In situ generation of redox active peptides driven by selenoester mediated native chemical ligation

Article abstract of DOI:10.1039/c4cc03835e

Redox active peptides are generated through selenoester mediated native chemical ligation (NCL). Distinct nanostructures, such as nanotubes to nanofibrillar architectures, were observed for self-assembling soft materials. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03835e

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In situ generation of redox active peptides driven
by selenoester mediated native chemical ligation†
Cite this: Chem. Commun., 2014,
50, 11397
Dnyaneshwar B. Rasale, Indrajit Maity and Apurba K. Das*
Received 20th May 2014,
Accepted 29th July 2014
DOI: 10.1039/c4cc03835e
www.rsc.org/chemcomm
Redox active peptides are generated through selenoester mediated stiffening of a hydrogel via thioester mediated NCL reaction.10
native chemical ligation (NCL). Distinct nanostructures, such as nano- Messersmith and coworkers described a strategy that forms
tubes to nanofibrillar architectures, were observed for self-assembling covalently cross-linked polymer hydrogels.11 In general, the
soft materials.
conceptual approach for NCL is based on the reaction between
two unprotected peptides, one bearing C-terminal thioester and
In nature, complex soft materials are created through hierarchical another N-terminal cysteine residue based peptide. The sulfhydryl
self-assembly of nanoscale biomolecules.1 Low molecular weight group of N-terminal cysteine residue undergoes trans-thioesterifi-
organic molecules are used in directed self-assembly, which further cation with C-terminal thioester12 and forms a thioester-linked
forms three-dimensional network structures. Various functional intermediate. A thioester-linked intermediate simultaneously
groups can be incorporated into the molecular structure2 to mimic and rapidly undergoes intramolecular S - N acyl transfer to
and develop biological processes in the laboratory. However, rational form a native amide bond.13
and universal molecular design remained a challenge to make them
Inspired by this chemoselective ligation reaction,14 our
suitable for biomaterial applications. Self-assembly of short peptides objective was to develop a simple and efficient approach that
has received significant attention because of their vast applica- can direct dynamic peptide self-assembly. To achieve this goal,
tions in drug delivery,3 tissue engineering4 and supramolecular we have synthesized compounds 1–4 with an N-terminal capped
electronics.5 Several physical stimuli,6 such as pH, temperature, with an aromatic naphthalene-2-methoxycarbonyl (Nmoc) group.
light and enzymes, are used to explore peptide self-assembly.7 The C-terminals of 1–4 are protected with phenyl selenoester, which
Redox active peptide self-assembly still remains an area of can readily undergo NCL reaction at room temperature with
interest because of its potential application in drug delivery. Redox N-terminal cysteine and N-terminal cysteine based peptide Cys-Gly.
reactions are prevalent in nature to regulate various biological Selenoesters are efficient acyl donors and are easier to synthesise
functions. Redox active8 nature mimicking dynamic self-assembly than the corresponding thioesters because of the better leaving
could be achieved using chemoselective native chemical ligation group characteristics of a selenolate versus a thiolate.15
(NCL) reactions. Peptide self-assembly could be explored through
L-Cysteinylglycine, a pro-oxidant, is generated from extra-
native chemical ligation reaction because (i) this method is cellular glutathione through the catalytic activity of an enzyme
orthogonal, (ii) self-assembly through NCL reaction is particu- g-glutamyltransferase.16 Cys-Gly is known to be a highly reactive
larly interesting for the development of controlled dynamic self- metabolite that is directly related to induce the oxidative stress17 in
assembly and (iii) the presence of free sulfhydryl groups in cells. Lin et al. reported that higher cysteine glycine levels were
peptides that can offer multiple applications after NCL reactions. marginally associated with an increased risk of breast cancer in
NCL reactions are the most revolutionary method for the women.18 Here, we have used Cys-Gly dipeptide in the development
complete or semi synthesis of proteins.9 In addition to its useful of dynamic peptide self-assembly through the NCL reaction. The
application in protein synthesis, NCL reactions can be used as selenoester easily undergoes NCL reaction with Cys-Gly dipeptide,
effective and efficient methods for the development of soft which could be an assay in further clinical research.
biomaterials. Collier et al. reported an excellent method for the
The NCL reaction proceeds through the thioester-linked
intermediate where acyl transfers from Se - S15 are followed
by intramolecular S - N acyl transfer to give a peptide bond
(Fig. 1). Compounds 1–4 (20 mmol LÀ1) were dissolved in 100 mL of
ethanol and Cys or Cys-Gly (20 mmol LÀ1) was dissolved in 900 mL
of phosphate buffer (pH = 8, 100 mmol LÀ1). 900 mL of Cys (C) or
Department of Chemistry, Indian Institute of Technology Indore, Indore 452017,
India. E-mail: apurba.das@iiti.ac.in
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc03835e
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Fig. 1 Selenoester mediated native chemical ligation. Ligated products
were formed upon the NCL of selenoesters 1–4 with Cys-Gly and cysteine
at pH B 8. NmYCG self-assembled in its reduced form while oxidized
NmFC(SePh)G (sulfur linked with selenophenol) and (NmFC)2 self-assembled
to form self-supporting soft materials.
Fig. 2 (A) Dynamic self-assembly of (NmFC)2. NCL reaction of compound
NmFSePh 2 with cysteine gives NmFC under inert atmosphere and sub-
sequently formed dynamic (NmFC)2 gel upon exposure to air. (B) Visual
changes of reaction progress initially mixing of compound NmYSePh 1
(20 mmol LÀ1) with Cys-Gly (20 mmol LÀ1) in phosphate buffer at pH 8.
(i) Milky white solution at 5 min, (ii) turbid at 10 min, (iii) colorless solution at
15 min, (iv) colorless viscous solution at 30 min and (v) self-supporting gel
formed after 60 min. (C) HPLC trace analysis of a representative ligation of
compound 1 with Cys-Gly with corresponding ESI-MS of ligated product after
1 h and 2 with Cys-Gly and cysteine after 1 h with corresponding ESI-MS of
ligated products.
Cys-Gly (CG) was added to the reaction vial containing com-
pounds 1 to 4 (entries 1–8). The reaction vial was left undis-
turbed, and self-supporting gels were observed after 1 h of
reaction for entries 1, 2 and 6. The ligation reactions of Nmoc
(Nm) capped amino acid based selenoesters with Cys or Cys-Gly
was monitored with reverse phase high performance liquid
chromatography (HPLC), and the corresponding products were
analyzed by ESI-MS (Fig. 2C and Fig. S4–S30, ESI†). We also
monitored the visual changes of solution 1 upon mixing with
dipeptide Cys-Gly in phosphate buffer (Fig. 2B). The gels were
stable in the pH range of 4–10, and the minimum gelation
concentration for NmYCG, NmFC(SePh)G and (NmFC)2 gels
was 5 mmol LÀ1. Gel melting temperature (Tgel) was also measured
by the test tube inversion method at different concentrations of
gelators. Tgel was observed as 70 1C, 71 1C and 65 1C for NmYCG,
NmFC(SePh)G and (NmFC)2 gels, respectively, at 20 mmol LÀ1
(Table S1, ESI†) concentration.
Table 1 Native chemical ligation under inert as well as air conditions
CGa
Entry Substrate and Cb inert atm.c [%]
Conversion in
Conversion
in airc [%]
Geld
G
1
2
1
2
CG
CG
499 NmYCG
499 NmYCG
22.64/76e NmFCG/ 77.36 NmFC(SePh)G G
NmFC(SePh)G
3
4
5
6
7
8
3
4
1
2
3
4
CG
CG
C
C
C
499 NmLCG
65.79 NmVCG
499 NmYC
94.31 NmFC
499 NmLC
499 NmVC
499 (NmLCG)2
63.35 (NmVCG)2
95.09 (NmYC)2
84.90 (NmFC)2
499 (NmLC)2
79.28 (NmVC)2
S
S
S
G
S
S
The self-assembly of small peptides bearing N-terminal
naphthalene moieties has been reported by several groups.19
Compound 1 gives 499% ligated peptide with Cys-Gly and
gives 499% with cysteine (Table 1). Thus, NCL reaction with
selenoesters is the easiest and simplest way to synthesise peptides.
However, the thioesters with similar amino acid sequences (com-
pounds 5–8) yielded poor conversion (Fig. S2 and S3, ESI†) of peptides
(Table S2, ESI†) and were unable to form gel under similar conditions.
The control experiment with Nmoc capped valine selenoester 3 with
alanine confirms the requirement of a C-terminal cysteine group in
ligation reaction with selenoester (ESI†).
C
a
b
c
CG = Cys-Gly dipeptide. C = cysteine. The percentage conversions
d
were calculated by integrating the peak areas from HPLC. G = gel, S =
solution, Nm = Nmoc. Entry 2 yield in inert atm.: the combined yield
for NmFCG and NmFC(SePh)G is 98.64% (22.64% + 76%).
e
Typically, NCL reactions were carried out in the presence of However, entries 3–8 showed exactly reverse behavior to entry 1.
a reductant such as tris(2-carboxyethyl)phosphine (TCEP) and Oxidized forms of NCL products were observed for entries 3–8
dithiothreitol (DTT). TCEP or DTT helps to avoid the formation of in similar conditions to the NCL reaction of entry 1 (Table 1).
disulfides. Surprisingly, we have not used any reductant for the reaction A self-supporting gel for entry 1 was observed with 99% synthesized
of compound 1 with Cys-Gly (entry 1). The reduced form of NmYCG peptide NmYCG. Another self-supporting gel for entry 6 was also
was observed after 16 h in aerobic conditions (Fig. S4, ESI†).20 observed for the newly synthesized 84.9% oxidized peptide (NmFC)2.
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However, in the case of entry 2, a dipeptide Cys-Gly reacted with double rings and aromatic side chains appears in the range of
compound 2 and formed the corresponding tripeptide, NmFCG. 310–330 nm in solution (Fig. S40, ESI†). NmYCG gel showed an
NmFCG further reacted with cleaved selenophenol and formed a emission peak at 337 nm and a broad emission peak at 461 nm,
sulfur–selenium bond (Fig. 1). The resulting product NmFC(SePh)G indicating efficient overlapping of naphthalene double rings
turned into a self-supporting gel. Our data indicate that reduced and higher order p–p stacking aggregation in gel phase medium
and oxidized forms of ligated peptides can self-assemble and lead (Fig. S41, ESI†). Similarly, NmFC(SePh)G and (NmFC)2 gels showed
to self-supporting gels.21 The self-assembly of ligated products emission peaks at 337 nm and 334 nm, respectively. The broad
drives self-selection and self-organization into the reduced and emission peak at 465 nm suggests a higher order aggregate state for
oxidized form of peptides.
NmFC(SePh)G gel (Fig. S41, ESI†). Here, the aromatic naphthalene
The disassembly phenomenon of (NmFC)2 was observed ring plays a pivotal role in the self-assembly process via p–p
using a reductant TCEP (40 mmol LÀ1) after 2 h, which further stacking interactions.
explores gelators as versatile candidates for drug delivery (Fig. 2A).
Wide angle powder X-ray diffraction studies were performed
The dynamic gel–sol reversal behaviour of (NmFC)2 was monitored to examine the self-assembly of NmYCG, NmFC(SePh)G and
by HPLC and rheology measurements (Fig. 3A and Fig. S37, ESI†). (NmFC)2 in gel phase (Fig. S42–S44, ESI†). The diffraction peak
The HPLC analysis of (NmFC)2 after the addition of TCEP showed appeared at 2y = 18.131, 19.531, and 17.991 with corresponding
an 87% conversion of NmFC after 6 h of reaction. The rheology d spacing of 4.88 Å, 4.5 Å and 4.9 Å for NmYCG, NmFC(SePh)G,
experiment showed that the gel starts to break after 1 h of TCEP and (NmFC)2 dried gels, respectively. These values indicate the
addition. Gel formation for (NmFC)2 upon NCL reaction was spacing between two peptides within the b-sheet structures.
indicated by time sweep experiment (Fig. S36, ESI†). The disulfide The reflection peak appeared at 2y = 23.141, 24.461, and 24.161
reduction leads to the gel–sol transition of (NmFC)2, which was also with the corresponding d spacing values of 3.84 Å, 3.64 Å, and
monitored with an oscillatory time sweep experiment after the 3.68 Å for dried gels of NmYCG, NmFC(SePh)G and (NmFC)2,
addition of TCEP (Fig. S37, ESI†).
respectively.24 These values clearly show the p–p stacking inter-
Circular dichroism (CD) was used to elucidate peptide action among the self-assembled peptides. The self-assembly
conformation in a gel phase medium (Fig. 3B). The CD spectrum mechanism is shown in Fig. S31 (ESI†).
of NmYCG (entry 1) shows a positive peak at 208 nm and a negative
A time resolved fluorescence study was employed to investigate
peak at 221 nm, corresponding to the coexistence of a random-coil the higher order aggregation of the fluorophore groups (Nmoc) of
and b-sheet conformation.22 However, CD spectra of NmFC(SePh)G NmYCG, NmFC(SePh)G and (NmFC)2 gels. We measured fluores-
and (NmFC)2 in gel state show characteristics of a twisted conforma- cence decay traces of the gels at an excitation of 376 nm, and the
tion. The two negative peaks at 222 nm and 205 nm indicate the emission was monitored at 470 nm. The average lifetime of 1.14 ns
twisted conformation of self-assembled peptides, which may be for NmYCG, 3.39 ns for NmFC(SePh)G and 1.06 ns for (NmFC)2
attributed to the supramolecular ordering of peptide molecules.23 were observed (Table S4 and Fig. S45, ESI†). The average fluores-
The CD spectrum of gel (NmFC)2 upon treatment with TCEP cence lifetime of the gel samples indicates a denser aggregated
indicates the change in the conformation of the reduced nanofibrous network in the gel state.
disassembled peptide (Fig. S38, ESI†). Fourier transform infra-
Transmission electron microscopy (TEM) was used to char-
red spectroscopy (FTIR) was also used to support the secondary acterize the morphology of the self-assembled architectures in
structures of self-assembled peptides (Table S3, ESI†). The ligated gel phase. The nanotubular25 structures were observed for a gel
peptide NmYCG shows peaks at 1640 cmÀ1 and 1688 cmÀ1, which formed by NmYCG (entry 1) with an average diameter of
correspond to the turn type structures.
150 nm. The average inner diameter of nanotubes was observed
Fluorescence spectra were recorded to determine the mole- to be 20 nm in the TEM image (Fig. 4B). However, gels formed
cular arrangement of NmYCG, NmFC(SePh)G and (NmFC)2 in by NmFC(SePh)G (entry 2) and (NmFC)2 (entry 6) showed
gel phase. The characteristic emission peak for naphthalene entangled nanofibrillar networks with diameters ranging from
10 to 60 nm (Fig. S47, ESI†). The atomic force microscopy (AFM)
studies of the gels also showed nanofibrillar morphology (Fig. 4).
The NmYCG gel exhibited highly aligned nanostructures in gel
phase medium with a height of 3 to 6 nm (Fig. 4A). However, the
gel formed by NmFC(SePh)G showed elongated thin nanofibrous
morphology with an average width of 2 nm. The AFM analysis of
(NmFC)2 indicated that nanofibers originated from thick fibers.
The AFM result for gel–sol transition indicates the breaking of
nanofibers, which leads to the disassembly of the self-assembled
gel (NmFC)2 (Fig. S46, ESI†).
In summary, we have demonstrated selenoester mediated
native chemical ligation to explore dynamic redox active peptide
self-assembly. Selenoester mediated native chemical ligation is a
rapid ligation process over thioester mediated native chemical
ligation. Cysteine and Cys-Gly peptides have provided mechanistic
Fig. 3 (A) Real time HPLC analysis for (NmFC)2 formation upon exposure
to air followed by the addition of TCEP at 90% showed conversion back to
the reduced form of NmFC. (B) CD spectra of self-assembled peptides
(i) NmYCG, (ii) NmFC(SePh)G and (iii) (NmFC)2 (concentration = 2 mmol LÀ1
)
formed upon NCL reaction at pH B 8.
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insight into the NCL driven self-assembly process. We have also
shown the redox active dynamic peptide gels, which are formed
via the oxidation and reduction of the Nmoc-protected peptide
synthesized via NCL reactions. Here, gel–sol transition is highly
dependent on the oxidation and reduction of cysteine and
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AKD sincerely acknowledges CSIR, New Delhi, India, for
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