Efficient, traceless semi-synthesis of α-synuclein labeled with a fluoro phore/thioamide FRET pair

Article abstract of DOI:10.1055/s-0033-1339853

We have shown that thioamides can be incorporated into proteins through semi-synthesis and used as probes to monitor structural changes. To date, our methods have required the presence of a cysteine at the peptide ligation site, which may not be present in the native peptide sequence. Here, we present a strategy for the semi-synthesis of thioproteins using homocysteine as a ligation point with subsequent masking as methionine, making the ligation 'traceless'. Georg Thieme Verlag Stuttgart, New York.

Full text of DOI:10.1055/s-0033-1339853

2454▌
LETTER
letter
Efficient, Traceless Semi-Synthesis of α-Synuclein Labeled with a Fluoro-
phore/Thioamide FRET Pair
Semi-Synthesis of Labeled α-Synuclein
Rebecca F. Wissner, Anne M. Wagner, John B. Warner, E. James Petersson*
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA
Fax +1(215)5732112; E-mail: ejpetersson@sas.upenn.edu
Received: 01.08.2013; Accepted after revision: 29.08.2013
Abstract: We have shown that thioamides can be incorporated into
proteins through semi-synthesis and used as probes to monitor
structural changes. To date, our methods have required the presence
of a cysteine at the peptide ligation site, which may not be present
in the native peptide sequence. Here, we present a strategy for the
semi-synthesis of thioproteins using homocysteine as a ligation
point with subsequent masking as methionine, making the ligation
‘traceless’.
Key words: proteins, peptides, thiols, chromophores, bioorganic
chemistry
Rebecca F. Wissner (near right) earned a B.S. degree in Chemistry
in 2009 from New York University, working with Prof. Paramjit Aro-
ra. She is a graduate student in Prof. Petersson’s group, studying pro-
tein folding using semi-synthetic proteins.
Fluorescence spectroscopy is a powerful technique for
studying protein dynamics and stability. Distance-depen-
dent chromophore interactions, such as Förster resonance
energy transfer (FRET) and photoinduced electron trans-
fer (PET) are routinely employed for monitoring protein
conformation in real time.1 We have recently shown that
a peptide backbone thioamide, a single-atom substitution
of the carbonyl oxygen with sulfur, can quench a variety
of natural and unnatural amino acids.2 Unlike commonly
used fluorescence quenchers, thioamides are sufficiently
small that they can be placed at nearly any position of the
protein sequence without significantly altering the sec-
ondary structure.3 Our laboratory has developed semi-
synthetic methods to incorporate these minimally perturb-
ing fluorescence probe pairs into full-length proteins.
Backbone thioamides cannot currently be installed into a
protein by means of ribosomal expression. Therefore, we
incorporate thioamides into full-length proteins using na-
tive chemical ligation (NCL), a fragment condensation re-
action that typically takes place between peptides bearing
a C-terminal thioester and an N-terminal cysteine, to form
a native amide bond.4 Expressed protein ligation (EPL), a
variant of NCL, allows us to express a large portion of the
protein in E. coli cells, to which we can append a small
synthetic thioamide-containing fragment with high effi-
ciency.5
Anne M. Wagner (far left) graduated in 2005 with a B.S. in Chemis-
try from Mount Holyoke College, working with Prof. Megan Núñez.
She received her Ph.D. from the University of Pennsylvania in 2013
for her work on protein functionalization.
John B. Warner (far right) received a B.S. in Chemistry from Clark-
son University in 2008, working with Prof. Silvana Andreescu. He is
a graduate student in Prof. Petersson’s group, developing methods for
multi-functionalizing proteins.
E. James Petersson (near left) was educated at Dartmouth College,
where he worked in the labs of David Lemal. He then studied under
Dennis Dougherty at the California Institute of Technology as an NIH
Predoctoral Fellow. After obtaining his Ph.D. in 2005, he worked as
an NIH Postdoctoral Fellow at Yale University with Alanna Schepa-
rtz. He was appointed as Assistant Professor in the Department of
Chemistry at the University of Pennsylvania in 2008 and in the Bio-
chemistry and Molecular Biophysics group in the Perelman School of
Medicine in 2013.
that eventually mature into β-sheet rich fibrils.7 Despite
recent developments of new methods to study αS dynam-
ics, the molecular details underlying its aberrant oligo-
merization remain poorly understood.8 Recently, we
combined unnatural amino acid mutagenesis with native
chemical ligation to install the FRET pair, p-cyanophenyl-
alanine (Cnf) and a thioamide, into full-length αS for mis-
folding studies.9 This FRET system provides reliable
distance measurements over a range of 8–30 Å within a
protein’s three-dimensional structure. Using our double-
labeled constructs, we were able to demonstrate that we
can monitor conformational changes in monomeric αS us-
ing urea or trimethylamine oxide (TMAO) to denature or
compact the protein, respectively.
Misfolding of the abundant neuronal protein α-synuclein
(αS) has been implicated in the pathogenesis of several
debilitating neurodegenerative disorders.6 Under physio-
logical conditions, αS is soluble, monomeric, and intrinsi-
cally disordered. In its pathological conformation, αS
monomers associate with each other to form oligomers
Despite agreement of our data with other studies of αS in
TMAO, we were somewhat concerned about the presence
of the non-native Cys residues in our labeled αS con-
structs.10 Cys mutants of αS display enhanced aggregation
kinetics and altered fibril morphology as a result of inter-
SYNLETT 2013, 24, 2454–2458
Advanced online publication: 20.09.2013
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9
3
6
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5
2
1
4
1
4
3
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2
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DOI: 10.1055/s-0033-1339853; Art ID: ST-2013-R0734-L
© Georg Thieme Verlag Stuttgart · New York

LETTER
Semi-Synthesis of Labeled α-Synuclein
2455
molecular disulfide bond formation.11 Although we per- dure for the preparation of Fmoc-Asp′(Ot-Bu)-nitrobenz-
formed the previous FRET studies with our Cys- otriazole 3 follows a previous report by Shallaby and is
containing double-labeled constructs in the presence of a illustrated in Scheme 2.17 Coupling of 4-nitro-1,2-phenyl-
reducing agent, it is nonetheless possible that some enediamine to Fmoc-Asp(Ot-Bu)-OH was achieved using
amount of disulfide formation occurred during our exper- isobutyl chloroformate (IBCF) to generate 1 in 89%
iments.
yield.20 Amide 1 was then added to a solution of P4S10 and
anhydrous Na2CO3 at 0 ºC. Selective thionation of the Asp
backbone carbonyl was completed within one hour to af-
ford compound 2 in 79% yield.21 Intramolecular diazoni-
um cyclization of thioamide 2 gave the activated
benzotriazole 3 in 67% yield.22 Compound 3 can be isolat-
ed from the final reaction by precipitation with ice-cold
water in sufficient purity for peptide coupling reactions.
Since the activated benzotriazoles are subject to degrada-
tion through hydrolysis and intramolecular nucleophilic
attack by the carbonyl oxygens, it is best to minimize han-
dling of compound 3 prior to peptide coupling.
While many laboratories carry out protein synthesis with
subsequent radical desulfurization of Cys to form Ala, this
strategy is not viable for us as desulfurization of the thio-
amide would also occur.12 Methods developed in our lab-
oratory enable us to generate double-labeled αS for
misfolding studies that does not contain cysteine at the li-
gation site. It has been shown that NCL can be performed
with an N-terminal homocysteine (Hcs) in place of Cys.
Hcs can then be selectively methylated to yield methio-
nine at the ligation site.13 Hcs-mediated ligation was pre-
viously restricted to short C-terminal peptides in which
Hcs could be installed using solid-phase peptide synthesis
(SPPS). We have recently established that we can use E.
coli aminoacyl transferase (AaT) to deliver disulfide-pro-
tected Hcs [S-(thiomethyl)homocysteine, Hcm] to the N-
terminus of a large expressed protein fragment.14 In E. coli
cells, AaT transfers Phe, Leu, or Met from an aminoacyl
tRNA to the N-terminus of a protein.15 By using a modi-
fied methionine aminoacyl tRNA synthetase enzyme
(Met*RS), we can generate Hcm-tRNA in situ, which
serves as a viable substrate for AaT.16 Once Hcm is trans-
ferred to the N-terminus of a protein, it is easily deprotect-
ed by a reducing agent to form Hcs for ligation. By
combining unnatural amino acid mutagenesis with EPL at
homocysteine (which is converted into methionine), we
can incorporate our minimal FRET pair into αS in a trace-
less manner that minimizes unnecessary synthesis of pep-
tide fragments composed of natural amino acids. A
retrosynthetic analysis of our target protein, Ac-
αSFAsp′2Cnf39, is shown in Scheme 1. The prime symbol
is used to denote a backbone thioamide bond in an amino
acid represented by the standard three-letter code, and αSF
represents a construct with all of the native Tyr residues
(39, 125, 133, and 136) mutated to Phe to prevent chromo-
phore cross talk.
NO2
O
H
P4S10
Fmoc
O
N
1) NMM, IBCF
N
H
Fmoc-Asp(Ot-Bu)-OH
Na2CO3
2)
NH2
NH2
1
O
O2N
NH2
NO2
NO2
S
H
S
H
N
NaNO2, H+
Fmoc
O
Fmoc
N
N
N
N
H
NH2
O
N
O
O
2
3
Scheme 2 Synthesis of aspartyl thioamide precursor 320
Although, we have previously used on-resin methods to
prepare thioesters for NCL, we find that off-resin activa-
tion is simplest for short peptides. The N-terminal thio-
amide-containing fragment of αS was synthesized using
standard Fmoc-based SPPS procedures with the exception
of direct acylation of Val3 by benzotriazole 3 (i.e., no ac-
tivating agents were added). Thiopeptide 4 was synthe-
sized on 2-chlorotrityl resin, cleaved under mild
Previously, we have shown that fluorenylmethoxycarbon-
yl (Fmoc)-protected thiocarboxybenzotriazoles can be
used with conventional Fmoc-protected amino acids to
synthesize thioamide-containing peptides.5a,9 The proce-
Scheme 1 Retrosynthetic analysis of Ac-αSFAsp′2Cnf39
© Georg Thieme Verlag Stuttgart · New York
Synlett 2013, 24, 2454–2458

2456
R. F. Wissner et al.
LETTER
conditions (10% AcOH) to retain the Asp side-chain pro-
tecting group, and then activated using benzotriazol-1-
yloxytripyrrolidinophosphonium hexafluorophosphate
(PyBOP) to form the mercaptopropionate thioester 5
(Scheme 3). Epimerization of the α-carbon of the C-termi-
nal phenylalanine residue was eliminated by reducing the
PyBOP activation time to 25 minutes.18 Following acidic
deprotection of Asp′(Ot-Bu)2, the thioester peptide 6 was
isolated by HPLC (5.3 μmol, 5% yield) and its identity
was confirmed by MALDI MS analysis.
O
OR1
O
O
H
H
R2
Ac N
N
N
N
H
H
Figure 1 Ac-αSFAsp′2Cnf39 synthesis. Left: Functionalization of
αSF6–140Cnf39 by (a) cleavage of His11 tag at Factor Xa site; (b) attach-
ment of Hcm by AaT-catalyzed modification; (c) ligation of an N-ter-
minal thioester peptide (6), and; (d) conversion of Hcs to Met by
methylation to form Ac-αSFAsp′2Cnf39 (11). Top right: MALDI MS
analysis of full length 11. Calcd m/z [M + H]+ 14480.1, found:
14479.6. Middle right: Trypsinized fragment corresponding to resi-
dues 1–6 of Ac-αSFAsp′2Cnf39 confirms methylation. Calcd m/z [M +
H]+ 828.34; found: 828.46. The asterisk indicates the expected mass
of the 1–6 fragment with unmethylated Hcs at position 5. Calcd m/z
[M + H]+ 814.39. Bottom right: Trypsinized fragment corresponding
to residues 33–43 of Ac-αSFAsp′2Cnf39 confirms Cnf incorporation.
Calcd m/z [M + H]+ 1189.66; found: 1189.85. The asterisk indicates
the expected mass of the 33–43 fragment with Tyr at position 39.
Calcd m/z [M + H]+ 1180.65.
S
O
PyBOP, DIPEA,
HS(CH2)2CO2Me
S
4: R1 = t-Bu, R2 = OH
5: R1 = t-Bu, R2 = S(CH2)2CO2Me
6: R1 = H, R2 = S(CH2)2CO2Me
TFA
Scheme 3 Synthesis of thioamide-containing thioester peptide 6 for
native chemical ligation
The expressed C-terminal Cnf-containing αS fragment for
NCL was generated as depicted in Figure 1. AaT selec-
tively modifies the α-amine of proteins bearing a lysine or
arginine as the N-terminal residue. Prior to this work, we
demonstrated that Met5Lys6 could be used as a point of
disconnection in the synthesis of αS.14 To minimize per-
turbation of native αS dynamics resulting from Cnf incor-
poration, we chose to replace the similarly sized Tyr or
Phe at position 39. A plasmid containing the C-terminal
protein fragment (αSF6–140) with a TAG codon installed at
position 39 was prepared. An N-terminal His11 tag and a
Factor Xa proteolysis site precede the αS gene. E. coli
cells were transformed with two plasmids, one containing
the Cnf-selective mutant synthetase (CnfRS) with tRNA
and the other containing HisTag-αSF6–140TAG39.19 Follow-
ing an initial growth period, protein expression was in-
duced by adding Cnf and isopropyl β-D-1-
thiogalactopyranoside (IPTG) to the culture medium. The
expressed protein, HisTag-αSF6–140Cnf39 (7), was purified
by Ni-affinity chromatography and subsequently cleaved
by Factor Xa to yield αSF6–140Cnf39 (8). Following purifi-
cation by ion-exchange chromatography, the truncated
protein was incubated with AaT, Met*RS, E. coli total
tRNA, Hcm, and ATP for two hours resulting in complete
formation of αSF5–140Hcm5Cnf39 (9). αSF5–140Hcm5Cnf39
was subjected to an additional round of ion-exchange
chromatography prior to use in the NCL reaction. Typi-
cally, about 1 μmol of pure 9 is obtained from a 1 L pro-
tein expression.
phine, 2% thiophenol, at pH 7.5]. Disulfide deprotection
of the Hcm residue was observed by MALDI MS within
five minutes. The ligation reaction was allowed to pro-
ceed for 24 hours prior to buffer exchange into methyla-
tion reaction buffer (20 mM Tris, pH 8.6). Ac-
αSFAsp′2Hcs5Cnf39 (10) was converted to Ac-
αSFAsp′2Cnf39 (11), with native Met5, by a short treatment
(10 min) with 3000 equivalents of MeI. Allowing the re-
action to proceed for longer periods of time led to S-alkyl-
ation of the thioamide (data not shown). Ac-
αSFAsp′2Cnf39 was isolated from the NCL reaction by
HPLC and characterized by MALDI MS, PAGE gel, and
UV–Vis and spectroscopy (Figure 1 and Supporting In-
formation). The conversion over the two-step liga-
tion/methylation sequence was 41%. MALDI MS
analysis of trypsin-digested 11 confirmed selective alkyl-
ation of Hcs to form Met in the purified product.
Using a similar double-labeled construct containing cys-
teine at the ligation site (Ac-αSFPhe′4Cys9Cnf39), we ob-
served thioamide quenching of Cnf after compaction of
the protein in high concentrations of TMAO.9 Since the
Ser-to-Cys mutation in this protein constituted a small de-
viation from the native αS protein sequence, we sought to
investigate this phenomenon using our newly synthesized
Ac-αSFAsp′2Cnf39 construct (Asp′2 used here because la-
beling at Phe′4 would not be compatible with our ligation
strategy using Hcs). To correct for any changes in Cnf flu-
orescence that are not due to the presence of the thio-
amide, an oxoamide control protein (αSFCnf39) was also
The ligation reaction was initiated by incubation of 9 with
1.5 equivalents of Ac-MetAsp′ValPhe-SR (6) in degassed
ligation buffer [6 M guanidinium hydrochloride, 0.2 M
sodium phosphate, 20 mM tris(2-carboxyethyl)phos-
Synlett 2013, 24, 2454–2458
© Georg Thieme Verlag Stuttgart · New York

LETTER
Semi-Synthesis of Labeled α-Synuclein
2457
HRMS (NIH RR-023444), MALDI MS (NSF MRI-0820996), and
NMR (NIH RR-022442).
prepared for fluorescence experiments. In buffer without
TMAO, thioamide quenching of Cnf in Ac-αSFAsp′2Cnf39
(i.e., FRET) was minimal. In accordance with our previ-
ous observations, thioamide quenching of Cnf increased
as a function of TMAO concentration (Figure 2, left axis).
To assign distance constraints based on these measure-
ments, the measured quenching efficiency (EQ) values
were converted to distances using Förster theory. In 2 M
and 4 M TMAO, RFRET was calculated as 15.4 Å and 12.6
Å, respectively (Figure 2, right axis). Taken together with
our previous results, these data indicate that the N-termi-
nal region of αS undergoes significant compaction in high
TMAO concentrations. Furthermore, our new results con-
clusively demonstrate that the observed quenching is not
due to Cys-mediated dimerization of αS.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.SnuIpofoiprmntgirStanoIuifpog
r
t
iornat
References and Notes
(1) Royer, C. A. Chem. Rev. 2006, 106, 1769.
(2) (a) Goldberg, J. M.; Batjargal, S.; Petersson, E. J. J. Am.
Chem. Soc. 2010, 132, 14718. (b) Goldberg, J. M.; Speight,
L. C.; Fegley, M. W.; Petersson, E. J. J. Am. Chem. Soc.
2012, 134, 6088. (c) Goldberg, J. M.; Wissner, R. F.; Klein,
A. M.; Petersson, E. J. Chem. Commun. 2012, 48, 1550.
(3) (a) Miwa, J. H.; Pallivathucal, L.; Gowda, S.; Lee, K. E.
Org. Lett. 2002, 4, 4655. (b) Miwa, J. H.; Patel, A. K.;
Vivatrat, N.; Popek, S. M.; Meyer, A. M. Org. Lett. 2001, 3,
3373. (c) Reiner, A.; Wildemann, D.; Fischer, G.; Kiefhaber,
T. J. Am. Chem. Soc. 2008, 130, 8079. (d) Choudhary, A.;
Raines, R. T. ChemBioChem 2012, 12, 1801.
(4) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776.
(5) (a) Batjargal, S.; Wang, Y. J.; Goldberg, J. M.; Wissner, R.
F.; Petersson, E. J. J. Am. Chem. Soc. 2012, 134, 9172.
(b) Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249.
(6) Galvin, J. E.; Lee, V. M. Y.; Trojanowski, J. Q. Arch.
Neurol. 2001, 58, 186.
(7) (a) Uversky, V. N.; Li, J.; Fink, A. L. J. Biol. Chem. 2001,
276, 10737. (b) Auluck, P. K.; Caraveo, G.; Lindquist, S.
Annu. Rev. Cell Dev. Biol. 2010, 26, 211. (c) Wood, S. J.;
Wypych, J.; Steavenson, S.; Louis, J. C.; Citron, M.; Biere,
A. L. J. Biol. Chem. 1999, 274, 19509.
(8) Drescher, M.; Huber, M.; Subramaniam, V. ChemBioChem
2012, 13, 761.
(9) Wissner, R. F.; Batjargal, S.; Fadzen, C. M.; Petersson, E. J.
J. Am. Chem. Soc. 2013, 135, 6529.
Figure 2 Refolding assay. Left: Monomeric double-labeled αS (Ac-
αSFAsp′2Cnf39) is mixed with TMAO to induce compaction and an in-
crease in quenching efficiency (EQ). Right: EQ (blue dashed line) of
Ac-αSFAsp′2Cnf39 determined at varying concentrations of TMAO.
Interchromophore distance (RFRET, green solid line) computed using
Förster theory as described in Supporting Information.
(10) Ferreon, A. C. M.; Moosa, M. M.; Gambin, Y.; Deniz, A. A.
Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 17826.
(11) (a) Pivato, M.; De Franceschi, G.; Tosatto, L.; Frare, E.;
Kumar, D.; Aioanei, D.; Brucale, M.; Tessari, I.; Bisaglia,
M.; Samori, B.; de Laureto, P. P.; Bubacco, L. PLoS One
2012, 7, e50027. (b) Zhou, W.; Freed, C. R. J. Biol. Chem.
2004, 279, 10128.
By combining unnatural amino acid mutagenesis with
EPL at Met, we can install our minimal Cnf/thioamide
FRET probe pair into αS without introducing a mutation
at the ligation site. We have demonstrated that selective
alkylation of Hcs can be performed in the presence of a
thioamide, rendering our new ligation strategy compatible
with the minimal probe pairs that we have developed. The
αS refolding studies presented here demonstrate how this
methodology can be used to study protein folding dynam-
ics, though these techniques are by no means limited to
αS. Over 20,000 proteins in the PDB contain MetArg or
MetLys motifs that can be used as points of disconnection
in their retrosynthetic analyses.14 We are currently work-
ing to expand these methods even further by combining
EPL at Met with multiple ligation strategies. This method-
ology will allow us to install thioamide-containing syn-
thetic fragments in the middle of a large protein target in
a traceless and efficient manner.
(12) Hendrickson, T. L.; Imperiali, B. Biochemistry 1995, 34,
9444.
(13) (a) Tam, J. P.; Yu, Q. T. Biopolymers 1998, 46, 319.
(b) Saporito, A.; Marasco, D.; Chambery, A.; Botti, P.;
Monti, S. M.; Pedone, C.; Ruvo, M. Biopolymers 2006, 83,
508. (c) Aussedat, B.; Fasching, B.; Johnston, E.; Sane, N.;
Nagorny, P.; Danishefsky, S. J. J. Am. Chem. Soc. 2012, 134,
3532. (d) Pachamuthu, K.; Schmidt, R. R. Synlett 2003, 659.
(14) Tanaka, T.; Wagner, A. M.; Warner, J. B.; Wang, Y. J.;
Petersson, E. J. Angew. Chem. Int. Ed. 2013, 52, 6210.
(15) (a) Suto, K.; Shimizu, Y.; Watanabe, K.; Ueda, T.; Fukai, S.;
Nureki, O.; Tomita, K. EMBO J. 2006, 25, 5942.
(b) Watanabe, K.; Toh, Y.; Suto, K.; Shimizu, Y.; Oka, N.;
Wada, T.; Tomita, K. Nature (London) 2007, 449, 867.
(16) Link, A. J.; Vink, M. K. S.; Agard, N. J.; Prescher, J. A.;
Bertozzi, C. R.; Tirrell, D. A. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 10180.
Acknowledgment
(17) Shalaby, M. A.; Grote, C. W.; Rapoport, H. J. Org. Chem.
1996, 61, 9045.
(18) Nagalingam, A. C.; Radford, S. E.; Warriner, S. L. Synlett
2007, 2517.
This work was supported by funding from the University of Penn-
sylvania, including a grant from the Institute on Aging, and the Na-
tional Institutes of Health (NIH NS081033 to EJP). Instruments
supported by the National Science Foundation and NIH include:
© Georg Thieme Verlag Stuttgart · New York
Synlett 2013, 24, 2454–2458

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R. F. Wissner et al.
LETTER
(19) Taskent-Sezgin, H.; Chung, J.; Patsalo, V.; Miyake-Stoner,
S. J.; Miller, A. M.; Brewer, S. H.; Mehl, R. A.; Green, D.
F.; Raleigh, D. P.; Carrico, I. Biochemistry 2009, 48, 9040.
(20) Synthesis of α-N-Fmoc-L-aspartate-2-amino-5-
nitroanilide (1): Fmoc-Asp(Ot-Bu)-OH (2.67 g, 6.50
mmol) was dissolved in THF (35 mL) under argon flow and
the solution was cooled to –10 °C in a NaCl/ice (1:3) bath.
N-Methylmorpholine (NMM, 1.43 mL, 13 mmol) and
isobutyl chloroformate (IBCF, 0.85 mL, 6.5 mmol) were
added dropwise with stirring. After 15 min, 4-nitro-o-
phenylenediamine (1.0 g, 6.5 mmol) was added and the
reaction was allowed to proceed with stirring under argon
flow at –10 °C for 2 h. The reaction was then allowed to
proceed for an additional 6 h with stirring at r.t. The reaction
mixture was dried by rotary evaporation, resuspended in
DMF (20 mL), and then poured into sat. KCl solution (200
mL). The precipitated product was filtered and washed with
cold H2O. The precipitate was then dissolved in minimal
EtOAc and purified over a silica gel column in hexanes–
EtOAc (3:2) to afford 1 as a yellow solid in 88.9% yield; Rf
0.5 (hexanes–EtOAc, 1:1). 1H NMR (500 MHz, CDCl3): δ =
8.1 (s, 1H), 8.0 (s, 1H), 7.93 (dd, J = 2.5, 9.0 Hz, 1 H), 7.74
(dd, J = 3.6, 7.5 Hz, 2 H), 7.57 (dd, J = 3.4, 7.4 Hz, 2 H),
7.36–7.41 (m, 2 H), 7.26–7.30 (m, 2 H), 6.62 (d, J = 9.0 Hz,
1 H), 5.98 (d, J = 8.2 Hz, 1 H), 4.73 (s, H) 4.63 (br s, 1 H),
4.45–4.53 (m, 2 H), 4.19 (t, J = 6.3 Hz, 1 H), 2.97 (dd, J =
monitored by TLC. After approximately 1 h, the reaction
was filtered through Celite® (Sigma-Aldrich) and dried by
rotary evaporation. The crude reaction material was
dissolved in EtOAc and purified over a silica gel column in
hexanes–EtOAc (1:1) to afford 2 as a yellow foam (2.46 g,
78.6% yield); Rf 0.7 (hexanes–EtOAc, 1:1). 1H NMR (500
MHz, CDCl3): δ = 9.84 (br s, 1 H), 8.07 (br s, 1 H), 7.96 (d,
J = 8.4 Hz, 1 H), 7.76 (d, J = 6.9 Hz, 2 H), 7.52 (dd, J = 7.3,
19.6 Hz, 2 H) 7.39–7.43 (m, 2 H), 7.28–7.32 (m, 2 H), 6.56
(d, J = 9.0 Hz, 1 H), 6.06 (d, J = 8.2 Hz, 1 H), 5.07 (br s, 1
H), 4.84 (br s, 2 H), 4.37 (br s, 2 H), 4.21–4.13 (m, 1 H),
3.18–3.05 (m, 2 H), 1.45 (s, 9 H). 13C NMR (125 MHz,
CDCl3): δ = 204.9, 172.1, 157.2, 149.6, 144.3, 142.2, 139.0,
128.8, 128.1, 126.5, 125.9, 125.8, 122.8, 121.0, 115.6, 83.5,
68.4, 58.4, 47.8, 41.6, 28.9. HRMS (ESI): m/z [M + H]+
calcd for C29H31N4O6S: 563.196; found: 563.197.
(22) Synthesis of α-N-Fmoc-L-thioaspartatenitrobenzo-
triazolide (3): Compound 2 (1.00 g, 1.78 mmol) was added
to glacial acetic acid diluted with 5% H2O (25 mL). NaNO2
(0.16 g, 2.23 mmol) was added in small portions over 5 min
with constant stirring at r.t. After 30 min, the reaction was
quenched by the addition of ice water (500 mL). The
resulting pale orange precipitate 3 was filtered, washed
extensively with ice water, and allowed to dry under
vacuum. The final product was characterized and used in
peptide synthesis without any further purification; Rf 0.9
(hexanes–EtOAc, 1:1). 1H NMR (500 MHz, CDCl3): δ =
9.64 (s, 1 H), 8.46 (d, J = 8.6 Hz, 1 H), 8.33 (d, J = 8.7 Hz,
1 H), 7.77–7.78 (m, 2 H), 7.59–7.64 (m, 2 H) 7.38–7.44 (m,
2 H), 7.29–7.35 (m, 2 H), 6.45–6.52 (m, 1 H), 6.23 (d, J =
8.7 Hz, 1 H), 4.48–4.54 (m, 1 H), 4.34–4.41 (m, 1 H), 4.21–
4.26 (m, 1 H), 3.07–3.15 (m, 1 H), 2.86–2.97 (m, 1 H), 1.42
(s, 9 H). 13C NMR (125 MHz, CDCl3): δ = 206.7, 169.3,
156.4, 150.6, 149.9, 144.6, 142.3, 132.8, 128.7, 128.0,
126.0, 123.2, 122.6, 121.0, 113.6, 83.4, 68.2, 59.0, 48.1,
41.9, 28.9. HRMS (ESI): m/z [M + Na]+ calcd for
4.6, 17.2 Hz, 1 H), 2.72–2.80 (m, 1 H), 1.46 (s, 9 H). 13
C
NMR (125 MHz, CDCl3): δ = 172.5, 170.8, 157.3 , 149.0,
144.4, 142.3, 139.6, 128.8, 128.2, 125.9, 125.4, 124.5,
121.7, 121.1, 115.9, 83.6, 68.3, 52.7, 48.2, 38.3, 29.1.
HRMS (ESI): m/z [M + H]+ calcd for C29H31N4O7: 547.219;
found: 547.218.
(21) Synthesis of α-N-Fmoc-L-thioaspartate-2-amino-5-
nitroanilide (2): P2S5 (2.47 g, 5.56 mmol) and anhyd
Na2CO3 (0.589 g, 5.56 mmol) were stirred in THF (30 mL)
at r.t. under argon flow until a clear yellow solution was
obtained. After cooling the solution to 0 °C on ice, 1 (3.04 g,
5.56 mmol) was added, and the reaction was carefully
C29H27N5NaO6S: 596.158; found: 596.158.
Synlett 2013, 24, 2454–2458
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