S
1
Supp
orting
Information for
Remote Control of Mechanochemical Reactions Under Physiological
Conditions Using Biocompatible Focused Ultrasound
Yuxing Yao,
1,‡
Molly E. McFadden,
1,‡
Stella M. Luo,
1
Ross W. Barber,
1
Elin Kang,
1
Avinoam
Bar
-
Zion,
1
Cameron A. B. Smith,
1
Zhiyang Jin,
2
Mark Legendre,
1
Bill Ling,
1
Dina Malounda,
1
Andrea
Torres,
1
Tiba Hamza,
1
Chelsea E. R. Edwards,
1
Mikhail G. Shapiro,
1,2,3,
* and Maxwell J. Robb
1,
*
1. Division of Chemistry and Chemical
Engineering, California Institute of Technology, Pasadena,
California 91125, United States; 2. Division of Engineering and Applied Science, California Institute
of Technology, Pasadena, California 91125, United States; 3. Howard Hughes Medical Institute,
P
asadena, California 91125, United States
‡
These authors contributed equally
*Maxwell J. Robb and
Mikhail G. Shapiro
Email:
mrobb@caltech.edu; mikhail@caltech.edu
Table of Contents
I. General Experimental Details and Methods
................................
................................
...............
S
2
II. Supplementary Fig
ures
................................
................................
................................
.............
S
4
III. Synthetic Details
................................
................................
................................
.......................
S
9
IV. General Procedure for Ultrasonication Experiments with an Immersion Probe
.....................
S
12
V. General Procedure for Experiment with Focused Ultrasound (FUS)
................................
......
S
12
VI. Characterization of Aminocoumarin Release Using Photoluminescence Spectroscopy
.......
S
13
VII. Characterization of Camptothecin Release Using Liquid Chromatography
..........................
S
21
VIII. Cytotoxicity Experiments
................................
................................
................................
......
S
25
IX. References
................................
................................
................................
.............................
S
25
X.
1
H and
13
C NMR Spectra
................................
................................
................................
........
S
26
S
2
I.
General Experimental Details and Methods
Reagents from commercial sources were used without further purification unless otherwise stated. Dry
dichloromethane
was obtained from a Pure Process Technology solvent purification system. All reactions
were performed under a N
2
atmosphere unless specifie
d otherwise. Column chromatography on silica gel
was performed on a Biotage Isolera system using SiliCycle SiliaSep HP flash cartridges.
NMR spectra were recorded using a 400 MHz Bruker Avance III HD with Prodigy Cryoprobe. All
1
H NMR
spectra are reported in δ units, parts per million (ppm), and were measured relative to the signals for
residual chloroform (7.26 ppm) or toluene (2.08 ppm) in deuterated solvent. All
13
C NMR spectra were
measured in deuterated solvents and are repor
ted in ppm relative to the signals for chloroform (77.16 ppm).
Multiplicity and qualifier abbreviations are as follows: s = singlet, d = doublet, t = triplet, q = quartet, dd =
doublet of doublets, dq = doublet of quartets, ABq = AB quartet, m = multiplet,
br = broad.
High resolution mass spectra (HRMS) were obtained via direct injection on an Agilent 1260 Infinity II
Series
HPLC coupled to a 6230 LC/TOF system in electrospray ionization (ESI+) mode
.
Polymer molar mass and dispersity was
determined on a GP
C system with an Agilent pump (G7110B),
equipped with an autosampler (G7129A), a Wyatt DAWN 8 multi
-
angle laser light scattering detector (λ
=
658.9nm), a Wyatt Optilab refractive index detector (RI) (
λ
=
658nm), and an Agilent PL Aquagel
-
OH
MIXED
-
H column
. Aqueous buffer was prepared containing 0.2
M NaNO
3
with 200 ppm NaN
3
. Filtered
aqueous buffer was used as the eluent at
a
flow rate of 0.3 mL/min at 25
°
C. The data were analyzed using
Wyatt Astra 7 with dn/dc = 0.153 mL/g to obtain
average molar masses
(
M
w
and
M
n
) for each PMSEA
polymer reported
.
Photoluminescence spectra were recorded on a Shimadzu RF
-
6000 spectrofluorophotometer using a
quartz microcuvette (Starna Cells 18F
-
Q
-
10
-
GL14
-
C, 10 x 2 mm). Excitation and emission slit widths were
5 nm and 3 n
m, respectively.
A T
ecan Spark microplate reader was also used to acquire
photoluminescence spectra
in some experiments
with 96
-
well microplate (Costar). Excitation and emission
slit widths were
both
5 nm
.
High
-
Performance Liquid Chromatography (HPLC) meas
urements were performed with an Agilent Eclipse
Plus C18 column (959961
-
902) or a C8 column (993967
-
906) using an in
-
line UV
-
vis detector and
acetonitrile
/water as the eluent.
S
onication
experiments
with an immersion probe
were performed using a 500
W
att V
ibra Cell 505 liquid
processor (20 kHz) equipped with a 0.5
-
inch diameter solid probe (part #630
-
0217), sonochemical adapter
(part #830
-
00014), and a Suslick reaction vessel made by the Caltech glass shop (analogous to vessel
#830
-
00014 from Sonics and Mat
erials).
LCMS measurements were performed with an Agilent 6140 Series Quadrupole LCMS Spectrometer
System equipped with a
n
Agilent Eclipse Plus C18 column using an in
-
line UV
-
vis detector and
acetonitrile/water
as the eluent.
Compounds
DA
-
1, DA
-
2,
1
-
CoumNH
2
, and
2
-
CoumNH
2
were synthesized as reported previously
(1)
.
Internal standard
4
-
methyl
-
2
-
oxo
-
2
H
-
chromen
-
7
-
yl acetate
(
IS
)
was prepared according to the
literature
(2)
.
Monomer
2
-
methylsulfinyl ethyl acrylate (MSEA) was prepared according to the literature
(3)
.
All polymer solutions were dialyzed against deionized water for at least 5
cycles
to remove any residual
small molecule in the reaction mixture to prevent any
aminocoumarin
(
CoumNH
2
)
or
camptothecin
(
CPT
)
from affecting functional assays.
S
3
Gas vesicle
s
(GVs)
were produced and purified
from cyanobacteria
Anabaena flos
-
aquae
as described
previously
(4, 5)
.
GvpC
was removed from the GVs by urea treatment followed by dialysis process.
As a
quality assurance step
to confirm successful GvpC removal
,
pressure
-
dependent
optical density (OD)
measurements were performed for each sample at 0
–
7
bar
to match previously rep
orted measurements
using an echoVis Vis
-
NIR light source coupled with an STS
-
VIS spectrometer (Ocean Optics) and a
176.700
-
QS sample chamber (Hellma Analytics). GV concentrations were measured
as OD at 500 nm
using a spectrophotometer (NanoDrop ND
-
1000, Th
ermo Scientific)
. OD
500
= 1 corresponds to 114 pM of
GV particles
(4, 5)
.
Both polymer solution and GV suspension
were
placed in a vacuum environment at
0.5 atm
overnight to
remove transiently trapped air bubbles before FUS treatment.
GV suspension and polymer solution were
gently mixed to desired concentration without introducing observable air bubbles in a 2
mL
m
icrocentrifuge
tube.
T
he total volume of sample
w
as
800
μ
L. Th
e
m
icrocentrifuge
tube was stabilized on a homemade
holder which positions the center of
the
sample to be at the focus of a focused ultrasound transducer
(Pre
c
ision Acoustics, H115)
operating at 330 kHz (fundamental frequency) or 916 kHz (third harmonic)
.
All cell lines were ordered from American Type Culture Collection.
S
4
II.
Supplementary Figures
Chart S
1
. Structures of all compounds used in this
study.
N
O
O
O
H
H
O
O
Br
Me
Me
O
O
Me
Br
Me
O
Ph
Me
OH
D
A
-1
N
O
O
O
H
H
O
O
Br
Me
Me
O
O
Me
Me
Me
O
Ph
Me
OH
D
A
-2
O
O
Me
O
Me
O
IS
O
N
O
O
O
O
Me
Me
Br
O
O
Me
Me
Br
Me
O
NH
O
O
O
O
Ph
Me
1
-C
o
u
m
N
H
2
O
N
O
O
O
O
Me
Me
Br
O
O
Me
Me
Me
Me
O
NH
O
O
O
O
Ph
Me
2
-C
o
u
m
N
H
2
1
-C
PT
2
-C
PT
O
N
O
O
O
O
Me
Me
O
O
Me
Me
Me
O
O
O
Ph
Br
O
O
S
Me
O
Br
O
O
S
Me
O
PMSEA
-C
PT
O
O
N
N
O
O
Et
C
o
n
tr
o
l
-C
PT
HO
O
N
N
O
O
Et
C
PT
H
H
H
H
H
H
n
O
N
O
O
O
O
Me
Me
O
O
Me
Me
Me
O
O
O
Ph
Me
Br
O
O
S
Me
O
O
O
N
N
O
O
Et
H
H
n
O
N
O
O
O
O
Me
Me
O
O
Me
Me
Me
O
NH
O
O
O
O
Ph
Me
Br
O
O
S
Me
O
Br
O
O
S
Me
O
n
PMSEA
-C
o
u
m
N
H
2
C
o
n
tr
o
l
-C
o
u
m
N
H
2
H
H
O
N
O
O
O
O
Me
Me
O
O
Me
Me
Me
O
NH
O
O
O
O
Ph
Me
Me
Br
O
O
S
Me
O
H
H
n
N
O
O
O
H
H
O
O
Br
Me
Me
O
O
Me
Br
Me
O
Ph
Me
O
O
O
O
N
N
O
O
Et
N
O
O
O
H
H
O
O
Br
Me
Me
O
O
Me
Me
Me
O
Ph
Me
O
O
O
O
N
N
O
O
Et
n
n
S
5
Figure S
1
.
T
emperature
increase above baseline
of an 800 μL water sample
inside
a plastic
microcentrifuge
tube under
330
kHz or 916 kHz
FUS
.
The t
emperature probe was inserted
into the
tube
with the
probe tip
positioned at the
tube center
.
Continuous
-
wave
FUS was
applied
at 1.
9
MPa peak
negative pressure
(PNP)
,
10 s on
/
20 s off, 4 repetitions
, corresponding to a spatial
peak
-
temporal
average
acoustic
intensity
(I
spta
)
of 40 W/cm
2
.
Note that our I
spta
value differ
s
from the literature
(6)
because
we considered the time average acoustic intensity with 33% duty cycle.
Figure S
2
.
Release of aminocoumarin
(
CoumNH
2
) from
PMSEA
-
CoumNH
2
(2 mg/mL,
M
n
= 260 kg/mol,
Ð
=
1.47)
increases
as a function of temporal exposure to biocompatible
FUS
(330 kHz, 3.6 W/cm
2
, 1.47
MPa PNP,
4.5%
duty cycle) in
the
presence of GVs (1.4 nM).
Release of
CoumNH
2
does not occur i
n
the absence of GVs even after 30 min of insonation.
0
50
100
150
0
10
20
30
40
Ti
m
e
(
s
)
T
e
m
p
e
r
a
t
u
r
e
i
n
c
r
e
m
e
n
t
(
°
C
)
330 kHz, 1.9 MPa, 40 W/cm
2
916 kHz, 1.9 MPa, 40 W/cm
2
S
6
Figure S
3
.
No significant increase in PL intensity is observed from chain
-
end functionalized
polymer
Control
-
CoumNH
2
(2 mg/mL,
M
n
= 244 kg/mol,
Ð =
1.51) upon exposure to biocompatible
FUS
(330
kHz, 3.6 W/cm
2
, 1.47 MPa PNP,
4.5%
duty cycle
, 10 min
) in
the
presence or absence of GVs (1.4 nM).
S
7
Figure S
4
.
Release of c
amptothecin (
CPT
) is observed following the thermally
-
induced retro
-
Diels
–
Alder
reaction of
1
-
CPT
and subsequent dilution in polar protic solvent. Comparison of partial
1
H NMR spectra
of (a) pristine
1
-
CPT
, (b)
1
-
CPT
after
heating for
24 h at 100 °C, and (c) maleimide
M1
indicates significant
retro
-
Diels
–
Alder
reaction
occurs thermally
.
Subsequent analysis
by
LCMS
with in
-
line UV
-
vis absorption
spectroscopy of (d) the NMR solution of
1
-
CPT
after
hea
t
ing for
24 h at 100 °C diluted into 3:1
acetonitrile
/
methanol
,
and (e) pure
CPT
standard supports that
CPT
is released from thermally
-
activated
1
-
CPT
in polar protic solvent. Good agreement with the
CPT
standard is observed in retention time
,
mass
-
to
-
charge ratio
,
and absorption
spectrum
.
S
8
Figure S
5
.
Analysis of
thermally activated
1
-
CPT
(24 h at 100 °C in toluene
-
d
8
)
immediately
after
dilution
in either (a) aprotic
acetonitrile,
or (b) 3:1
acetonitrile
/
methanol
using
LCMS
with in
-
line UV
-
vis absorption
spectroscopy
.
The
amo
unt
of
CPT
relative to remaining unreacted
1
-
CPT
is significantly greater after
dilution in protic solvent consistent with
the expected reactivity
of the furfuryl carbonate
.
Figure S
6
.
No release of camptothecin (
CPT
) from
PMSEA
-
CPT
(4 mg/mL,
M
n
= 319 kg/mol,
Ð
=
1.47)
is observed in aqueous solution, even after 2 months
in
ambient conditions.
The same sample volume
was used for each injection.
S
9
III.
Synthetic Details
((3aR,4R,7S,7aS)
-
2
-
(2
-
((2
-
bromo
-
2
-
methylpropanoyl)oxy)ethyl)
-
7
-
(1
-
(((((S)
-
4
-
ethyl
-
3,14
-
dioxo
-
3,4,12,14
-
tetrahydro
-
1H
-
pyrano[3',4':6,7]indolizino[1,2
-
b]quinolin
-
4
-
yl)oxy)carbonyl)oxy)ethyl)
-
1,3
-
dioxo
-
6
-
phenoxy
-
1,2,3,3a,7,7a
-
hexahydro
-
4H
-
4,7
-
epoxyisoindol
-
4
-
yl)methyl
2
-
bromo
-
2
-
methylpropanoatebromo
-
2
-
methylpropanoate (1
-
CPT).
A flame
-
dried 25 mL round bottom flask was
charged with
4
-
dimethylaminopyridine (DMAP)
(544 mg, 4.43 mmol) and camptothecin (302 mg, 0.867
mmol). The
flask
was evacuated and refilled wit
h N
2
three times, after which 5 mL
of
dichloromethane
was
added.
P
hosgene (15 wt% in
toluene
, 700
μ
L, 0.981 mmol) was added to the off
-
yellow suspension and
the reaction was allowed to stir at room temperature. After
1 h
the reaction had become homogenous
and
dark red, and
DA
-
1
(
ref.
1)
was added as a solution in 5 mL
dichloromethane
. The reaction continued to
stir for 20 h, after which it was quenched with sat. NH
4
Cl (8 mL). The products were extracted into
dichloromethane
(3 x 5 mL) and the combined organic layers were washed with brine (5 mL), dried with
sodium sulfate
, and filtered. The
crude mixture was
purified
via silica gel chromatography (0
–
10%
methanol
/
dich
loromethane
)
,
followed by separation of
the diastereomers
by reverse
-
phase HPLC (70%
acetonitrile/water
). The
title
product
(single diastereomer)
was
isolated
as
a
light yellow solid
(84 mg, 27%)
.
TLC (5%
methanol
/
dichloromethane
):
R
ƒ
= 0.55
1
H NMR
(400 MHz, CDCl
3
) δ:
8.38 (s, 1 H), 8.07 (d,
J
= 8.5 Hz, 1H), 7.91 (d,
J
= 8.1 Hz, 1H), 7.84
–
7.76 (m,
1H), 7.68
–
7.59 (m, 1H), 7.36 (s, 1H), 7.25
–
7.18 (m, 2H), 7.13
–
7.05 (m, 1H), 6.90
–
6.81 (m, 2H), 5.69 (d,
J
= 17.3 Hz, 1H), 5.49 (q,
J
= 6.5 Hz, 1H), 5.39 (d
,
J
= 17.2 Hz, 1H), 5.26 (s, 2H), 4.98 (s, 1H), 4.66 (A
b
q,
Δν
AB
= 104 Hz,
J
AB
= 12.5 Hz, 2H), 3.91
–
3.79 (m, 2H), 3.76
–
3.72 (m, 2H), 3.25
–
3.14 (m, 1H), 3.05
–
2.94
(m, 1H), 2.33 (dq,
J
= 14.9, 7.4 Hz, 1H), 2.18 (dq,
J
= 14.7 Hz, 7.5 Hz, 1H), 1.94 (s, 3H), 1.
91 (s, 3H), 1.70
(s, 3H), 1.68 (s, 3H), 1.53 (d,
J
= 6.6 Hz, 3 H), 1.01 (t,
J
= 7.4 Hz, 3H).
13
C{
1
H} NMR (101 MHz, CDCl
3
) δ:
173.5, 172.6, 171.2, 171.1, 167.3, 162.2, 157.3, 154.4, 152.9, 152.4,
148.6, 146.5, 145.5, 131.4, 131.0, 130.0, 129.3, 128.7, 128.4
, 128.23, 128.19, 125.9, 120.6, 119.4, 101.2,
95.8, 90.2, 88.3, 78.2, 71.8, 67.3, 63.6, 62.2, 55.6, 55.5, 51.4, 50.1, 47.8, 36.9, 32.0, 30.75, 30.71, 30.5,
30.4, 15.7, 7.7.
HRMS (ESI, m/z):
calcd for [C
48
H
46
Br
2
N
3
O
14
+
] (M+H)
+
1046.1341, found
1046.1355
.
N
N
O
O
O
OH
Et
1
)
Ph
o
sg
e
n
e
,
D
MAP
,
C
H
2
Cl
2
,
rt
,
1
h
2
)
D
A
-1
,
rt
,
2
0
h
N
O
O
O
H
H
O
O
Br
Me
Me
O
O
Me
Br
Me
Ph
O
Me
O
O
O
O
N
N
O
O
27%
Et
1
-C
PT
S
10
((3aR,4R,7S,7aS)
-
2
-
(2
-
((2
-
bromo
-
2
-
methylpropanoyl)oxy)ethyl)
-
7
-
(1
-
(((((S)
-
4
-
ethyl
-
3,14
-
dioxo
-
3,4,12,14
-
tetrahydro
-
1H
-
pyrano[3',4':6,7]indolizino[1,2
-
b]quinolin
-
4
-
yl)oxy)carbonyl)oxy)ethyl)
-
1,3
-
dioxo
-
6
-
phenoxy
-
1,2,3,3a,7,7a
-
hexahydro
-
4H
-
4,7
-
epoxyisoindol
-
4
-
yl)methyl pivalate (2
-
CPT).
A
flame
-
dried 25 mL round bottom flask was charged with DMAP (336 mg, 2.75 mmol) and camptothecin
(192 mg, 0.552 mmol). The
flask
was evacuated and refilled with N
2
three times, after which 5 mL
of
dichloromethane
was added.
P
ho
sgene (15 wt % in
toluene
, 430
μ
L, 0.602 mmol) was added to the off
-
yellow suspension and the reaction was allowed to stir at room temperature. After
1 h
the reaction had
become homogenous and dark red, and
DA
-
2
(
ref
.
1)
was added as a solution in 5 mL
of
dichloromethane
.
The reaction continued to stir for 18 h, after which it was quenched with sat. NH
4
Cl (8 mL). The products
were
extracted into
dichloromethane
(3 x 5 mL) and the combined organic layers were washed with brine
(5 mL), dried with Na
2
SO
4
, and filtered.
The crude mixture was purified via silica gel chromatography
(0
–
10%
methanol
/
dichloromethane
)
,
followed by separation of the diastereomers by reverse
-
phase HPLC
(70%
acetonitrile/water
).
The title product (single diastereomer) was isolated as a light yellow solid (
50 mg
,
28
%).
TLC (5%
methanol
/
dichloromethane
):
R
ƒ
= 0.57
1
H NMR (400 MHz, CDCl
3
) δ:
8
.39 (s, 1H), 8.07 (d,
J
= 8.5 Hz, 1H), 7.92 (dd,
J
= 8.3, 1.3 Hz, 1H), 7.81
(ddd,
J
= 8.4, 6.9, 1.2 Hz, 1H), 7.69
–
7.62 (m, 1H), 7.35 (s, 1H), 7.25
–
7.19 (m, 2H), 7.13
–
7.07 (m, 1H), 6.85
(dd,
J
= 7.6, 1.6 Hz, 2H), 5.70 (d,
J
= 17.2 Hz, 1H), 5.50 (q,
J
= 6.5
Hz, 1H), 5.41 (d,
J
= 17.3 Hz, 1H), 5.28
(s, 2H), 4.93 (s, 1H), 4.56 (A
b
q, Δν
AB
= 120 Hz,
J
AB
= 12.7 Hz, 2H), 3.90
–
3.79 (m, 2H), 3.73
–
3.63 (m, 2H),
3.26
–
3.13 (m, 1H), 3.04
–
2.92 (m, 1H), 2.35 (dq,
J
= 14.9, 7.4 Hz, 1H), 2.19 (dq,
J
= 14.8, 7.5 Hz), 1.71 (s,
3H), 1.69 (s, 3H), 1.54 (d,
J
= 6.6 Hz, 3H), 1.21 (s, 9H), 1.01 (t,
J
= 7.5 Hz, 3H).
13
C{
1
H}
NMR (101 MHz, CDCl
3
) δ:
177.8, 173.5, 172.6, 171.2, 167.3, 162.1, 157.3, 154.5, 153.0, 152.5,
148.8, 146.6, 145.4, 131.4, 131.0, 130.1, 129.4, 128.7, 128.4, 128.24, 128.22, 125.9, 120.8, 119.4, 101.3,
95.7, 90.2, 88.6, 78.2, 71.8, 67.4, 62.3, 62.2, 55.5,
51.4, 50.1, 47.9, 39.0, 36.9, 32.1, 30.53, 30.46, 27.2,
15.7, 7.8.
HRMS (ESI, m/z):
calcd for [C
49
H
49
BrN
3
O
14
+
] (M+H)
+
982.2392, found 982.2408
.
1
)
Ph
o
sg
e
n
e
,
D
MAP
,
C
H
2
Cl
2
,
rt
,
1
h
2
)
D
A
-2
,
rt
,
1
8
h
N
O
O
O
H
H
O
O
Br
Me
Me
O
O
Me
Me
Me
Ph
O
Me
O
O
O
O
N
N
O
O
28%
Et
2
-C
PT
N
N
O
O
O
OH
Et