The chemical ligation of selectively S-acylated cysteine peptides to form native peptides via 5-, 11- and 14-membered cyclic transition states

Article abstract of DOI:10.1039/c003234d

Cysteine and C-terminal cysteine peptides are selectively S-acylated at 0-20 °C by N-(Pg-α-aminoacyl)benzotriazoles to give N-Pg-S-acyl-isodi-, -isotri-, and -isotetra-peptides isolated in good yields. N-Fmoc-S-acyl-isopeptides are Fmoc deprotected to aff

Full text of DOI:10.1039/c003234d

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The chemical ligation of selectively S-acylated cysteine peptides to form
native peptides via 5-, 11- and 14-membered cyclic transition states†
Alan R. Katritzky,*a Nader E. Abo-Dya,a,b Srinivasa R. Talaa and Zakaria K. Abdel-Samiib
Received 19th February 2010, Accepted 23rd March 2010
First published as an Advance Article on the web 7th April 2010
DOI: 10.1039/c003234d
Cysteine and C-terminal cysteine peptides are selectively S-
acylated at 0–20 ◦C by N-(Pg-a-aminoacyl)benzotriazoles
to give N-Pg-S-acyl-isodi-, -isotri-, and -isotetra-peptides
isolated in good yields. N-Fmoc-S-acyl-isopeptides are Fmoc
deprotected to afford free S-acyl-isopeptides isolated in high
yields. S-Acyl-isodi-, S-acyl-isotetra-, and S-acyl-isopenta-
peptides undergo chemical ligation; migration of the cysteine
S-acyl groups to the N-terminal amino acids via 5-, 11-,
and 14-membered transition states giving the corresponding
native di-, tetra-, and penta-peptides. By contrast, the S-
acyl-isotripeptide prefers intermolecular acylation from one
molecule to another over an 8-membered intramolecular tran-
sition state. The developed methodology allows convenient
isolation of stable, unprotected S-acyl cysteine peptides in-
cluding the first isolation of S-acyl-isopeptides, which should
facilitate the investigation of ligation by physical organic
chemistry techniques.
We now report the following novel findings (i) selective acyla-
tion by N-acylbenzotriazoles under mild conditions of cysteine-
containing peptides into isolated S-acyl isopeptides, including
examples containing free hydroxyl and/or carboxyl groups; (ii)
solution phase Fmoc group deprotection of N-Fmoc-S-acyl
isotri-, isotetra-, and isopenta-peptides possessing free carboxyl
groups; (iii) microwave-assisted chemical ligations (1 h, 50 ◦C)
of these S-acyl isopeptides to form native peptides via 11- and
14-membered ligation transition states without use of auxiliaries.
Synthesis of cysteine dipeptides
N-(Protected-a-aminoacyl)-benzotriazoles 1a–d (see Table 1) were
prepared in 75–90% yield from the corresponding N-protected
amino acids following our published one-step procedure.9a
N-Protected cysteine dipeptides 2a–d were synthesized in 74–
98% yields by peptide coupling reactions of N-(Pg-a-aminoacyl)-
benzotriazoles 1a–d with L-cysteine in aqueous acetonitrile
(MeCN–H2O, 7 : 3) in the presence of Et3N for one hour at 20 ◦C
(Scheme 1, Table 1).9a Novel dipeptides 2a–d were characterized
by 1H, and 13C-NMR spectroscopy and elemental analysis.
Peptides and proteins play vital roles in almost all biological and
physiological processes in living organisms.1 In native chemical
ligation (NCL), chemo selective reactions of a C-terminal thioester
and an N-terminal cysteine residue form a native peptide bond
at the ligation site, a powerful technique for the synthesis of
peptides and proteins.2,3 Contemporary syntheses of peptides and
proteins now incorporate chemo selective peptide ligation in an
ever expanding manner.4 The classical NCL method needs an
N-terminal cysteine residue on a peptide or glycopeptide fragment.
Thiol auxiliary ligation5 and sugar-assisted ligation6 can overcome
this limitation, but need auxiliaries and can sterically hinder the
ligation.
Scheme 1 Preparation of N-Pg-dipeptides 2a–d from N-protected
(a-aminoacyl)benzotriazoles 1a–d and L-cysteine.
S-Acyl peptides have helped elucidate biophysical, structural
and other properties of S-acylated proteins of mammalian cells.7
While peptides lacking free hydroxyl or amino groups can be
efficiently S-acylated by acyl chlorides;8 specific S-acylation of
cysteine-containing peptides has remained a synthetic challenge,
given the lability of the thioester bond.7 We demonstrated previ-
ously that peptide coupling of N-(Pg-a-aminoacyl)benzotriazoles
with unprotected amino acids in MeCN–H2O occurs with preser-
vation of chirality.9
Synthesis of S-acyl isopeptides
S-Acyl isodipeptides 3a,b were synthesized by peptide coupling
reactions between N-(Pg-a-aminoacyl)benzotriazoles 1a,b and
L-cysteine hydrochloride in aqueous acetonitrile (MeCN–H2O,
10 : 1) in the presence of one equivalent of Et3N for one hour
at RT in 35–65% yields (Scheme 2).9a Novel S-acyl isodipeptide
hydrochloride salts 3a,b were characterized by 1H, 13C-NMR
Table 1 Preparation of N-Pg-dipeptides 2a–d
Reactant 1 R1
Product 2
Yield of 2 (%)a
aCenter for Heterocyclic Compounds, Department of Chemistry, Univer-
sity of Florida, Gainesville, FL, 32611-7200, USA. E-mail: katritzky@
chem.ufl.edu; Fax: +1 352-392-9199; Tel: +1 352-39-0554
1a
1b
1c
1d
CH2Ph
CH2SMe
H
N-Fmoc-L-Phe-L-Cys-OH (2a) 98
N-Fmoc-L-Met-L-Cys-OH (2b) 85
N-Fmoc-L-Gly-L-Cys-OH (2c) 84
bDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Zagazig University, Zagazig, 44519, Egypt
CH(OH)Me N-Z-L-Thr-L-Cys-OH (2d)
74
† Electronic supplementary information (ESI) available: Experimen-
tal details, characterization and HPLC-MS analysis. See DOI:
10.1039/c003234d
a Isolated yield.
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Scheme 4 Fmoc deprotection of S-acyl isotripeptides 4b,4c.
is not necessary in this procedure.16 The S-acyl isotripeptides 5a
and 5b with a free amino group are needed for the chemical ligation
Scheme 2 Preparation of S-acyl isodipeptides 3a,b.
1
studies. The S-acyl isotripeptides 5a,b were characterized by H,
spectroscopy and elemental analysis. This is the first isolation
of hydrochloride salts of S-(a-aminoacyl)cysteines, the unstable
intermediates formed after transthioesterification during native
chemical ligation.10
and 13C-NMR, and elemental analysis.
N-(N-Fmoc-Glycyl-leucyl)benzotriazole 6, prepared by our
published procedure,9c reacted with cysteine in acetonitrile–water
(3 : 1) in the presence of one equivalent of triethylamine to give
Fmoc-Gly-L-Leu-L-Cys-OH 7 in 88% yield. The S-acylation of
7 by Z-Ala-Bt 1g in acetonitrile–water in the presence of one
equivalent of KHCO3 gave S-acyl isotetrapeptide 8 in 75% yield.
Fmoc deprotection of 8 was achieved using DBU in dry THF to
give amino-unprotected S-acylated isotetrapetide 9 in 90% yield
(Scheme 5). Compounds 6–9 were each characterized by 1H, and
13C-NMR spectroscopy and by elemental analysis.
S-Acylation of cysteine-containing dipeptides 2a–d (each
containing a free carboxyl group) by treatment with N-
◦
acylbenzotriazoles 1a,e,g,h at 0–5 C in the presence of KHCO3
afforded S-acyl isotripeptides 4a–d in 72–87% yields (Scheme 3,
Table 2). Peptide 2d, containing a threonine residue with an
unprotected hydroxyl group was thus selectively S-acylated to 4d.
Scheme 3 S-Acylation of dipeptides 2a–d to form isotripeptides 4a–d.
Scheme 5 Preparation of mono free amino S-acyl-isotetrapeptide 9.
Classical methods for N-Fmoc-deprotection utilize a mild base
such as 20% piperidine,11 required in excess to deprotonate the flu-
orene and scavenge liberated dibenzofulvene (DBF). This method
is not suitable for solution phase peptide synthesis due to low
volatility of the solvent used and the solvent dependant reversible
reaction of dibenzofulvene with piperidine.12 Sheppeck II et al.,
used thiols as dibenzofulvene scavengers in the deprotection of
N-Fmoc-protected amines.13
We now report a new facile and efficient procedure for Fmoc
deprotection of S-acyl isopeptides possessing a free carboxylic
group and a labile thioester functionality. When N-Fmoc-S-acyl
isopeptides 4b,4c in dry THF were treated with two equivalents
of DBU, DBF liberated out, and peptide precipitated as a di-
DBU salt of carbamate, carboxylate. DBU carbamate salts are
known.14,15 After decantation, acidification of the precipitate with
2 N HCl caused evolution of carbon dioxide. Adjustment of the pH
to 5 precipitated the unprotected S-acyl isopeptides 5a,5b in 88–
90% yields (Scheme 4). Protection of carboxylic group in peptides
Treatment of Gly-L-Cys(Z-L-Ala)-OH (5b) with N-(N-Fmoc-
glycyl-leucyl)benzotriazole 6 afforded S-acyl isopentapeptide 11
as shown in Scheme 6. 1H-NMR and HPLC-MS confirmed
compound 11 and isolated in 80% yield. Fmoc deprotection of
11 was achieved using DBU in dry THF to give mono free amino
S-acyl isopentapetide 12 characterized by 1H-NMR spectroscopy
and HPLC-MS (Scheme 6, ESI).
Chemical ligations
S-Acyl isodipeptides 3a,b underwent classical S to N acyl transfer
at 20 ◦C in acetonitrile–water (3 : 1), in the presence of triethy-
lamine as expected to give native cysteine dipeptides 2a,b in 80–
89% yields (Scheme 7). These S to N acyl transfers involve a
5-membered transition state due to proximity of the amino group
in 3a,b to the thioester functionality. Native cysteine dipeptides
Table 2 Preparation of S-acyl isotripeptides 4a–d
Dipeptide 2
RCOBt used
Product 4
Yield of 4 (%)a
2a
2b
2c
2d
1e
1f
1g
1a
Fmoc-L-Phe-L-Cys(4-Me-Ph)-OH (4a)
Fmoc-L-Met-L-Cys(Z-L-Leu)-OH (4b)
Fmoc-L-Gly-L-Cys(Z-L-Ala)-OH (4c)
Z-L-Thr-L-Cys(Fmoc-L-Phe)-OH (4d)
78
83
87
72
a Isolated yield.
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Scheme 6 Preparation of mono free amino S-acyl isopentapeptide 12.
Scheme 7 Native chemical ligation of S-acyl isodipeptides 3a,b to give
native dipeptides 2a,b.
1
2a,b were characterized by H, and 13C-NMR spectroscopy and
elemental analysis.
We next investigated long range chemical ligations which require
eight-, eleven- and fourteen-membered ring transition states for
intramolecular nucleophilic attack. Literature peptide ligations
via◦direct aminolysis of thioesters are usually accomplished at
37 C, during more than 48 h,5 we now show that reaction was
significantly accelerated by microwave irradiation. When S-acyl
isotripeptide 5b was suspended in NaH2PO4/Na2HPO4 at pH 7.8
and irradiated with microwave at 50 watts and 50 ◦C for one hour,
the product was shown by HPLC-MS analysis (ESI) to consist
mainly of the disproportionation product 14 (>85%) together with
a minor amount of ligation product 13 (3%) (Scheme 8, Table 3).
This indicates that intermolecular aminolysis of thioester is faster
than 8-membered ring intramolecular attack.
Scheme 8 Chemical ligation of mono free amino isotripeptide 5b.
By contrast, when S-acyl isotetrapeptide 9 was suspended in
NaH2PO4/Na2HPO4 at pH 7.8 and irradiated with microwave at
50 watts and 50 ◦C for one hour, subsequent HPLC-MS analysis
(ESI) confirmed the major product to be 15 formed by the desired
ligation (Scheme 9, Table 3). In this case the intramolecular
Scheme 9 Chemical ligation of mono free amino isotetrapeptide 9.
11-membered ring nucleophilic attack by the amino group on the
thioester was evidently faster than the intermolecular attack.
Table 3 Ligated native peptides formed by chemical ligation
Isolated
Entry
Ligated peptide
Retention time/min
[M + H]+ founda
Yield (%)b
Purity (%)c
1
2
3
Z-Ala-Gly-Cys-OH, 13
Z-Ala-Gly-Leu-Cys-OH, 15
Z-Ala-Gly-Leu-Gly-Cys-OH, 16
27.91
21.75
24.19
384.1
497.1
554.2
ndd
95
90
ndd
67
61
a HPLC-MS (ESI), b crude yield, c based on 1H-NMR, d not determined.
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Scheme 10 Chemical ligation of mono free amino isopentapeptide 12.
Again, when S-acyl isopentapeptide 12 was suspended in
NaH2PO4–Na2HPO4 at pH 7.8 and irradiated with microwave
at 50 watts and 50 ◦C for one hour. HPLC-MS analysis (ESI) con-
firmed the major product to be 16, produced by the desired ligation
(Scheme 10, Table 3). This result indicates that intramolecular 14-
membered ring nucleophilic attack by the amino group on the
thioester in 12 was also faster than intermolecular attack by one
of molecule of 12 on another.
Haase and Seitz recently demonstrated that an internal cysteine
residue can accelerate thioester based peptide ligation up to 25
fold providing indirect evidence for S- to N-acyl transfer via 8,
11, 14, 17, 20, or 23-membered transition states to form native
peptides (in 3–56% hplc yields).17 Our results show that 11- and 14-
membered transition states allow easy intramolecular ligation to
form native peptides, but an 8-membered transition state reaction
is disfavoured.
In conclusion, we have demonstrated (i) selective S-acylation,
in good yields and under mild conditions, of cysteine peptides
having free hydroxyl and/or carboxyl groups; (ii) solution phase
Fmoc group deprotection of N-Fmoc-S-acyl isotri-, isotetra- and
isopenta-peptides having free carboxyl groups in 15 min with
DBU and (iii) microwave assisted chemical ligation involving the
migration of cysteine S-acyl groups in 9 and 12 to N-terminal
amino acids in these peptides via 11- and 14-membered transition
states.
As in native chemical ligation, these chemical ligations take
place utilizing a single cysteine, without the use of auxiliaries. S-
Acyl isopentapeptide 12 was converted into native pentapeptide
16 as major product (Table 3). Isotetrapeptide 9 gave native
tetrapeptide 15 as major product (Table 3) and characterized by
HPLC-MS. However, isotripeptide 5b gave a mixture of the desired
ligated product 13 (3%) together with disproportionation product
14 (>85%) of intermolecular trans-acylation. This indicates that,
intermolecular trans-acylation is favoured over an 8-membered
cyclic transition state, whereas intramolecular chemical ligation
reactions are preferred when they are produced via 5-, 11- and
14-membered transition states.
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