Long-range chemical ligation from N→N acyl migrations in tryptophan peptides via cyclic transition states of 10- to 18-members

Article abstract of DOI:10.1002/chem.201400125

Chemical ligations to form native peptides from N→N acyl migrations in Trp-containing peptides via 10- to 18-membered cyclic transition states are described. In this study, a statistical, predictive model that uses an extensive synthetic and computational approach to rationalize the chemical ligation is reported. N→N acyl migrations that form longer native peptides without the use of Cys/Ser/Tyr residues or an auxiliary group at the ligation site were achieved. The feasibility of these traceless chemical ligations is supported by the N-C bond distance in N-acyl isopeptides. The intramolecular nature of the chemical ligations is justified by using competitive experiments and theoretical calculations. Going the distance: Chemical ligations to form native peptides from N→N acyl migrations in Trp-containing peptides via 10- to 18-membered cyclic transition states are described (see scheme; Bn: benzyl; Z: benzyloxycarbonyl). The ligations are achieved without the use of Cys/Ser/Tyr residues or an auxiliary group. This new approach represents a significant development in the field.

Full text of DOI:10.1002/chem.201400125

DOI: 10.1002/chem.201400125
Full Paper
&
Peptide Synthesis
Long-Range Chemical Ligation from N!N Acyl Migrations in
Tryptophan Peptides via Cyclic Transition States of 10- to 18-
Members
Suvendu Biswas,[a] Roger Kayaleh,[a] Girinath G. Pillai,[a, b] Christopher Seon,[a] Ian Roberts,[a]
Vadim Popov,[a] Khalid A. Alamry,[c] and Alan R. Katritzky*[a, c]
Abstract: Chemical ligations to form native peptides from
N!N acyl migrations in Trp-containing peptides via 10- to
18-membered cyclic transition states are described. In this
study, a statistical, predictive model that uses an extensive
synthetic and computational approach to rationalize the
chemical ligation is reported. N!N acyl migrations that
form longer native peptides without the use of Cys/Ser/Tyr
residues or an auxiliary group at the ligation site were ach-
ieved. The feasibility of these traceless chemical ligations is
supported by the NꢀC bond distance in N-acyl isopeptides.
The intramolecular nature of the chemical ligations is justi-
fied by using competitive experiments and theoretical calcu-
lations.
Introduction
and problematic, because the extraneous groups in the ligated
product can be challenging to remove.[11–15]
Native chemical ligation (NCL), first reported by Wieland et al.[1]
and later developed by Kent and co-workers,[2] is a chemoselec-
tive and regioselective reaction of a peptide thioester and a ter-
minal Cys residue of another peptide that gives a native amide
bond at the ligation site through a rapid S!N acyl transfer via
a cyclic transition state (TS). This is one of the best techniques
for the synthesis of peptides and semisynthesis of proteins,
takes place in aqueous solution, and is becoming widely
used.[3–5] In biomedical research, NCL has been used in the syn-
thesis of the cancer protein NY-ESO-1,[6] cytochrome b562,[7]
and dendrimers, monodisperse macromolecules with highly
branched three-dimensional architectures.[8] Although still of
great use, NCL is limited by the need for an N-terminal Cys res-
idue at the ligation site to afford a peptide containing an inter-
nal Cys and by the low abundance of Cys in globular proteins
(1.7% of the residues).[9–11] To overcome the limitation of the
low abundance of Cys, great effort has gone into developing
thiol auxiliary groups. However, subsequent ligations were
found to be hard to complete, because of steric hindrance,[12,13]
There are a number of approaches to circumvent the need
for an N-terminal Cys residue at the ligation site in classical
NCL. These techniques include 1) the use of an auxiliary group
followed by removal of that group after ligation,[16–18] 2) NCL
followed by the conversion of penicillamine into Val,[17]
3) sugar-assisted NCL,[19–21] 4) Cys-free “direct aminolysis”,[22]
and 5) traceless Staudinger ligation.[23] However, the develop-
ment of new ligation methods is still an important area of re-
search in order to access modified peptides and proteins.
In an attempt to address these limitations, our group has
devoted considerable effort toward developing alternative
techniques. We have reported ligations of S-acylated Cys-con-
taining peptides to form the corresponding native peptides
through long-range chemical ligations via 8- to 19-membered
transition states.[24–28] This methodology utilizes the selective S-
acylation of Cys-containing peptides by N-acyl benzotriazoles
followed by microwave-assisted high-yielding chemical liga-
tions of the resulting S-acyl isopeptides under mild conditions
and with no auxiliary groups. However, the low abundance of
Cys in the natural peptide sequence remains an obstacle. To
address this challenge, we reported recently the chemoselec-
tive O!N acyl migration of O-acyl serines via 5-, 8-, and 11-
membered transition states,[29] and we successfully demon-
strated long-range O!N acyl migrations of O-acyl Tyr units via
10- to 18-membered transition states.[30]
[a] Dr. S. Biswas, R. Kayaleh, G. G. Pillai, C. Seon, I. Roberts, Dr. V. Popov,
Prof. A. R. Katritzky
Department of Chemistry, University of Florida
Gainesville, FL 32611 (USA)
E-mail: katritzky@chem.ufl.edu
[b] G. G. Pillai
To the best of our knowledge, N!N acyl migration for the
synthesis of native peptides has not been explored. We discov-
ered recently the first examples of successful chemoselective
N!N acyl migration involving Trp-containing isopeptides via
10-, 11-, and 12-membered cyclic transition states.[31] These
chemical ligations were achieved by migration of the N-pepti-
Department of Chemistry, University of Tartu, Tartu 50411 (Estonia)
[c] Prof. K. A. Alamry, Prof. A. R. Katritzky
Department of Chemistry, King Abdulaziz University
Jeddah 21589 (Saudi Arabia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201400125.
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doyl unit of a Trp isopeptide
unit to produce natural peptides.
They utilize neither Cys nor Ser
residues, nor an auxiliary group
at the ligation site. However, this
methodology still needs to be
fully developed and explored by
examining the following factors:
1) the range of cyclic transition
states, 2) the best conditions for
the ligation step, and 3) the ef-
fects of substituents in the
amino acid residue and rationali-
zation of the relative abundance
of ligated product. The purpose
of this work is to identify the
structural features controlling
the ligation and to correlate the
Scheme 1. Synthesis of isodipeptide 4 and isotripeptides 9a–c.[31] Bn: benzyl; Boc: tert-butoxycarbonyl; Bt: benzo-
triazole; Cbz: benzyloxycarbonyl; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DIPEA: N,N-diisopropylethylamine.
variation in reactivity of N-acyl peptides. We have documented
the synthetic and computational investigation of N-acyl iso-
peptide ligations to form native peptides from nonterminal Trp
residues via 10- to 18-membered cyclic transition states.
peptides 9a–c, which were prepared by the usual coupling
and deprotection protocol (Scheme 1) under DMF/piperidine
conditions, N!N acyl migration occurred via a 10- to 12-mem-
bered cyclic TS to give Cbz-protected tripeptides 10a–c (44.4,
71.4, and 99.1%, respectively) as the ligation products
(Scheme 2). This N!N acyl chemical ligation occurs in Trp, one
of the important natural amino acids present in peptides and
proteins. This encouraged us to explore the area of chemical li-
gation by developing a general, high-yielding, and feasible
pathway to the synthesis of Trp-containing peptides via liga-
tion techniques.
Results and Discussion
Intermediate isodipeptide 4 was synthesized and served as the
starting material for the investigation of N!N acyl migration
via 10- to 18-membered cyclic transition states. In this study,
we aim to investigate a novel chemical ligation strategy for
N!N acyl transfer by developing a general methodology and
a computational model to predict the feasibility of N!N acyl
migration in peptide synthesis. Compound 4 was coupled with
the benzotriazolides of dipeptides 12a–o and tripeptides 17a–
c of a-, b-, or g-amino acids to afford isotetrapeptides 14a–o
and isopentapeptides 19a–c, which were required for the liga-
tion studies involving 13- to 18-membered cyclic transition
states. To enhance the migration rates, possibly by lowering
the steric hindrance at the ligation sites, we placed Gly, b-, and
g-amino acid units within the isopeptides in order to study the
feasibility of N!N acyl migrations in 13- to 18-membered
cyclic transition states. A statistical model was generated by
using conformation analysis and molecular descriptors.
Scheme 2. Chemical ligation of N-acyl isopeptides 9a–c in DMF/piperi-
dine.[31]
Preliminary results on N!N acyl migrations via 10- to 12-
membered cyclic TSs
Feasibility of N!N acyl migrations via a 13-membered
cyclic TS
N-acylation of the indole nitrogen atom in Trp was challenging
but was achieved when Boc-protected Trp (1) was treated with
Cbz-Ala-Bt (2) in MeCN in the presence of a strong base, such
as DBU, which resulted in the Boc-protected N-acyl isodipep-
tide 3 (80%; Scheme 1). Subsequent Boc deprotection was
conducted by using 4n HCl in dioxane solution to afford the
hydrochloride salt of the unprotected isodipeptide 4 (91%).
In our previous studies,[31] we tried chemical ligation experi-
ments on 4 and observed that chemical ligation via a 7-mem-
bered cyclic TS was not favored in either aqueous buffer or
basic DMF/piperidine conditions. However, in the longer iso-
The starting isotetrapeptides 14a–d for the N!N acyl transfer
via a 13-membered TS were prepared by a straightforward
coupling reaction. N-Acyl benzotriazoles are advantageous re-
agents to construct peptides, peptidomimetics, and peptide
conjugates.[32–34] Compound 4 and four different Boc-protected
dipeptide benzotriazolides 12a–d were first coupled in MeCN/
DIPEA to afford the Boc-protected isotetrapeptides 13a–d. No
chromatography was needed, and compounds 13 were puri-
fied by acidic and basic workups. Boc deprotection of the Trp-
containing isotetrapeptides in 4n HCl/dioxane afforded the
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HCl salts of the unprotected isotetrapeptides 14a–d
(Scheme 3). The amino acids Gly, Ala, Phe, and Pro
were chosen for the isotetrapeptide sequences to
enable a comparative study on the effect of the sub-
stituents at the chemical ligation site for the given
13-membered cyclic TS.
Table 1. Chemical ligation of N-acyl isopeptides 14a–d via 13-membered TSs.
Compd
Cyclic
TS
size
Yield
[%]
Relative area [%][b,c]
Product characterization[d]
ligated peptide (LP)
SM
LP
BA
(tR)
(tR)
(tR)
LP
[M+H]+[e]
14a
14b
14c
14d
13
13
13
13
87
88
84
84
39.11
(41.3)
27.20
(46.3)
94.99
(40.2)
83.05
(45.7)
60.89
(46.3)
72.80
(56.5)
5.01
(46.8)
16.95
(54.6)
0.00
0.00
0.00
0.00
15a
15b
15c
15d
614.3
Initially, the chemical ligation experiments were
carried out for 14a under aqueous conditions
(pH 7.4, 1m buffer strength, microwave irradiation,
508C, 50 W, 3 h) to produce the expected ligation
products. However, the expected ligation product
was observed in relatively low yield (2%). The ligation
experiments were then switched to basic piperidine/
DMF conditions (Scheme 4). To our delight, this re-
sulted in the expected ligation products, which, in
some cases, were produced in almost quantitative
yield (Table 1). In the cases of Phe- and Pro-contain-
ing isotetrapeptides 14c and 14d, the relative abun-
628.3
704.3
654.3
[a] Total crude yield of products isolated. [b] Determined by HPLC-MS (semiquantita-
tive). 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 (and corrected for starting ma-
terial). [c] SM: starting material; LP: ligated peptide; BA: bisacylated product; tR: reten-
tion time. [d] By HPLC-MS. [e] Found.
dances of ligated peptides 15c and 15d were 5 and 17%, re-
spectively. This result was expected, because it was anticipated
that the chemical ligation would be less feasible, due to steric
hindrance by the CH2Ph group in the case of 14c or, for 14d,
as a result of the Pro residue inducing a turn in the peptide
chain, which would result in too large a distance between the
reaction sites. HPLC/MS, with (ꢀ)ESI-MS/MS, confirmed the for-
mation of the ligated products 15a–d, because these produce
different MS fragmentation patterns from those of the initial
isotetrapeptides 14a–d. The relative abundances of the crude
ligated mixtures were analyzed by HPLC (Table 1).
Feasibility of N!N acyl migra-
tions via a 14-membered cyclic
TS
Coupling reactions between 4
and four different Boc-protected
dipeptide benzotriazolides 12e–
h were performed to study N!
N acyl transfer via a 14-mem-
bered TS. Boc deprotection of
tryptophan tetrapeptides 13e–h
in 4n HCl/dioxane gave the HCl
salts of the unprotected tetra-
peptides 14e–h, which were
chosen as potential substrates
Scheme 3. Synthesis of isotetrapeptides 14a–d for ligation studies through 13-membered TSs.
for the ligation study via a 14-
membered TS (Scheme 5). The
amino acids Gly, Ala, b-Ala, Phe,
and Pro and other amino acids
in the isotetrapeptide sequence
were chosen to enable a compa-
rative study on the effect of
chemical ligation between the
given 14-membered cyclic TSs
and the 13-membered cyclic TSs.
Chemical ligation via a 14-
membered cyclic TS was investi-
gated by subjecting isotetrapep-
tides 14e–h to microwave irradi-
ation at 508C and 50 W for 3 h
under basic piperidine/DMF con-
ditions (Scheme 6). The reaction
mixtures were cooled, the sol-
Scheme 4. Chemical ligation of N-acyl isopeptides 14a–d through 13-membered TSs.
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However, in the case of 14g
(Phe at the N terminus), the liga-
tion product was observed in
only 30% yield, and only 3% of
the ligated product 15h was ob-
served in case of 14h (Pro at the
N terminus). This is consistent
with our findings for 13-mem-
bered TSs for ligation products
with similar amino acids. HPLC/
MS, with (ꢀ)ESI-MS/MS, con-
firmed that the ligated products
15e–h each produced different
MS fragmentation patterns from
those of the initial isotetrapepti-
des 14e–h. The relative abun-
dances of the crude ligated mix-
tures as analyzed by HPLC are
shown in Table 2.
Scheme 5. Synthesis of isotetrapeptides 14e–h for ligation studies through 14-membered TSs.
Feasibility of N!N acyl migra-
tions via a 15-membered cyclic
TS
The initial isotetrapeptides for
the N!N acyl transfer via 15-
membered TSs were prepared
by following a similar protocol
to that in Scheme 5. Four differ-
Scheme 6. Chemical ligation of N-acyl isopeptides 14e–h through 14-membered TSs.
Table 2. Chemical ligation of N-acyl isopeptides 14e–h via 14-membered TSs.
ent Boc-protected dipeptide
benzotriazolides 12i–l were first
coupled with compound 4 in
MeCN/DIPEA to afford the Boc-protected isotetrapep-
tides 13i–l. Boc deprotection in 4n HCl/dioxane
gave the HCl salts of the free isotetrapeptides 14i–
l (Scheme 7).
Compd
Cyclic
TS
Yield
[%][a]
Relative area [%][b,c]
Product characterization[d]
ligated peptide (LP)
SM
LP
BA
size
(tR)
(tR)
(tR)
LP
[M+H]+[e]
Chemical ligation via a 15-membered cyclic TS was
investigated under similar conditions to those in
Scheme 4. The abundance of the expected ligation
products was low in most cases (Scheme 8). In the
case of the proline-containing isotetrapeptide 14l,
only 1% of the ligated product 15l was observed.
This result was probably due to a Pro-induced turn in
the peptide chain. HPLC/MS, with (ꢀ)ESI-MS/MS, con-
firmed that the ligated products 15i–l each produced
different MS fragmentation patterns from those of
the initial isotetrapeptides 14i–l. The relative abun-
dances of the crude ligated mixtures, as analyzed by
HPLC, are shown in Table 3.
14e
14 f
14g
14h
14
14
14
14
83
88
87
84
11.06
(39.3)
1.75
(48.0)
69.50
(40.3)
97.37
(44.8)
88.94
(45.7)
98.25
(56.5)
30.50
(47.3)
2.63
0.00
0.00
0.00
0.00
15e
15 f
15g
15h
628.3
642.3
718.3
668.3
(55.0)
[a] Total crude yield of products isolated. [b] Determined by HPLC-MS (semiquantita-
tive). 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 (and corrected for starting ma-
terial). [c] SM: starting material; LP: ligated peptide; BA: bisacylated product; tR: reten-
tion time. [d] By HPLC-MS. [e] Found.
vent was removed under reduced pressure, and the ligation
mixtures (1.0 mgmlꢀ1 in methanol) were analyzed by HPLC/
Feasibility of N!N acyl migrations via a 16- to 18-mem-
MS. The NH2 site of the unprotected N-acyl isotetrapeptides
14e–h attacked intramolecularly the amide carbonyl carbon
atom (C=O) linked to the indole nitrogen atom of Trp via a 14-
membered cyclic TS (Scheme 4) to give the ligation peptides
15e–h. Formation of the expected ligation products in the
cases of Gly and Ala (in 15e and 15 f) was almost quantitative.
bered cyclic TS
N!N acyl transfer via a 16- to 18-membered TS would facili-
tate the synthesis of longer peptides. Coupling reactions be-
tween 4 and three different Boc-protected tripeptide benzo-
triazolides, 17a–c, in MeCN in the presence of DIPEA (3 equiv)
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tion, 508C, 50 W, 3 h) to produce
the expected ligation products.
Ligation did not occur for 16-
membered
cyclic
transition
states (we recovered mainly the
starting material 19a). Com-
pounds 19b and 19c were mi-
crowave irradiated in basic pi-
peridine/DMF conditions (508C,
50 W, 3 h), and the reaction mix-
tures were analyzed by HPLC/
MS, which showed significant
amounts of ligated products
(Scheme 10). The abundance of
the expected ligation products
from 19b was 31% and from
19c was 21%. HPLC/MS, with
(ꢀ)ESI-MS/MS, confirmed that
the ligated products 20b and
20c each produced different MS
fragmentation patterns from
those of the initial isotetrapepti-
des 19b and 19c. The abundan-
ces of the crude ligated mixtures
as analyzed by HPLC are shown
in Table 4. On combining all of
our experimental results, we ob-
served that the intramolecular
N!N acyl migration is highly fa-
Scheme 7. Synthesis of isotetrapeptides 14i–l for ligation studies through 15-membered TSs.
Scheme 8. Chemical ligation of N-acyl isopeptides 14i–l in DMF/piperidine.
Table 3. Chemical ligation of N-acyl isopeptides 14i–l via 14-membered TSs.
vored
for
medium-ring-size
cyclic transition states, whereas
there is a decrease in the expect-
ed ligation products for larger-ring-size cyclic transi-
tion states.
Compd
Cyclic
TS
size
Yield
[%][a]
Relative area [%][b,c]
Product characterization[d]
ligated peptide (LP)
SM
LP
BA
(tR)
(tR)
(tR)
LP
[M+H]+[e]
Isolation of ligated product
14i
14j
14k
14l
15
15
15
15
85
90
86
87
8.29
91.71
(43.8)
46.40
(54.1)
4.13
(47.4)
0.86
(55.9)
0.00
0.00
0.00
0.00
15i
15j
15k
15l
642.3
(37.7)
53.60
(45.4)
95.87
(40.7)
99.14
(45.4)
The formation of ligated product 15 f from com-
pound 14 f was further confirmed by isolation with
semi-preparative HPLC and characterization by 1H
and 13C NMR spectroscopy, elemental analysis, and
656.3
732.3
682.3
1
analytical HPLC. The H NMR spectra showed a clear
difference in the d=11.00–7.00 ppm range (Figure 1).
The appearance of the new peak in the case of 15 f
[a] Total crude yield of products isolated. [b] Determined by HPLC-MS (semiquantita-
tive). 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 (and corrected for starting ma-
terial). [c] SM: starting material; LP: ligated peptide; BA: bisacylated product; tR: reten-
tion time. [d] By HPLC-MS. [e] Found.
1
at d=10.85 ppm in the H NMR spectrum (which was
absent in 14 f) indicated the formation of the desired
ligation product. This peak at d=10.85 ppm is a typi-
cal NH proton peak from the indole ring in a Trp
moiety and clearly confirmed the intramolecular N!
at 208C gave the Boc-protected N-acyl isopentapeptides 18a–
c (Scheme 9). Compounds 18 were purified by acidic and basic
workups, and no chromatography was required. The HCl salts
of the unprotected isopentapeptides 19a–c were achieved
upon Boc deprotection of N-acyl isopentapetides 18a–c in 4n
HCl/dioxane.
N acyl migration of Z-alanine to the N terminus to form native
peptide 15 f (Scheme 6).
Competitive ligation experiments
To further support the intramolecular nature of the chemical li-
gation of unprotected isopeptides 14a–o and 19a–c, we stud-
ied the chemical ligation of isotetrapeptide 14 f in the pres-
The chemical ligation experiments were performed first for
19a under basic piperidine/DMF conditions (microwave irradia-
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ence of 20 equivalents of dipep-
tide 22 (H-Gly-Gly-OMe) under
the same reaction conditions as
those of Scheme 6 (Scheme 11).
HPLC-MS analysis of the isolated
crude product (Scheme S72 in
the Supporting Information)
confirmed the formation of 20%
of the desired ligation product
15 f with a retention time at
48.0 min, along with 80% of the
starting material 14 f with a reten-
tion time at 56.5 min. None of
the bisacylated product 16 f was
observed. It was also observed
that there is no Cbz-protected
tripeptide 23 (which is the N-acy-
lated product of dipeptide 22
through an intermolecular liga-
tion pathway) in the HPLC-MS
analysis. This competitive experi-
ment supports the hypothesis
that the N!N acylation is intra-
molecular rather than intermolec-
ular.
Scheme 9. Synthesis of isopentapeptides 19a–c for ligation studies.
Scheme 10. Chemical ligation of N-acyl isopeptides 19a–c through 16- to 18-membered TSs.
Table 4. Chemical ligation of N-acyl isopeptides 19a–c in DMF/piperidine.
Computational analysis
Internal chemical ligations can be
considered mechanistically as intramolecular nucleo-
philic reactions between the amide carbonyl carbon
atom (C=O) linked to the indole nitrogen atom of Trp
and the unprotected N terminus (the distance be-
tween these two reactive termini is represented as
b(NꢀC)). A cyclic transition state is attained for this
transformation, and molecular structure is therefore
an important factor. The lowest energy conformer fa-
cilitates N!N acyl transfer due to the formation of
an amide bond in the ligated peptide. We applied
techniques previously employed[27,35,36] for similar li-
gation reactions including a full conformation search
followed by scoring of the conformers based on the
spatial distances between the reactive termini, b(Nꢀ
C). The full conformation search considered both ro-
tatable bonds and the phenyl ring of twenty-four re-
Compd
Cyclic
TS
size
Yield
[%][a]
Relative area [%][b,c]
Product characterization[d]
ligated peptide (LP)
SM
LP
BA
(tR)
(tR)
(tR)
LP
[M+H]+[e]
19a
19b
19c
16
17
18
83
86
87
100
0
0.00
0.00
0.00
20a
20b
20c
–
(49.4)
69.40
(53.9)
79.30
(53.8)
(NA)
30.60
(66.9)
20.70
(67.5)
739.3
753.3
[a] Total crude yield of products isolated. [b] Determined by HPLC-MS (semiquantita-
tive). 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 (and corrected for starting ma-
terial). [c] SM: starting material; LP: ligated peptide; BA: bisacylated product; tR: reten-
tion time; NA: no ligated product. [d] By HPLC-MS. [e] Found.
lated compounds, 9a to 20c (including Leu/Val-containing iso-
peptides, which were used for prediction purposes), and the
analysis was performed by using the MMX force field,[37] in
PCMODEL v.9.3 software.[38] The best preorganized conformer
for each compound, with the b(NꢀC) values, is shown in
1
Figure 1. Difference in the H NMR spectrum of isolated ligated peptide 15 f
Scheme 11. Competitive chemical ligation of N-acyl isopeptide 14 f in DMF/
(left-hand side) relative to the starting material 14 f (right-hand side).
piperidine.
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In general, the feasibility of ligation depends on
the spatial distance between the reactive sites, steric
hindrance, and hydrogen-bond distances.[27] The cal-
culated data with steric energy and heat of formation
for all twenty-four compounds 9a to 20c (including
Leu/Val-containing isopeptides, which were used for
prediction purposes) are given in Table S2 in the Sup-
porting Information. We extended our studies by
using the data in Table 5 to design a predictive
model with statistical techniques to correlate the rel-
ative abundance (ligated product%) with the quanti-
tative structure–activity/property relationship (QSAR/
QSPR).[45] The genetic algorithm linear regression
method was performed by using QSARINS soft-
ware,[46] which establishes a correlation between the
dependent variable (property/response is the relative
abundance of the ligated peptide) and the independ-
ent variables (molecular descriptors or factors). The
quality of the regression is reflected in the numerical
values of statistical parameters, including the coeffi-
cient of determination (R2), the standard error (s), the
Fisher criterion (F), the Student’s test (t), and the
cross-validation coefficient of the determination
(R2CV). These statistical parameters for the model are
shown in Table 6, the structure–activity relationship is
given by Equation (2) (in which g1 and g2 are b(NꢀC)
and the Balaban index, respectively), and the regres-
Table 5. Computational data for preorganized (conformer) compounds 9a to 20c.
Compd TS
size[a]
Sequence
Relative
b(NꢀC) Balaban
abundance[b] [ꢁ]
index
9a
9b
9c
14a
14b
14c
14d
–
10
11
12
H-Gly-Trp(Cbz-Ala)-OBn
H-b-Ala-Trp(Cbz-Ala)-OBn
H-Gaba-Trp(Cbz-Ala)-OBn
44.40
71.40
99.10
60.89
72.80
5.012
16.95
NA
4.013
1.437
1.444
1.447
1.454
1.456
1.244
1.253
1.455
1.448
1.444
1.443
1.221
1.236
1.437
1.426
1.430
1.428
1.198
1.218
1.417
1.405
1.281
1.231
1.206
3.171
3.010
3.245
3.328
4.845
3.995
3.253
3.188
2.996
2.978
3.731
4.526
3.233
3.555
3.126
3.655
4.045
4.324
3.887
3.562
4.911
3.743
4.090
13-G H-Gly-Gly-Trp(Cbz-Ala)-OBn
13-A H-Ala-Gly-Trp(Cbz-Ala)-OBn
13-F
13-P H-Pro-Gly-Trp(Cbz-Ala)-OBn
13-V H-Val-Gly-Trp(Cbz-Ala)-OBn
13-L
14-G H-Gly-b-Ala-Trp(Cbz-Ala)-OBn
14-A H-Ala-b-Ala-Trp(Cbz-Ala)-OBn
14-F
14-P H-Pro-b-Ala-Trp(Cbz-Ala)-OBn
14-V H-Val-b-Ala-Trp(Cbz-Ala)-OBn
14-L
15-G H-Gly-Gaba-Trp(Cbz-Ala)-OBn
15-A H-Ala-Gaba-Trp(Cbz-Ala)-OBn
15-F
15-P H-Pro-Gaba-Trp(Cbz-Ala)-OBn
15-V H-Val-Gaba-Trp(Cbz-Ala)-OBn
15-L
16
H-Phe-Gly-Trp(Cbz-Ala)-OBn
–
H-Leu-Gly-Trp(Cbz-Ala)-OBn
NA
14e
14 f
14g
14h
–
88.94
98.25
30.50
2.630
NA
H-Phe-b-Ala-Trp(Cbz-Ala)-OBn
–
H-Leu-b-Ala-Trp(Cbz-Ala)-OBn
NA
14i
14j
14k
14l
–
91.71
46.40
4.130
0.861
NA
H-Phe-Gaba-Trp(Cbz-Ala)-OBn
–
H-Leu-Gaba-Trp(Cbz-Ala)-OBn
H-Ala-Pro-Ala-Trp(Cbz-Ala)-OBn
H-Ala-Pro-b-Ala-Trp(Cbz-Ala)-OBn 30.60
NA
0
20a
20b
20c
17
18
H-Ala-Pro-Gaba-Trp(Cbz-Ala)-OBn 20.70
[a] The transition state size and amino acid code for the N-terminal residue. [b] The
relative abundance of the ligated product. NA: Test compound for model validation.
sion plot is shown in Figure 3. With reference to the generated
model, the spatial distance b(NꢀC) has 85% correlation with
the relative abundance, and the Balaban index has 71% corre-
lation with the relative abundance, results that are shown as
Table 5. For understanding of the colinearity between the cal-
culated 127 3D molecular descriptors by using PADEL descrip-
tors and the relative abundance of the ligated peptide, the
highly contributing descriptors, that is, the spatial distance,
b(NꢀC), and the Balaban index, are shown in Table 5. The Bala-
ban index estimates the isomeric discrimination ability of the
peptide.[39] Two of the preorganized 3D conformers with the
highest and lowest b(NꢀC) values for the 14-membered-TS
compounds 14 f and 14h are shown in Figure 2a and b, re-
spectively (3D representations for all TSs are given in Table S1
in the Supporting information). These results confirms that the
b(NꢀC) value is highly dependent on the amino acid sequence
chosen for the peptide chain.
To correlate the spatial distance, b(NꢀC), and the stabilities
of selected conformers, quantum chemical calculations were
carried out at the DFT/def-SVP level[40–42] of theory by using
Turbomole Software.[43,44] To determine whether these results
are due to the congestion offered by the steric energy, we cal-
culated those energy values by using molecular mechanics.
Steric energy can also influence the relative abundance of the
ligated product; thus, we determined the steric energy to pri-
oritize the conformers, in addition to the spatial distance, b(Nꢀ
C). The steric energy of the preorganized conformers (DES) is
derived as the difference between the global minimum energy
(Eglobal) and the energy of the best preorganized conformer
(Econformer), as shown in Equation (1).
Figure 2. a) Preorganized 3D representation with b(NꢀC) value for com-
pound 14 f. b) Preorganized 3D representation with b(NꢀC) value for com-
pound 14h.
DES ¼ EglobalꢀEconformer
ð1Þ
Chem. Eur. J. 2014, 20, 1 – 11
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Experimental validation of the model
Table 6. Statistical model for the relative abundance of the ligated pep-
tide.
To test the statistical model, three further compounds, 14m–o
(with Val at the N terminus), were synthesized by using a similar
protocol (Scheme 12). The amino acids Gly, b-Ala, and Gaba
were chosen for the isotetrapeptide sequence to afford a sys-
tematic investigation into the feasibility of chemical ligation
for the 13- to 15-membered cyclic TSs.
Factor[a]
Coefficient[b]
s[c]
t[d]
R2 =0.93, F=106.1, s=9.92, n=2, N=18, R2cvloo =0.90, R2adj =0.88
I
g1
g2
intercept
distance b(NꢀC)
Balaban index
0.1439
ꢀ36.143
134.304
ꢀ0.626
42.50
37.50
0.406
[a] The molecular descriptor based on the BMLR stepwise model. [b] Co-
efficients of the respective factors including the intercept. [c] Standard
error. [d] Student’s t test (t criterion).
After the ligation experiment was carried out under similar
conditions to those used previously (Scheme 12), the crude re-
action mixture was analyzed by HPLC-MS. The expected ligated
products 15m–o each produced different MS fragmentation
patterns from those of the starting isotetrapeptides 14m–o,
and the relative abundances of the ligated peptides are shown
in Table 8. The experimental and predicted abundances of the
peptides were not a quantitative match, but a qualitative cor-
relation was observed. These results indicate that the 13- and
14-membered cyclic TSs are more feasible than the 15-mem-
bered cyclic TS. This was consistent with our findings with all
other compounds for the 13- to 15-membered cyclic TSs
(Table 7).
The chemical ligation through an intramolecular N!N acyl
transfer to form the native peptides was highly favorable. In-
tramolecular chemical ligation occurs smoothly in smaller (10-
to 12-membered cyclic TS) isotripeptides. It is also highly fa-
vored in medium-sized (13- to 15-membered cyclic TS) isote-
trapeptides, but it is highly dependent on the peptide se-
quence and spatial distance, b(NꢀC). If the terminal amino
acids are Phe and Pro, the large steric bulk of the substituents
on those amino acids force the two reactive sites apart. If the
Figure 3. Correlation plot for the relative abundance model of ligated prod-
uct in Table 7.
Table 7. Experimental and predicted relative abundances of ligated products.
Compd Cyclic TS size[a] Ligated peptide sequence Exptl[b] Calcd[c] Diff.
regression plots in Figures S1 and S2 in the Support-
ing Information.
9a
9b
9c
14a
14b
14c
14d
–
10
11
12
Cbz-Ala-Gly-Trp-OBn
Cbz-Ala-b-Ala-Trp-OBn
Cbz-Ala-Gaba-Trp-OBn
44.40
71.40
99.10
60.89
72.80
5.012
16.95
NA
45.67
79.50
85.98
78.14
75.41
7.90
24.03
77.29
77.57
85.79
86.31
29.28
2.56
1.27
8.1
Relative abundance ¼ 0:1439
þððg1 ꢁ ꢀ36:143 ꢂ 0:62Þ þ ðg2 ꢁ 134:304 ꢂ 0:4ÞÞ
13.12
17.25
2.61
2.88
7.08
NA
ð2Þ
13-G
13-A
13-F
13-P
13-V
13-L
14-G
14-A
14-F
14-P
14-V
14-L
15-G
15-A
15-F
15-P
15-V
15-L
16
Cbz-Ala-Gly-Gly-Trp-OBn
Cbz-Ala-Ala-Gly-Trp-OBn
Cbz-Ala-Phe-Gly-Trp-OBn
Cbz-Ala-Pro-Gly-Trp-OBn
Cbz-Ala-Val-Gly-Trp-OBn
Cbz-Ala-Leu-Gly-Trp-OBn
Cbz-Ala-Gly-b-Ala-Trp-OBn
Cbz-Ala-Ala-b-Ala-Trp-OBn
Cbz-Ala-Phe-b-Ala-Trp-OBn
Cbz-Ala-Pro-b-Ala-Trp-OBn
Cbz-Ala-Val-b-Ala-Trp-OBn
Cbz-Ala-Leu-b-Ala-Trp-OBn
Cbz-Ala-Gly-Gaba-Trp-OBn
Cbz-Ala-Ala-Gaba-Trp-OBn
Cbz-Ala-Phe-Gaba-Trp-OBn
Cbz-Ala-Pro-Gaba-Trp-OBn
Cbz-Ala-Val-Gaba-Trp-OBn
Cbz-Ala-Leu-Gaba-Trp-OBn
Cbz-Ala-Ala-Pro-Ala-Trp-OBn
In this present study, the percentage of ligated
peptide increases with a) shorter spatial b(NꢀC) dis-
tance and b) higher Balaban index.
–
NA
NA
14e
14 f
14g
14h
–
88.94
98.25
30.50
2.630
NA
NA
91.71
46.40
4.130 14.84
0.861
NA
NA
0
3.15
11.94
1.22
0.07
NA
By using the Equation (2) model, the relative abun-
dance of ligated product for the twenty-four com-
pounds, 9a to 20c (including Leu/Val-containing iso-
peptides, which were used for prediction purposes),
78.21
62.17
79.21
59.83
–
NA
14i
14j
14k
14l
–
12.5
13.43
10.71
6.579
NA
was calculated. The cross-validation coefficient (R2
)
cv
was very close to the regression coefficient (R2),
which resembles the quality of the model. To our de-
light, the experimental relative abundances of the li-
gated peptides for compounds 9a, 14b, 14c, 14e,
14g, 14h, and 20b were found to be very close to
the predicted values. The experimental relative abun-
dances of ligated product for the remaining com-
pounds were close to the predicted values, and the
results are shown in Table 7.
7.44
49.96
60.10
5.31
30.19
14.29
–
NA
20a
20b
20c
5.31
0.41
6.41
17
18
Cbz-Ala-Ala-Pro-b-Ala-Trp-OBn 30.60
Cbz-Ala-Ala-Pro-Gaba-Trp-OBn 20.70
[a] The transition state size and amino acid code for the N-terminal residue. [b] Experi-
mental relative abundance of ligated peptide. NA: Test compound for model valida-
tion. [c] Predicted relative abundance of ligated peptide based on the model.
&
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8
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Acknowledgements
We thank the University of Flori-
da and the Kenan foundation,
and we also acknowledge King
Abdulaziz University for technical
and financial support of this
work. We thank Dr. C. D. Hall, Mr.
Wang, and G. Valuvenko for
helpful discussions. G.G.P. is
grateful to the European Social
Fund under project 1.2.0401.09-
0079 supported by graduate
school “Functional materials and
technologies”.
Scheme 12. Synthesis of 14m–o and chemical ligation to validate the statistical model.
Table 8. Chemical ligation of N-acyl isopeptides 14m–o in DMF/piperi-
Keywords: acyl migration · chemical ligation · peptides ·
transition states · tryptophan
dine.
Compd Cyclic Yield Relative area [%][b,c] Product characterization[d]
TS
size
[%][a] SM
LP
(tR)
BA
(tR)
ligated peptide (LP)
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14o
13
14
15
83
88
87
2.50
(48.1) (57.1)
1.80
(46.8) (57.2)
91.65 8.35
(47.2) (57.6)
97.5
0.00 15m
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656.3
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We have demonstrated an efficient and convenient synthesis
of novel N-acyl isopeptides containing Trp residues. The subse-
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migration was favored through a long-range 10- to 18-mem-
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an auxiliary group at the ligation site. A statistical model was
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Received: January 12, 2014
Published online on && &&, 0000
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FULL PAPER
Going the distance: Chemical ligations
to form native peptides from N!N acyl
migrations in Trp-containing peptides
via 10- to 18-membered cyclic transition
states are described (see scheme; Bn:
benzyl; Z: benzyloxycarbonyl). The liga-
tions are achieved without the use of
Cys/Ser/Tyr residues or an auxiliary
&
Peptide Synthesis
S. Biswas, R. Kayaleh, G. G. Pillai, C. Seon,
I. Roberts, V. Popov, K. A. Alamry,
A. R. Katritzky*
&& – &&
Long-Range Chemical Ligation from
N!N Acyl Migrations in Tryptophan
Peptides via Cyclic Transition States of
10- to 18-Members
group. This new approach represents
a significant development in the field.
Chem. Eur. J. 2014, 20, 1 – 11
www.chemeurj.org
11
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ