Supporting Information for

Hydrogel biomaterials that stiffen and soften on demand reveal that

skeletal muscle stem cells harbor a mechanical memory

Christopher M. Madl,* Yu Xin Wang, Colin A. Holbrook, Shiqi Su, Xuechen Shi, Fitzroy J. Byfield,
Gwendoline Wicki, Iris A. Flaig, Helen M. Blau*

Helen M. Blau, Christopher M. Madl

Email: [email protected], [email protected]

This PDF file includes:

Supporting text
Figures S1 to S10
SI References

1
Supporting Information Text

1. Supporting Note 1

Our hydrogel culture platforms were designed to mimic key biophysical aspects of the cell
microenvironment during the process of stem cell activation following tissue damage. After injury,
the multinucleated muscle fiber degenerates, leaving a hollow tube of basal lamina ECM, a
structure that has been termed a “ghost fiber” (1). Thus, as MuSCs break quiescence and
activate, they remain attached to the basal lamina, even though the injury has disrupted the cell-
cell contacts with the muscle fiber that exist in resting tissue. As the residual ECM tube is a quasi-
two-dimensional (2D) environment, culturing MuSCs on the 2D surface of hydrogels whose
stiffness resembles that of the muscle ECM is a reasonable model system for the early stages of
muscle repair when cell-ECM adhesions dominate. The composition of the ECM is also tightly
regulated during muscle repair, serving as an important signal that regulates myogenic
progression in MuSCs (2–4). The early stages of muscle repair are characterized by high
expression of laminin isoforms (3), and our previous work has confirmed that culturing MuSCs on
laminin-functionalized surfaces promotes MuSC expansion in vitro and engraftment in vivo (5, 6).
Thus, the hydrogels used in this study were functionalized with laminin to best mimic the
dominant ECM composition during MuSC activation. This simplified system enables causal
relationships to be more readily determined due to the limited number of variables, but this
advantage comes at the expense of the complex and heterogeneous environment present during
muscle regeneration in vivo. Future work may leverage 3D organotypic culture systems (7) in
combination with advanced biomaterials approaches to more completely model MuSC-mediated
regeneration in vitro.

2. SI Materials and Methods

2.1. Materials

Precursors and solvents for chemical synthesis were purchased from Sigma-Aldrich,
Fisher Scientific, Acros Organics, or Tokyo Chemical Industry (TCI) and used without further
purification, unless otherwise noted. Multi-arm poly(ethylene glycol) (PEG) precursor materials
were purchased from JenKem Technology USA. Cell culture reagents were purchased from
Thermo Fisher Scientific, unless otherwise noted.

2.2. Synthesis of hydrogel precursors

Detailed synthetic protocols are provided in the supporting information and in our
previously published work (6). Small molecule precursors bicyclo[6.1.0]nonyne N-
hydroxysuccinimide (BCN-NHS) and azido ortho-nitrobenzyl ester N-hydroxysuccinimide (N3-
oNB-NHS) were prepared following known synthetic routes (8–11). An amine reactive
photocaged ODIBO precursor (pODIBO-NHS) was prepared by adapting known synthetic routes,

2
as described below in the Detailed Synthetic Procedures (12). Azide-functionalized PEGs were
prepared by reacting 8-arm PEG-amine with activated NHS ester azides under standard amide
coupling conditions, as previously described (6, 8, 13). BCN- and pODIBO-functionalized sulfated
PEGs were prepared by sequentially reacting 4-arm or 8-arm PEG-amine with cysteic acid and
BCN-NHS/p-ODIBO-NHS as in our previous work (6). All synthesized PEGs were dialyzed
against MilliQ-grade water and lyophilized prior to use. Azide-functionalized laminin-111 was
prepared by reacting purified laminin with N3-PEG-NHS following our previous protocols for
laminin functionalization (5, 6).

2.3. Hydrogel characterization

Azide- and BCN-containing PEG precursors were separately dissolved in phosphate
buffered saline (PBS) to the desired polymer concentration, thoroughly mixed by pipetting, added
to cylindrical silicone molds (8 mm diameter × 0.8 mm thickness), and allowed to crosslink at
37°C for 30 minutes. The resulting hydrogels were equilibrated in PBS for at least 1 hour before
mechanical testing. For dynamically softening or stiffening hydrogels, following equilibration, the
hydrogels were exposed to 365 nm light (~450 mW/cm2) for 4 minutes and returned to PBS to
equilibrate for an additional hour. An ARG2 rheometer and an 8 mm parallel plate geometry were
used to perform oscillatory rheology. Samples were strained to 5% and frequency sweeps from
0.1 to 10 Hz were collected. The shear modulus (G) was taken to be the average storage
modulus (G’) over the linear range centered at 1 Hz, following our established protocol (5, 6).
Shear moduli were converted to Young’s moduli (E) by assuming Poisson’s ratio for PEG gels is
~0.5.
The spatial heterogeneity of the hydrogel stiffness was assessed by AFM scans using a
Nanowizard 4 system (Bruker) mounted on a Leica DMI 6000 B microscope. AFM probes with a 1
μm diameter SiO2 particle attached (Novascan Technologies, Boone, IA) and a spring constant of
0.063 N/m were used to indent the hydrogels at a frequency of 1 Hz. Prior to the measurements,
the deflection sensitivity of the AFM probe was calibrated with a petri dish in PBS. The maximum
force applied to the hydrogels was 10 nN. All measurements were conducted at room
temperature in PBS. Each experimental condition included three hydrogels, with 2-3 regions
scanned per gel to create a stiffness map. Each map had a size of 10x10 μm and contained 8x8
data points. The roughness of the hydrogels was assessed by measuring the contact point of the
probe for each measurement in the map. The analysis of AFM force curves was performed using
JPK Data Processing software and fitted to the Hertz model for a sphere.
The mass swelling ratio of the hydrogels was measured by casting the gels as described
above, allowing the gels to swell overnight, and then weighing the gels in their hydrated state on
an analytical balance. The gel samples were then frozen at -80°C, lyophilized overnight, and re-
weighed in their dry state. The mass swelling ratio is the ratio of water present in the swollen gels
to the weight of the dry gel components.

3
 The laminin content in the hydrogels was measured by incorporating fluorescently
labeled laminin into the hydrogels. Laminin-PEG-N3 was reacted with a 50-fold molar excess of
Sulfo-Cyanine5-NHS ester (Lumiprobe) for 8 hours at room temperature with agitation. The
labeled laminin was then dialyzed (20 kDa MWCO) against PBS to remove unreacted dye. The
Cy5-labeled laminin-PEG-N3 was used to fabricate gels as described above, which were then
equilibrated in PBS before light exposure and imaging. The gels were imaged on a Zeiss LSM
900 confocal microscope. Soft static gels containing labeled laminin were exposed to the same
dose of UV light as the softening and stiffening gels to account for any changes in fluorescence
due to photobleaching of the fluorophore.

2.4. Muscle stem cell isolation

All animal protocols were approved by the Stanford University Administrative Panel on
Laboratory Animal Care (APLAC) and experiments were performed in compliance with the
institutional guidelines of Stanford University. Myogenin Sun1-GFP reporter mice were generated
following our published protocol (6) by crossing Gt(ROSA)26Sortm5(CAG-Sun1/sfGFP)Nat mice (JAX
#021039) (14) with Myogtm1.1(cre/ERT2)Lepr (JAX #025668) (15). Young (~2 mo) C57BL/6 mice were
purchased from The Jackson Laboratory, and aged (~22 mo) C57BL/6 mice were obtained from
the National Institute on Aging and housed for at least 2 additional months prior to use. Muscle
stem cells were isolated from the hindlimb muscles of mice following our published procedures (5,
6, 16). Briefly, muscles were digested with collagenase and dispase on a gentleMACS Octo
Dissociator (Miltenyi) and filtered through a 45 μm cell strainer. The cell suspension was then
incubated with PE-Cy7 conjugated antibodies against CD45, CD11b, CD31, and Sca1 (BD
Biosciences; lineage markers, Lin) to label non-muscle cell types and integrin-α7-PE (Ablab) and
CD34-Alexa647 (BD Biosciences) to label MuSCs. The labeled cells were sorted by FACS to
deplete non-muscle lineage cells and enrich MuSCs (Lin-, integrin-α7+, CD34+).

2.5. Muscle stem cell culture and analysis

PEG hydrogels for MuSC culture were formed directly in glass bottom 24-well plates
(Cellvis) that were first functionalized with (3-azidopropyl)trimethoxy silane (8) as described in our
published protocol (6). Sterile PEG precursor solutions in PBS were maintained on ice to slow
gelation. PEG-sulfo-BCN or PEG-sulfo-[4-BCN/4-pODIBO] and laminin-PEG-N3 (100 μg/mL final
concentration) were mixed together, followed by addition of azide-functionalized PEGs. The
PEG-laminin mixture (200 μL/well) was pipetted into the well plate, and the plate was then
centrifuged at 2000×g for 10-15 minutes at room temperature to provide a uniform coating of
hydrogel. The plates were incubated for 30 minutes at 37°C and then equilibrated in cell culture
medium at 37°C prior to cell seeding. Freshly isolated MuSCs were immediately seeded onto
hydrogels at a density of 2,000 cells/well for day 7 endpoints and 15,000 cells/well for day 1 and
day 3 timepoints. These cell concentrations were chosen to minimize cell-cell contact and

4
maximize cell-substrate interactions throughout the course of the experiment. Following our
previous protocol (6), MuSCs were cultured in FluoroBrite DMEM with 15% FBS, L-glutamine,
sodium pyruvate, non-essential amino acids, penicillin/streptomycin, and 2.5 ng/mL FGF-2. For
experiments using the myogenin Sun1-GFP reporter MuSCs, the medium was supplemented
daily with 4-hydroxytamoxifen (1 μM). Media was replaced on days 3 and 5. For
mechanotransduction inhibitor experiments, cells were treated with DMSO vehicle, rhosin (30
μM), NSC23766 (50 μM), or verteporfin (4 μM) at the time of seeding and refreshed on days 1
and 2 of culture before being washed out at day 3. The concentrations of inhibitors were selected
based on previously published results indicating efficacy without impairing cell adhesion or
viability (17–19). For dynamic hydrogel softening and stiffening experiments, gels were exposed
to 365 nm light (~450 mW/cm2) for four minutes. For EdU incorporation experiments, F-ara-EdU
was added to the culture medium on day 7, 6 hours prior to fixation, at a final concentration of 10
μM.

2.6. Immunofluorescence analysis of MuSC fate

MuSCs on hydrogels were fixed with 4% paraformaldehyde in PBS. For general
immunofluorescence analysis, the fixed cells were permeabilized with PBS plus 0.25% (v/v)
Triton X-100 (PBST) and incubated with blocking buffer (5% bovine serum albumin (BSA) and 5%
goat serum (GS) in PBST). Samples were subsequently incubated with primary antibodies
against Pax7 (Developmental Studies Hybridoma Bank, 2 μg/mL), MyoD (Santa Cruz
Biotechnology, clone G-1, 1:200), myogenin (Santa Cruz Biotechnology, clone D-10, 1:200), YAP
(Santa Cruz Biotechnology, clone 63.7, 1:200), and Ki67 (Cell Signaling Technology, clone D3B5,
1:400) in dilution buffer (2.5% BSA and 2.5% GS in PBST). Samples were washed with PBST
and then incubated with secondary antibodies (AF488 goat anti-mouse IgG2a, AF488 goat anti-
mouse IgG3, Cy3 goat anti-mouse IgG2b, and AF 647 goat anti-mouse IgG1, Jackson
ImmunoResearch, 1:500; Alexa Fluor Plus 405 goat anti-rabbit IgG, Thermo Fisher Scientific,
1:250) in dilution buffer. Samples were washed with PBST and stained with DAPI and TRITC-
phalloidin, if desired, prior to imaging. To label EdU, after immunostained reporter cells had been
imaged, cells were stained with AF488-azide (Lumiprobe) via a copper-catalyzed click reaction
(19, 20), which simultaneously quenched the GFP reporter signal, enabling selective visualization
of the EdU+ cells. For Rho and Rac GTPase activity staining, a previously published protocol was
followed with some modifications (18, 21). After permeabilization with PBST, fixed cells were
blocked with 10% GS in PBST for 1 hour, followed by incubation with GST-tagged GTPase
binding proteins (RHO-RBD at 50 μg/mL for RhoA and PAK-PBD at 25 μg/mL for Rac1,
Cytoskeleton Inc.) for an additional hour. Cells were washed with PBS and then incubated with
anti-GST antibody (Thermo Fisher, A-5800, 1:300) for 1 hour. After washing with PBS, the cells
were then incubated with secondary antibody (AF647 goat anti-rabbit IgG, Jackson
ImmunoResearch, 1:500) and DAPI for 1 hour, before washing with PBS and PBST. All samples

5
were imaged on a Zeiss AxioObserver inverted microscope with a 7-channel ZEISS Colibri 7 light
source for multichannel fluorescence imaging. Tiled image regions were collected near the center
of the gels. Images were deconvolved and stitched using our previously published GPU-
accelerated image processing pipeline (6, 22). For quantitative analysis, images were segmented
and analyzed using either Cell Profiler or the CellSeg pipeline (23).

2.7. Single cell RNA sequencing and bioinformatics analysis

Cells cultured on hydrogels were washed with PBS and detached from the substrates by
treatment with TrypLE Express and manual pipetting. The cells were collected by centrifugation
and fixed using the Cell Fixation kit from Parse Biosciences. Single cell libraries for RNA
sequencing were prepared based on a SPLiT-seq approach (24) using the Evercode Mini WT
and Evercode WT kits from Parse Biosciences, following the manufacturer's protocols. The
SPLiTseq approach uses combinatorial barcoding to uniquely tag RNA transcripts from individual
cells that precludes the need to use microfluidic based approaches to physically isolate the
individual cells. This enables cells to be collected at different time points and from different
experimental conditions, stored, and then processed together into a single sequencing library to
mitigate potentially confounding batch effects. Library quality was validated using an Agilent
Bioanalyzer 2100 at the Stanford Genomics Facility. Sequencing was performed by Novogene on
an Illumina NovaSeq 6000 system. Sequencing reads were mapped to the mouse reference
genome and correlated with single cell barcodes using the Parse Biosciences analysis pipeline.
Cells were sequenced to an average read depth of 180k reads/cell. Downstream analysis of
transcriptomic data was performed using Scanpy (25), and RNA velocity analysis was performed
using scVelo (26). For the heatmaps presented in Figures 5E and G, the data were subsampled
using scSampler to aid in data visualization while preserving the diversity of cell types present
(27).

2.8. Effect of dihydrotetrazine photooxidation on cell viability

Human primary lung fibroblasts, MRC-5 (ATCC #CCL-171), were used as a model
adherent cell type to assess the cytotoxicity of photocatalyzed dihydrotetrazine oxidation. MRC-
5s were cultured in high glucose DMEM, supplemented with 10% fetal bovine serum (FBS) and
1% penicillin/streptomycin (Pen/Strep). For live/dead fluorescence imaging, FluoroBrite DMEM
(Life Technologies) supplemented with 10% FBS, 1% Pen/Strep and 1% L-Glutamine (Life
Technologies) was used. MRC-5s were maintained at 37°C and 5% CO2. Cells were passaged
after reaching 90% confluency, using a 1:10 dilution of 0.5% Trypsin-EDTA in PBS. To fabricate
hydrogels for cytotoxicity analysis, 4-arm PEG-sulfo-BCN and cell adhesive RGD-N3 peptides
(500 μM final concentration) were mixed together, followed by addition of 8-arm azide-
functionalized PEGs. For experiments using the tethered IRDye 700DX, the IRDye700DX-PEG-
N3 was added prior to the addition of the 8-arm PEG-azide. The PEG mixture (22 μL/well) was

6
pipetted into an azide-functionalized 12-well plate (prepared as described previously (6)), and
then a 12 mm glass coverslip that had been passivated with Sigmacote (Sigma-Aldrich) was
placed on top of the PEG mixture droplet. The plate was then incubated at 37°C for 30 minutes to
allow the gels to crosslink. Gels were then incubated for 1 hour in PBS containing 10% Pen/Strep
and 1x Antioxidant Supplement (Sigma-Aldrich, 1000x), allowing swelling and removal of the top
coverslip. After several washes with PBS to remove excess antibiotic, cells resuspend in
FluoroBrite DMEM culture medium containing 1x antioxidant were seeded at 25,000 cells/well.
Cells were allowed to adhere for 24 hours before the phototoxicity experiments were performed.
For conditions testing soluble methylene blue, media containing 10 μM methylene blue was
added to the cells prior to light illumination. For conditions testing tethered IRDye 700DX, cells
were seeded either on gels that contained no dye or gels with 20 μM tethered IRDye 700DX. In
experiments assessing toxicity of light-triggered stiffening, gels were illuminated with a light
emitting diode (LED) at 660 nm (methylene blue) or 680 nm (IRDye 700DX), placed underneath
the gel at a distance of 2 cm. Culture medium was changed immediately after illumination. 24
hours after dye/light exposure, cells were incubated in calcein-AM (1 µM) and ethidium
homodimer (1 µM) diluted in FluoroBrite DMEM for 30 minutes. The staining solution was
replaced with fresh FluoroBrite DMEM, and the samples were imaged. Four images per gel were
captured with a Keyence inverted fluorescence microscope (BZ X-710) at 4x magnification, using
the GFP (Ex: 470/40, DM 495, BA 525/50, OP-87763) and the TRITC (Ex: 545/25, DM 565, BA
605/70, OP-87764) filter sets, as well as in brightfield. A manual count of green cells (alive) and
red nuclei (dead) was performed to calculate the cell viability ratio.

2.9. Hydrogel stiffening by dihydrotetrazine photooxidation

Hydrogels were prepared by first mixing 4-arm PEG-sulfo-BCN and IRDye700DX-PEG-
N3 (20 μM final concentration) before adding 8-arm PEG-[4-N3/4-dHTz] to facilitate crosslinking.
The molar ratio of BCN to azide plus dihydrotetrazine was maintained at 1:1. Gels were
crosslinked in 8 mm diameter, 0.8 mm thick silicone molds for 30 minutes at 37°C. Gels were
then equilibrated in PBS for at least 1 hour at 37°C prior to rheological testing. For stiffening
conditions, the gels were exposed to 680 nm LED light for 10 minutes, before being returned to
PBS for at least 1 hour at 37°C for equilibration. An ARG2 rheometer and an 8 mm parallel plate
geometry were used to perform oscillatory rheology, following the procedure described in the
main text.

2.10 Serum stability of dihydrotetrazine materials

mPEG-dHTz was dissolved to a concentration of 10 mM in FluoroBrite DMEM containing
10% FBS and maintained at 37°C for 24 hours. Stability was assessed by measuring the UV-
visible light spectra of the solution immediately upon dissolution and after 1 hour, 2 hours, 3
hours, and 24 hours.

7
2.11. Statistical analysis
At least 3 independent replicates were used for each experiment. Statistical analysis was
performed using GraphPad Prism 8. For population level data, data sets were assumed to follow
normal distributions, and the following analyses were used: comparisons between two
experimental groups with a single varying parameter were performed using two-tailed Student’s t-
tests and comparisons among more than two experimental groups with a single varying
parameter were performed using one-way analysis of variance (ANOVA) with Tukey post-hoc
testing. For single cell immunofluorescence data, the data were not normally distributed, so
comparisons among more than two experimental groups with a single varying parameter were
performed using nonparametric Kruskal-Wallis tests with Dunn’s multiple comparisons
corrections. Corrected p-values of less than 0.05 were considered statistically significant.

8
3. Detailed Synthetic Procedures

3.1. General Considerations

All reagents were purchased from Sigma-Aldrich, Fisher Scientific, Acros Organics, or Tokyo
Chemical Industry (TCI) and used without further purification, unless otherwise noted. Multi-arm
PEG starting materials were purchased from JenKem Technology USA. NMR spectroscopy was
performed on Varian Mercury 400 MHz or Varian Inova 500 MHz spectrometers in the Stanford
University NMR Facility. Chemical shifts were referenced to the residual solvent peak. UV-visible
light spectroscopy was performed using a NanoDrop 2000 spectrophotometer (Thermo).

3.2. Synthesis of BCN-NHS

The amine reactive BCN precursor BCN-NHS was prepared following
a known synthetic route (8–10), and was published previously (6). 1H

NMR (500 MHz, CDCl3) δ ppm 4.45 (d, J = 8.3 Hz, 2H), 2.84 (s, 4H),
2.27 (m, 6H), 1.56 (m, 2H), 1.50 (m, 1H), 1.06 (m, 2H). 13C NMR (126
MHz, CDCl3) δ ppm 168.70, 151.59, 98.66, 70.32, 28.93, 25.44, 21.28,
20.67, 17.13. Rf 0.50 (1:1 hexanes:EtOAc).

3.3. Synthesis of (3-azidopropyl)trimethoxysilane

(3-azidopropyl)trimethoxysilane was synthesized following a known
synthetic route (8), and was published previously (6). 1H NMR (500 MHz,
CDCl3) δ ppm 3.58 (s, 9H), 3.27 (t, J = 7.0 Hz, 2H), 1.71 (m, 2H), 0.70 (m,
2H). 13C NMR (126 MHz, CDCl3) δ ppm 53.69, 50.58, 22.42, 6.29.

3.4. Synthesis of Photocleavable Linker (N3-oNB-NHS)

The ortho-nitrobenzyl photocleavable linker was prepared following a known synthetic route (10,
11), and was published previously (6). 1H NMR (500 MHz, CDCl3) δ ppm 7.59 (s, 1H), 7.00 (s,
1H), 6.49 (q, J = 6.4 Hz, 1H), 4.17 (t, J = 6.1 Hz, 2H), 3.97 (s,
3H), 3.33 (m, 2H), 2.89 (t, J = 7.3 Hz, 2H), 2.85 (br. s, 4H),
2.46 (m, 2H), 2.29 (quin, J = 6.7 Hz, 2H), 1.90 (quin, J = 7.0
Hz, 2H), 1.62 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ ppm 171.34, 169.03, 168.06, 154.01, 146.97,
139.76, 133.18, 109.25, 108.13, 68.44, 67.35, 56.26, 50.41,
31.13, 27.50, 25.54, 24.09, 21.95.

9
3.5. Synthesis of Photo-SPAAC Linker (pODIBO-NHS)
The photo-activatable strained cyclooctyne (pODIBO) precursor was synthesized by adapting
previously published synthetic routes for the pODIBO core structure (28, 29).

3.5.1. 3-((tert-butyldimethylsilyl)oxy)benzaldehyde

3-hydroxybenzaldehyde (15 g, 123 mmol, 1 eq.) and imidazole (8.36 g, 123 mmol, 1 eq.) were
added to a 1 L round bottom flask with a stir bar and dissolved in anhydrous dichloromethane
(400 mL). tert-butyldimethylsilyl chloride (22.22 g, 147 mmol, 1.2 eq.) was added. The flask was
purged with nitrogen, and the reaction was allowed to proceed at room temperature for 3 h. The
mixture was filtered to remove the white precipitate, concentrated by rotary evaporation, and
purified by silica flash chromatography, eluting with 10:1 hexanes:ethyl acetate. Fractions
containing the desired product were identified by TLC, combined, and concentrated in vacuo to
afford the product as a faint yellow oil (27.26 g, 115 mmol, 93.5% yield). 1H NMR (500 MHz,
CDCl3) δ ppm 9.96 (s, 1H), 7.48 (dt, J = 7.5, 1.3 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.34 (dd, J =
2.6, 1.6 Hz, 1H), 7.11 (m, 1H), 1.00 (s, 9H), 0.23 (s, 6H). 13C NMR (126 MHz, CDCl3) δ ppm
192.07, 156.36, 137.88, 130.05, 126.52, 123.54, 119.83, 25.58, 18.17, -4.46. Rf 0.58 (10:1
hexanes:EtOAc).

10
3.5.2. (3-((tert-butyldimethylsilyl)oxy)phenyl)methanol

3-((tert-butyldimethylsilyl)oxy)benzaldehyde (22.0 g, 93 mmol, 1 eq.) was added to a 500 mL

round bottom flask with a stir bar and dissolved in anhydrous diethyl ether (150 mL). The flask
was purged with nitrogen and cooled on in an ice bath. Lithium aluminum hydride (4.06 g, 107
mmol, 1.15 eq.) was added to a separate 150 mL round bottom flask and suspended in
anhydrous diethyl ether (125 mL). This flask was also purged with nitrogen and cooled in an ice
bath. The LiAlH4 suspension was added dropwise to the stirring mixture of starting material via
cannula transfer, maintaining both mixtures on ice. After addition was complete, the reaction
mixture was allowed to warm to room temperature and react for an additional 30 minutes. The
reaction mixture was then returned to the ice bath to cool. The reaction was quenched by slow,
dropwise addition of water (4 mL), followed by 15% aqueous sodium hydroxide (4 mL) and water
(12 mL). The mixture was allowed to warm to room temperature and stirred for 15 minutes.
Magnesium sulfate was added, and the mixture was stirred for an additional 15 minutes. The
mixture was filtered, and the solids were washed with diethyl ether. The combined solutions were
concentrated in vacuo to afford the product as a colorless oil (16.73 g, 70 mmol, 75.3% yield). 1H
NMR (500 MHz, CDCl3) δ ppm 7.22 (t, J = 7.8 Hz, 1H), 6.95 (dq, J = 7.6, 0.7 Hz, 1H), 6.86 (m,
1H), 6.77 (m, 1H), 4.63 (s, 2H), 1.88 (s, 1H), 1.00 (s, 9H), 0.21 (s, 6H). 13C NMR (126 MHz,
CDCl3) δ ppm 155.83, 142.48, 129.46, 119.71, 119.18, 118.56, 65.08, 25.64, 18.15, -4.43. Rf
0.18 (10:1 hexanes:EtOAc).

11
3.5.3. tert-butyl(3-((4-(tert-butyl)phenoxy)methyl)phenoxy)dimethylsilane

(3-((tert-butyldimethylsilyl)oxy)phenyl)methanol (16.0 g, 67 mmol, 1 eq.), 4-tert-butylphenol (10.08

g, 67 mmol, 1 eq.), and triphenylphosphine (17.6 g, 67 mmol, 1 eq.) were added to a 1 L round
bottom flask with a stir bar and dissolved in anhydrous tetrahydrofuran (350 mL). The flask was
purged with nitrogen and cooled in an ice bath. Diisopropyl azodicarboxylate (13.57 g, 67 mmol,
1 eq.) was added dropwise to the stirring reaction mixture. After reacting for 5 minutes on ice, the
reaction mixture was allowed to warm to room temperature and react for an additional 30
minutes. The reaction mixture was concentrated by rotary evaporation and purified by silica flash
chromatography, eluting with 10:1 hexanes:ethyl acetate. Fractions containing the product were
concentrated by rotary evaporation, filtered through silica gel, eluted with hexanes, and
concentrated in vacuo to afford the product as a colorless oil (19.7 g, 53 mmol, 79.1% yield). 1H
NMR (500 MHz, CDCl3) δ ppm 7.32 (m, 2H), 7.25 (t, J = 7.8 Hz, 1H), 7.04 (dq, J = 7.6, 0.7 Hz,
1H), 6.93 (m, 3H), 6.81 (m, 1H), 5.03 (s, 2H), 1.32 (s, 9H), 1.00 (s, 9H), 0.20 (s, 6H). 13C NMR
(126 MHz, CDCl3) δ ppm 156.45, 155.83, 143.53, 138.81, 129.49, 126.24, 120.19, 119.45,
119.08, 114.28, 69.69, 34.06, 31.55, 25.67, 18.19, -4.47. Rf 0.73 (10:1 hexanes:EtOAc); Rf 0.23
(hexanes).

12
3.5.4. 3-(tert-butyl)-9-((tert-butyldimethylsilyl)oxy)dibenzo[b,f]cyclopropa[d]oxocin-1(7H)-
one

Aluminum chloride (2.54 g, 19 mmol, 1 eq.) was suspended in anhydrous dichloromethane (450
mL) in a 1 L round bottom flask with a stir bar. A solution of tetrachlorocyclopropene (3.43 g, 19
mmol, 1 eq.) in anhydrous dichloromethane (5 mL) was added to the stirring suspension, and the
flask was purged with nitrogen. After reacting at room temperature for 15 minutes, the mixture
was cooled in a dry ice/isopropanol bath. A solution of tert-butyl(3-((4-(tert-
butyl)phenoxy)methyl)phenoxy)dimethylsilane (7.00 g, 19 mmol, 1 eq.) in anhydrous
dichloromethane (20 mL) was added dropwise to the reaction mixture over the course of 10
minutes. The mixture was allowed to react in dry ice/isopropanol for 3 h, warm to room
temperature, and react for an additional 30 minutes. The reaction was quenched by addition of
5% aqueous hydrochloric acid (150 mL), and the organic layer was separated. The organic layer
was washed with brine, dried over magnesium sulfate, filtered, and concentrated by rotary
evaporation. The residue was purified by silica flash chromatography, eluting with
dichloromethane to 50:1 dichloromethane:methanol. Fractions containing the desired product
were combined and concentrated in vacuo to afford the product as an amorphous light yellow
solid (2.24 g, 5.3 mmol, 27.9% yield). A second run of the reaction using 3.35 g (9.0 mmol) of
starting material afforded 1.40 g (3.3 mmol, 36.7% yield) of purified product. 1H NMR (500 MHz,
CDCl3) δ ppm 7.97 (d, J = 1.7 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.52 (dd, J = 8.4, 1.8 Hz, 1H),
7.22 (d, J = 8.5 Hz, 1H), 6.99 (m, 2H), 5.27 (d, J = 11.7 Hz, 1H), 4.79 (d, J = 12.5 Hz, 1H), 1.37
(s, 9H), 1.02 (s, 9H), 0.28 (s, 6H). 13C NMR (126 MHz, CDCl3) δ ppm 162.25, 160.63, 159.77,
147.99, 140.95, 135.57, 132.26, 131.18, 130.99, 128.14, 122.29, 121.92, 120.65, 117.88, 116.65,
78.62, 34.51, 31.25, 25.50, 18.19, -4.33. Rf 0.43 (50:1 DCM:MeOH).

13
3.5.5. 3-(tert-butyl)-9-hydroxydibenzo[b,f]cyclopropa[d]oxocin-1(7H)-one

3-(tert-butyl)-9-((tert-butyldimethylsilyl)oxy)dibenzo[b,f]cyclopropa[d]oxocin-1(7H)-one (3.6 g, 8.56

mmol, 1 eq.) was added to a 100 mL round bottom flask with a stir bar and dissolved in
anhydrous tetrahydrofuran (40 mL). A solution of tetrabutylammonium fluoride (1 M, 8.57 mL, 1
eq.) in tetrahydrofuran was added, and the reaction mixture was stirred at room temperature for
30 minutes. The reaction was quenched by addition of saturated aqueous ammonium chloride
(50 mL). The mixture was diluted with dichloromethane (250 mL), and the organic layer was
separated. The aqueous layer was extracted with dichloromethane (2 × 25 mL), and the
combined organic layers were washed with brine (100 mL), dried over magnesium sulfate,
filtered, and concentrated by rotary evaporation. The residue was purified by silica flash
chromatography, eluting with 30:1 dichloromethane:methanol. Fractions containing the desired
product were identified by TLC, combined, and concentrated in vacuo to afford the product as a
white solid (1.63 g, 5.32 mmol, 62.1% yield). 1H NMR (500 MHz, DMSO-d6) δ ppm 10.72 (br. s,
1H), 7.75 (m, 2H), 7.60 (dd, J = 8.5, 2.4 Hz, 1H), 7.26 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 2.4 Hz,
1H), 6.98 (dd, J = 8.3, 2.4 Hz, 1H), 5.29 (d, J = 12.2 Hz, 1H), 4.81 (d, J = 12.2 Hz, 1H), 1.32 (s,
9H). 13C NMR (126 MHz, DMSO-d6) δ ppm 161.53, 159.89, 150.92, 147.19, 144.18, 141.25,
140.28, 135.04, 130.67, 129.47, 122.21, 117.97, 116.88, 115.99, 115.63, 78.11, 34.20, 31.02. R f
0.24 (30:1 DCM:MeOH).

14
3.5.6. tert-butyl 3-(2-((3-(tert-butyl)-1-oxo-1,7-dihydrodibenzo[b,f]cyclopropa[d]oxocin-9-
yl)oxy)ethoxy)propanoate

3-(tert-butyl)-9-hydroxydibenzo[b,f]cyclopropa[d]oxocin-1(7H)-one (1.63 g, 5.32 mmol, 1 eq.),

hydroxy-PEG1-t-butyl ester (1.11 g, 5.83 mmol, 1.1 eq.), and triphenylphosphine (1.84 g, 7.02
mmol, 1.3 eq.) were added to a 150 mL round bottom flask and dissolved in anhydrous
tetrahydrofuran (60 mL). The flask was purged with nitrogen and cooled in an ice bath.
Diisopropyl azodicarboxylate (1.19 g, 5.88 mmol, 1.1 eq.) was added dropwise to the stirring
reaction mixture. After reacting for 5 minutes on ice, the reaction mixture was allowed to warm to
room temperature and react for an additional 30 minutes. The reaction mixture was concentrated
by rotary evaporation and purified by silica flash chromatography, eluting with 1:1 hexanes:ethyl
acetate. Fractions containing the product were identified by TLC, combined, and concentrated in
vacuo to afford the product as a yellow oil (2.23 g, 4.66 mmol, 87.6% yield). 1H NMR (400 MHz,
CDCl3) δ ppm 7.96 (m, 2H), 7.52 (dd, J = 8.6, 2.5 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.06 (m, 2H),
5.28 (d, J = 12.0 Hz, 1H), 4.80 (d, J = 12.2 Hz, 1H), 4.22 (m, 2H), 3.87 (m, 2H), 3.81 (t, J = 6.4
Hz, 2H), 2.53 (m, 2H), 1.46 (s, 9H), 1.36 (s, 9H). Rf 0.34 (1:1 hexanes:EtOAc).

3.5.7. 3-(2-((3-(tert-butyl)-1-oxo-1,7-dihydrodibenzo[b,f]cyclopropa[d]oxocin-9-
yl)oxy)ethoxy)propanoic acid

tert-butyl 3-(2-((3-(tert-butyl)-1-oxo-1,7-dihydrodibenzo[b,f]cyclopropa[d]oxocin-9-
yl)oxy)ethoxy)propanoate (2.23 g, 4.66 mmol, 1 eq.) was dissolved in 15 mL dichloromethane
and added to a 50 mL round bottom flask with a stir bar. Trifluoroacetic acid (6 mL) was added to
the stirring solution, and the reaction was allowed to proceed at room temperature for 2 h. The
reaction mixture was washed with water (2 × 15 mL) and brine (15 mL), dried over magnesium
sulfate, filtered, and concentrated in vacuo to afford the product as a white solid (1.07 g, 2.53
mmol, 54.3% yield). 1H NMR (500 MHz, DMSO-d6) δ ppm 7.85 (d, J = 8.5 Hz, 1H), 7.78 (d, J =
2.7 Hz, 1H), 7.64 (dd, J = 8.5, 2.4 Hz, 1H), 7.35 (d, J = 2.4 Hz, 1H), 7.30 (d, J = 8.5, 1H), 7.18
(dd, J = 8.5, 2.7 Hz, 1H), 5.37 (d, J = 12.2 Hz, 1H), 4.88 (d, J = 12.5 Hz, 1H), 4.24 (m, 2H), 3.76
(m, 2H), 3.68 (t, J = 6.2 Hz, 2H), 2.47 (t, J = 6.3 Hz, 2H), 1.33 (s, 9H).

15
3.5.8. 2,5-dioxopyrrolidin-1-yl 3-(2-((3-(tert-butyl)-1-oxo-1,7-
dihydrodibenzo[b,f]cyclopropa[d]oxocin-9-yl)oxy)ethoxy)propanoate (pODIBO-NHS)

3-(2-((3-(tert-butyl)-1-oxo-1,7-dihydrodibenzo[b,f]cyclopropa[d]oxocin-9-yl)oxy)ethoxy)propanoic
acid (1.07 g, 2.53 mmol, 1 eq.) was suspended in anhydrous acetonitrile (25 mL) in a 50 mL
round bottom flask with a stir bar. N-hydroxysuccinimide (0.44 g, 3.82 mmol, 1.5 eq.), and N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.73 g, 3.82 mmol, 1.5 eq.) were
sequentially added to the stirring mixture, and the flask was purged with nitrogen. The reaction
was allowed to proceed overnight at room temperature under a nitrogen atmosphere. The
reaction mixture was filtered to remove a white precipitate and concentrated by rotary evaporation
to remove the acetonitrile. The residue was dissolved in dichloromethane (50 mL), washed with
water (2 × 50 mL) and brine (50 mL), dried over magnesium sulfate, filtered, and concentrated in
vacuo to afford the product as a white solid (1.11 g, 2.14 mmol, 84.6% yield). 1H NMR (500 MHz,
CDCl3) δ ppm 7.94 (m, 2H), 7.50 (dd, J = 8.5, 2.7 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.06 (m, 2H),
5.27 (d, J = 12.2 Hz, 1H), 4.78 (d, J = 12.2 Hz, 1H), 4.24 (m, 2H), 3.94 (t, J = 6.2 Hz, 2H), 3.89
(m, 2H), 2.92 (t, J = 6.2 Hz, 2H), 2.83 (br. s, 4H), 1.35 (s, 9H). 13C NMR (126 MHz, CDCl3) δ ppm
168.90, 166.61, 161.81, 160.28, 152.59, 147.87, 143.90, 142.09, 140.49, 135.37, 130.96, 130.55,
121.87, 117.76, 117.36, 116.91, 114.56, 78.82, 69.41, 67.75, 65.98, 34.50, 32.25, 31.27, 25.52.
Rf 0.47 (20:1 DCM:MeOH).

16
3.6. Synthesis of Dihydrotetrazines
Dihydrotetrazine compounds were prepared based on known synthetic routes (30, 31).

3.6.1. 6-(6-(pyridine-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridine-3-amine

2-cyanopyridine (3.00 g, 28.8 mmol, 2 eq.), 5-amino-2-cyanopyridine (1.71 g, 14.3 mmol, 1 eq.),
and hydrazine monohydrate (5 mL) were added to a 2-neck 100 mL round bottom flask with a stir
bar. The flask was equipped with a reflux condenser and purged with nitrogen. The reaction
mixture was brought to reflux under a nitrogen atmosphere, and the reaction was allowed to
proceed for 5 h. The reaction mixture was allowed to cool to room temperature, and then ice-cold
water (50 mL) was added. The solid was broken up into a powder with a spatula and collected by
filtration. The filtered solid was washed with ice cold water (50 mL), dried on the filter, and further
dried under vacuum. The crude product was redissolved in acetone and concentrated onto
deactivated silica gel1 in vacuo. A slurry of the adsorbed product in dichloromethane was added
to a wet-packed flash silica column. The desired product was eluted using a gradient of
dichloromethane to 20:1 dichloromethane:methanol. Fractions containing the desired product
were identified by thin layer chromatography, combined, and concentrated in vacuo to afford the
product as an orange solid (1.42 g, 5.6 mmol, 39.4% yield). 1H NMR (500 MHz, DMSO-d6) δ
ppm 8.73 (s, 1H), 8.67 (s, 1H), 8.62 (d, J = 4.9 Hz, 1H), 7.94 (m, 3H), 7.66 (d, J = 8.5 Hz, 1H),
7.51 (dd, J = 6.5, 5.2 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 5.90 (s, 2H). 13C NMR (126 MHz, DMSO-

1
Deactivated silica gel was prepared following a published protocol (32). Silica gel (100 g) was
added to a 500 mL recovery flask, suspended in anhydrous chloroform (200 mL), and cooled in
an ice bath. The flask was purged with nitrogen, and ethyltrichlorosilane (5.1 g, 4.2 mL) was
added dropwise via syringe, swirling the flask to mix. The suspension was allowed to return to
room temperature and allowed to stand at room temperature overnight with occasional swirling to
mix. The silica gel was filtered on a Buchner funnel and washed with chloroform (2 × 200 mL)
and methanol (5 × 200 mL). The silica gel was transferred to a 500 mL recovery flask and dried
by rotary evaporation. Deactivated silica gel was stored at room temperature.

17
d6) δ ppm 148.55, 147.50, 146.69, 146.65, 146.63, 137.33, 134.16, 134.08, 125.16, 121.85,
120.79, 120.28. Rf 0.36 (20:1 DCM:MeOH).

3.6.2. 3,6-di(pyridine-2-yl)-1,4-dihydro-1,2,4,5-tetrazine
The symmetric dipyridinyl dihydrotetrazine was synthesized as a by-product of the
reaction to produce the asymmetric aminopyridinyl dihydrotretrazine (see section
1.2.1). During silica column chromatography, fractions containing the symmetric
product were identified by thin layer chromatography, combined, and concentrated
in vacuo to afford the product as an orange solid (0.77 g, 3.2 mmol). 1H NMR (500
MHz, DMSO-d6) δ ppm 8.99 (s, 2H), 8.64 (m, 2H), 7.95 (m, 4H), 7.53 (ddd, J = 7.3,
4.9, 1.2 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ ppm 148.63, 147.23, 146.30,
137.45, 125.38, 121.05. Rf 0.67 (20:1 DCM:MeOH).

18
3.6.3. 5-oxo-5-((6-(6-(pyridine-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridine-3-
yl)amino)pentanoic acid

6-(6-(pyridine-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridine-3-amine (1.28 g, 5.0 mmol, 1 eq.)

and glutaric anhydride (1.13 g, 10 mmol, 2 eq) were added to a 100 mL round bottom flask with a
stir bar and dissolved in anhydrous tetrahydrofuran (35 mL). The flask was equipped with a reflux
condenser and purged with nitrogen. The mixture was brought to reflux under a nitrogen
atmosphere, and the reaction was allowed to proceed for 20 h. The reaction mixture was cooled
to room temperature and concentrated to ~10 mL by rotary evaporation. The concentrated
mixture was cooled in an ice bath and then diluted with 40 mL ice cold diethyl ether to fully
precipitate the product. The solid was collected by vacuum filtration, washed with cold ether (2 ×
50 mL), dried on the filter, and further dried under vacuum to afford the product as a dark orange
solid (1.38 g, 3.8 mmol, 74.3% yield). 1H NMR (500 MHz, DMSO-d6) δ ppm 12.11 (br. s, 1H),
10.37 (s, 1H), 8.93 (s, 1H), 8.88 (s, 1H), 8.82 (d, J = 4.9 Hz, 1H), 8.16 (dd, J = 8.7, 2.3 Hz, 1H),
7.93 (m, 3H), 7.52 (m, 1H), 2.43 (t, J = 7.4 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.83 (quin, J = 7.3
Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ ppm 174.11, 171.57, 148.58, 147.29, 146.34, 146.07,
141.39, 138.88, 137.37, 137.25, 126.64, 125.28, 121.36, 120.94, 35.31, 32.89, 20.18.

19
3.6.4. 2,5-dioxopyrrolidin-1-yl 5-oxo-5-((6-(6-(pyridine-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-
yl)pyridine-3-yl)amino)pentanoate (“pyridyl-dHTz-NHS”)

5-oxo-5-((6-(6-(pyridine-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridine-3-yl)amino)pentanoic acid
(1.36 g, 3.7 mmol, 1 eq.), N-hydroxysuccinimide (0.85 g, 7.4 mmol, 2 eq.), and N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (2.13 g, 11.1 mmol, 3 eq.) were added
to a 50 mL round bottom flask with a stir bar and dissolved in anhydrous DMF (15 mL). The flask
was purged with nitrogen, and the reaction was allowed to proceed for 2 h at room temperature.
The reaction mixture was diluted into 150 mL dichloromethane and washed with 1 M aqueous
lithium chloride (2 × 150 mL) and brine (150 mL). The organic phase was concentrated onto
deactivated silica gel in vacuo. The adsorbed crude product was loaded as a slurry in 10%
acetone in hexanes onto a wet-packed silica flash column. The product was eluted with a
gradient of 10%-90% acetone in hexanes. Fractions containing the desired product were
identified by thin layer chromatography, combined, and concentrated in vacuo to afford the
product as an orange solid (1.18 g, 2.5 mmol, 68.6% yield). 1H NMR (500 MHz, DMSO-d6) δ
ppm 10.42 (s, 1H), 8.93 (s, 1H), 8.87 (s, 1H), 8.83 (d, J = 1.7 Hz, 1H), 8.63 (d, J = 4.1 Hz, 1H),
8.16 (dd, J = 8.7, 1.8 Hz, 1H), 7.94 (m, 3H), 7.53 (m, 1H), 2.82 (s, 4H), 2.79 (t, J = 7.2 Hz, 2H),
2.53 (t, J = 7.4 Hz, 2H), 1.96 (quin, J = 7.3 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ ppm
173.23, 170.71, 169.24, 149.06, 146.96, 146.82, 146.50, 141.93, 139.40, 137.89, 137.85, 127.19,
125.76, 121.84, 121.42, 35.00, 32.79, 25.93, 20.27. Rf 0.80 (70% acetone in hexanes).

20
3.7. Synthesis of Functionalized PEGs
3.7.1. 8-arm PEG-N3, 8-arm PEG-[4-N3/4-oNB-N3], 4-arm PEG-Sulfo-BCN
The detailed synthetic protocols and characterization of 8-arm PEG-N3, 8-arm PEG-[4-N3/4-oNB-
N3], and 4-arm PEG-Sulfo-BCN have been previously published (6). The 1H NMR spectra of the
final products are reproduced below.
PEG
CDCl3

a
b

b water

7 6 5 4 3 2 1 0
Chemical Shift (ppm)

1H NMR (500 MHz, CDCl3) characterization for 8-arm PEG-N3.

21
 b
d
a c e
f
g
h
j
k i d
a,g
a
CDCl3
PEG c,k
h
f b
j

2.5 2.0 1.5

Chemical Shift (ppm)
7.5 7.0 6.5
a
Chemical Shift (ppm)

CDCl3 i

3.25
Chemical Shift (ppm)

4.25 4.00
Chemical Shift (ppm)

8 7 6 5 4 3 2 1 0
Chemical Shift (ppm)

1H NMR (500 MHz, CDCl3) characterization for 8-arm PEG-[4-N3/4-oNB-N3].

22
 d,e,f

PEG
h
i g
c
j,k b water
m a
a
l c,d c,d
e,f e,f 2.50 2.25 2.00 1.75 1.50
Chemical Shift (ppm)

i k b a

4.75 4.50 4.25 4.00 3.25 3.00

Chemical Shift (ppm) Chemical Shift (ppm) 1.25 1.00
Chemical Shift (ppm)
CDCl3

l m h

9 8 7 6 5 4 3 2 1 0
Chemical Shift (ppm)

1H NMR (500 MHz, CDCl3) characterization for 4-arm PEG-Sulfo-BCN.

23
3.7.2. 8-arm PEG-Sulfo-[4-BCN/4-pODIBO]
8-arm PEG-Sulfo-Boc

8-arm PEG-amine (1.00 g, MW ~ 20 kDa, 0.40 mmol -NH2, 1 eq.) was added to a 50 mL round
bottom flask with a stir bar and dissolved in anhydrous dimethylformamide (10 mL). In a separate
vial containing a stir bar, Boc-L-cysteic acid (430.9 mg, 1.60 mmol, 4 eq.) was dissolved in
anhydrous dimethylformamide (10 mL). After complete dissolution of the Boc-L-cysteic acid,
HATU (608.4 mg, 1.60 mmol, 4 eq.) was added and allowed to dissolve. N,N-
diisopropylethylamine (697 μL, 4.00 mmol, 10 eq.) was added, and the reaction was allowed to
proceed for 10 minutes at room temperature. The activated Boc-L-cysteic acid solution was
added dropwise to the stirring PEG solution, and the reaction was allowed to proceed overnight at
room temperature. The reaction mixture was diluted with water (60 mL) and dialyzed against
water (MWCO 2 kDa, 4 × 4 L, 4°C). The solution was flash frozen in liquid nitrogen and
lyophilized to afford the product as a slightly yellow solid (1.14 g, 0.052 mmol, 95% yield). 1H
NMR characterization is included below.

24
 PEG
b
c a
a
g d,e a a

CDCl3

c e

4.50 3.25 3.00

Chemical Shift (ppm) Chemical Shift (ppm)

f g b water

10 9 8 7 6 5 4 3 2 1 0
Chemical Shift (ppm)
1H NMR (500 MHz, CDCl3) characterization for 8-arm PEG-Sulfo-Boc.

25
8-arm PEG-Sulfo-Amine

8-arm PEG-Sulfo-Boc (1.13 mg, MW ~ 22 kDa, 0.051 mmol) was dissolved in dichloromethane
(12.5 mL) and added to a 50 mL round bottom flask with a stir bar. While stirring,
triisopropylsilane (625 μL), water (625 μL), and trifluoroacetic acid (12.5 mL) were sequentially
added. The reaction was allowed to proceed for 4 h at room temperature. The reaction mixture
was concentrated by rotary evaporation, and the residue was added dropwise to ice cold diethyl
ether (80 mL) to precipitate the PEG. The PEG was collected by centrifugation, washed with cold
ether (2 × 50 mL), and dried under a stream of compressed air. The solid was re-dissolved in
water (20 mL) and dialyzed against water (MWCO 2 kDa, 4 × 4 L, 4°C). The solution was flash
frozen in liquid nitrogen and lyophilized to afford the product as a white solid (895 mg, 0.042
mmol, 82% yield). 1H NMR characterization is included below.

26
 PEG
a
DMSO-d6
b,c
e
d

c b
a

4.00 3.00 2.75

Chemical Shift (ppm) Chemical Shift (ppm)
d

9.0 8.5 8.0 7.5

Chemical Shift (ppm)

9 8 7 6 5 4 3 2 1 0
Chemical Shift (ppm)
1H NMR (500 MHz, DMSO-d6) characterization for 8-arm PEG-Sulfo-amine.

27
8-arm PEG-Sulfo-[4-BCN/4-pODIBO]

BCN-NHS (8.24 mg, 0.028 mmol, 0.75 eq.) and pODIBO-NHS (14.7 mg, 0.028 mmol, 0.75 eq.)
were added to a 25 mL round bottom flask containing a stir bar. 8-arm PEG-Sulfo-amine (100
mg, MW ~ 21.2 kDa, 0.038 mmol -NH2, 1 eq.) was dissolved in anhydrous DMF (2 mL) and
added to the flask. After complete dissolution of the reagents, N,N-diisopropylethylamine (39.4
μL, 0.226 mmol, 6.0 eq.) was added, and the reaction was allowed to proceed overnight at room
temperature. The reaction mixture was diluted to 20 mL with water and dialyzed against water
(MWCO 2 kDa, 3 × 4 L, 4°C). The solution was flash frozen in liquid nitrogen and lyophilized to
afford the product as a white solid. The ratio of conjugated BCN:pODIBO was estimated to be
~1:1.0 based on integration of the corresponding 1H NMR signals. 1H NMR characterization is
included below.

28
 a
b g
c,d b
e a
a
f c,d c,d
e,f e,f
b
e l
c,d g h
i d,e,f
f k
j b a
l
a
f
e
d c a
c
PEG

b,h CDCl3 d,f,g

3.00 2.75 2.50 2.25 2.00 1.75 1.50
c Chemical Shift (ppm)
e
a

b a
8.00 7.75 7.50 7.25 7.00
Chemical Shift (ppm)

g 1.25 1.00
k
Chemical Shift (ppm)
i j
e b
c

d
5.25 5.00 4.75 4.50 4.25 4.00
Chemical Shift (ppm)
3.25 3.00
CDCl3
Chemical Shift (ppm)

f a

10 9 8 7 6 5 4 3 2 1 0
Chemical Shift (ppm)

1H NMR (500 MHz, CDCl3) characterization for 8-arm PEG-Sulfo-[4-BCN/4-pODIBO].

29
3.7.3. mPEG-dHTz

Methoxy-PEG-amine (100 mg, MW ~ 2kDa, 0.05 mmol -NH2, 1 eq.) and pyridyl-dHTz-NHS (34.8
mg, 0.075 mmol, 1.5 eq.) were added to a 25 mL round bottom flask with a stir bar and dissolved
in anhydrous dimethylformamide (3 mL). N,N-diisopropylethylamine (34.8 μL, 0.20 mmol, 4 eq.)
was added, and the reaction was allowed to proceed overnight at room temperature. The PEG
was precipitated by dropwise addition to ice cold diethyl ether (45 mL) and collected by
centrifugation. The supernatant was decanted, and the pellet was washed with cold ether (25
mL), dried under a stream of compressed air, and re-dissolved in dichloromethane (5 mL). The
solution was washed with water (2.5 mL) and brine (2.5 mL) and concentrated to ~ 2 mL. The
product was precipitated by dropwise addition to ice cold diethyl ether (45 mL) and collected by
centrifugation. The supernatant was decanted, and the pellet was washed with cold ether (2 × 25
mL), dried under a stream of compressed air, and re-dissolved in water (5 mL). The solution was
flash frozen in liquid nitrogen and lyophilized to afford the product as an orange solid (85 mg,
0.036 mmol, 72.3% yield). 1H NMR characterization is included below.

30
 b
a c PEG
d o

e
f
g
n i h
m k
o j
l

CDCl3 k m
d,g
l
a e,f,i b
c h
j n water

9 8 7 2.50 2.25 2.00

Chemical Shift (ppm)
Chemical Shift (ppm)
CDCl3

10 9 8 7 6 5 4 3 2 1 0
Chemical Shift (ppm)

1H NMR (500 MHz, CDCl3) characterization for mPEG-dHTz.

31
3.7.4. 8-arm PEG-[4-N3/4-dHTz]

8-arm PEG-amine (400 mg, MW ~ 10 kDa, 0.32 mmol -NH2, 1 eq.), azidoacetic acid NHS ester
(34.8 mg, 0.176 mmol, 0.55 eq.), and pyridyl-dHTz-NHS (148.6 mg, 0.32 mmol, 1 eq.) were
added to a 25 mL round bottom flask with a stir bar and dissolved in anhydrous
dimethylformamide (4 mL). N,N-diisopropylethylamine (223 μL, 1.28 mmol, 4 eq.) was added,
and the reaction was allowed to proceed overnight at room temperature. The PEG was
precipitated by dropwise addition to ice cold diethyl ether (45 mL) and collected by centrifugation.
The supernatant was decanted, and the pellet was broken up with a spatula and washed with
cold ether (25 mL), dried under a stream of compressed air, and re-dissolved in dichloromethane
(5 mL). The solution was washed with water (2 × 2.5 mL) and brine (2.5 mL). The product was
precipitated by dropwise addition to ice cold diethyl ether (45 mL) and collected by centrifugation.
The supernatant was decanted, and the pellet was washed with cold ether (2 × 25 mL), dried
under a stream of compressed air, and re-dissolved in water (6 mL). The solution was flash
frozen in liquid nitrogen and lyophilized to afford the product as an orange solid (304 mg, 0.026
mmol, 65% yield). The ratio of conjugated dHTz:azide was estimated to be 1:1.47 based on
integration of the corresponding 1H NMR signals. 1H NMR characterization is included below.

32
 l
j PEG
m k
n i h
g
f
b a
e
d
a c
b a
e,f,i d,g CDCl3
k m
b l
a h
c b water
j n

10 9 8 7 2.50 2.25 2.00

Chemical Shift (ppm)
Chemical Shift (ppm)
CDCl3

10 9 8 7 6 5 4 3 2 1 0
Chemical Shift (ppm)

1H NMR (500 MHz, CDCl3) characterization for 8-arm PEG-[4-N3/4-dHTz].

33
3.7.5. IRDye700DX-PEG-Azide

A solution of Azide-PEG-Amine (MW ~ 3.5 kDa, 18 mg/mL) was prepared in anhydrous DMF.
The PEG solution (50 μL, 0.9 mg, 1 eq.) was added to a vial containing IRDye700DX-NHS (0.5
mg, 1 eq.) and mixed by pipetting. Excess N,N-diisopropylethylamine (1 μL, ~22 eq.) was added
and mixed by pipetting. The reaction was transferred to a fresh microcentrifuge tube and agitated
at 500 rpm overnight at room temperature. The reaction mixture was diluted with 250 μL MilliQ-
grade water and dialyzed against MilliQ-grade water (MWCO 0.5-1 kDa, 4 × 1 L, 4°C). The
dialyzed solution was flash frozen in liquid nitrogen and lyophilized to afford the product as a
blue/turquoise solid in quantitative yield. A stock solution of the dye conjugate was prepared by
dissolving the product in MilliQ-grade water to 10 mM. MALDI-TOF mass spectral and UV-Vis-
NIR spectral characterization are included below.

34
 1.00
Normalized Count (a.u.)

0.75 4429.85

0.50

0.25

0.00
2000 3500 5000 6500 8000
Mass (m/z)

MALDI-TOF mass spectrometry for IRDye700DX-PEG-azide.

Copy of IRDye700DX Absorbance
1.0
Absorbance (a.u.)

0.5

0.0
300 400 500 600 700 800
Wavelength (nm)

UV-Vis-NIR absorption spectrum for IRDye700DX-PEG-azide.

35
Supporting Figures

Fig. S1. UV light exposure alone does not alter cell fate. (A) The proliferative fraction and (B)
progenitor fraction of cells at day 7 is not significantly altered by exposure to the same dose of UV
light used in the softening and stiffening hydrogel experiments. Data are presented as mean ±
s.d. *p<0.05, ***p<0.001, ns = not significant.

36
 No i t nm
3.4

Relative Elastic
Modulus
1 m
0.9

Fig. S2. Spatial stiffness and swelling characterization for softening hydrogels. (A)
Representative heat map of AFM-measured relative stiffness for 10 μm x 10 μm areas of oNB-
based hydrogels before and after exposure to 365 nm light. (B) Consistent with the bulk
rheological characterization, exposure to light results in a ~3-fold decrease in elastic modulus,
with measured values consistent between independent samples. (C) Samples exhibit locally
homogeneous stiffness over 10 μm x 10 μm areas, as demonstrated by standard deviations of
the elastic moduli that comprise less than 3% of the absolute value of the mean stiffness of the
sample. (D) Samples also exhibit spatial homogeneity over larger length scales, as indicated by
consistent average moduli measured at disparate regions of the same gel. (E) The surface of the
gels does not exhibit buckling due to swelling post-irradiation, as the standard deviation of the
surface height from AFM measurements does not differ before and after light exposure. (F) Bulk
swelling ratio measurements confirm the gels do not substantially swell after softening. Data are
presented as mean ± s.d. ***p<0.001, ns = not significant.

37
 So t Sti

50 m

Laminin Cy5

No i t nm

50 m

Laminin Cy5

Fig. S3. Laminin presentation is consistent across hydrogel conditions. (A) Representative
confocal micrographs of fluorescently labeled laminin-PEG-azide incorporated in static soft and
stiff hydrogels. (B) The laminin content, as measured by the relative fluorescence intensity, does
not significantly vary between the soft and stiff hydrogels. (C) Representative confocal
micrographs of fluorescently labeled laminin-PEG-azide incorporated in softening (oNB-based)
hydrogels with and without exposure to 365 nm light. (D) The laminin content, as measured by
the relative fluorescence intensity, does not significantly vary after light-triggered softening. Data
are presented as mean ± s.d. ns = not significant.

38
 yo eni a tor ti ation ar er

Fig. S4. Single cell data for immunofluorescence staining of the myogenic factors (A) Pax7 and
(B) MyoD and the activation markers (C) Ki67 and (D) phosphorylated p38 MAP kinase (P-p38).
The dashed lines represent the fluorescence intensity threshold used to determine cells staining
positive for these markers, as reported in Fig. 2C-F.

39
Fig. S5. Photo-SPAAC triggered stiffening is stable under cell culture conditions. (A) Reaction
schematic of the photo-SPAAC reaction. (B) Idealized schematic of PEG-based hydrogel

40
networks that can be stiffened on demand by light exposure via photo-SPAAC. (C,D) UV-visible
light spectroscopy analysis of pODIBO-functionalized PEGs dissolved in DMEM plus 10% FBS
and maintained at 37°C for 3 weeks does not show any noteworthy decomposition of the
pODIBO. (E,F) After 3 weeks in in DMEM plus 10% FBS at 37°C, pODIBO is still able to undergo
rapid photoactivation, as observed in UV-visible light spectroscopy analysis upon exposure to 365
nm light. (G) PEG hydrogels containing pODIBO groups and excess azides maintain their initial
stiffness over the course of 1 week under MuSC culture conditions (DMEM plus 15% FBS at
37°C) as well as their ability to stiffen on demand by exposure to 365 nm light. Data are
presented as mean ± s.d. ***p<0.001, n.s. = not significant.

41
 H

Fig. S6. Photooxidation-mediated tetrazine ligation stiffens hydrogels in a cell-compatible manner

but is not stable under cell culture conditions. (A) Reaction schematic of the photo-triggered

42
tetrazine ligation reaction. (B) Idealized schematic of PEG-based hydrogel networks that can be
stiffened on demand by light exposure via photo-catalyzed tetrazine ligation. (C) Photooxidation
by soluble methylene blue resulted in substantial cell death, while (D) photooxidation by hydrogel
tethered IRDye 700DX resulted in no appreciable loss in cell viability. (E) UV-Vis spectroscopy
analysis of photooxidation of PEG-conjugated dihydrotetrazine (dHTz) to tetrazine (Tz) upon
exposure to 680 nm LED light for varying amounts of time. (F) Reaction kinetics of dHTz
photooxidation. (G) Hydrogels containing dHTz, conjugated IRDye 700DX, and excess BCN
groups stiffen upon exposure to 680 nm LED light. (H) UV-Vis spectroscopy analysis of PEG-
conjugated dHTz revealed spontaneous oxidation to tetrazine (Tz) upon exposure to serum-
containing cell culture medium, indicating this chemistry is not suitable for multi-day cell culture
experiments.

43
 No i t nm
3.7

Relative Elastic
Modulus
1 m
0.9

H
No i t nm

50 m

Laminin Cy5

Fig. S7. Characterization of pODIBO hydrogels pre- and post- stiffening. (A) Representative heat
map of AFM-measured relative stiffness for 10 μm x 10 μm areas of pODIBO-based hydrogels
before and after exposure to 365 nm light. (B) Consistent with the bulk rheological
characterization, exposure to light results in a ~3-fold increase in elastic modulus, with measured
values consistent between independent samples. (C) Samples exhibit locally homogeneous
stiffness over 10 μm x 10 μm areas, as demonstrated by standard deviations of the elastic moduli
that comprise less than 3% of the absolute value of the mean stiffness of the sample. (D)
Samples also exhibit spatial homogeneity over larger length scales, as indicated by consistent
average moduli measured at disparate regions of the same gel. (E) The surface of the gels does
not exhibit buckling due to de-swelling post-irradiation, as the standard deviation of the surface
height from AFM measurements does not differ before and after light exposure. (F) Bulk swelling
ratio measurements indicate a moderate degree of de-swelling after light exposure due to the

44
addition of new crosslinks in the network. (G) Representative confocal micrographs of
fluorescently labeled laminin-PEG-azide incorporated in stiffening (pODIBO-based) hydrogels
with and without exposure to 365 nm light. (H) The laminin content, as measured by the relative
fluorescence intensity, does not significantly vary after light-triggered stiffening. Data are
presented as mean ± s.d. ***p<0.001, ns = not significant.

45
 Sin le ell N Se en in

Pax7 Myod1 Mki67 Myog Myh3 Acta1

2.5 4 3
3 5
3

MAP2
MAP2
MAP2

MAP2

MAP2

MAP2
0 0 0 0 0 0
MAP1 MAP1 MAP1 MAP1 MAP1 MAP1

Sin le ell rotein mm no l ore en e

Pax7 MyoD Ki67 MyoG Size
4 6 4
4 4
MAP2

MAP2

MAP2
MAP2

MAP2
1 2
2 2
2
MAP1 MAP1 MAP1 MAP1 MAP1

Fig. S8. UMAP projections overlaid with expression levels for (A) myogenic genes for single cell
RNA sequencing analysis and (B) cell fate markers for single cell protein immunofluorescence
analysis.

46
 Sin le ell N Se en in Sin le ell rotein mm no l ore en e

o n ed o n ed
1.0 1.0

Relative Density

Relative Density
0.8 0.8

0.6 0.6
MAP2

MAP2
So t

So t
0.4 0.4

0.2 0.2

0.0 0.0
1.0 1.0

Relative Density

Relative Density
0.8 0.8
MAP2

MAP2
0.6 0.6
Sti

Sti
0.4 0.4

0.2 0.2

0.0 0.0
MAP1 MAP1 MAP1 MAP1

Fig. S9. Young (2 mo) and aged (>24 mo) cells distribute into the same clusters as a function of
substrate stiffness, demonstrated here by similar distributions on the UMAP projections,
indicating that aged MuSCs are still capable of mechanosensing similar to young MuSCs at day 7
of culture, by both (A) single cell RNA sequencing and (B) single cell protein
immunofluorescence.

47
 Fraction of Total Cells

Stiff+
Rho

Soft
Soft
Soft

Soft
Soft

Stiff

Stiff
Soft

Stiff

Stiff
Stiff
Stiff
Young Aged Young Aged Young Aged

Day 1 Day 3 Day 7

Fig. S10. (A) UMAP projection of single cell RNA sequencing data with annotated cell fate
clusters. (B) Fraction of cells in each cell fate cluster as a function of time point and experimental
condition. Similar trends are observed between soft and stiff culture substrates for both young
and aged MuSCs. Treatment with Rho inhibitor (Rhoi) for 3 days on stiff substrates results in a
distribution of cells more similar to cells cultured on soft substrates than stiff substrates at day 3.

48
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