Synthesis and characterization of a photocleavable collagen-like peptide

Article abstract of DOI:10.1039/c7ob02198d

A 34-amino acid long collagen-like peptide rich in proline, hydroxyproline, and glycine, and with four photoreactive N-acyl-7-nitroindoline units incorporated into the peptide backbone was synthesized by on-resin fragment condensation. Its circular dichroism supports a stable triple helix structure. The built-in photochemical function enables the decomposition of the peptide into small peptide fragments by illumination with UV light of 350 nm in aqueous solution. Illumination of a thin film of the peptide, or a thin film of a photoreactive amino acid model compound containing a 5-bromo-7-nitroindoline moiety, with femtosecond laser light at 710 nm allows for the creation of well-resolved micropatterns. The cytocompatibility of the peptide was demonstrated using human mesenchymal stem cells and mouse embryonic fibroblasts. Our data show that the full-length peptide is cytocompatible as it can support cell growth and maintain cell viability. In contrast, the small peptide fragments created by photolysis are somewhat cytotoxic and therefore less cytocompatible. These data suggest that biomimetic collagen-like photoreactive peptides could potentially be used for growing cells in 2D micropatterns based on patterns generated by photolysis prior to cell growth.

Full text of DOI:10.1039/c7ob02198d

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: A. Ornelas, K. N.
Williams, K. Hatch, A. Paez, A. C. Aguilar, C. C. Ellis, N. Tasnim, S. Ray, C. Dirk, T. Boland, B. Joddar, C. Li
and K. Michael, Org. Biomol. Chem., 2018, DOI: 10.1039/C7OB02198D.
This is an Accepted Manuscript, which has been through the
Volume 14 Number
1 7 January 2016 Pages 1–372
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Page 1 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
Synthesis and Characterization of a Photocleavable Collagen-Like Peptide
Alfredo Ornelas,1 Kaitlyn N. Williams,1 Kevin A. Hatch,2 Aurelio Paez,4 Angela C. Aguilar,2
1
Cameron C. Ellis,3 Nishat Tasnim,4 Supriyo Ray,1 Carl W. Dirk, Thomas Boland,4,5 Binata
Joddar,4,5 Chunqiang Li,2,5* and Katja Michael1,5*
1Department of Chemistry and Biochemistry, The University of Texas at El Paso, 500 West
University Avenue, El Paso, TX 79968, USA
2Department of Physics, The University of Texas at El Paso, 500 West University Avenue, El
Paso, TX 79968, USA
3Deaprtment of Biological Sciences, The University of Texas at El Paso, 500 West University
Avenue, El Paso, TX 79968, USA
4Department of Metallurgical, Materials and Biomedical Engineering, The University of Texas at
El Paso, 500 West University Avenue, El Paso, TX 79968, USA
5Border Biomedical Research Center, The University of Texas at El Paso, 500 West University
Avenue, El Paso, TX 79968, USA
*Corresponding Authors: kmichael@utep.edu; cli@utep.edu
Abstract
A 34 amino acid long collagenꢀlike peptide rich in proline, hydroxyproline, and glycine, and
with four photoreactive Nꢀacylꢀ7ꢀnitroindoline units incorporated into the peptide backbone was
synthesized by onꢀresin fragment condensation. Its circular dichroism supports a stable triple
helix structure. The builtꢀin photochemical function enables the decomposition of the peptide
into small peptide fragments by illumination with UV light of 350 nm in aqueous solution.
Illumination of a thin film of the peptide, or a thin film of a photoreactive amino acid model
compound containing a 5ꢀbromoꢀ7ꢀnitroindoline moiety, with femtosecond laser light at 710 nm
allows for the creation of wellꢀresolved micropatterns. The cytocompatibility of the peptide was
demonstrated using human mesenchymal stem cells and mouse embryonic fibroblasts. Our data
show that the fullꢀlength peptide is cytocompatibe as it can support cell growth and maintain cell
viability. In contrast, the small peptide fragments created by photolysis are somewhat cytotoxic
and therefore less cytocompatible.
These data suggest that biomimetic collagenꢀlike
photoreactive peptides could potentially be used for growing cells in 2D micropatterns based on
patterns generated by photolysis prior to cell growth.
Introduction
Collagen and collagenꢀderived materials have been used in tissue engineering applications, as
these biomaterials form soft hydrogels that mimic the extracellular matrix providing structural
support in which cells can grow, differentiate, and proliferate. The extracellular matrix is
geometrically and topologically inhomogeneous,1, 2 which are factors that modulate cell polarity
and function.3 In the laboratory, collagen and gelatin can selfꢀassemble in aqueous buffers
resulting in hydrogels consisting of protein fibrils that form a porous mesh microstructure. The
size of the fibrils, the density of the mesh, and the mechanical properties can be controlled by
varying the pH, temperature, and ionic strength at which the hydrogel is prepared.4 For all of the
reasons above collagens are attractive materials for biofabrication. In tissue engineering,
biofabrication strategies typically include the development of scaffolds or composite constructs,
1

Organic & Biomolecular Chemistry
Page 2 of 25
View Article Online
DOI: 10.1039/C7OB02198D
which exhibit tissue mimetic hierarchical features. Often the constructs include living cells or
cell aggregates, bioactive molecules, or biomaterials that are printed and used for tissue
fabrication.5 A list of the various techniques to print these cellꢀladen constructs has been
published recently.6 While a plethora of technologies exist, the most common ones are extrusion
pens,7 inkjet,8 electrospray,9 laserꢀinduced forward transfer,10 and photopolymerization, for
example the polymerization of poly(ethylene oxide) and poly(ethylene glycol)dimethacrylate to
generate a hydrogel.11 All have been adapted from the additive manufacturing field. For 2D cell
patterning, a common technique is the patterning of scaffolds or cell adhesive domains via micro
contact printing, in which a pattern is being etched into a silicon master, which is then used as a
master for the printing process.12 Another common technique for 2D cell patterning is inkjet
printing. For example, a singleꢀlayer cellular micropattern was fabricated by printing an ink of
primary embryonic hippocampal neurons in the pattern of a ring, with a 0.5 mm wide rim, 2.5
mm across, on a bioꢀpaper substrate made from rat tail Type I collagen.13 While the 2D and 3D
patterning processes described above rely on depositing cells on or into natural or unnatural
polymers whose structures cannot be manipulated, we envisioned a new approach, i.e. a
collagenꢀlike peptide with unique properties, allowing for its siteꢀspecific decomposition at the
microscale, creating short peptide fragments with potentially different properties than the bulk
material. We hypothesized that the macroscopic material made from a collagenꢀlike peptide with
incorporated photoreactive moieties could be photolytically decomposed when illuminated at
precise microscopic locations creating micropatterns.
Here we report a novel collagenꢀlike peptide, with builtꢀin photoreactive Nꢀacylꢀ7ꢀnitroindoline
moieties and its characterization. We proposed that micropatterns could potentially be
introduced into a biomimetic macroscopic bulk material by a photodegradation method, which
could potentially guide tissue growth in specific 2D or even 3D patterns in the future. Recently,
we showed the ability of such a photoreactive peptide (1) to undergo photolysis by a twoꢀphoton
absorption process.14 Its twoꢀphoton photolysis products obtained by irradiation of a
concentrated film had been verified by mass spectrometry,14 but the synthesis of peptide 1 had
not been detailed out, and no further chemical or biological characterization had been conducted.
Here we present the synthesis of this photoreactive 34 amino acid long collagenꢀlike peptide (1)
consisting of five hexapeptide repeats that are separated by the unnatural amino acid 5ꢀ
carboxylic acidꢀ7ꢀnitroindoline (Figure 1), and the synthesis of the photoreactive amino acid
building block. We also present studies on its secondary structure, its photolytic properties under
UV light in an aqueous solution, its suitability for the generation of micropatterns using
femtosecond laser photolysis, and its cytocompatibility with mesenchymal stem cells as well as
mouse embryonic fibroblasts. In addition, we show that a 5ꢀbromoꢀ7ꢀnitroindoline based amino
acid, which can be easily synthesized from commercially available starting materials, also
undergoes photolysis by a twoꢀphoton absorption process, and that it can serve as a model
compound for more complex photoreactive 7ꢀnitroindolineꢀbased compounds with respect to
patterning.
2

Page 3 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
Figure 1. Photoreactive target peptide 1 with collagenꢀlike repeating units and nitroindoline
moieties (red) built into the peptide backbone. Photolytic cleavage occurs at the Nꢀpeptidylꢀ7ꢀ
nitroindoline amide bonds indicated with arrows.
In designing a collagenꢀlike peptide whose bulk material can potentially be modified by
photolysis at precise locations, the following factors were considered: a) the amino acid
components and length of the peptide; b) the photochemistry; and c) the synthetic strategy to
access such a material. Collagens are major components in many extracellular matrices, and
they play central roles in all phases of wound healing, including cell proliferation, remodeling,
hemostasis, and inflammation.15 To mimic the properties of collagen and other collagenꢀ
16ꢀ19
mimicking peptides, which are typically about 30 amino acids long,
we chose five
hexapeptide repeats rich in glycine, proline, and hydroxyproline, with a glycine residue at every
third position within the hexapeptide repeat. Since the ability to undergo photolytic cleavage
into small peptide fragments was a required property, the design of the target peptide included
four 7ꢀnitroindoline moieties, which can be introduced via the photoreactive building block N-
(FmocꢀGly)ꢀ5ꢀcarboxylic acidꢀ7ꢀnitroindoline20, 21 in solid phase peptide synthesis (SPPS) using
the Fmoc/tBu strategy.22 In previous work we have shown that this building block is suitable for
the installation of a photoreactive moiety at the Cꢀterminus of peptides by SPPS, which can be
photochemically converted into aliphatic or aromatic peptide thioesters and peptide
hydrazides.20, 21 The photochemical properties of Nꢀacylꢀ7ꢀnitroindoline in an inert organic
solvent in the presence of water, alcohols, or ammonia were first discovered more than 40 years
ago and resulted in the acylation of these nucleophiles, producing carboxylic acids, esters, and
amides.23 Mechanistic studies of the underlying photochemistry suggest that upon light
activation of the Nꢀacylꢀ7ꢀnitroindoline 2, a nitronic anhydride intermediate 3 is formed,24, 25
possibly by a sigmatropic rearrangement.26 In an inert organic solvent the nitronic anhydride 3
can either acylate a nucleophile, e.g. water, and produce a carboxylic acid and 7ꢀnitroindoline 4
(Scheme 1, path A),24, 27 or form a carboxylic acid and nitrosoindole 5 in a photoredox reaction
(path B, 100% water). The latter has been exploited for the photorelease of caged amino acids.28
Which of the two paths predominates is solventꢀdependent,24 and is also influenced by the
presence or absence of acid. For example, under acidic conditions, path B seems to be preferred,
presumably due to protonation of the acyl oxygen of the nitronic anhydride intermediate 3.26
Other Nꢀacylꢀ7ꢀnitroindoline derivatives, with a bromo,23, 29ꢀ33 nitro,34, 35 or a carboxamido
substituent20, 21, 36 at position 5 of the indoline ring, also undergo photoacylation (path A) with a
number of different nucleophiles, including water, in inert organic solvents such as
dichloromethane (DCM), acetonitrile, tetrahydrofuran, pyridine, N,Nꢀdimethylformamide,
N,N,N’,N’ꢀtetramethylurea, dimethylsulfoxide, and Nꢀmethylpyrrolidone. With respect to
photoreactive peptide 1, photolysis into small heptapeptide fragments with Cꢀterminal glycine
residues may occur by either pathway depending on the reaction conditions. We have recently
shown that the photolysis of peptide 1 can also be accomplished by a twoꢀphoton absorption
process.14 In that experiment, a highly concentrated thin film of peptide 1 in water was irradiated
with femtosecond laser light at 710 nm. The massꢀspectrometric analysis of the photolysis
products showed that all expected peptide fragments formed, and that the photolysis had
occurred via path A (Scheme 1) producing peptide fragments with Nꢀterminal 7ꢀnitroindoline
moieties.14
3

Organic & Biomolecular Chemistry
Page 4 of 25
View Article Online
DOI: 10.1039/C7OB02198D
Scheme 1. Light activation of an Nꢀacylꢀ7ꢀnitroindoline and its solventꢀdependent
decomposition. A low water content favors path A, while a high water content favors path B.
Results and Discussion
Due to the five repeating hexapeptide sequences separated by four photoreactive moieties in
peptide 1, it lends itself to being synthesized by solid phase peptide synthesis (SPPS) using onꢀ
resin peptide fragment condensation. The photoreactive glycine building block 6 can be
synthesized in seven steps from commercially available starting materials.20, 21 It was a key
component in the synthesis of the protected photoreactive peptide segment 7 (Scheme 2), which
was preꢀprepared by SPPS on diphenyldiazomethane resin37 for the fragment condensation.
Since the attachment of the first amino acid to diphenyldiazomethane resin does not rely on
activation with a coupling reagent, this resin is highly suitable for the recovery of unreacted
amino acid excess, particularly when it is a precious building block such as the photoreactive
glycine derivative 6. The coupling of the remaining Fmocꢀprotected amino acids and the
removal of Fmoc groups were accomplished under standard conditions using an uronium
coupling reagent and Nꢀhydroxybenzotriazole (HOBt) in the presence of N,Nꢀ
diisopropylethylamine (DIPEA), and piperidine, respectively.22 The protected peptide acid 7
was cleaved from the resin with a dilute solution of trifluoroacetic acid in DCM. Scheme 3
shows the assembly of the photoreactive target peptide 1. First, peptide 8 was synthesized by
SPPS on Rink Amide resin followed by four onꢀresin fragment condensations using the preꢀ
prepared photoreactive peptide 7. Global deprotection and cleavage from the resin was
accomplished with 95% trifluoroacetic acid (TFA), and purification of the peptide by reversed
phase chromatography. Through a combination of semiꢀautomatic FmocꢀSPPS and peptide
fragment condensation, a 41% overall yield of peptide 1 was obtained. The peptide was
characterized by mass spectrometry, UVꢀVIS spectrophotometry, fluorescence spectroscopy, and
circular dichroism (CD). The peptide’s ability to undergo photolytic cleavage with UV light in
an aqueous solution was also investigated. Furthermore, in order to obtain preliminary cell
toxicity information the peptide’s ability to support the lateral growth of human mesenchymal
stem cells, and its effect on the viability of mouse embryonic fibroblasts were studied.
4

Page 5 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
+
N
_
N
diphenyl-
diazo-
methane
resin
HOOC
a, b
N
O
NO2
NHFmoc
6
O
O
N
diphenyl-
diazo-
methane
resin
NO2
FmocHN
O
c, d, e
O
O
tBu
Hyp Pro
N
diphenyl-
diazo-
methane
resin
Pro Pro
Fmoc
Gly
Gly
NO2
f
COOH
tBu
N
Pro Pro Gly Hyp Pro Gly
Fmoc
NO2
7
Scheme 2. SPPS of the protected photoreactive peptide 7 on diphenyldiazomethane resin.
a) 6, DCM; b) TFA/DCM; c) 20% piperidine/DMF; d) FmocꢀaaꢀOH, HBTU, HOBt,
DIPEA, DMF (2×); e) Ac2O, DIPEA, DMF; f) 3% TFA/DCM.
5

Organic & Biomolecular Chemistry
Page 6 of 25
View Article Online
DOI: 10.1039/C7OB02198D
MeO
MeO
O
O
FmocHN
Nle NH
Rink
Amide
resin
MeO
MeO
a, b, c
O
O
tBu
NH
Pro Pro Gly Hyp Pro Gly
Fmoc
Nle NH
8
Rink
Amide
resin
a, d, c
4x
MeO
a
MeO
Gly
O
O
tBu
NH
O
Pro Pro
NO2
Hyp Pro
Gly
Nle NH
Rink
Amide
resin
tBu
N
O
Gly Pro Hyp
Pro Pro
O2N
Gly
tBu
Hyp Pro
N
O
Pro Pro
NO2
Gly
Gly
tBu
N
O
Gly Pro Hyp
Pro Pro
Gly
O2N
Hyp Pro
tBu
N
H
Pro Pro
Gly
Gly
e
1
Scheme 3. Synthetic strategy for the preparation of photoreactive peptide 1 by on resin fragment
condensation. a) 20% piperidine/DMF; b) FmocꢀaaꢀOH, HBTU, HOBt, DIPEA, DMF (2×); c)
Ac2O, DIPEA, DMF; d) 7, TBTU, HOBt, NMM, DMF; e) TFA/H2O/TIS 95:2.5:2.5.
6

Page 7 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
Circular dichroism and thermal stability of peptide 1
The farꢀUV CD spectra of peptide 1 show the typical signature of a triple helix (Figure 2a). The
overall structure closely resembles the triple helices observed for collagen peptides that consist
of naturally occurring amino acids.38ꢀ40 Increasing the temperature from 20˚C to 70˚C in 2˚C
increments and then followed by 30˚C to 70˚C in 10˚C steps show practically no loss of structure
(Figure 2a). Figures 2b and 2c show the mean molar elipticity at [θ]200 and [θ]222, respectively,
with the rise in temperature. There is minimal change in intensity both at 200 nm and 222 nm
indicating that the secondary structure of the peptide is stable in the temperature range studied.
Unlike other triple helical peptides of similar length,38, 41, 42 peptide 1 contains four units of the
unnatural amino acid 5ꢀcarboxylic acidꢀ7ꢀnitroindoline, which could be responsible for its
unusual stability. Unlike proteins that consist predominantly of αꢀhelices or βꢀsheets, the CD
spectra of collagen and gelatin have a strong minimum at approximately 200 nm which can be
attributed to random coil structures and a positive peak at 222 nm, which is often not a
pronounced maximum. Both are typical features of the CD spectra of collagen.43 Importantly,
the collagenꢀlike structure formed by peptide 1 is very stable at 37 ˚C, which is highly significant
as it enhances its utility for biological applications and its potential usefulness for cell adherence
and growth.
(a)
(b)
(c)
Figure 2. Secondary structure determination of the triple helical peptide using farꢀUV CD. a)
The change in color from blue to red denotes the rise in temperature from 20 ˚Cꢀ 70 ˚C; b) Mean
molar ellipticity at 200 nm at each temperature from 20 ˚Cꢀ 70 ˚C; c) Mean molar ellipticity at
222 nm at each temperature from 20 ˚Cꢀ 70 ˚C.
Photolysis of an aqueous solution of peptide 1 using UV light, and LCꢀMS analysis
In order to study whether or not peptide 1 is capable of undergoing photolysis at 350 nm light
irradiation, an aqueous solution of 1 was illuminated for 5 min. The mass spectrometric analysis
showed that starting material 1 was completely consumed. The three major peaks observed in
the mass spectrum correspond to the Nꢀterminal (9), middle (10), and Cꢀterminal (11) peptide
fragments. Peptide fragments 10 and 11 contain a 7ꢀnitrosoindole moiety, which is in
accordance with Corrie’s solventꢀdependence study (Scheme 1, path B).24 However, a small
peak that corresponds to a hexapeptide fragment with a nitroindoline (12) was also observed in
this mass spectrum, suggesting that under the reaction conditions photolysis occurred by two
pathways, albeit the expected reaction path (Scheme 1, path B) predominated. The peptide
7

Organic & Biomolecular Chemistry
Page 8 of 25
View Article Online
DOI: 10.1039/C7OB02198D
mixture was also subjected to liquid chromatographyꢀmass spectrometry (LCꢀMS) analysis. The
data show that the photolysis of peptide 1 was complete; i.e., no large peptide fragments with
intact photoreactive moieties were found. Also, peptides 9 – 12 are major photolysis products,
with peptides 9 – 11 being most prominent. However, the total ion count (TIC) chromatogram
appears to be complex and is consistent with the presence of peptide conformers, and several
additional compounds. MS/MS analysis of three of these compounds suggests the presence of an
unusual peptidylꢀnitroindole and two peptidylꢀnitrosoindolines (Electronic Supplementary
Information).
H-Pro-Pro-Gly-Hyp-Pro-Gly-OH
9
NO
H
N
Pro-Pro-Gly-Hyp-Pro-Gly-NH2
11
O
NO
H
N
Pro-Pro-Gly-Hyp-Pro-Gly-OH
10
O
NO2
H
N
Pro-Pro-Gly-Hyp-Pro-Gly-NH2
12
O
Figure 3. HR ESI MS after irradiation of peptide 1 with 350 nm light in water. Peptide 9: m/z
[M+H]+ calcd. 537.2672, obs. 537.2671; 10: m/z [M+H]+ calcd. 709.2946, obs. 709.2919; 11:
m/z [M+Na]+ calcd. 730.2925, obs. 730.2918; 12: m/z [M+H]+ calcd. 726.3211, obs. 726.3202.
Generation of a micropattern by twoꢀphoton absorption chemistry
In order to demonstrate the suitability of peptide 1 as a material for the generation of precise
micropatterns using 710 nm femtosecond laser light, a nearly dry film of compound 1 was
illuminated in a tree pattern with about 10 ꢀm wide features. The illumination occurred through
a mask of a tree in the optical path. This mask was made from a cover glass slip with a dark
colored tape; only the tree, approximately 1 cm wide, was transparent. Only laser light that
passed through the mask reached the film of the photoreactive peptide 1 in form of the tree
pattern. Initially, the illuminated areas fluoresce (Figure 4 AꢀC), which triggers the photolysis
resulting in nonꢀfluorescent decomposition products such as peptides 9, 12, and other previously
described photolysis products.14 Upon removal of the mask only the background consisting of
intact peptide 1 fluoresces, and the tree appears dark (Figure 4, D). When viewed under a
regular white light microscope, the light background color of compound 1, and the orange color
8

Page 9 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
of the photolysis products is clearly visible (Figure 4 E). The fact that the photolysis of peptide 1
only occurs at sites of illumination, and does not propagate through lightꢀprotected areas of the
material, suggests that peptide 1 could potentially be suitable for photolithography and tissue
engineering applications, such as matrix guided cell growth in distinct micropatterns.44
Figure 4. (A) Initial fluorescent tree pattern generated by twoꢀphoton excitation of the collagenꢀ
like peptide 1 with femtosecond laser at 710 nm light through a tree mask at time 0 min; (B)
Fluorescent tree pattern after 10 min of irradiation, fluorescence decay is observed; (C) Tree
pattern after 30 min of irradiation, fluorescence is barely visible due to progressive photolysis to
nonꢀfluorescent products; (D) After removal of mask and immediate recording of the
fluorescence only the background fluoresces while the tree appears dark; (E) Tree under white
light.
Photolysis of Nꢀ(Fmocꢀglycyl)ꢀ5ꢀbromoꢀ7ꢀnitroindoline (13) by a twoꢀphoton absorption
mechanism
An important question was whether Nꢀacylꢀ7ꢀnitroindolines other than those with a carboxamido
group at position 5 of the aromatic ring (as present in peptide 1), or with a methoxy group at
46
position 4,45,
also have the ability to undergo photolysis by a twoꢀphoton absorption
mechanism. Using the photoreactive amino acid 13 (Figure 5) as a model compound, we
investigated whether its photolysis could be achieved by a twoꢀphoton absorption mechanism
using a femtosecond laser at 710 nm, and whether Nꢀacylꢀ5ꢀbromoꢀ7ꢀnitroindolines can undergo
localized photolysis within a macroscopic film creating a specific micropattern. The oneꢀphoton
absorption properties and release of carboxylic acids caged as Nꢀacylated 7ꢀnitroindolines have
been reported in the literature.23,25,27,36 However, the photolysis of Nꢀacylꢀ7ꢀnitroindolines has
never been investigated in the context of biopolymers, macroscopic materials, and thin films.
After irradiation of a concentrated film of 13 with the femtosecond laser at varying laser powers
ranging from 100 mW to 200 mW the formation of orange/brown spots indicates the formation
of 5ꢀbromoꢀ7ꢀnitroindoline (14) at the different irradiated sites. The observed color change was
the first indication of a successful photolysis (Figure 5).
9

Organic & Biomolecular Chemistry
Page 10 of 25
View Article Online
DOI: 10.1039/C7OB02198D
1 mm
O
HN
FmocHN
N
O2N
O2N
Br
14
13
Br
Figure 5. Images of a yellow film of Nꢀ[fluorenylmethyloxycarbonylꢀglycyl]ꢀ5ꢀbromoꢀ7ꢀ
nitroindoline (13) before (a) and after (b) irradiation with a femtosecond laser at 710 nm at
several locations with varying laser power (b). The photolysis produces orange/brown colored 5ꢀ
bromoꢀ7ꢀnitroindoline (14) and colorless fluorenylmethyloxycarbonylꢀglycine.
Compound 13 exhibits a weak but measurable fluorescence, which is associated with its
photochemical conversion into the nonꢀfluorescent compound 14. This unique property was
exploited to investigate whether the photolysis occurs by a twoꢀphoton absorption mechanism
using a fluorescence microscope. All the images produced were collected and analyzed using
ImageJ37 to measure the fluorescence intensity of individual channels. For each irradiated spot, a
stack of images was created. An area was chosen within the image, and the average fluorescence
intensity in each of the green and red channels was recorded. This measurement was performed
in the same region of interest for every image in the stack, each taken one minute apart. A
minimum fluorescence threshold was approximated for each image stack, and the fluorescence
intensity normalized accordingly. A plot of these normalized intensities over time at varying
laser powers from a single sample of 13 is shown in Figure 6. As it can be observed, the kinetics
of the photoreaction are proportional to the intensity of the laser as the fluorescence decay is the
fastest with the highest laser power (200 mW) and the slowest with the weakest laser power (100
mW).
10

Page 11 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
Figure 6. Fluorescence decay plots of compound 13 with exponential fitting at varying laser
power.
These fluorescence decay plots may be modeled using an exponential decay regression line of
the form F t ꢂ Feꢃꢄꢅ, where β is the fluorescence decay rate. This was done using the curve
ꢀ ꢁ
fitting capabilities in Matlab. As mentioned before the fluorescence decay measured is directly
proportional to the kinetics of the reaction. Therefore the fluorescence decay is equal to the rate
of the reaction at every specific point and varying laser power. Since this photoreaction is
occurring as a result of twoꢀphoton absorption, the rate of the reaction is directly proportional to
the probability of twoꢀphoton absorption as shown in Eq. (1) where I is the excitation laser
power.
ꢆ ∝ ꢇꢈ
Eq. 1 Probability of twoꢀphoton absorption, rate of reaction is proportional to the intensity of the
laser squared.
Plotting a graph (Figure 7) where the x axis is the ln(I) and the yꢀaxis corresponds to the ln(β) for
each of the laser intensities employed and their corresponding decay rates produces a linear
graph where the slope of the reaction has to be 2 to prove that a twoꢀphoton absorption process
occurred. After plotting the graph, the slope observed was 1.996 (Figure 7), which clearly shows
that compound 13 was photoꢀcleaved via a twoꢀphoton absorption process.
Figure 7. Double log plot of reaction rate vs. laser intensity for photoreactive compound 13.
11

Organic & Biomolecular Chemistry
Page 12 of 25
View Article Online
DOI: 10.1039/C7OB02198D
After irradiation the sample shown in Figure 5b was dissolved and analyzed by HRꢀESIꢀTOFꢀ
MS, which showed the formation of 5ꢀbromoꢀ7ꢀnitroindoline 14 (m/z [M+H]+ calcd. 242.9769
and 244.9749, obs. 242.9784 and 244.9765, but not 5ꢀbromoꢀ7ꢀnitrosoindole. The result is in
accordance with Corrie’s photolysis data, which show that the photolysis pathway depends on
the water content of the sample (Scheme 1). He demonstrated that a similar Nꢀacylꢀ7ꢀ
nitroindoline in an inert organic solvent with only 1% water photolyzed to the nitroindoline.24
Unlike our photolysis experiment of peptide 1 in water resulting predominantly in nitrosoindoles
(Figure 3), compound 13 was irradiated as a highly concentrated, viscous film using nonꢀ
anhydrous DMF as a solvent. This film contained small quantities of water, similar to Corrie’s
reaction conditions in Scheme 1, path A, allowing for the expected photoꢀhydrolysis to take
place.
To test whether compound 13 could serve as an Nꢀacylꢀnitroindolineꢀcontaining model
compound for the generation of micropatterns, an image of the logo of the University of Texas at
El Paso (“UTEP”) was projected on a thin film of compound 13 through a mask in the optical
path of 710 nm femtosecond laser light. The experiment was conducted in a similar manner as
described for the tree patterns shown in Figure 4. Initially, the illuminated areas fluoresce
(Figure 8, left), which is associated with the photolysis to produce the nonꢀfluorescent
nitroindoline 14. Upon removal of the mask only the background consisting of intact compound
13 fluoresces, and the logo appears dark (Figure 8, middle). When viewed under a regular white
light microscope (Figure 8 right), the yellow background is due to the presence of compound 13,
and the orangeꢀbrown micropattern consists of compound 14.
50 ꢀm
Figure 8. Left: Fluorescent logo of the University of Texas at El Paso generated by excitation
of the photoreactive amino acid 13 with a femtosecond laser at 710 nm through a “UTEP” mask;
middle: The letters of the logo underwent photolysis to the nonꢀfluorescent nitroindoline 14.
After removal of the mask the background fluoresces; right: UTEP logo under white light.
Cytocompatibility
The cytocompatibility of peptide 1 was studied using human mesenchymal stem cells in a
qualitative cell proliferation assay, and mouse embryonic fibroblasts in a Live/Dead assay. The
latter assay also included the photolysis products of peptide 1, and collagen as a control. Figure
9 illustrates that peptide 1 is not cytotoxic to cultured human mesenchymal stem cells as the cells
showed viability and confluence within 24 hours of culture. Furthermore, peptide 1 promotes
cell adhesion and migration. The blue line in the right image of Figure 9 denotes the edge of the
immobilized peptide material from where undried/uncoated remnant liquid was aspirated prior to
cell seeding. As the rim does not have any of peptide 1 immobilized, cells did not migrate,
adhere or proliferate in that area, showing their preference to growing on the peptideꢀcoated
surface over the plastic of the well.
12

Page 13 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
50 ꢀm
Figure 9. Human mesenchymal stem cells, preꢀstained with PKH26, a cell membrane stain, and
seeded atop collagenꢀlike peptide 1 preꢀimmobilized onto tissue culture grade wells. Left:
Images taken from center of the well. Right: cells grew only where the peptide sheet was
present.
The cytocompatibility of peptide 1 and its photolysis products was also assessed using mouse
embryonic fibroblasts by a Live:Dead cytotoxicity assay.47 Figure 10A–C depicts confocal
microscopy images of these cells cultured in wells coated with (A) peptide 1; (B) peptide 1
irradiated with UV light; and (C) collagen as a control. The green fluorescence indicates live
cells, and the red fluorescence indicates dead cells. The fibroblasts retained their viability and
expressed their normal spindle shaped morphology48 on the peptide coated (A) and collagen
coated (C) surfaces. However, on the surface coated with the irradiated peptide (B), the
fibroblasts do not show spindle shaped morphology, and most of the cells are dead.
To interpret the results of the Live/Dead cytotoxicity assay,47 it is well known that live cells are
distinguished by the presence of endogenous intracellular esterase activity, determined by the
enzymatic conversion of the nonfluorescent cellꢀpermeant calcein AM to the intensely
fluorescent calcein (green). Although the polyanionic dye calcein is well retained within live
cells producing an intense uniform green fluorescence, it is also important to note the healthy
morphology of the cells. In Figure 10, fibroblasts with the healthy spindleꢀshaped morphology
are observed only in (A) and (C), but not in (B) where the liveꢀstained cells appear rounded and
shrunk in their morphology. Moreover, in (B) we observe cells coꢀstained with both fluorescent
dyes, green and red. The red fluorescence is conferred by the ethidium homodimerꢀ1 (EthDꢀ1),
which enters cells with damaged membranes and undergoes a significant enhancement of
fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead
cells. EthDꢀ1 cannot cross the intact plasma membrane of live cells. Therefore the presence of
both stains, green and red, and the absence of normal spindle shaped morphology in cells imaged
in (B) imply that these cells are in the process of dying due to the presence of a cytotoxic surface
caused by the photolysis products of peptide 1. Although a similar number and morphologies of
live cells are noted in (A) and (C), more dead cells are noted in the collagen coated controls (C)
compared with the surface coated with peptide 1 (A). Since a solution of peptide 1 was
deposited onto the wells in several layers followed by drying it may have created a bulge with,
13

Organic & Biomolecular Chemistry
Page 14 of 25
View Article Online
DOI: 10.1039/C7OB02198D
which may have caused an overall smaller number of cells adhering onto this surface. However,
the cells that did attach retained their viability and morphology implying that peptide 1 created a
nonꢀcytotoxic surface (A). In the future, we would need to improve the method for creating
smooth and leveled peptide surfaces for tissue engineering applications. In conclusion, the
photolysis products of peptide 1 show a certain cytotoxicity when compared to the intact peptide
1 and collagen, making peptide 1 a potential candidate for patterned 2D cell growth.
In principle, a number of different compounds could be responsible for the cytotoxic effect
observed in Figure 10 (B). Previously, it was shown that the photolysis of a concentrated film of
peptide 1 results in a mixture of peptidylꢀ7ꢀnitroindolines.14 Therefore, the irradiation of a dry
but not anhydrous film of peptide 1 with UV light is expected to produce predominantly peptides
9, 12, and [5ꢀcarboxylic acidꢀ7ꢀnitroindoline]ꢀProꢀProꢀGlyꢀHypꢀProꢀGly (17). With the limited
cytotoxicity data at hand, the cytotoxic effect observed cannot be ascribed to a particular
compound. In general, depending on the water content of the peptide sample at the time of
irradiation, several peptidylꢀ7ꢀnitroindolines and peptidylꢀ7ꢀnitrosoindoles are possible
photolysis products with potential effects on cell viability. The cytotoxicity of the individual
compounds is not known.
(A)
(B)
(C)
Figure 10. In the wells of a 96 well plate, mouse embryonic fibroblasts were seeded atop a
surface coated with (A) nonꢀirradiated peptide 1; (B) irradiated peptide 1 resulting in its photoꢀ
decomposition; and (C) collagen. Shown are representative merged images captured using
bright field and fluorescence, where the green fluorescence depicts live cells and the red
fluorescence indicates dead cells. The cells appear healthy and express their characteristic
spindle shaped morphology atop the surface coated with peptide 1 (A). On the collagen coated
surface (C), cells also express their ideal morphology, however, numerous dead cells are also
noted. On the surface coated with peptide 1 that was irradiated with UV light prior to cell
seeding (B), the cells are not found to express their ideal morphology and appear rounded. Also,
very few live cells are observed.
Experimental
Reagents and Solvents
Fmoc amino acid derivatives were purchased from Anaspec Inc., BACHEM or ChemꢀImpex
Intl. Inc. Diphenyldiazomethane resin and Rink Amide resins were obtained from BACHEM
and Novabiochem, respectively. The coupling reagents HBTU and TBTU were purchased from
14

Page 15 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
Anaspec Inc. and Novabiochem, respectively. HOBt, piperidine, and reserpine were obtained
from Sigma Aldrich. Tetrakis(triphenylphosphine)palladium(0), Nꢀmethylaniline, Nꢀ
methylmorpholine, TIS, and tetrachloroꢀpꢀbenzoquinone were obtained from Acros. 5ꢀBromoꢀ7ꢀ
nitroindoline, thionyl chloride, and Ultramark were acquired from Alfa Aesar. Solvents, DIPEA,
TFA, and bromophenol blue were obtained from Fisher Scientific. CDCl3 and DMSOꢀd6 were
purchased from Acros and Cambridge Isotope Laboratories, respectively.
Thin layer
chromatography was performed on silica gel 60 F254 on aluminum (Merck). Column
chromatography was performed on silica gel 60, 230ꢀ400 mesh from Natland International Corp.
Instrumentation
Peptides were synthesized semiꢀautomatically using a Tribute peptide synthesizer from Protein
Technologies, Inc. (USA). Reversed phase chromatography was performed on a Fast Protein
Liquid Chromatography (FPLC) system in an AKTA Purifier from GE Healthcare Life Sciences.
Superfrost microscope slides were obtained from Fisher Scientific (USA). UVꢀVIS absorption
spectra were measured on a Shimadzu UVꢀ3101PC UVꢀVISꢀNIR scanning spectrophotometer
using quartz cuvettes of 1 cm path length. Fluorescence spectra were measured on a Shimadzu
RFꢀ6000 spectrofluorophotometer using standard quartz cuvettes of 1 cm path length at rt. The
fluorescence quantum yield was not determined. The excitation and emission bandwidths were 5
nm, and the Raman scattering of the solvent was subtracted. 1H NMR and 13C NMR were
recorded on a JEOL ECAꢀ600 (600 MHz) or a Bruker Avance III HD (400 MHz).
Tetramethylsilane was used as an internal standard. Mass spectrometry was performed on a high
resolution JEOL Accu TOF mass spectrometer using an Electrospray Ionization (ESI) source or
a High Resolution QExactive Plusꢀmass spectrometer from Thermo Fisher Scientific. The
photolysis of peptide 1 in aqueous solution was performed in a Rayonet RPR200 photochemical
reactor (USA) equipped with 16 UV lamps (350 nm). FarꢀUV Circular Dichroism (CD) studies
were conducted using a Jascoꢀ1500 spectropolarimeter connected to a Peltier temperature
controller.
Synthesis
Peptide 1
The photoreactive peptide 1 was synthesized from hexapaptide 8, which was elongated by
repeated coupling of preꢀprepared peptide 7 by on resin fragment condensation. Hexapeptide 8
was synthesized on Rink Amide resin (loading capacity 0.62 mmol/g). The resin (0.059 mmol,
96 mg) was swollen in DCM (3 mL) for 30 min and washed 5 × with DMF. The resin’s Fmoc
group was removed with 20% piperidine in DMF (3 mL) under shaking for 15 min followed by
washing with DMF (5 × 10 mL). FmocꢀGlyꢀOH (0.059 mmol, 18 mg, 1 equiv.), HBTU (0.059
mmol, 23 mg), HOBt (0.059 mmol, 8 mg) and DIPEA (0.118 mmol, 21 ꢀL) were dissolved in
DMF (0.12 M, 0.5 mL), immediately added to the resin and mixed for 45 min, then washed with
DMF (5 × 5 mL) followed by capping with 10% Ac2O, 5% DIPEA in DMF (3 mL, 15 min). The
reason for using only ~ 1 equiv. of the first amino acid was to achieve an incomplete loading to
reduce peptide aggregation during SPPS. Fmoc removal was accomplished as described before.
The loading of the first amino acid onto the resin was 75% as determined by the quantification of
dibenzofulvene by UVꢀVIS spectrophotometry (290 nm, ε5253, 1 cm. path length). The next five
amino acids [FmocꢀProꢀOH, FmocꢀHyp(tBu)ꢀOH, FmocꢀGlyꢀOH, FmocꢀProꢀOH, and Fmocꢀ
ProꢀOH] were coupled by dissolving 5 equiv. of the amino acid derivative (0.30 M), 5 equiv.
HBTU, 5 equiv. HOBt and 10 equiv. of DIPEA in DMF, which was immediately added to the
15

Organic & Biomolecular Chemistry
Page 16 of 25
View Article Online
DOI: 10.1039/C7OB02198D
resin, stirred for 15 min, doubleꢀcoupled with half the quantities of the previous coupling step to
furnish the resinꢀbound hexapaptide 8. After capping and removal of the Nꢀterminal Fmoc group
as previously described, the peptide was further elongated by peptide fragment condensation (4
×) with the photoreactive hexamer 7 (Scheme 3). For the fragment condensation, a solution of 7
(0.062 mmol, 62 mg, 2.5 equiv.), TBTU, 0.062 mmol, 20 mg), HOBt (0.062 mmol, 8 mg), and
NMM 0.125 mmol, 14 ꢀL) in NMP (0.10 M, 0.60 mL) was added to the resin and stirred for 9ꢀ
11 hours. Coupling reactions were monitored with the bromophenol blue and chloranil tests,49, 50
and capping and removal of the Nꢀterminal Fmoc group was performed as described before. The
full length peptide 1 was cleaved off the resin with simultaneous sideꢀchain deprotection using
95% TFA, 2.5% TIS, 2.5% water, 5 mL, 3 h, and the resin was washed twice with TFA. The
crude peptide was concentrated under vacuum to a glassy film and precipitated with cold diethyl
ether. The solution was centrifuged and the peptide pellet was washed several times with cold
diethyl ether. The crude peptide 1 was dissolved in water and preꢀpurified by size exclusion
chromatography on Superdex by isocratic elution with water. Final purification (48%) was
achieved by reversed phase FPLC (Source) using a gradient of 10ꢀ35% (solvent A: 2% CH3CN,
and 0.1% TFA in H2O; solvent B: 85% CH3CN, and 0.1% TFA in H2O. After lyophilization 75
mg of peptide 1 (49% with respect to the loading of the Cꢀterminal amino acid) was obtained as
a pale yellow solid. ESI TOFꢀMS m/z [M+2H]2+ calcd. 1685.7205 (most abundant species), obs.
1685.7175; [M+3H]3+ calcd. 1123.8061 (monoisotopic species), obs. 1123.8211. λmax (H2O, 29.7
ꢀM) = 251 and 332 nm, ε251 = 14297 Mꢀ1 cmꢀ1, ε332 = 2384 Mꢀ1 cmꢀ1. λem (H2O, 29.7 ꢀM, excited
at 332 nm) = 397 nm.
Nꢀ(Fmocꢀglycyl)ꢀ5ꢀcarboxylic acidꢀ7ꢀnitroindoline (6)
The photoreactive glycine building block 6 suitable for SPPS was synthesized from nitroindoline
derivative 1520 via glycine derivative 16 by Pd(0) catalyzed deallylation35 (Scheme 4). In a
round
bottom
flask,
compound
16
(1.15
mmol,
0.61
g)
and
tetrakis(triphenylphosphine)palladium (0.12 mmol, 0.13 g) were dissolved in anhydrous
tetrahydrofuran (THF, 10 mL) under argon. Nꢀmethylaniline (11.50 mmol, 1.25 mL) was added
to the solution, which immediately turned dark red. The reaction was monitored by TLC until the
starting material was consumed (1h). THF was removed under reduced pressure and the
remainder was dissolved in ethyl acetate and washed extensively with a 1M HCl solution (10 ×
50 mL), followed by water (5 × 50 mL), brine (2 × 50 mL) and dried over magnesium sulfate.
Ethyl acetate was removed under reduced pressure to obtain an orange solid (0.56 g,
1
quantitative) and no further purification was required. Rf = 0.18 (MeOH/DCM 5:95). H NMR
(400 MHz, 295 K, DMSOꢀd6) δ 13.44 (s, 1 H, COOH); 8.10 (s, 1 H, H6); 8.08 (s, 1 H, H4); 7.90
3
(d, 2 H, 3J = 7.4 Hz, FmocꢀArH); 7.78 (t, 1 H, NH); 7.75 (d, 2 H, J = 7.4 Hz, FmocꢀArH); 7.42
(t, 2 H, FmocꢀArH); 7.34 (t, 2 H, FmocꢀArH); 4.33ꢀ4.30 (m, 4 H, FmocꢀCH2, H2, H2’); 4.25 [t, 1
H, 3JFmoc (CH/CH2) = 7.0 Hz, Fmoc (benzylic)]; 4.10 (d, 2 H, 3JH /NH = 6.0 Hz, Ηα, Ηα ); 3.29 (t, 2
α
3
H, JH2/H3 = 8.1 Hz, H3) ppm; 13C NMR (100 MHz, 295 K, DMSOꢀd6) δ 168.2, 156.4, 143.8,
140.7, 139.3, 137.6, 136.5, 129.3, 127.6, 127.0, 125.2, 123.4, 120.0, 65.7, 48.8, 46.6, 43.6, 28.3
ppm. ESIꢀTOFꢀMS m/z [MꢀH]ꢀ calcd. 486.1301; obs. 486.1327. λmax (DCM, 41.1 ꢀM) = 268,
301 and 324 nm, ε268 = 18701 Mꢀ1 cmꢀ1, ε301 = 5092 Mꢀ1 cmꢀ1, ε324 = 1935 Mꢀ1 cmꢀ1. λem (DCM,
82.1 ꢀM, excited at 324 nm) = 392 nm.
16

Page 17 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
Scheme 4.
Synthesis of the photoreactive amino acid building block 6: a) SOCl2, toluene,
70oC; b) Pd(PPh3)4, Nꢀmethylaniline, THF.
Peptide 7
Diphenyldiazomethane resin (loading capacity 0.7 mmol/g, 0.99 mmol, 1.421 g) was swollen in
10 mL DCM in a peptide synthesis vessel for 20 min. The photoreactive building block 6 (0.995
mmol, 0.485 g) was dissolved in a DCM/DMF mixture (2:1) (0.20 M, 5 mL), added to the resin
and in a peptide synthesizer shaken for 5 h. Upon completion of the coupling, the excess of
compound 6 was recovered. The reason for using only ~ 1 equiv. of the first amino acid was to
achieve an incomplete loading to reduce peptide aggregation during SPPS. After coupling of
compound 6, the resin was washed with DMF (5 × 10 mL), capped with 5 mL DCM/HOAc (1:1)
for 1h, and washed with DMF (5 × 10 mL). Fmoc removal was accomplished with 5 mL of 20%
piperidine in DMF for 15 min followed by washing with DMF (5 × 10 mL). The loading of the
first amino acid onto the resin was 47% as determined by the quantification of dibenzofulvene by
UVꢀVIS spectrophotometry (290 nm, ε5253, 1 cm. path length). FmocꢀProꢀOH (4.97 mmol,
1.677 g), HBTU (4.97 mmol, 1.885 g) and HOBT (4.97 mmol, 0.671 g) were dissolved in DMF
(1 M, 5 mL) and DIPEA (9.9 mmol, 1.73 mL) was added just before the solution was poured into
the reaction vessel with the resin. The peptide reaction vessel was shaken for 15 min, followed
by DMF washings (5 × 10 mL) and double coupling of the amino acid, with half the amounts of
the previous coupling for 15 min. Capping was performed with 10% Ac2O and 5% DIPEA in
DMF (10 mL, 10 min). Using the same protocol, the peptide was elongated with the remaining
amino acids in the order FmocꢀHyp(tBu)ꢀOH, FmocꢀGlyꢀOH, FmocꢀProꢀOH, FmocꢀProꢀOH,
except for that the Nꢀterminal Fmoc group was not removed. Each coupling was monitored by
the bromophenol blue test,50 and chloranil test (preferred for Nꢀterminal proline).49 Peptide 7
was cleaved off the resin by repeated treatment with 3% TFA in DCM (10 mL) for 3 min until it
was no longer visible in TLC (15 repetitions), then washed with fresh DCM (2 × 10 mL) and
MeOH (2 × 10 mL). All peptideꢀcontaining fractions were combined, concentrated under
reduced pressure to approximately 5 mL and precipitated with cold diethyl ether to obtain the
crude photoreactive hexamer 7. The suspension was centrifuged and the yellow pellet was
washed several times with cold diethyl ether. The crude peptide was purified by silica column
chromatography using a gradient of 5% MeOH to 10% MeOH in DCM to isolate peptide 7
(0.380 g) in 81% yield with respect to the loading of the first amino acid on the resin. Rf = 0.10
(5% MeOH in DCM). HRꢀESIꢀTOFꢀMS m/z [M+H]+ calcd.: 1005.4358; obs. 1005.4334;
[M+Na]+ calcd. 1027.4178; obs. 1027.4150. λmax (DCM, 99.5 ꢀM) = 267 and 327 nm, ε267
=
10303 Mꢀ1 cmꢀ1, ε327 = 1103 Mꢀ1 cmꢀ1 λem (DCM, 99.5 ꢀM, excited at 327 nm) = 391 nm.
.
NꢀFmocꢀglycylꢀ5ꢀbromoꢀ7ꢀnitroindoline (13)
This photoreactive amino acid was synthesized by acylation of commercially available 5ꢀbromoꢀ
7ꢀnitroindoline with Fmoc protected glycine chloride generated in situ using a procedure similar
to a published method.29 5ꢀBromoꢀ7ꢀnitroindoline (2.00 mmol, 0.49 g, 1 equiv.) and FmocꢀGlyꢀ
17

Organic & Biomolecular Chemistry
Page 18 of 25
View Article Online
DOI: 10.1039/C7OB02198D
OH (3.00 mmol, 0.89 g) were suspended in anhydrous toluene (10 mL) under argon. The
o
mixture was stirred at 70 C for 15 minutes. Then thionyl chloride (8.00 mmol, 0.58 mL) was
added dropwise which resulted in a clear solution after 30 min. The reaction was monitored by
TLC until near completion (24 h). The solution was then diluted with ethyl acetate (250 mL) and
washed with a saturated solution of NaHCO3 (3 × 150 mL), water (3 x 150 mL) and brine (2 x
100 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under
reduced pressure. Compound 13 was purified by silica column chromatography (EtOAc:Hex
1
1:1); 0.940 g, 90% of an orange solid. Rf 0.18 (EtOAc:Hex 1:2); H NMR (600 MHz, 295 K,
3
3
CDCl3) δ 7.76 (s, 1H, H6); 7.75 (d, 2H, J = 7.5 Hz, Fmoc, arom.); 7.59 (d, 2H, J = 7.5 Hz,
Fmoc, arom.); 7.53 (s, 1H, H4); 7.38 (t, 2H, Fmoc, arom.); 7.29 (t, 2H, Fmoc, arom.); 5.82 (t,
1H, NH); 4.36 (d, 2H, 3JCH/CH2 = 7.2 Hz, FmocꢀCH2); 4.22ꢀ4.17 [m, 5H, Hα, Hα’, H2, H2’, Fmoc
(benzylic)]; 3.22 (t, 2H, 3JH2/H3 = 8.0 Hz, H3, H3’). 13C NMR (150 MHz, 295 K, CDCl3) δ 167.3,
156.7, 144.0, 141.6, 141.1, 138.7, 133.5, 132.2, 128.0, 127.4, 125.8, 125.5, 120.3, 117.4, 67.6,
49.1, 47.3, 44.5, 29.2. HRꢀESIꢀTOFꢀMS m/z [M+NH4]+ calcd. 539.0930 and 541.0913, obs.
539.0904 and 541.0937; [M+Na]+ calcd. 544.0484 and 546.0467, obs. 544.0489 and 546.0463.
λmax (CHCl3, 76.6 ꢀM) = 257 and 342 nm, ε257 = 8968 Mꢀ1 cmꢀ1, ε342 = 651 Mꢀ1 cmꢀ1. λem
(CHCl3, 23.7 ꢀM, excited at 342 nm) = 383 nm, and 633 nm (weak), broadly emitted across the
visible.
Allyl Nꢀ(Fmocꢀglycyl)ꢀ5ꢀcarboxylateꢀ7ꢀnitroindoline (16)
5ꢀAllyl carboxylateꢀ7ꢀnitroindoline (15)20 was reacted with FmocꢀGlyꢀOH in the presence of
SOCl2 similar to a reported procedure.29 FmocꢀGlyꢀOH (4.00 mmol, 1.189 g) and derivative 15
(2.00 mmol, 0.496 g, 1 equiv.) were suspended in anhydrous toluene (15 mL) under argon and
o
warmed up to 70 C. SOCl2 (10.00 mmol, 0.73 mL) was added dropwise, After 60 min a clear
solution was obtained. The reaction was monitored by TLC until near completion (40 h). The
solution was diluted with ethyl acetate (200 mL) and washed with a saturated solution of
NaHCO3 (3 × 100 mL), water (3 × 100 mL) and brine (2 × 100 mL). The organic layer was
dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. Product 16 was
purified by silica column chromatography (EtOAc:Hex 2:1). An orange solid was obtained,
0.981 g, 93% .Rf = 0.13 (EtOAc:hexanes 1:1). 1H NMR (600 MHz, 296 K, CDCl3) δ 8.34 (s, 1 H,
H6); 8.08 (s, 1 H, H4); 7.75 (d, 2 H, 3J = 7.5 Hz, Fmoc, arom.); 7.59 (d, 2 H, 3J = 7.5 Hz, Fmoc,
arom.); 7.38 (t, 2 H, Fmoc, arom.); 7.30 (t, 2 H, Fmoc, arom.); 6.06ꢀ5.99 (m, 1 H, CH2=CHꢀ);
3
5.81 (t, 1 H, NH); 5.42 (dd, 1 H, Jtrans = 17.2 Hz, 2J = 1.3 Hz, Allyl, olef. trans); 5.32 (dd, 1 H,
3
3Jcis = 10.4 Hz, Allyl, olef. cis); 4.84 (d, 2 H, Jaliph./olef. = 5.2 Hz, Allyl, aliph.); 4.37 (d, 2 H,
3
3JCH/CH2 = 6.9 Hz, FmocꢀCH2); 4.28 (t, 2 H, JH2/H3 = 8.1 Hz, H2); 4.22ꢀ4.19 (m, 3 H, Fmoc
(benzylic), Hα, Hα'); 3.29 (t, 2 H, H3). 13C NMR (150 MHz, 296 K, CDCl3) δ 167.3, 163.9,
156.3, 143.7, 141.2, 140.1, 137.5, 136.7, 131.6, 129.4, 127.7, 127.2, 127.1, 125.1, 125.0, 120.0,
119.1, 67.3, 66.3, 49.0, 47.0, 44.3, 28.7. HRꢀESIꢀTOFꢀMS m/z [M+Na]+ calcd. 550.1590, obs.
550.1597; [M+K]+ calcd. 566.1330, obs. 566.1333. λmax (DCM, 37.9 ꢀM) = 258, 267 and 324
nm, ε258 = 19461 Mꢀ1 cmꢀ1, ε267 = 17445 Mꢀ1 cmꢀ1, ε324 = 2192 Mꢀ1 cmꢀ1. λem (DCM, 75.9 ꢀM,
excited at 324 nm) = 390 nm, tailing weakly through the visible.
Circular Dichroism (CD) of peptide 1 and thermal stability measurements
FarꢀUV CD experiments were conducted to study the secondary structure of peptide 1. The
measurements were carried out using a 20 µM solution of the lyophilized peptide 1 in 15 mM
NaCl and 10 mM Na/K buffer (pH 6.8). The sample was placed into a quartz cuvette of 1 mm
18

Page 19 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
path length (Jasco) and heated from 20°C ꢀ 70°C to characterize its thermal stability. At each
temperature, the sample was equilibrated for 4 min prior measurement of the spectrum. CD scans
were performed at a range of 196ꢀ240 nm with three accumulations of data at each temperature
to improve the S/N ratio. Each spectrum underwent 11 point SavitzkyꢀGolay smoothing to
minimize high frequency noise. The experimentally estimated ellipticities (θobs) were converted
to mean molar ellipticity [θ] using the formula: [θ] = (θobs)/ncl where l is the path length of the
cuvette; c is the concentration of the peptide, and n is the number of stereogenic centers in the
peptide.
Photolysis of an aqueous solution of peptide 1 at 350 nm and LCꢀMS/MS analysis
The photoreactive peptide 1 (2 mg) was dissolved in 2 mL of HPLC grade water (pH 7.3; 0.29
mM), placed into a plastic microcentrifuge tube and irradiated with ultraviolet light at 350 nm in
a Rayonet photoreactor for 5 min at room temperature, followed by ESIꢀTOF mass spectrometric
analysis of the crude reaction mixture. The photolysis products of peptide 1 were separated on
an Aqua C18 column (5µm, 125Å, porous silica, Phenomenex) selfꢀpacked to a length of 25 cm,
into a New Objective PicoTip Emitter (PF360ꢀ100ꢀ15ꢀNꢀ5. Tip 15 ± 1µm). The Liquid
Chromatography (LC) was performed on an UltiMate 3000 Dionex RSLCnano UHPLC (Thermo
Fisher Scientific). The column was equilibrated in 95% Solvent A (100% H2O, 0.1% formic
acid), 5% Solvent B (90% acetonitrile, 10% H2O, 0.1% formic acid), and maintained at a
constant flow rate of 0.3 µL/min throughout the run. A 5 µL volume of the irradiated photolyzed
peptide 1 sample at a concentration of 1 µg/µL was loaded onto the column attached to a
Nanospray Flex Ion Source (Thermo Fisher Scientific) for 5 min before beginning the gradient.
Elucidation gradient of the photolyzed products consisted of an increase to 99% Solvent B over
31 min. A plateau of 99% Solvent B was maintained for 5 min, then brought down to 5%
Solvent B for a 20ꢀmin reꢀequilibration with a total run time of 62 min. MS/MS data acquisition
of the separated peptides was achieved using a Q Exactive Plus Hybrid QuadrupoleꢀOrbitrap
Mass Spectrometer (Thermo Fisher Scientific). The Q Exactive Plus was set to fragment the top
10 ions at full scan range of 300ꢀ1600 m/z (resolution of 70,000, AGC target 1e6) and ddꢀMS2
(resolution at 17,500, AGC target at 1e5). The total ion chromatogram (TIC), mass spectra of
individual eluting peptides, and MS/MS data can be viewed in the Electronic Supplementary
Information.
Generation of a tree micropattern in a film of peptide 1
The photoreactive collagenꢀlike peptide 1 (1 mg) was dissolved in 3 ꢀL of water and placed on a
microscope slide. The solution was left to dry at room temperature in the dark for 12 h affording
a thin film of 1. The sample was covered with a coverslip and immediately used for the
photolysis experiment. The tree mask was placed between the objective lens and the polygonal
galvanometer to ensure the tree image was projected on the sample at the focal plane. The
sample was irradiated under the twoꢀphoton microscope using 710 nm light with excitation laser
output power of 200 mW. The delivered laser power at the sample location is 10% of this value,
i.e., 20 mW. Upon excitation, the photoreactive peptide 1 emits a weak fluorescence, which
decreases over time. Fluorescence images were recorded at 0, 10 and 30 min using a
combination of red and green photomultiplier tubes. After removal of the mask another
fluorescence image as well as an image using a white light microscope were recorded.
19

Organic & Biomolecular Chemistry
Page 20 of 25
View Article Online
DOI: 10.1039/C7OB02198D
Laser setꢀup
The details of our inꢀhoused developed videoꢀrate twoꢀphoton microscope was described in
Reference.51 The light source is a modeꢀlocked Ti:Sapphire laser (Maitai HP, 690ꢀ1040 nm, 100
fs, 80 MHz, Newport, Santa Clara, CA). We have used 710 nm light to achieve twoꢀphoton
excitation of Nꢀacylꢀnitroindoline moieties.
The homeꢀbuilt xꢀy scanner (polygon,
galvanometer) has a 30 frames/s scanning rate. The laser power at the sample site is varied by
rotating a halfꢀwave plate in front of a polarizer. The fluorescence signal from the sample are
detected in three spectral channels with photomultiplier tubes (PMTs, R3896, Hamamatsu,
USA): red (570ꢀ616 nm, FF01ꢀ593/46, Semrock, USA), green (500ꢀ550 nm, FF03ꢀ525/50,
Semrock, USA), and blue (417ꢀ477 nm, FF02ꢀ447/60, Semrock, USA). The outputs of these
three PMTs are fed into red/green/blue channels of a frame grabber (Solios eA/XA, Matrox,
Quebec, Canada). Twoꢀdimensional images in the xꢀy plane are acquired through a homeꢀbuilt
software program. Each frame has 500×500 pixels. Each final static image is an average of 30
frames. In the UTEP logo written experiment a photomask was placed at the intermediate image
plane in the optical path, and the logo pattern was projected onto the objective lens focal plan to
partially block the illumination light for pattern formation.
Twoꢀphoton excitation and photolysis of NꢀFmocꢀglycylꢀ5ꢀbromoꢀ7ꢀnitroindoline (13)
The photoreactive amino acid 13 (2 mg) was dissolved in 2 ꢀL DMF to give a 1.92 M solution
which was placed on a microscope slide. The solution was left to dry at room temperature in the
dark for 30 min affording an approximately 70 m thick film of compound 13. The sample was
covered with a coverslip and immediately used for the photolysis experiment. Several spots
within the sample were chosen for irradiation with the femtosecond laser at 710 nm, and each
spot was irradiated with a specific excitation laser power (100 mW, 125 mW, 150 mW, 175 mW,
or 200 mW). Upon excitation, the photoreactive amino acid 9 emits a weak fluorescence, which
was collected using a combination of red and green photomultiplier tubes. The excitation
triggers the photolysis of the glycine’s amide bond, producing nonꢀfluorescent Fmocꢀglycine and
5ꢀbromoꢀ7ꢀnitroindoline (14). As the photolysis progresses, the number of fluorescent molecules
decreases, and consequentially a decrease in average fluorescence intensity at the irradiation site
is measured. An image was taken every minute at each location and the fluorescence profile was
tracked throughout the reaction. The location of each irradiated spot was recorded before a new
spot was irradiated with a new laser excitation power.
Culture and passaging of human Mesenchymal Stem Cells (HMSC)
Human adipose derived mesenchymal stem cells were obtained from Lonza at passage 3 (Lonza,
Allendale, NJ, USA) and cultured according to the manufacturer’s recommendations.
Certification was obtained and kept on file to ensure that the purchased HMSC were verified to
be of the correct lineage and uncontaminated by other cell types or organisms. For the HMSC
culture, a complete growth medium, specifically MSCGM HMSC growth BulletKit™ medium
(Lonza), was used for maintaining the HMSC in an undifferentiated state. Prior to cell seeding,
Tꢀ75 culture flasks were coated with 0.1% gelatin (Sigma Aldrich, St. Louis, MO, USA) and
incubated (37°C, 1 h). After this, the cell suspension in medium was transferred to a gelatin
coated Tꢀ75 flask and incubated for 1 h (37°C, 5% CO2 and 95% RH). Prior to cell culture, the
gelatin solution used for coating of the flasks was aspirated. After 70% confluency in culture
was attained, cells were trypsizined and passaged for further experiments. The normal
20

Page 21 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
morphology and phenotype of the cultured cells were confirmed by comparison with published
images of HMSC Cells preꢀstained with PKH26,52 a red fluorescent membrane staining dye
(Sigma) following the manufacturer’s protocols.
Human Mesenchymal Stem Cell (HMSC) growth on peptide 1
For testing the cytocompatibility of peptide 1, ~1mg was dissolved in 100 µL of distilled water
and coated atop 24 wells of a tissue culture treated polystyrene dish and dried under a sterile
laminar flow hood for 10ꢀ15 min prior to culture. HMSC were seeded atop this layer and
cultured for 24 hours after which they were imaged using fluorescence microscopy (Zeiss
Axiovision).
Mouse embryonic fibroblasts
Mouse embryonic fibroblasts, Sandos inbred mouse (SIM)ꢀderived 6ꢀthioguanineꢀ and ouabainꢀ
resistant (STO) and growth inactivated by treatment with mitomycinꢀC (MITCꢀSTO, Passage 6),
were obtained from ATCC (https://www.atcc.org/; ATCC 56ꢀX) and were employed to assess
the cytocompatibility of peptide 1, the photolysis products of peptide 1, and collagen. These
53, 54
cells are feeder cells to support the growth of other cells such a stem cells
They have been
treated with mitomycinꢀC and will not replicate, and thus serve as an ideal resource for
cytotoxicity testing.48
Live/Dead cytotoxicity assay of mouse embryonic fibroblasts on peptide 1, photolyzed peptide 1
and collagen
The peptide surfaces were prepared by dissolving 750 ꢀg of peptide 1 in 5 ꢀL of
water/dimethylfomamide (1:1) and pipetting 5×1 ꢀL of this solution, 1 ꢀL at a time on top of
each other, letting the solvent evaporate each time, in a well of a 96 well plate. For the irradiated
peptide surface, a second well was prepared in the same manner, and the dried peptide film was
then exposed to 365 nm of a 4W UV lamp for 3 h. A color change from pale yellow to orange
was clearly visible indicating the photodecomposition of peptide 1 into its photolysis products.
Collagen coated wells were prepared using a Rat Tail Collagen I solution (in 0.02 N acetic acid,
concentration range 3 to 4mg/mL, SigmaꢀAldrich). The collagen solution (1 mL) was diluted to
a working concentration of 0.01% using sterile, tissue culture grade d.d. water. Next, the wells
were coated with 100 µL of this diluted solution and the protein was allowed to bind overnight at
2–8°C. Next, the excess fluid was removed from the coated surface and allowed to dry
overnight. Prior to adding cells, the collagenꢀcoated wells were rinsed with 1X PBS (3 times).
About 15,000 cells were seeded per well (96 well plate, NalgeneꢀNunc, ThermoꢀFisher
Scientific) along with 100 µL of the complete growth medium on various substrates, including a)
peptide 1, b) the irradiated peptide 1; and c) collagen. For cell culture, the cells were cultured in
a humidified incubator (37ꢁ°C, 5% CO2) using Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin, all from
SigmaꢀAldrich. The 96ꢀwell plate was then placed back into the incubator to allow the cells to
acclimate with the sample and kept undisturbed for 36 h. After this, the spent media was
changed and the wells supplemented with fresh DMEM. At this point the wells were imaged to
confirm cell adhesion onto the wells. Once cell adhesion was confirmed, the DMEM media was
removed and the wells incubated with 100ꢁµL of the preꢀwarmed cytotoxicity reagent (Marker
Gene Technologies Inc. Live:Dead Cytotoxicity Assay Kit Green/Red Staining).47 The cells
were then placed back into the incubator for 45ꢁmin. After incubation, the cells in wells were
21

Organic & Biomolecular Chemistry
Page 22 of 25
View Article Online
DOI: 10.1039/C7OB02198D
washed using 1X PBS and imaged using light and confocal microscopy (ZEISS LSM 700
Confocal, Germany).
Conclusions
A photoreactive 30mer collagenꢀlike peptide (1) with four photoreactive Nꢀpeptidylꢀ7ꢀ
nitroindoline moieties was synthesized, and its photochemical and photophysical properties were
studied. The temperature dependent circular dichroism spectra of this peptide provide the
evidence of its folding into a triple helix similar to other collagenꢀmimicking peptides reported in
the literature, and show an unusual structural stability, possibly due to the four units of the
unnatural amino acid 5ꢀcarboxylic acidꢀ7ꢀnitroindoline in the peptide sequence. When an
aqueous solution of peptide 1 is irradiated at 350 nm, photolysis occurs at all photoreactive
amide bonds producing mainly the expected Nꢀpeptidylꢀ7ꢀnitrosoindoles. When a thin film of
peptide 1 is irradiated with a femtosecond laser at 710 nm through a patterned mask,
micropatterns consisting of the photolysis products of peptide 1 are generated. The ability of Nꢀ
glycylꢀ5ꢀbromoꢀ7ꢀnitroindoline to also undergo photolysis by a twoꢀphoton absorption
mechanism using a femtosecond laser at 710 nm and its suitability for generating wellꢀresolved
micropatterns was established. Since Nꢀglycylꢀ5ꢀbromoꢀ7ꢀnitroindoline is easily accessible from
commercially available starting materials, in some aspects it can be used as a model compound
for more complex Nꢀacylꢀ7ꢀnitroindolines such as peptide 1. Qualitative cytocompatibility
studies suggest that peptide 1 is not toxic to cells, as human mesenchymal stem cells were able to
grow on a surface coated with that peptide, and mouse embryonic fibroblasts remained viable.
In contrast, a surface coated with the photolysis products of peptide 1 exhibited a certain
cytotoxicity to the fibroblasts. These data suggest that the photoreactive collagenꢀlike peptide 1
has potential as a new biomimetic material that could be used for 2D patterned cell growth based
on patterns generated by laser photolysis because of different cytotoxicity properties of peptide 1
when compared to its photolysis products.
Conflict of Interests
There are no conflicts of interest to declare.
Acknowledgements
This work was supported by the National Science Foundation, grant# DMRꢀ1205302. Circular
Dichroism data presented in this article was obtained with an instrument purchased with support
from the National Institute of General Medical Sciences of the National Institutes of Health
under linked award numbers RL5GM118969, TL4GM118971, and UL1GM118970. Optical
experiments were supported by the National Institute of General Medical Sciences under grant#
1SC2GM103719, and by National Science Foundation under grant# DBIꢀ1429708. This work
was also supported by an interdisciplinary research grant of the University of Texas at El Paso
(UTEP), Office of Research and Sponsored Projects. We thank Dr. Armando Varela for the cell
images and acknowledge access to the LCꢀMS of the Biomolecule Analysis Core Facility at
UTEP’s Border Biomedical Research Center supported by the National Institute on Minority
Health and Health Disparities, grant# 2G12MD007592.
22

Page 23 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
Electronic Supplementary Information
13
Mass spectra of compounds 1, 6, 7, 13, and 16; 1H and C NMR spectra of compounds 6, 13,
and 16, and FPLC of compounds 1 and 7; LCꢀMS analysis of the photolysis products of
compound 1 and MS/MS data of selected components.
References
1.
2.
3.
C. Frantz, K. M. Sewart and V. M. Weaver, J. Cell Sci., 2010, 123, 4195ꢀ4200.
D. Han and P. I. Gouma, Nanomedicine, 2006, 2, 37ꢀ41.
F. Berthiaume, P. V. Moghe, M. Toner and M. L. Yarmush, FASEB J. , 1996, 10, 1471ꢀ
1484.
4.
5.
M. Achilli and D. Mantovani, Polymers, 2010, 2, 664ꢀ680.
J. Groll, T. Boland, T. Blunk, J. A. Burdick, D. W. Cho, P. D. Dalton, B. Derby, G.
Forgacs, Q. Li, V. A. Mironov, L. Moroni, M. Nakamura, W. Shu, S. Takeuchi, G.
Vozzi, T. B. Woodfield, T. Xu, J. J. Yoo and J. Malda, Biofabrication, 2016, 8, 013001.
B. R. Ringeisen, R. K. Pirlo, P. K. Wu, T. Boland, W. Sun, Q. Hamid, Y. Huang and D.
B. Chrisey, MRS Bulletin, 2013, 38, 834.
6.
7.
8.
9.
L. Ouyang, R. Yao, Y. Zhao and W. Sun, Biofabrication, 2016, 8, 035020.
T. Xu, J. Jin, C. Gregory, J. J. Hickman and T. Boland, Biomaterials, 2005, 26, 93.
A. Abeyewickreme, A. Kwok, J. R. McEwan and S. N. Jayasinghe, Integr Biol (Camb),
2009, 1, 260ꢀ266.
10.
N. R. Schiele, D. T. Corr, Y. Huang, N. A. Raof, Y. Xie and D. B. Chrisey,
Biofabrication, 2010, 2, 032001.
11.
12.
13.
B. Dhariwala, E. Hunt and T. Boland, Tissue Eng, 2004, 10, 1316ꢀ1322.
M. Fritz and M. Bastmeyer, Methods Mol Biol, 2013, 1018, 247ꢀ259.
T. Xu, C. A. Gregory, P. Molnar, X. Cui, S. Jalota, S. B. Bhaduri and T. Boland,
Biomaterials, 2006, 27, 3580.
14.
K. A. Hatch, A. Ornelas, K. N. Williams, T. Boland, K. Michael and C. Li, Biomed. Opt.
Express, 2016, 7, 4654ꢀ4659.
15.
16.
17.
18.
19.
M. S. Agren, ed., Functional Biomaterials, Elsevier Ltd., Amsterdam, 2016.
S. M. Yu, Y. Li and D. Kim, Soft Matter, 2011, 7, 7927ꢀ7938.
Y. Li and S. Yu, Curr. Opin. Chem. Biol., 2013, 17, 968ꢀ975.
V. HernandezꢀGordillo and J. Chmielewski, Biomaterials, 2014, 35, 7363ꢀ7373.
J. Xiao, Biophysical Characterization of Collagen Mimic Peptides, Springer, Singapore,
1 edn., 2017.
20.
21.
22.
T. J. Hogenauer, Q. Wang, A. K. Sanki, A. J. Gammon, C. H. Chu, C. M. Kaneshiro, Y.
Kajihara and K. Michael, Org. Biomol. Chem., 2007, 5, 759ꢀ762.
A. Pardo, T. J. Hogenauer, Z. Cai, J. A. Vellucci, E. M. Castillo, C. W. Dirk, A. H. Franz
and K. Michael, ChemBioChem, 2015, 16, 1884ꢀ1889.
W. C. Chan and P. D. White, eds., Fmoc solid phase peptide synthesis: a practical
approach, Oxford University Press, Oxford, 2000.
23.
24.
B. Amit, D. A. BenꢀEfraim and A. Patchornik, J. Am. Chem. Soc., 1976, 98, 843ꢀ844.
J. Morrison, P. Wan, J. E. T. Corrie and G. Papageorgiou, Photochem. Photobiol. Sci.,
2002, 1, 960ꢀ969.
23

Organic & Biomolecular Chemistry
Page 24 of 25
View Article Online
DOI: 10.1039/C7OB02198D
25.
26.
A. D. Cohen, C. Helgen, C. G. Bochet and J. P. Toscano, Org. Lett., 2005, 7, 2845ꢀ2848.
J. E. Mendez, N. J. Westfall, K. Michael and C. W. Dirk, Trends Photochem. Photobiol.,
2012, 14, 75ꢀ91.
27.
28.
G. Papageorgiou, D. Ogden, G. Kelly and J. E. T. Corrie, Photochem. Photobiol. Sci.,
2005, 4, 887ꢀ896.
G. Papageorgiou, D. C. Ogden, A. Barth and E. E. T. Corrie, J. Am. Chem. Soc., 1999,
121, 6503ꢀ6504.
29.
30.
S. Pass, B. Amit and A. Patchornik, J. Am. Chem. Soc., 1981, 103, 7674ꢀ7675.
K. Vizvardi, C. Kreutz, A. S. Davis, V. P. Lee, B. J. Philmus, O. Simo and K. Michael,
Chem. Lett., 2003, 32, 348ꢀ349.
31.
O. Simo, V. P. Lee, A. S. Davis, C. Kreutz, P. H. Gross, P. R. Jones and K. Michael,
Carbohydr. Res., 2005, 340, 557ꢀ566.
32.
33.
C. M. Kaneshiro and K. Michael, Angew. Chem. Int. Ed., 2006, 45, 1077ꢀ1081.
A. Hassner, D. Yagudayev, T. K. Pradhan, A. Nedelman and B. Amit, Synlett, 2007,
2405ꢀ2509.
34.
35.
C. Helgen and C. G. Bochet, J. Org. Chem., 2003, 68, 2483ꢀ2486.
J.ꢀL. Débieux, A. Cosandey, C. Helgen and C. G. Bochet, Eur. J. Org. Chem., 2007,
2073–2077.
36.
37.
K. C. Nicolaou, B. S. Safina and N. Winssinger, Synlett, 2001, SI, 900ꢀ903.
P. H. Chapman and D. Walker, J. Chem. Soc. Chem. Commun., 1975, DOI:
C39750000690, 690ꢀ691.
38.
39.
40.
A. V. Persikov, Y. Xu and B. Brodsky, Protein Sci., 2004, 13, 893ꢀ902.
T. Koide, Philos. Trans. R. Soc. Lond. B Biol. Sci., 2007, 362, 1281ꢀ1291.
S. Frank, R. A. Kammerer, D. Mechling, T. Schulthess, R. Landwehr, J. Bann, Y. Guo,
A. Lustig, H. P. Bachinger and J. Engel, J. Mol. Biol., 2001, 308, 1081ꢀ1089.
E. Leikina, M. V. Mertts, N. Kuznetsova and S. Leikin, Proc. Natl. Acad. Sci. U.S.A.,
2002, 99, 1314ꢀ1318.
41.
42.
43.
44.
45.
F. W. Kotch and R. T. Raines, Proc. Natl. Acad. Sci. USA, 2006, 103, 3028ꢀ3033.
R. Gopal, J. S. Park, C. H. Seo and Y. Park, Int. J. Mol. Sci., 2012, 13, 3229ꢀ3244.
B. Joddar, A. T. Guy, H. Kamiguchi and Y. Ito, Biomaterials, 2013, 34, 9593ꢀ9601.
M. Matsuzaki, G. C. R. EllisꢀDavies, T. Nemoto, Y. Miyashita, M. Iino and H. Kasai,
Nature Neurosci., 2001, 4, 1086ꢀ1092.
46.
47.
G. Papageorgiou and J. E. T. Corrie, Tetrahedron, 2000, 56, 8197ꢀ8205.
B. J. Kroesen, G. Mesander, J. G. t. Haar, T. H. The and L. d. Leij, J. Immunol. Methods,
1992, 156, 47ꢀ54.
48.
T. Murakami, I. Saitoh, E. Inada, M. Kurosawa, Y. Iwase, H. Noguchi, Y. Terao, Y.
Yamasaki, H. Hayasaki and M. Sato, Cell Med., 2013, 6, 75ꢀ81.
T. Christensen, Acta Chem. Scand. B 1979, 33, 763ꢀ766.
V. Krchňák, J. Vágner, P. Šafár and M. Lebl, Collect. Czech. Chem. Commun., 1988, 53,
2542ꢀ2548.
49.
50.
51.
52.
53.
Y. Acosta, Q. Zhang, A. Rahaman, H. Ouellet, C. Xiao, J. Sun and C. Li, Biomed. Opt.
Express, 2014, 5, 3990ꢀ4001.
B. Joddar, S. A. Kumar and A. Kumar, Cell Biochem. Biophys., 2017, DOI:
10.1007/s12013ꢀ017ꢀ0828ꢀz. [Epub ahead of print].
Y. Zhou, H. Mao, B. Joddar, N. Umeki, Y. Sako, K.ꢀI. Wada, C. Nishioka, E. Takahashi,
Y. Wang and Y. Ito, Sci. Rep., 2015, 5, 11386.
24

Page 25 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB02198D
54.
B. Joddar, C. Nishioka, E. Takahashi and Y. Ito, J. Mat. Chem. B, 2015, 3, 2301ꢀ2307.
25