Ligations of N-acyl tryptophan units to give native peptides via 7-, 10-, 11- and 12-membered cyclic transition states

Article abstract of DOI:10.1039/c3ob27421g

N-Acyl tryptophan isopeptides undergo acyl transfer in chemical ligations via 7-, 10-, 11- and 12-membered cyclic transition states to yield natural peptides, representing the first examples of successful isopeptide ligations from N-acyl tryptophan units.

Full text of DOI:10.1039/c3ob27421g

Organic &
Biomolecular Chemistry
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Ligations of N-acyl tryptophan units to give native
peptides via 7-, 10-, 11- and 12-membered cyclic
transition states†
Cite this: Org. Biomol. Chem., 2013, 11,
1594
Received 13th December 2012,
Accepted 11th January 2013
Vadim Popov,a Siva S. Pandaa and Alan R. Katritzky*a,b
DOI: 10.1039/c3ob27421g
www.rsc.org/obc
N-Acyl tryptophan isopeptides undergo acyl transfer in chemical
In an alternative approach, our group developed24–27 liga-
ligations via 7-, 10-, 11- and 12-membered cyclic transition states tions of S-acylated cysteine peptides to form native peptides
to yield natural peptides, representing the first examples of suc- through a diversity of transition states with 8- to 19-membered
cessful isopeptide ligations from N-acyl tryptophan units.
rings. This methodology requires no auxiliary groups and
enables the selective S-acylation of cysteine peptides by N-acyl-
benzotriazoles in good yields and under mild conditions fol-
lowed by microwave-assisted chemical ligations of the S-acyl
isopeptides. However, ligation through an 8-membered tran-
sition state can be a challenge and the low abundance of
cysteine remains an obstacle. We recently reported the chemo-
selective O- to N-acyl migration of O-acyl serines via 5-, 8- and
11-membered transition states.28
We now demonstrate the first N- to N-acyl migration invol-
ving tryptophan isopeptides migrations via 7-, 10-, 11-, and 12-
membered transition states. These chemical ligations have
been achieved by migration of an N-peptidoyl unit of a trypto-
phan isopeptide unit to produce natural peptide and utilize
neither cysteine residue nor an auxiliary group at the ligation
site.
We synthesized the intermediate mono-isodipeptide 4 to
study the N-acyl migration from the indole nitrogen to the N-
terminal group of tryptophan amino acid sequence via a 7-
membered transition state and also to serve as starting
material to study the possibility of N- to N-acyl migration via
10-, 11- and 12-membered cyclic transition states. Compound
4 on coupling with α-, β- or γ-amino acids gave the starting
mono-isotripeptides (9a–c) needed for the ligation studies
involving these large transition states. To enhance migration
rates, we used a glycine unit at the N-terminus of mono-isotri-
peptide 9a and β- and γ-amino acid units in mono-isotripep-
tides 9b and 9c respectively.
A recent important advance in peptide chemistry has been the
development of orthogonal ligation methods which provide a
powerful strategy to synthesize macromolecular peptides1–6
difficult to obtain by conventional peptide synthesis. The
chemical basis of such ligations is a regiospecific coupling of a
C-terminal electrophile of one peptide with a N-terminal
nucleophile of a second peptide, without any protection or
activation step. The peptide segments can be synthetic1–4 or
biosynthetic in origin.5,6 Many organic reactions have been
enabled by ligations via a variety of chemical linkages, includ-
ing amide,7 thioester,8 thiazolidine,9 oxaproline,10 oxime,11
hydrazone,12 and thioether moieties.13
Native chemical ligation (NCL), while of great importance,
is subject to limitations including (i) the requirement of a N-
terminal cysteine residue at the ligation site to afford a peptide
containing an internal cysteine, and (ii) the low abundance of
cysteine in human proteins (1.7% of the residues).14–17 In
attempts to overcome the limitation of low abundance of
cysteine, considerable effort has been devoted to developing
auxiliary thiol groups, but their use in ligations was found: (i)
difficult to complete due to steric hindrance18–23 and (ii) pro-
blematic since extraneous groups present in the ligated
product, can be difficult to remove.18–23 Another strategy
involves the conversion of the cysteine into a serine residue
after NCL,16,17 but this requires post-NCL modifications after
NCL peptide synthesis.
Boc-protected tryptophan was treated with benzyl bromide
in presence of Hunig’s base in DMF to obtain (S)-benzyl
2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanoate (2)
in 92% yield. The N-acylation of the indole of compound 2 was
carried out by react with Cbz-Ala-Bt in presence of DBU in
aCenter for Heterocyclic Compounds, Department of Chemistry, University of Florida,
Gainesville, FL 32611-7200, USA. E-mail: katritzky@chem.ufl.edu;
Fax: +1 352-392-9199; Tel: +1 352-392-0554
bDepartment of Chemistry, King Abdulaziz University, Jeddah, 21589 Saudi Arabia
†Electronic supplementary information (ESI) available: 1H, 13C NMR, CHN/
HRMS for all compounds and HPLC chromatograms and ESI-MS spectra of lig-
ation studies. See DOI: 10.1039/c3ob27421g
acetonitrile to afford Boc-protected mono-isodipeptide
3
(78%), which on deprotection with dioxane–HCl solution
afforded unprotected mono-isodipeptide 4 (90%) (Scheme 1).
1594 | Org. Biomol. Chem., 2013, 11, 1594–1597
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Scheme 1 Synthesis of N-acyl tryptophan isodipeptide 4.
Chemical ligation via a 7-membered cyclic transition state ligation mixture under basic condition (DMF, piperidine, MW
was investigated by subjecting mono-isodipeptide 4 to micro- 50 °C, 50 W, 3 h) which disclosed a small amount (2%) the
wave irradiation at 50 °C, 50 W irradiation power for 3 h using ligated product 5, together with a major peak corresponding to
1 M NaH2PO4/Na2HPO4 phosphate buffer to maintain pH 7.4 4.
(Scheme 2). The reaction was allowed to cool to room tempera-
Unprotected mono-isodipeptide 4, on coupling with benzo-
ture and acidified with 2 N HCl to pH = 1. The mixture was triazolide of Boc protected α-, β- or γ-amino acids 7a–c at room
extracted with ethyl acetate (3 × 20 mL), the combined organic temperature in DMF in the presence of 2 equiv. of triethyl-
extracts were dried over MgSO4 and the solvent was removed amine gave mono-isotripeptides 8a–c in good yields. Com-
under reduced pressure. The ligation mixture was weighed and pounds 8a–c on deprotection with dioxane–HCl solution
then a solution in methanol (1 mg mL−1) was analyzed by afforded unprotected mono-isotripeptides 9a–c in good yields,
HPLC-MS.
which were fully characterized by 1H, 13C NMR and CHN/
HPLC-MS analysis indicates the ligation did not take place HRMS analysis (Scheme 3).
in aqueous conditions (pH 7.3, 1 M buffer strength, MW
When compounds 9b–c were subjected to ligation
50 °C, 50 W, 3 h). We also examined the HPLC-MS of the (Scheme 4) under aqueous conditions, (pH 7.3, 1 M buffer
Scheme 2 Study of the feasibility of N→N acyl migration via a 7-membered cyclic transition state.
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Scheme 3 Synthesis of mono-isotripeptides 9a–c.
Scheme 4 Study of the feasibility of N→N acyl migrations via a 10-, 11- and 12-membered cyclic transition states.
Table 1 Chemical ligations of N-acyl isotripeptides 9a–c in buffer
Relative areaa,b (%)
Product characterization by HPLC-MS
Ligated
peptide (LP)
Transacylation
product (TA)
Total crude
yield (%)
of products
isolated
Cyclic
TS size
[M + H]+
[M + H]+
React
React
LP (RT)
TA (RT)
LP
found
TA
found
9a
9b
9c
10
11
12
nd
87
92
nd
29.1 (59.2)
6.3 (59.5)
nd
10a
10b
10c
nd
571.2
585.2
11a
11b
11c
nd
776.1
790.1
36.9 (49.9)
86.4 (48.8)
33.9 (65.52)
7.3 (65.4)
a Determined by HPLC-MS semiquantitative. The area of ion-peak resulting from the sum of the intensities of the [M + H]+ and [M + Na]+ ions of
each compound was integrated (corrected for starting material). b LP = ligated peptide, TA = transacylation product.
strength, MW 50 °C, 50 W, 3 h), we obtained the desired (44%), 10b (71%) and 10c (99%) were obtained via 10-, 11- and
ligated products 10b (29%) and 10c (6%) via 11- and 12-mem- 12-membered cyclic transition state observed when 9a–c were
bered transition states (Scheme 4, Table 1). However 10a subjected to ligation reaction under microwave irradiation in
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Table 2 Chemical ligation of N-acyl isotripeptides 9a–c in DMF–piperidine
Relative areaa,b (%)
Product characterization by HPLC-MS
Ligated
peptide (LP)
Transacylation
product (TA)
Total crude
yield (%)
of products
isolated
Cyclic
TS size
[M + H]+
found
[M + H]+
React
React
LP (RT)
TA (RT)
LP
TA
found
9a
9b
9c
10
11
12
92
91
90
36.3 (48.2)
28.6 (48.2)
0.9 (45.2)
44.4 (57.3)
71.4 (57.1)
99.1 (54.2)
19.2 (63.2)
0.00
0.00
10a
10b
10c
557.1
571.1
585.1
11a
11b
11c
762.1
—
—
a Determined by HPLC-MS semiquantitative. The area of ion-peak resulting from the sum of the intensities of the [M + H]+ and [M + Na]+ ions of
each compound was integrated (corrected for starting material). b LP = ligated peptide, TA = transacylation product.
piperidine–DMF at 50 °C, 50 W for 3 h (Scheme 4, Table 2). 10 J. P. Tam and Z. Miao, J. Am. Chem. Soc., 1999, 121, 9013.
HPLC-MS indicated the formation of desired intramolecular 11 J.-S. Fruchart, H. Gras-Masse and O. Melnyk, Tetrahedron
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Tryptophan isopeptides with α-, β-, or γ-amino acid units were
synthesized and acyl migration form indole nitrogen to terminal
NH2 was studied under microwave irradiation. Intramolecular
acyl transfer through 10-, 11- and 12-membered transition states
was favoured over 7-membered transition state and acyl
migration occur more readily in basic non-aqueous media rela-
tive to aqueous buffered conditions. This observations form a
starting point for development of a ligation method.
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