73
143.9, 135.4, 131.9, 131.2, 128.4, 126.7, 126.3, 126.2, 124.3, 122.6, 122.4, 122.1 (d, J = 273.5
Hz), 122.1 (d, J = 273.7 Hz), 121.6, 121.2, 121.0, 119.9 (q, J = 32.5 Hz), 113.7 (d, J = 16.2 Hz),
99.5 (t, J = 26.5 Hz), 77.1, 76.4, 69.7, 69.6, 69.6, 69.5, 68.7, 66.8, 57.0, 50.4, 49.4, 46.0, 44.0,
32.7, 31.7, 30.8, 27.7, 27.7, 26.9, 26.6, 26.5

19
F NMR (471 MHz, Methanol–d4) δ –59.75 (s, 3F), –59.78 (s, 3F), –104.24 (m, 2F), –107.76 (q,
J = 11.6 Hz, 2F)

HRMS (ESI–TOF) m/z calcd. for C71H66Cl2F10IrN12O12+ ([M + H]+) 1731.3764, found
1731.3824.

S74
Lenalidomide–PEG0–Ir (6)

F3C
F PF6
Me N
O
N F
O N Ir
F
HN N
O HN N
66
O F
F3C

A stirred solution of Ir-G2-Acid (13) (5 mg, 11.41 µmol) in THF (0.5 mL) was cooled to 0 °C
before the addition of TCBC (1.77 µL, 11.41 µmol) and Et3N (1.27 µL, 9.13 µmol) under N2. The
reaction mixture was stirred for 1 h before the addition of lenalidomide (3 mg, 4.56 µmol) and
DMAP (0.8 mg, 6.84 µmol). The reaction mixture was slowly warmed to room temperature and
left to stir for 24 h. Upon completion, the reaction mixture was evaporated under reduced pressure,
resuspended in minimal DMSO, and purified via preparative HPLC (gradient elution: 30 to 100%
MeCN/H2O (0.1% formic acid)) to give Lenalidomide-PEG0-Ir (6) as a yellow solid (2.2 mg,
36%)

1
H NMR (500 MHz, d1–CDCl3): δ 10.34 (d, 1H), 9.41 (s, 2H), 8.39 (t, 2H), 8.06 (dd, 1H), 8.03
(s, 1H), 7.96 (t, 2H), 7.68 (t, 1H), 7.64 (d, 1H), 7.68 (s, 1H), 7.51 (d, 1H), 7.41, (d, 2H), 7.30 (t,
1H), 7.26 (d, 1H), 6.59 (m, 2H), 5.56 (d, 2H), 5.08 (dd, 1H), 4.83 (dd, 1H), 4.54 (d, 1H), 3.26 (t,
3H), 2.64 (s, 3H), 2.50 (m, 1H), 1.93 (m, 6H).

HRMS (ESI–TOF): m/z calcd. for C51H35F10IrN7O4 ([M]+) 1192.22205, found 1192.22195.

UHPLC: Agilent 1290 Infinity 2 C18 UHPLC, gradient: 5 – 95% MeCN/H2O (0.1% formic acid)
over 10 minutes, 1 mL/min, 250 nm): tr = 5.680 min

S75
S76
Rapamycin–PEG3–azide (20)

Me
OH
O
O
Me
OMe O
N
20
H

O O
Me H
OMe
O OH O
Me O N3
O O O O
Me OMe Me Me

Azido–PEG3–carboxylic acid (79.0 mg, 0.32 mmol), 2, 4, 6–trichlorobenzoyl chloride (78.0 mg,
0.32 mmol), and triethylamine (48.5 mg, 48 mmol) were combined in 1.5 mL anhydrous THF and
stirred for one hour at room temperature. Then, rapamycin (146.3 mg, 0.16 mmol) and DMAP
(19.5 mg, 0.16 mmol) dissolved in 1 mL anhydrous THF was added dropwise. The mixture was
then stirred overnight. The mixture was then subjected to silica chromatography (20% to 100%
EtOAc/Hexanes over 14 CV, 25 g SiO2, 25 mL/min) to afford the product as a colorless resin. The
residue was further purified by silica chromatography (25 g SiO2 HP, 25 mL/min, 90% EtAc/Hex,
7 CV) to afford the product as a colorless solid (61 mg, 33%).

Compound isolated as an undetermined mixture of rotamers

1
H NMR (500 MHz, d1–CDCl3): δ 6.36–6.27 [m, 1.5H, including 6.32 (dd, J = 14.8, 10.7 Hz,
part of H19)], 6.27–6.19 [m, 1H, including 6.23 (dd, J = 14.8, 10.2 Hz, part of H20)], 6.19–6.01 [m,
2H, including 6.15 (dd, J = 14.5, 10.6 Hz, part of H21), 6.07 (dd, J = 15.1, 10.2 Hz, part of H21)],
5.96–5.80 [m, 1.5H, including 5.92 (1H, d, J = 10.9 Hz, part of H18), 5.83 (d, J = 11.0 Hz, part of
H18)], 5.53–5.42 [m, 1.5H, including 5.48 (dd, J = 15.3, 8.5 Hz, part of H22)], 5.41–5.30 [m, 1.5 H,
including 5.39 (d, J = 5.39 (d, J = 10.1 Hz, part of H30), 5.34 (d, J = 9.9 Hz, part of H30)], 5.28–
5.15 [m, 1H, including 5.21 (d, J = 5.7 Hz, part of H2)], 5.13–5.02 [m, 1.5H, including 5.09 (m,
part of H34)], 4.76 (s, 1H, part of C10–OH), 4.66–4.31 [m, 2H, including 4.62 (td, J = 11.2, 10.2,
4.9 Hz, part of H40)], 4.29–3.95 [m, 2H, including 4.24 (d, J = 4.6 Hz, part of H2), 4.13 (d, J = 5.4
Hz, part of H28)], 3.88–3.71 [m, including 3.83 (dp, J = 8.4, 3.9, 2.7 Hz, part of H14), 3.75 (d, J =

S77
5.4 Hz, part of H27)], 3.71–3.62 [m, 4H, including 3.64 (d, J = 6.5 Hz, part of H27)], 3.62–3.53 [m,
18H, including 3.60 (m, part of H16)], 3.52–3.44 [m, 2H, including 3.50 (d, J = 14.3 Hz, part of
H6)], 3.38–3.19 [m, 16H, including 3.31 (s, part of C52–CH3), 3.26 (s, part of C51–CH3), 3.24 (m,
part of H31)], 3.17–3.01 [m, 7H, including 3.09 (m, part of H39), 3.07 (s, part of C50–CH3)], 2.65–
2.56 [m, 2H, including 2.61 (m, part of H25)], 2.56–2.47 [m, 5H, including 2.55 (t, J = 6.6 Hz),
2.50 (m, part of H33)], 2.38–2.18 [m, 9H, including 2.25 (m, part of H23)], 2.13–2.00 [m, 2.5H,
including 2.01 (d, J = 13.4 Hz, part of H39)], 1.98–1.98 [m, 3H, including 1.96 (m, part of H41),
1.94 (dt, J = 14.3, 5.7 Hz, part of H11)], 1.88–1.75 [m, 3H, including 1.85 (m, part of H35), 1.81
(dd, J = 13.9, 7.7 Hz)], 1.75–1.62 [m, 10H, including 1.72 (m, part of H4), 1.70 (s, part of C47–
CH3), 1.66 (m, part of H5)], 1.62–1.48 [m, 11H, including 1.59 (s, part of C44–CH3), 1.54 (m, part
of H12)], 1.45–1.29 [m, 8H, including 1.42 (m, part of H5), 1.40 (m, part of H4), 1.33 (m, part of
H37)], 1.28–1.09 [m, 8H, including 1.25 (m, part of H13), 1.25 (ddd, J = 14.8, 12.2, 3.8 Hz, part of
H41), 1.13 (m, part of H24)], 1.09–1.03 [m, 3H, including 1.05 (t, J = 5.0 Hz, part of H36)], 1.03–
0.94 [m, 10H, including 1.01 (d, J = 6.8 Hz, part of C49–CH3), 0.99 (d, J = 6.6 Hz, part of C45–
CH3)], 0.94–0.89 [m, 4H, including 0.92 (d, J = 6.5 Hz, part of C46–CH3)], 0.89–0.85 [m, 4H,
including 0.88 (d, J = 6.6 Hz, part of C43–CH3)], 0.85–0.81 [m, 5H, including 0.84 (d, J = 6.8 Hz,
part of C49–CH3)], 0.80–0.68 [m, 4H, including 0.79 (d, J = 6.6 Hz, part of C43–CH3), 0.77 (dd, J
= 23.6, 11.4 Hz, part of H38)]

13
C NMR (126 MHz, d1–CDCl3): 14.6 and 214.3 (C26), 208.1 and 207.6 (C32), 196.2 and 193.2
(C9), 171.0, 169.5 and 169.2 (C1), 166.6 and 165.8 (C8), 140.3 and 139.6 (C22), 136.4 and 136.0
(C29), 135.9 (C17), 133.3 and 133.1 (C20), 130.1 and 129.9 (C21), 129.1 (C18), 126.5 and 126.4 (C19),
126.4 and 126.0 (C30), 98.6 and 98.4 (C10), 86.4 and 84.7 (C27), 84.2 and 84.0 (C16), 80.7 and 80.7
(C39), 76.8 (C28), 76.3 (C40), 75.3 (C34), 70.5, 70.5, 70.4, 70.2, 69.9, 66.5, 67.6 and 67.0 (C14), 58.8
and 58.8 (C51),57.5 and 57.4 (C52), 55.8 (C50), 56.2 and 51.1 (C2), 50.5, 46.5 and 46.0 (C31), 44.1
(C6), 41.5 (C25), 40.7–39.4 [m, including 40.1 (C24)], 39.0 (C15), 38.2 (C36), 35.9 and 35.7 (C38),
35.3, 34.8 (C23), 33.8 (C11), 33.2 (C35), 32.7 (C37), 31.2–30.8 [m, including 31.1 (C13)], 29.6 (C41),
27.0 (C3), 26.9 (C12), 25.1 (C5), 21.4 and 21.3 (C45), 20.6 (C4), 16.2 and 16.1 (C43), 15.8 (C49), 15.7
(C43), 13.4 and 13.0 (C47), 13.4 (C46), 10.2 and 10.1 (C44)

S78
HRMS (ESI–TOF) m/z calcd. for C60H98N5O172+ ([M + NH4]+) 1160.6957, found 1160.6954.

S79
Rapamycin–PEG3–Ir (7)

CO2—
F3C
F
OMe
Me
Me N
Me
N F
O Ir
O F
OH
Me N
OMe O Me
N N
Me
H N N
O F
O N F3C
O O CO2H
H
Me OMe
O OH O O O
Me 7
O O
Me OMe Me Me

To an 8 ml vial was added rapamycin–PEG3–azide (20) (5.7 mg, 5.0 µmol), iridium–alkyne 3 (5.5
mg, 5.0 µmol), CuSO4 (2.5 mg, 10 µmol), Sodium ascorbate (9.9 mg, 50 µmol), and 1.0 ml of
1:1 t–BuOH:H2O. The reaction was let to stir over night at 35 °C in the dark. The crude mixture
was evaporated onto 1.0 g silica gel and purified by normal phase chromatography (1% MeOH to
20% MeOH/DCM over 12 CV, 25 g SiO2, 25 mL/min), followed by reverse phase HPLC (20–
100% MeCN/H2O with 0.1% formic acid) to give product 8 as a yellow film (4.2 mg, 37%).

Compound isolated as an undetermined mixture of rotamers

1
H NMR (800 MHz, d4–MeOD): δ 9.04 (s, 1H), 8.89 (s, 1H), 8.34 (1H, d, J = 2.8 Hz), 8.33 (1H,
d, J = 2.8 Hz), 8.08 (1H, d, J = 5.8 Hz), 8.06 (1H, d, J = 5.8 Hz), 8.04 (d, J = 2.1 Hz), 7.83 (1H, d,
J = 5.6 Hz), 7.80 (1H, d, J = 5.8 Hz), 7.49–7.47 (1H, m), 6.83–6.68 (m, 2H), 6.54–6.37 [m, 1H,
including 6.42 (dd, J = 14.8, 11.1 Hz, part of H19)], 6.35–6.20 [m, 1H, including 6.26 (dd, J = 14.8,
10.6 Hz, part of H20)], 6.19–6.11 [m, 1H, including 6.14 (dd, J = 15.1, 10.6 Hz, part of H21)], 6.10–
5.92 [m, 1H, including 6.09 (d, J = 9.6 Hz, part of H18)], 5.82 (ddd, J = 10.6, 7.0, 2.3 Hz, 2H),
5.59–5.40 [m, 1H, including 5.44 (dd, J = 15.0, 9.7 Hz, part of H22)], 5.33–5.15 [m, 1H, including
5.20 (dd, J = 10.4, 1.7 Hz, part of H30)], 5.15–5.04 [m, 1H, including 5.07 (dq, J = 8.2, 4.7 Hz, part
of H34)], 5.05–4.99 [m, 1H, including 5.02 (m, part of H2)], 4.63–4.53 [m, 4H, including 4.58 (m,
S80
part of H40)], 4.48 (s, 2H), 4.37–4.16 [m, 1.5H, including 4.19 (m, part of H28)], 4.16–4.01 [m,
including 4.07 (dd, J = 6.3, 4.5 Hz, part of C27), 4.09 (m, part of H14)], 3.88–3.83 (m, 2H), 3.80–
3.61 [m, 3H, including 3.67 (m, part of H16), 3.59–3.49 (m, 9H), 3.36–3.29 [m, 5H, including 3.34
(s, part of C52–CH3), 3.32 (m, part of H31), 3.31 (m, part of H6)], 3.26–3.21 [m, 3H, including 3.25
(s, part of C51–CH3)], 3.20–3.14 [m, including 3.20 (s, 3H), 3.19 (m, part of H39)], 3.13–3.08 [m,
3H, including 3.11 (s, part of C50–CH3)], 2.82–2.74 [m, 1H, including 2.78 (dt, J = 17.6, 3.3 Hz,
part of H33)], 2.55–2.48 [m, 3H, Including 2.52 (t, J = 6.2 Hz), 2.50 (m, part of H25)], 2.47–2.36
[m, 1H, including 2.41 (m, dddd, J = 17.5, 8.7, 5.2 Hz, part of H33)], 2.36–2.25 [m, 1.5H, including
2.27 (dpd, J = 16.6, 6.5, 2.9 Hz, part of H23)], 2.23–2.14 [m, including 2.19 (m, part of H3)], 2.14–
2.03 [m, 2H, including 2.10 (dt, J = 13.5, 6.6 Hz, part of H11)], 2.02–1.93 [m, 1.5H, including 1.99
(m, part of H13), 1.97 (ddd, J = 13.7, 11.4, 4.0 Hz, part of H15)], 1.92–1.85 [m, 2H, including 1.88
(d, J = 15.3 Hz, part of H41), 1.86 (m, part of H13)], 1.84–1.79 [m, 3H, including 1.82 (s, part of
C47–CH3)], 1.78–1.55 [m, 25H, including 1.78 (m, part of H35), 1.70 (s, 3H), 1.68 (s, 3H), 1.67 (s,
part of C44–CH3), 1.65 (s, 3H), 1.64 (s, 3H), 1.59 (m, part of H12)], 1.49–1.35 [m, 4H, including
1.44 (t, J = 12.5 Hz, part of H24), 1.38 (m, part of H37)], 1.35–1.23 [m, 6H, including 1.30 (m, part
of H41)], 1.22–1.05 [m, 4H, including 1.13 (m, part of H36), 1.13 (m, part of H24), 1.07 (dd, J = 5.6,
2.9 Hz part of H36)], 1.04–0.98 [m, 4H, including 1.01 (d, J = 6.6 Hz, part of C45–CH3)], 0.98–0.92
[including 0.94 (dd, J = 6.5, 1.7 Hz, part of C48–CH3)], 0.91–0.88 [including 0.90 (d, J = 6.4 Hz,
part of C46–CH3)], 0.87–0.80 [m, 6H, including 0.81 (d, J = 6.7 Hz, part of C49–CH3), 0.81 (d, J =
6.7 Hz, part of C43–CH3)], 0.80–0.69 [m, 1.5H, including 0.76 (dt, J = 25.0, 11.8, 7 Hz, part of
H38]

13
C NMR (500 MHz, d4–MeOD): δ 214.1 (C26), 209.3 (C32), 199.6 (C9), 173.0 (C53), 171.0, 170.8,
169.2 (C8), 168.8 (d, J = 6.4 Hz), 166.1 (dd, J = 258.9, 9.3 Hz), 163.9 (dd, J = 262.2, 13.3 Hz),
163.3, 162.9, 157.5, 157.4, 156.3 (m), 152.5 (m), 152.3, 152.0, 147.0 (m), 145.6, 140.5 (C22), 139.2
(C17), 138.2 (C29), 134.0 (C20), 132.4 (C21), 128.9 (C18), 128.4 (C19), 127.9 (m), 127.7, 127.4, 127.1
(C30), 125.8, 124.1, 124.0, 123.8, 123.3 (q, J = 273.1 Hz), 122.4–121.7 (m), 115.1 (t, J = 16.5 Hz),
100.6 (C10), 100.4 (d, J = 27.3), 86.5 (C27), 84.6 (C16), 82.1 (C39), 78.8, 78.0, 77.9 (C40), 77.3 (C28),
75.5 (C34), 71.4, 71.4, 70.4, 68.4 and 67.6 (C14), 58.2 (C51), 58.2, 57.9 (C50), 56.3 (C52), 52.8 (C2),
51.3 , 47.2 (C31), 45.5 (C6), 42.0 (C25), 41.8 (C15), 41.3 (C33), 41.2 (C24), 39.9 and 39.8 (C36), 37.1
(C23), 37.1 (C37), 36.4 (C11), 36.4, 35.1 (C35), 33.9 (C37), 32.2 and 31.1 (C13), 31.4, 30.9–30.5 [m,
S81
including 30.7 (C41)], 28.3, 27.8 (C3), 27.8 (C12), 27.7, 27.6, 27.2, 27.2, 26.0, 21.9 (C45), 21.8 (C4),
16.0 (C48), 16.0 (C43), 15.6 (C49), 14.3 (C47), 13.7 (C46), 10.9 (C44)

19
F NMR (400 MHz, d4–MeOD): δ –62.00 (s, 3F), –62.04 (s, 3F), –104.78 (m, 2F), –108.34 (m,
2F)

HRMS (ESI–TOF) m/z calcd. for C106H128F10IrN8O23+ ([M + H]+) 2263.8556, found 2263.8532.

S82
MM–206–PEG3–azide (21)

O
OH O N3
S O O O
S

Azide-PEG3-acid, CDI
O
NH
S F MeCN, rt, 4 h O
O NH
S F
O
F F 21
F F
F F
F F

Azido–PEG3–acid (5.3 mg, 0.021 mmol) and 1,1’–carbonyldiimidizole (CDI) (3.5 mg, 0.021
mmol) were dissolved in 0.5 ml acetonitrile at room temperature in an 8 ml dram vial equipped
with a stir bar. The mixture was stirred at room temperature under nitrogen for 20 minutes before
the addition of MM–206 (9.0 mg, 0.018 mmol). The reaction was then let to for 4 hours before
being concentrated under reduced pressure and dry loaded on silica for normal phase purification
(4 g SiO2, 0% EtOAc/hexanes to 40% EtOAc/hexanes) to afford the product (21) as an off white
solid (4.6 mg, 35%).

1
H NMR (500 MHz, d3–MeCN): δ 8.14 (dd, 1H, J = 6.8, 2.0 Hz), 7.95 (dd, 1H, J = 7.3, 2.7 Hz),
7.68–7.61 (m, 2H), 7.40–7.34 (m, 1H), 7.34–7.31 (m, 4H), 6.90 (s, 1H), 3.91 (t, 2H, J = 6.0 Hz),
3.68–3.64 (m, 2H), 3.64–3.61 (m, 2H), 3.60–3.53 (m, 6H), 3.32 (t, 2H, J = 5.0 Hz), 3.00 (t, 2H, J
= 6.0 Hz)

13
C NMR (500 MHz, d3–MeCN): δ170.4, 144.9, 133.1, 132.9, 130.8, 130.4, 130.0, 128.8, 128.6,
128.1, 126.6, 125.7, 123.9, 122.4, 70.8, 70.7, 70.7, 70.0, 66.8, 51.0, 35.4.

19
F NMR (400 MHz, d3–MeCN): δ -137.82– -138.01 (m, 2H), -147.66–-147.77 (m, 1H), -160.44
– -160.55 (m, 2H)

HRMS (ESI–TOF) m/z calcd. for C31H27F5N4NaO7S2+ ([M + Na]+) 749.1133, found 749.1146.

S83
MM-206-PEG3-Ir (11)
CO2—
F3C
F

Me N

O N F
HN Ir
N NH F
O N
O
N
O O
O F
S 3 N N F3C
N
CO2H
O
S NH
O
F 10
F

F
F
F

MM–206–PEG3–azide (4.0 mg, 0.0055 mmol) and IrG3–DBCO (7.5 mg, 0.0055 mmol) were
added to an 8 ml dram vial equipped with a stir bar and subsequently dissolved in 0.2 ml DMSO.
The mixture was stirred at room temperature under nitrogen in the dark for 1 hour at room
temperature before being directly injected onto a prep HPLC for reverse–phase purification (10–
100% ACN/H2O w/ 0.1% formic acid). HPLC fractions were combined and concentrated to afford
compound 10 as a dark yellow solid (1.1 mg, 11% yield). Compound was isolated as an
approximately 1:1 mixture of Regio isomers about the triazole linker.

HRMS (ESI–TOF) m/z calcd. for C92H71F15IrN11O14S22+ ([M + 2H]2+) 1047.7001, found
1047.7016

S84
 M + 2H

M + H + Na

M + 2Na
M+H

M + Na

HPLC trace: Vydac 218TP C18 HPLC, gradient: 0 – 90% MeCN/H2O (0.1% formic acid) over
60 minutes, 1 mL/min, 260 nm): tr = 38.3, 38.6 min

S85
PEG3-Diazarine-Amine (22)
22

An 8 mL scintillation vial was charged with 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzylamine

HCl (20.0 mg, 0.079 mmol). This was dissolved in 1 ml of DCM, and triethylamine (0.40 mmol)
and NHS-PEG3-N-Boc (21) (33.1 mg, 0.079 mmol) were added. The reaction was stirred at room
temperature for 12 hours. After completion, the reaction solution was concentrated under reduced
pressure, the residue dissolved in DMSO, and purified by normal phase chromatography (0-50%
EtOAc/Hexanes) to give the product (22) as a white solid (36.2 mg, 88% yield).

1
H NMR (500 MHz, d6-DMSO): δ 7.35 (d, 2H, J = 8.1 Hz), 7.17 (d, 2H, J = 8.0 Hz), 7.00 (bs,
1H), 6.00 (bs, 1H), 4.48 (d, 2H, J = 5.9 Hz), 3.78 (t, 2H, J = 5.7 Hz), 3.66 (dd, 2H, J = 6.1, 3.2
Hz), 3.61 (dd, 2H, J = 5.9, 3.3 Hz), 3.57-3.50 (m, 4H), 3.48 (t, 2H, J = 5.2 Hz), 3.29 (s, 2H), 2.56
(t, 2H, J = 5.7 Hz), 1.45 (s, 9H)

13
C NMR (125 MHz, d6-DMSO): δ 171.8, 155.9, 140.7, 127.9, 126.7, 125.4, 122.1 (q, J = 272.8,
70.4, 70.3, 70.2, 70.1, 67.2, 42.7, 42.5, 40.3, 36.9, 28.4, 70.4, 70.3, 70.2, 70.1. 67.2, 42.7, 42.5,
40.3, 36.9, 28.4

19
F NMR (376 MHz, d4-MeOD): δ -65.34 (s)

HRMS (ESI-TOF) m/z calcd. for C23H34F3N4O6+ ([M + H]+) 519.2425, found 519.2374.

Diazirine-PEG3-Desthiobiotin (23)

23
S86
 Me O
O
a.) 4M HCl,
O N
O N dioxane, rt, 1 h HN H
H NH O CF3
O CF3
b.) dethiobiotin, TEA O H
N N N
BocN N N DCM, rt, 12 h O
O
O

To an 8-mL vial was added of Diazirine N-Boc (0.013 mmol, 1 equiv.). The solid was then
dissolved in 1 ml of 4M HCl in dioxane and let to stir at rt for 1 hour. The reaction solution was
then concentrated under reduced pressure and taken up in 1.0 mL of DCM. To this solution was
added TEA (0.01 mmol, 8 equiv.) followed by NHS-desthiobiotin (0.02 mmol, 1.5 equiv.). The
reaction was let to stir at rt for 12 hour. After completion, the reaction solution was concentrated
under reduced pressure, taken up in DMSO and purified by reverse phase chromatography (0-90%
ACN/H2O) to give the product as a white solid (6.2 mg, 77% yield)

1
H NMR (500 MHz, d6-DMSO): d 7.42 (d, 2H, J = 8.4 Hz), 7.23 (d, 2H, J = 8.1 Hz), 4.44 (2H,
s), 3.86-3.80 (1H, m), 3.78 (t, 2H, J = 6.0 Hz), 3.73-3.68 (m, 1H), 3.65-3.61 (m, 6H), 3.60-3.57
(m, 2H), 3.53 (t, 2H, J = 5.5 Hz), 3.36 (t, 2H, J = 5.5 Hz), 2.52 (t, 2H, J = 6.0 Hz), 2.21 (t, 2H, J =
7.5 Hz), 1.64 (p, 2H, J = 7.4 Hz), 1.54-1.25 (m, 6H), 1.12 (d, 3H, J = 6.5 Hz)

13
C NMR (125 MHz, d6-DMSO): d 174.88, 172.68, 164.81, 141.21, 127.76, 127.23, 126.32 (d, J
= 1.3 Hz), 122.24 (q, J = 271.3), 70.19, 70.11, 69.91, 69.84, 69.20, 66.87, 56.00, 51.31, 42.07,
38.93, 36.29, 35.49, 29.34, 28.83, 25.78, 25.44, 14.23

19
F NMR (400 MHz, d4-MeOD): d -67.15 (s)

HRMS (ESI-TOF) m/z calcd. for C28H42F3N606+ ([M + H]+) 615.3112, found 615.3077

S87
Spectroscopic data

Ir-diolbppy (11)
Ir(dCO2HdFCF3ppy)2(diolbppy) (11) 1H NMR
8.90
8.90
8.38
8.38
8.06
8.05
7.85
7.84
7.83
7.83
7.54
6.83
6.82
6.81
6.80
6.80
6.80
6.78
6.78
5.89
5.88
5.87
5.87

1.65
1.64
CO2
CF3
Me F
Me
HO N

N
F
Ir
F
N
N
HO
Me F
Me CF3
CO2H

12.42
2.00
1.96
1.92
2.00
1.86
1.86

1.91

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 -4
f1 (ppm)

S88
 230
220
210
169.42
167.53
167.48

200
165.89

Me

HO
Me
HO
165.79

Me
164.75

190
Me
163.83
163.73

180
163.65

CF3
CF3
163.55
161.57

Ir

170
N
N
161.47
Ir- diolbppy (11) 13C NMR

CO2

155.69

CO2H
154.97

160
154.91

F
F

151.15
150.52

150
F
F

145.58
145.53

140
126.48
125.21
123.03

130
121.06
120.97
120.86

120
120.81
120.69
113.87

110
f1 (ppm)
113.73
99.34

100
99.12
98.91

90
80
70
69.52

60
50
40

29.74
30

29.61
20
10
0
-10

S89
Ir-diolbppy (11) 19F NMR

-104.49
-104.52
-104.52
-104.55
-104.57
-107.91
-107.94
-107.97
-59.79
CO2—
CF3
Me F
Me
HO N

N
F
Ir
F
N
N
HO
Me F
Me CF3
CO2H
6.0000

1.8966
1.9311

10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210
(ppm)

S90
 16
15
N3
O

14
13
O
O
N
H

12
O

11
10
O
S

9
8.73
O

-0.8335 8.72
8.70
8.00
NH2

-2.0000 7.98

8
-2.0042 7.90
7.88
Sulfonamide–PEG3–azide (17)

-1.5478

7
Sulfonamide–PEG3–azide (17) 1H NMR

6
(ppm)
3.59
3.58

5
3.58
3.57
3.57
3.55

4
3.55
-2.5631
3.54
-9.7156 3.52
-2.2116
-2.7337
3.45
3.44

3
3.43
3.42
3.39
3.38

2
3.37
3.35
3.32

1
0
-1
-2
-3
-4

S91
Sulfonamide–PEG3–azide (17) 13C NMR

165.75

147.03

137.58

128.27
126.04

70.25
70.23
70.14
70.08
69.71
69.23

50.43
O O
S
NH2
H
N
O
O O

N3
O

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10
(ppm)

S92
 9.04
9.04
8.90

14
8.90
8.54
8.35
8.11

13
8.10
8.09
8.08

12
8.00
7.90
7.84
7.83

11
7.83
7.82
7.82
7.81

10
0.93 7.80
0.96 7.50
1.29 7.49
2.02 6.81

9
1.15 6.80
1.00 6.80
0.94 6.79
Sulfonamide–PEG3–Ir (1)

3.46

8
6.79
1.03 6.78
0.94 6.78
Sulfonamide–PEG3–Ir (1) 1H NMR

Sulfonamide–PEG3–Ir (1) 13C NMR

0.86 6.77

7
0.87 6.76
1.86 6.76
6.76
5.87

6
1.95 5.86
5.86
5.85
5.85

5
5.84
2.11 5.84

f1 (ppm)
2.15 4.53
4.52

4
2.02 4.51
11.90 4.46
3.01 3.84

3
3.83
3.82
3.63
3.62
2.70

2
3.61
3.05
3.60
2.96
3.60
2.95
3.60

1
3.59
3.58
3.58
3.57

0
3.57
3.56
3.55
3.54
-1
3.53
3.52
3.22
1.70
-2

1.69
1.67
1.66
-3
-4
-5

S93
 230
220
210
170.83
170.17
168.94
168.88

200
168.72
167.27
167.19

190
165.22
165.13
165.03

180
163.30
162.83
157.59

170
157.48
156.37
152.53

160
152.32
152.07
147.70

150
147.05
147.00

Sulfonamide–PEG3–Ir (1) 19F NMR

145.70

140
138.92

129.02
127.91

130
127.69
127.48
127.27

120
126.63
125.69
124.46

110
124.15

f1 (ppm)
123.90
122.38

100
122.29
122.21
122.08

90
120.11
115.26
115.12

80
100.75
100.53
100.32

70
78.82
78.04
71.47
60

71.44
71.34
71.29
70.36
50

58.20
51.36
51.33
40

41.05

28.31
30

27.62
27.27
20
10
0
-10

S94
 10
0
-10
-20
-30
-40
-50
-62.04

-60
5.83
-62.07

-70
-80
-104.81
-104.83
-104.84

-90
-104.85
-104.86
-104.88

-100
-104.90

f1 (ppm)
2.00 -108.33
2.01 -108.34
-108.37

-110
-108.38
-108.40
-108.41

-120
-108.43
-108.44
-108.45

-130
-140
-150
-160
-170
-180
-190
-200
-210

S95
 16
15
O

14
N3

13
O

O
O

12
0.7970 11.49

11
N

Me
N

10
1.0000 9.89

N
N

9
Cl
O

S
Dasatinib–PEG3–azide (18)

NH
1.0992 8.23

8
7.41
7.41
N
Dasatinib–PEG3–azide (18) 1H NMR

1.1706 7.40
2.2679 7.40
NH

7
7.37
7.30
7.29
7.28
7.26

6
Me

1.1193

(ppm)
7.25
6.06

5
2.2900 4.18
2.4105 4.17
4.16

4
2.5846 4.14
3.66
14.0172
3.65
3.8693 3.63
3.60

3
0.9895 3.59
2.1400 3.58
3.56
2.3502 3.55

2
3.0816 3.55
3.54
3.2843 3.53
3.53
3.52

1
3.51
3.40
3.39
3.39
0
3.38
3.17
2.62
2.60
2.59
-1

2.57
2.56
2.54
2.41
2.24
-2
-3
-4

S96
 230
220
210
O

N3

200
O

190
O
O

180
171.59

170
165.64
N 163.05

Me
162.82

160
160.39
157.43

N
N

150
141.30
139.29

140
Cl

133.98
O

NH 132.90
129.51
128.66

130
127.49
N

126.13
Dasatinib–PEG3–azide (18) 13C NMR

NH

120
110
Me

(ppm)
100
90
83.11

80
70.26
70.20

70
70.15
70.13
69.73
66.50

60
61.88
56.42
52.84
50.44
50

44.04
40

35.16
30

26.06
20

18.78
10
0
-10

S97
 9.05
9.04
8.90
8.90

14
8.36
8.10
8.09

13
8.08
8.07
8.03
7.84

12
7.83
7.83
7.82
7.81

11
7.81
7.80
7.49
7.48

10
7.24
1.02 7.23
0.98 7.23

9
2.32 7.21
0.86 6.78
2.19 6.77
0.89 6.03

8
0.97 5.85
Dasatinib–PEG3–azide (18)

1.01 5.85
0.98 5.84
5.83
Dasatinib–PEG3–azide (18) 1H NMR

0.99

7
0.95 4.57
1.94 4.56
2.00 4.55
0.85 4.48

6
2.00 4.25
4.23
3.87

5
2.07 3.87
1.93 3.86
1.92 3.85

f1 (ppm)
2.10 3.70

4
6.18 3.69
10.09 3.68
3.12 3.66
1.71 3.65

3
3.34 3.65
2.02 3.64
2.82 3.64
3.13 3.64

2
2.72 3.59
2.92 3.58
3.01 3.58
3.16 3.57

1
3.56
3.56
3.55

0
3.55
3.54
3.54
3.53
-1 3.53
3.52
3.51
3.50
-2

3.21
2.77
2.69
2.68
-3

2.67
2.57
2.56
2.55
-4

2.45
2.31
1.71
1.70
-5

1.66
1.65

S98
 230
173.07
170.75
168.92

220
168.87
167.43
165.20
165.11

210
165.02
164.91
164.37

200
163.32
162.88
162.83

190
158.63
157.58
157.46

180
156.36
152.54
152.29

170
152.06
147.02
145.69

160
142.23
140.38
134.36

150
134.26
130.14
129.55
128.31

140
Dasatinib–PEG3–azide (18) 13C NMR

127.91
127.68
127.49

130
125.77
124.46
124.12

120
123.90
122.46
122.29

110
115.20

f1 (ppm)
115.06
100.76
100.55

100
100.33
84.01
78.81

90
78.02
73.86
71.46

80
71.41
71.39
70.38

70
67.59
64.40
62.26

60
58.22
57.47
53.84
50

51.39
51.35
44.62
40

35.93
28.27
27.71
30

27.28
27.26
25.66
20

18.75
10
0
-10

S99
Dasatinib–PEG3–azide (18) 19F NMR

-104.71
-104.74
-104.76
-104.79
-104.81
-108.26
-108.29
-108.31
-108.33
-108.34
-108.38
-61.97
-62.03
5.36

2.00
1.99

10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210
f1 (ppm)

S100
 9.78
8.42

16
7.29
7.28
7.27
7.24

15
7.23

Cl
7.22
7.21
7.20

O
6.87

14
6.85
4.51
4.51
4.50

HN

13
4.50

NH
4.49

Cl
4.48

N
4.48
4.05

12
O
4.04
4.03

N3

H
N
4.03
4.02

11
3.84
3.84
3.83
3.81
3.81

10
N
O
3.75
AT7519–PEG3–azide (19)

0.8249
3.75

O
3.73
3.73

9
3.72
AT7519–PEG3–azide (19) 1H NMR

3.71
O 3.70
O

0.7871 3.70
3.69

8
3.68
0.6760 3.60
3.59
0.8534 3.58

7
1.0000 3.58
3.57
0.7848 3.57
3.33
3.32

6
(ppm)
3.31
3.30
3.10
3.09

5
3.07
0.8698 3.07
3.06
0.9241 3.04
3.04

4
0.9554 2.72
2.2501 2.71
2.70
9.9945 2.69

3
2.0790 2.69
2.67
0.8476 2.66
0.8390 2.63
2.61

2
2.1710 2.61
2.0125 2.60
2.58
1.8798 2.57

1
2.56
2.56
2.54
2.54
2.53
0

2.53
2.00
1.99
1.98
-1

1.97
1.97
1.94
1.93
1.92
-2

1.91
1.91
1.45
1.44
-3

1.43
1.42
1.42
1.41
1.40
-4

1.39

S101
 230
Cl

220
O

210
HN

200
NH
Cl

190
O
N3

H
N

180
169.61

170
163.12

N
O

161.61

160
O

150
O
O
AT7519–PEG3–azide (19) 13C NMR

140
135.38
133.10
132.56
131.05

130
128.18
122.56
121.50

120
110
(ppm)
100
90
80
70.67
70.62
70.55

70
70.43
70.01
67.50

60
50
50.69
46.38
44.61
40 40.68

33.57
32.52
31.65
30
20
10
0
-10

S102
 9.06
9.06
8.91

14
8.91
8.37
8.36

13
8.36
8.34
8.11

12
8.10
8.09
8.08
8.04

11
7.85
7.84
7.84

10
7.83
1.06 7.82
AT7519–PEG3–Ir (5)

1.02 7.82

9
1.91 7.81
7.81
1.04
AT7519–PEG3–Ir (5) 1H NMR

7.50
2.05

8
7.50
1.00 7.49
2.03 7.48
4.93 7.48

7
1.72 7.47
7.46
7.45

6
1.88 6.80
6.78
2.58
6.78
2.00

5
6.76
1.88 5.87
1.20 5.86

f1 (ppm)
1.14 5.86

4
2.21 5.85
5.85
2.25
5.84

3
8.70
5.84
3.53 4.56
2.64 4.49

2
1.12 4.06
1.34 3.88
1.14 3.87
3.86

1
1.04
3.72
1.06
3.71
11.70 3.70

0
2.10 3.59
1.24 3.58
3.56
-1 3.32
3.31
3.31
3.31
-2

3.30
3.22
2.67
-3

2.66
1.71
1.71
-4

1.69
1.67
1.66
1.63
-5

1.60
1.58

S103
 230
220
210
200
168.46
166.09
165.47

190
165.30
164.67
164.57

180
162.63
162.53
160.61

170
160.57
160.41
155.64

160
155.22
AT7519–PEG3–Ir (5) 13C NMR

153.74

150
150.68
145.19
143.95

140
135.40
131.94

130
131.28
128.47

120
126.74
126.35
126.29

110
124.31

f1 (ppm)
123.24
123.20

100
122.67
122.46
121.27

90
121.06
121.03

80
120.12
119.86
113.76

70
113.63
99.55
99.34

60
77.18
76.40
69.72
50

69.64
69.61
40

69.58
68.71
66.81
30

57.00
50.41
49.41
20

46.00
44.08
32.77
10

31.70
30.86
0

27.76
27.70
26.99
-10

26.69

S104
26.54
AT7519–PEG3–Ir (5) 19F NMR

-104.19
-104.22
-104.24
-104.27
-104.29
-107.71
-107.74
-107.78
-107.81
-59.75
-59.78
6.11

2.07
2.03

10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210
f1 (ppm)

Lenalidomide–PEG0–Ir (6)
Lenalidomide–PEG0–Ir (6) 1H NMR

S105
 10.5
1.00 10.34

10.0
9.5
2.10 9.41

9.0
8.5
2.14 8.39

2.11 8.06
8.03

8.0
2.18 7.96
2.13 7.68
2.12 7.64
7.56

7.5
2.04 7.51
7.41
1.17 7.30

Rapamycin–PEG3–Ir (7)
7.26
1.03

7.0

Rapamycin–PEG3–Ir (7) 1H NMR

2.08 6.59

6.5
6.0
2.04 5.56

5.5
5.08

f1 (ppm)
5.0
1.08
4.83
1.14
1.13
4.54

4.5
4.0
3.5
3.12 3.26
3.0

3.21 2.64
2.50
2.5

1.27
2.0

5.95 1.93
1.5
1.0
0.5
0.0

S106
 9.04

16
9.04
8.89
8.89
8.34

15
8.34
8.33
8.33
8.09

14
8.08
8.06
8.06
8.04

13
8.04
7.80
7.79

12
7.48
7.48
7.47
7.46

11
7.46
5.82
5.82
5.82

10
0.99 4.48
1.00 3.86
1.02 3.86
0.99 3.85

9
1.01 3.85
0.97 3.68
0.97 3.67
3.67

8
0.99
1.00 3.67
1.03 3.66
1.03 3.34

7
2.06 3.34
1.04 3.32
0.97 3.29
1.00 3.25

6
0.95 3.20

f1 (ppm)
2.03 3.20
1.11 3.11
1.40 2.53

5
0.96 2.52
0.87 2.52
4.05 2.52
2.05 2.51

4
1.42 2.05
1.74 1.82
2.10 1.82
1.73

3
3.42
9.12 1.73
5.35 1.70
2
2.85 1.69
4.15 1.68
2.59 1.67
1.00 1.65
2.77 1.64
1

0.80 1.64
1.49 1.63
1.20 1.63
2.18 1.60
0

1.55 1.25
2.13 1.02
2.43 1.01
6.73 1.00
-1

24.87 0.94
3.81 0.94
6.35 0.93
4.09 0.93
-2

3.99 0.91
3.41 0.90
2.76 0.87
0.86
-3

5.91
1.54 0.82
0.81
0.81
-4

0.81

S107
 214.32
208.17

230
171.06
171.03
169.22

220
166.69
139.68
136.09

210
135.96
133.30
130.17
129.90

200
129.11
126.52
126.46

190
126.43
126.07
98.66

180
98.46
84.76
84.23

170
84.06
80.76
80.73

160
76.85
76.31
75.32

150
70.57
Rapamycin–PEG3–Ir (7) 13C NMR

70.53
70.47

140
70.29
69.93
67.09
66.59

130
58.82
57.52
57.49

120
56.26
55.83
51.19

110
f1 (ppm)
50.58
46.51
46.05

100
44.13
41.50
40.53

90
40.50
40.13
39.96

80
39.79
39.03
38.27

70
35.93
35.32
35.30

60
34.89
33.85
33.25
32.74
50

31.14
31.05
29.64
40

29.60
27.09
26.96
30

25.16
21.49
21.32
20

20.63
16.27
16.12
10

15.88
15.73
13.46
0

13.42
13.01
10.29
10.14

S108
-10
Rapamycin–PEG3–Ir (7) 19F NMR

-104.74
-104.77
-104.80
-108.30
-108.33
-108.35
-108.36
-62.00
-62.04

-76.92
3.01
3.00

2.15
2.13

10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210
f1 (ppm)

S109
 8.15

16
8.13
8.13
8.13

15
7.96
7.96

14
7.94
7.66
7.66

13
7.66
7.65
7.64

12
7.64
7.64
7.63

11
7.63
7.63
7.34

10
7.33
7.33
6.90

9
6.90
MM-206–PEG3–azide (21) 1H NMR

1.00 3.92
3.91

8
0.97
2.07 3.91
1.18 3.91

7
3.70 3.90
0.92 3.90
3.66

6
3.66

f1 (ppm)
3.65
3.63

5
3.62
3.62
1.91 3.62

4
1.96 3.61
2.06 3.59
6.03 3.59

3
1.89 3.59
1.92 3.58
3.58

2
3.58
3.57
3.57
1

3.57
3.56
3.56
0

3.55
3.55
3.55
-1

3.33
3.32
3.32
-2

3.32
3.31
3.31
-3

3.01
3.00
-4

2.99

S110
 230
220
210
200
190
180
170.44

170
160
150
144.90
133.18
MM-206–PEG3–azide (21) 13C NMR

140
132.97
130.83

130
130.41
130.05
128.85

120
128.64
128.12

110
f1 (ppm)
126.60
125.73

100
123.98
122.46

90
80
70.81
70.78

70
70.70
70.07
66.88

60
51.03
50
40

35.48
30
20
10
0
-10

S111
MM-206–PEG3–azide (21) 19F NMR

-137.85
-137.91
-137.97
-147.59
-147.63
-147.66
-147.69
-147.73

-160.39
-160.49
2.15

1.00

1.99
-65 -70 -75 -80 -85 -90 -95 -100 -105 -110 -115 -120 -125 -130 -135 -140 -145 -150 -155 -160 -165 -170 -175 -180 -185 -190 -195 -200
f1 (ppm)

S112
Diazirine-PEG3-NBoc (22) 1HNMR

S113
Diazirine-PEG3-NBoc (22) 13CNMR

S114
Diazirine-PEG3-NBoc (22) 19FNMR
4
3
5.
6
-

S115
Diazirine-PEG3-desthiobiotin (23) 1HNMR

S116
Diazirine-PEG3-desthiobiotin (23) 13CNMR

Diazirine-PEG3-Desthiobiotin (23) 19FNMR

5
1
.
7
6
-

S117
10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 -120 -130 -140 -150 -160 -170 -180 -190 -200 -210

S118
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