S1
Non-conventional fluorescent biogenic and
synthetic
polymers without aromatic
rings
Ruquan Ye,
a,b,c
Yuanyue
Liu,
d,e
Haoke Zhang,
a,c
Huifang Su,
a,c,f
Yilin Zhang,
g
Liguo Xu,
h
Rongrong Hu,
h
Ryan T.
K. Kwok,
a,c
Kam Sing Wong,
g
Jacky W.
Y.
Lam,
a,c
William A.
Goddard III
e
and Ben Zhong Tang
a,c,h,*
a
Department of Chemistry, Hong Kong Branch of Chinese National Engineering
Research Center for
Tissue Restoration
and Reconstruction, Institute for Advanced Study,
Institute of
Molecular Functional Materials, Division
of Biomedical Engineering,
Division of Life Science and
State Key Laboratory
of Molecular Neuroscience, The
Hong Kong University
of Science & Technology,
Clear Water Bay,
Kowloon,
Hong
Kong
b
Department of Chemistry, Rice University, 6100 Main Street, Houston,
Texas
77005,
USA
c
HKUST
Shenzhen
Research Institute,
No.
9 Yuexing
1st Road, South
Area, Hi-tech Park,
Nanshan, Shenzhen 518057, China
d
The Resnick Sustainability Institute,
e
Materials and Process Simulation Center and The
Resnick Sustainability Institute, California Institute of Technology, Pasadena, California
91125, United States
f
Sun Yat-sen University Cancer Center, State
Key Laboratory of
Oncology in South
China, Collaborative Innovation Center
for
Cancer
Medicine, Guangzhou
510060, China
g
Department of Physics, The Hong Kong University
of Science &
Technology, Clear
Water Bay,
Kowloon, Hong
Kong
Electronic
Supplementary
Material
(ESI)
for
Polymer
Chemistry.
This
journal
is
©
The
Royal
Society
of
Chemistry
2017
S2
h
Guangdong Innovative
Research Team, SCUT-HKUST Joint
Research
Laboratory, State
Key Laboratory of Luminescent Materials
and Devices,
South
China University of
Technology, Guangzhou 510640,
China
Email: tangbenz@ust.hk
S3
Table
of Content
I.
Experimental Methods
a)
General information..................................................................S4
b)
Experimental details..................................................................S4
c)
Characterization method.............................................................S7
II.
Supplementary Figures..................................................................S8
III.
Reference..................................................................................S22
S4
Experimental Methods
General information
Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Ile-OH,
Rink Amide-AM resin were
purchased
from GL Biochem
(Shanghai) Ltd.
N,N
-Diisopropylethylamine (99%;
DIPEA),
N,N,N′,N′
-tetramethyl-
O
-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU),
hydroxybenzotriazole (HOBT), Abeta,
L
-alanine,
L
-isoleucine,
L
-valine, NIPAM, NtBA,
mesitylene were
purchased from Meryer (Shanghai)
Chemical Technology Co., Ltd.
Papain was purchased
from Merck. Trifluoroacetic
acid (TFA), triisopropylsilane (TIPS),
BSA, GSH, PPRO, PALA, PLYS, azobisisobutyronitrile (AIBN),
potassium bromide
(FT-IR grade; KBr) were
purchased from Sigma-Aldrich. H
3
PO
4
(85%) and n-hexane
were purchased from VWR chemicals.
Tetrahydrofuran (THF), diethyl ether,
N,N
-
dimethylamide (DMF) were purchased
from RCI labscan Ltd. THF
was
distilled from
sodium benzophenone
ketyl under nitrogen immediately prior to use. Diethyl ether was
purified by passing through
an aluminum oxide column. NIPAM was recrystallized from
hexane/toluene. Milli-Q water was
supplied by Milli-Q Plus System (Millipore
Corporation, United States).
Synthesis of Oligopeptides (OALA, OVAL and OILE)
The synthetic procedure
was
followed
by a typical solid-phase peptide synthesis protocol
using Rink amide-AM resin
1
. Deprotection
of Fmoc group was carried
out
in 20%
piperidine in DMF for 15 mins
2. The
coupling of amino acids was
carried out
for 2.5 h
with molar
ratio of
Resin : Fmoc-protected
amino acid : HBTU
: HOBT
: DIPEA
=
1:4:4:4:8. Double coupling of
amino acid
was applied after
3
rd
position. For the last
S5
deprotection of Fmoc group, 30 mins
of
20% piperidine in DMF
2 and 20% piperidine
in THF
2 was carried out
to ensure the complete deprotection of Fmoc group. The resin
was then washed with DMF
3, ethanol
3, THF
3
and DCM
3, and
finally
air dried.
A cleavage cocktail of TFA/water/TIPS (96:2:2) was freshly
prepared
and
added
to
the
dried resin
for cleavage
of
oligopeptides.
After
1 h, the
cleavage cocktail
was
filtered and
the filtrate was added dropwise to cold ether (~45 mL) to precipitate the oligopeptides.
The precipitate was collected by centrifuge at 7000 rpm, and
further
resuspended in ~45
mL cold
ether, washed twice and dried
in vacuum. The crude
oligopeptide was
then
dissolved in minimal amount of TFA and
precipitate
in ~45 mL solvents.
After that,
the
transparent precipitate was dried in vacuum.
The process was
repeated
with solvents
including THF
3 and then water
3. The precipitate
must be vacuum-dried before
dissolution in
minimal amount of TFA, as otherwise an
extra amount of TFA would be
needed and it might result
in dissolution
of peptide in
THF/water.
The precipitate was
finally
dried
in
vacuum and stored
at –20
o
C before use.
Synthesis of PALA-SS
To a 100 mL round bottom flask was added
L
-alanine
(1.0
g; 11 mmol), HBTU (4.6 g;
12
mmol), HOBT (1.6 g; 12 mmol), DIPEA (4.2 mL;
24 mmol)
in
70 mL DMF. The
reaction was stirred at room temperature for 20 h until
all
the suspended
L
-alanine
consumed and
the solution turned transparent.
The
solution was
centrifuged
at 7000 rpm
and a transparent gel was
collected. The transparent gel was
further
dispersed in
50 mL
DMF,
sonicated
for
15 mins and
collected by centrifuge for 3 times. The extra DMF was
S6
removed by resuspending the gel
in 50 mL diethyl ether and centrifuging at 7000 rpm
for
10 mins.
The
precipitate
was collected and
dried in vacuum. The crude
polypeptide was
then dissolved in minimal
amount of TFA and precipitate in ~45 mL solvents. After that,
the precipitate was dried in vacuum. The process
was repeated with
solvents including
THF
3 and then
water
3. The precipitate
must be vacuum-dried before
dissolution in
minimal amount of TFA, as otherwise
an extra amount of TFA would be needed that
it
might result
in dissolution of polypeptide
in THF/water. The
precipitate
was finally dried
in vacuum and
stored at –20
o
C before use.
Synthesis of PALA-HT
PALA-HT was synthesized by acid-catalyzed
polycondensation as reported
2
. Briefly,
to a
100 mL round bottom flask was
added
L
-alanine
(1.67
g; 0.0188 mol) and H
3
PO
4
(0.06
mL; 0.94 mmol) in 60 mL mesitylene. The suspension
was
frozen by liquid nitrogen and
degassed 3 times with dry nitrogen. Then the
reaction was refluxed under
nitrogen for 5 h.
Water formed in the reaction was
removed
using a Dean-Stark trap
with
a reflux
condenser. Afterwards, the reaction was cooled to room temperature. The
solvent was
removed by filtration and
the precipitate
was washed
with
200 mL methanol and
200 mL
water.
The precipitate was
then dissolved in minimal amount of TFA and precipitated
from 100 mL
THF
three
times. The precipitate
was further washed
with water
3 by
sonication for 10 mins. The precipitate
was
collected
and
dried at 40
o
C under vacuum.
S7
Synthesis of PNIPAM and PNtBA
PNIPAM and PNtBA were synthesized
as reported
3, 4
. Briefly,
to a
100 mL two-neck
round bottom flask
was added monomer (15.7
mmol;
1.78 g NIPAM or 2.00 g NtBA)
and AIBN (0.125 mmol; 20.5
mg). The
flask was evacuated
under
vacuum and flushed
with dry nitrogen
for three times. Then
60 mL distilled THF
was injected and the reaction
was stirred at 70
o
C
for 4 h. Afterwards, the
reaction was stopped
and cooled
to
room
temperature. The THF solution was concentrated to ~10 mL under
reduced
pressure and
added dropwise to 150 mL bad solvents (n-hexane
for
PNIPAM
and
water
for PNtBA).
The precipitates were
collected by filtration.
The
precipitates were further dissolved in
minimal amount of THF and precipitated
from their
corresponding bad solvents
for
4
times. The precipitates were collected by filtration and dried under vacuum. The weight-
averaged molecular weights for PNIPAM
and PNtBA are 6320 and 8965 with PDI of
1.52 and 1.70,
respectively.
Characterization
FTIR spectra were recorded on a
PerkinElmer
16 PC FTIR spectrophotometer. Confocal
microscopy images were
performed on a Zeiss Laser Scanning Confocal Microscope,
LSM7 DUO.
The
images
were taken
using a 405 nm
laser
(4.0% power) with
a 429-485
nm filter. Solid state UV-vis absorbance and CD spectra were obtained on Chirascan
equipped with solid state
station under nitrogen.
The fluorescence quantum yields were
measured using a
calibrated integrating
sphere.
5, 6
A transparent KBr film
(1 cm in
diameter) was freshly prepared
by pressing ~60 mg 1 wt% of homogeneously-mixed
S8
peptides in KBr
at 10 ton for 1 min.
1
H and
13
C NMR
spectra were measured on a Bruker
ARX 400 NMR
spectrometer using TFA-
d
, CDCl
3
, DMSO-
d
6
as solvent and TFA (
δ
=
11.5), tetramethylsilane
(TMS;
δ
= 0), TMS (
δ
=
2.5) as
internal
reference, respectively.
High-resolution mass spectra (HRMS)
were recorded on a Finnigan MAT
TSQ
7000
Mass Spectrometer System operated in a MALDI-TOF mode. Weight-average molecular
weights and
polydispersities
(PDI) of the polymers were
estimated on a
Waters
gel
permeation chromatography (GPC) system using THF as eluent. Details about the
experimental setup can be
found in our previous publication
7
. Steady-state PL spectra and
fluorescence lifetimes of peptides were determined with a Hamamatsu C11367-11
Quantaurus-Tau time-resolved spectrometer.
Solution-state PL spectra of biomimetic
polymers were recorded on a
Perkin-Elmer
spectrofluorometer LS 55.
Supplementary Figures and Tables
Figure S1.
Luminescence
in nature.
(a) Normalized PL
emission of natural or synthetic
peptide and protein excited at 325 nm. (b) Physical appearance of natural
protein
containing aromatic amino acid without
extended conjugation under a
365 nm UV lamp.
(c) Physical
appearance of synthetic peptides
without aromatic
amino acid under a
365
nm UV lamp. The scale
bar is 5 mm.
S9
S10
Figure S2.
H-NMR spectra of (a) ALA and OALA
(b) PALA-SS and
PALA-HT. It is
noticeable that
a peak corresponding to guanidine structure (–N=C(N(CH
3
)
2
)
2
) at the N-
terminus is observed at
3.1 for
PALA-SS. (c) VAL
and OVAL; (d) ILE and OILE.
The
internal standard is TFA with
δ
= 11.5.
S11
Figure S3.
13
C-NMR spectra of (a) ALA and OALA; (b) PALA-SS and
PALA-HT; (c)
VAL and OVAL;
(d) ILE and OILE. The internal
standard is TFA marked with
asterisk.
S12
Figure S4.
MALDI-TOF
of
(a) OALA (b) OVAL
and
(c) OILE. The matrixes are
DHB
for OALA and
OVAL, and CHCA for OILE.
Figure S5.
Degree of polymerization and molecular
weight distribution of (a) PALA-HT
and (b) PALA-SS determined by MALDI-TOF
with DHB as matrix. The
degree of
polymerization for
PALA-HT is approximately calculated as (M-18)/71, as the end
groups are –NH
2
and –COOH. The degree
of polymerization of PALA-SS is