S- to N-Acyl transfer in S-acylcysteine isopeptides via 9-, 10-, 12-, and 13-membered cyclic transition states

Article abstract of DOI:10.1002/psc.2438

S-Acyl cysteine peptides containing α-, β- or γ-amino acid residues undergo long-range S- to N-acyl transfer to give analogs of native tripeptides and tetrapeptides containing additional carbon atoms in the chain. The ease of intramolecular S→N-acyl transfer relative to intermolecular transacylation is favored increasingly for 9<12<13~10-membered cyclic transition states; the observed order is explained on conformational and intermolecular interaction considerations.

Full text of DOI:10.1002/psc.2438

Research Article
Received: 8 May 2012
Revised: 5 July 2012
Accepted: 6 July 2012
Published online in Wiley Online Library: 12 October 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2438
S- to N-Acyl transfer in S-acylcysteine
isopeptides via 9-, 10-, 12-, and
13-membered cyclic transition states
Oleg Bol’shakov,a Judit Kovacs,a Mamta Chahar,a Khanh Ha,a
Levan Khelashvilia and Alan R. Katritzkya,b*
S-Acyl cysteine peptides containing a-, b- or g-amino acid residues undergo long-range S- to N-acyl transfer to give analogs of
native tripeptides and tetrapeptides containing additional carbon atoms in the chain. The ease of intramolecular S ! N-acyl
transfer relative to intermolecular transacylation is favored increasingly for 9 < 12 < 13 ~ 10-membered cyclic transition states;
the observed order is explained on conformational and intermolecular interaction considerations. Copyright © 2012 European
Peptide Society and John Wiley & Sons, Ltd.
Supporting information can be found in the online version of this article.
Keywords: S- to N-acyl transfer; peptides; cysteine; N-acylbenzotriazoles; S-acylation
peptides. Subsequently [29], we found that 8-membered transition
states are clearly disfavored, whereas the 11- and 14-membered
Introduction
Native chemical ligation (NCL) is an important two-step transfor-
mation of peptide and protein thioesters, comprising (i) chemo-
selective transthioesterification of a C-terminal thioester with
N-terminal cysteine moiety followed (ii) by S- to N-acyl rearrange-
ment to form a native amide bond [1–4]. Recent significant
achievements of NCL include the total chemical synthesis of both
post-translationally modified and unmodified proteins [5],
segmental isotopic labeling of proteins for NMR studies [6,7],
and the formation of peptide conjugates with macromolecules
such as peptide nucleic acid [8,9] and DNA [10]. NCL has been
particularly useful for assembling proteins that can probe biomo-
lecular processes in vitro [5,11,12] and in vivo [13–15].
transition states are relatively favored for ligations in S-acyl isopep-
tides containing non-terminal cysteine residues. The present work
studies ligation via other transition state sizes by replacing one or
more a-amino acid units by b- or g-aminoacyl units. We wish to
identify sequence and geometry requirements that enable long-
range acyl migration by study of ‘chemical ligations1’ of isotripep-
tides and isotetrapeptides containing a, b, or g-amino acid units
via 9-, 10-, 12- and 13-membered cyclic transition states.
Results and Discussion
The Feasibility of S-Acyl Monoisotripeptide2 7 to Undergo
S ! N-acyl Migration via 9-Membered Cyclic Transition State
One major limitation of NCL was its restriction to peptide chains
possessing cysteine at the N-terminus. The relative rarity of cyste-
ine (1.3% average content in proteins) has encouraged attempts
to overcome this limitation. The requirement of a cysteine residue
can be avoided by use of ligation auxiliaries [16,17], but these can
be difficult to remove, and synthetic strategies using removable
cysteine mimics can sterically hinder the ligation [18,19]. The
possibility of long-range acyl migration was demonstrated by
Kemp et al. utilizing a 4-mercapto-6-oxydibenzofuran template
for thiol capture [20–24], Wong group in sugar-assisted glycopep-
tide ligation [25], and Haase and Seitz utilized internal cysteine
residues to accelerate long-range thioester-based peptide
ligations but required relatively long reaction times (48–72 h)
and did not isolate or characterize ligation products [18].
We prepared starting monoisotripeptide 7 for study of S ! N-acyl
migration via 9-membered cyclic transition state as illustrated
*
Correspondence to: Alan R. Katritzky, Center for Heterocyclic Compounds,
Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA.
E-mail: katritzky@chem.ufl.edu
a Center for Heterocyclic Compounds, Department of Chemistry, University of
Florida, Gainesville, FL 32611-7200, USA
b Chemistry Department, King Abdulaziz University, Jeddah, 21589 Saudi Arabia
We previously demonstrated long-range S ! N-acyl shifts of
S-acylated cysteine peptides via 11- and 14-membered cyclic tran-
sition states [26,27]. Cysteine-containing peptides were selectively
S-acylated by N-acylbenzotriazoles [28] under mild reaction
conditions [26,27]. Deprotection of the resulting N-Fmoc-S-acyl
C-terminal cysteine tripeptides and tetrapeptides afforded S-acyl
isopeptides possessing free carboxyl groups that underwent micro-
wave-assisted chemical ligations (1 h, 50 ꢀC, at pH 8.2) to form native
1The term ‘chemical ligation’ of isopeptide is sometimes used for non-native
chemical ligations from S-acyl peptides to furnish native peptide analogs in
a stepwise approach. A cysteine-containing peptide is converted to an S-acyl
peptide, deprotected, and then transferred to a native peptide analog by a
subsequent S- to N-acyl migration. In contrast to native chemical ligation, this
isopeptide methodology allows the isolation and characterization of the S-acyl
peptides and migration of the S-acyl group in an independent step.
2The term ‘monoiso’ is used to indicate that the S atom has just one amino acid unit
attached to it; similarly, ‘diiso’ would indicate two amino acid units attached to S.
J. Pept. Sci. 2012; 18: 704–709
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

LONG-RANGE INTRAMOLECULAR S- TO N-ACYL TRANSFERS
Scheme 1. Chemical ligation of S-acyl monoisotripeptide 7.
in Scheme 1. Boc-protected b-alanine 1 was converted into
the corresponding benzotriazolide 2 by the standard method
[28]. L-Cysteine was reacted with the Boc-b-alanine benzotria-
zolide 2 in aqueous acetonitrile (MeCN/H2O, 7/3) containing
1 equiv of Et3N for 1 h at 20 ꢀC to give the Boc-b-alanyl-
cysteine dipeptide 4 (68%). Subsequent S-acylation of 4 with
Z-L-Ala-Bt 5 at room temperature in the presence of KHCO3
Chemical ligation via the 9-membered cyclic transition state
was attempted by microwave irradiation of a solution of 7 at
a 2 mM concentration in 0.4M NaH2PO4/Na2HPO4 buffer (pH = 8.2)
and acetonitrile (24 : 1) for 4 h at 50 ꢀC. However, HPLC-MS
(ESI) analysis of the reaction mixture (Scheme 1, Table 1)
revealed that the major product 9 arose from intermolecular
disproportionation 7 ! 9, thus indicating that intermolecular
aminolysis of one molecule thioester 7 by another (7 + 7 ! 9) is
favored over intramolecular attack through a 9-membered ring.
Ligated product 8 was also present (Table 1) but was formed in only
4% yield.
furnished the Boc-protected S-acyl monoisotripeptide
6
(70%), and Boc-group deprotection of 6 with dioxane satu-
rated with hydrochloric acid gas formed S-acyl monoisotripep-
tide 7 as its hydrochloride salt.
Table 1. Chemical ligation of S-acyl isopeptides 7, 17, 23, and 28
Cyclic
TS
Total
crude
yield (%)
of
Product and relative amounts
of each product (%)c,d
Product characterization by HPLC-MS
Structure
[M + H]+ found
TA product
size/b
(N-C)a
R
LM
LD
TA
Ligated peptide
monomer (LM)
Ligated peptide
dimer (LD)
Structure
[M + H]+
found
products
isolatedb
9/3.32
85
82
91
87
7 (5)
17 (3)
23 (0)
28 (1)
8 (4)
18 (12)
24 (0)
29 (5)
10 (0)
9 (91)
19 (17)
25 (54)
30 (17)
8
18
24
29
397
411
454
468
792
820
906
934
9
19
25
30
602
616
659
673
10/3.19
12/3.18
13/3.43
20 (68)
26 (46)
31 (77)
R, recovered reactant; LM, ligation monomer; LD, ligation dimer; TA, transacylation; TS, Transition state.
aThe distance between the terminal amino group and the thioester carbon atom.
bThe combined crude yield was calculated according to the following equation: combined crude yield = [(ligated peptide) + 2 ꢁ (transacylation
product)]/(starting material).
cDetermined by HPLC-MS semi-quantitative. 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.
dAmounts are corrected for LD = 2 mmol, LM = 1 mmol.
J. Pept. Sci. 2012; 18: 704–709 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

BOL’SHAKOV ET AL.
Demonstration of S ! N-Acyl Migration in S-Acyl
Monoisotripeptide 17 to Give Native Tripeptide Analog 18
via a 10-Membered Cyclic Transition State
Demonstration of S ! N-Acyl Migration in S-Acyl
Monoisotetrapeptide 22 to Give Native Tetrapeptide
Analog 23 via a 12-Membered Cyclic Transition State
To study chemical ligation via 10-membered cyclic transition
state, we coupled Boc-anhydride 11 with g–aminobutyric acid
12 following a literature procedure [29] (Scheme 2) and con-
verted the resulting Boc-g-aminobutyric acid 13 (87%) into the
corresponding novel Boc-aminoacylbenzotriazolide 14 (80%).
Coupling 14 with L-cysteine in CH3CN–H2O in the presence of
Et3N for 3 h at 20 ꢀC gave the Boc-protected cysteine dipeptide
15 (64%), which on S-acylation with Z-L-Ala-Bt 5 in aqueous
acetonitrile, in the presence of one equivalent of KHCO3 afforded
the S-acyl monoisotripeptide 16 (71%). Boc-group deprotection
of 16 using HCl(g) in dioxane gave amino-S-acylated monoisotri-
peptide 17 as its HCl salt.
To investigate chemical ligation, a suspension of 17 in
NaH2PO4/Na2HPO4 buffer at pH = 8.2 was subjected to micro-
wave irradiation at 50 ꢀC for 4 h. HPLC-MS (ESI) analysis revealed
the major reaction products as the expected ligation monomer
18, together with its dimer 20 as evidenced by m/z 820
[M + H]+. MS allows unambiguous distinction between the
ligation product 18 and starting tripeptide 17 (molecular ion
[M + H]+ : m/z = 411) and dimer 20. Small peptides containing a
cysteine residue easily dimerize to the corresponding disulfide
dimer in the absence of reducing agent [30]. The ratio of ligated
products (18 + 20) : intermolecular transacylation product 19 was
80 : 17 (Table 1). Demonstrating that intramolecular nucleophilic
attack by the amino group on the thioester group of 17 through
a 10-membered cyclic transition state is preferred over intermo-
lecular acylation between two molecules of 17 to give 19
(Scheme 2).
We investigated S ! N-acyl migration via 12-membered cyclic
transition state using S-acyl monoisotetrapeptide 22; coupling
N-terminal amino-unprotected S-acyl monoisotripeptide 7 with
Boc-Gly-Bt 21 afforded 22. Subsequent deprotection of the Boc
group in 22 then gave S-acyl monoisotetrapeptide 23 (Scheme 3).
The intramolecular S ! N-acyl migration experiment 23 ! 24
would proceed through a 12-membered-ring transition state
(Figure 1B).
The chemical ligation experiment was carried out at 2 mM
concentration of 23 under microwave irradiation at 50 ꢀC for
4 h. Examination of the crude product mixture by HPLC-MS
(ESI) revealed two major products in 54 : 46 ratio (Table 1).
The most abundant (+) ESI-MS peak m/z 929 [M + Na]+ was
the Na+ coordinated oxidized disulfide dimer 26 of the
pseudo native tetrapeptide analog 24 (MW 906) ion formed
by successful ligation of 23. The second major product was
the intermolecular transacylation product 25 with MS (ESI)
659 (Table 1). The mixture of 26 : 25 in 54 : 46 ratio was puri-
fied by semi-preparative HPLC that enabled the isolation and
the characterization of the native peptide analog 26. Ligated
product 26 was then further characterized by analytical HPLC
and High Resolution Mass Spectrometry (HRMS) analysis (Figure 2).
These results indicate that chemical long-range ligation (23! 24)
via a 12-membered cyclic transition state is feasible and could be
a promising approach for the synthesis of native peptide analogs.
It is also in agreement with Kemp’s work [23,24] for successful
S ! N-acyl shift in thiol capture ligations proceeding through
transition states with 12-membered rings (Figure 1A).
Scheme 2. Chemical ligation of S-acyl monoisotripeptide 17.
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LONG-RANGE INTRAMOLECULAR S- TO N-ACYL TRANSFERS
Scheme 3. Chemical ligation of S-acyl monoisotetrapeptide 23.
NaH2PO4/Na2HPO4 buffer at pH 8.2. HPLC-MS (ESI) analysis of
the crude ligation mixture revealed the successful ligation of 28
via a 13-membered cyclic transition state. Ligation product 29 was
the major component (82%) (molecular ion of the disulfide dimer
[M + H]+ m/z 934, versus 468 for the [M + H]+ molecular ion of the
starting S-(Z-Ala)tripeptide 28). The HPLC-MS (ESI) also confirmed
that substantial amount of 30 was formed by intermolecular transa-
cylation (Table 1, Scheme 4). Thus, the feasibility of long-range acyl
migration via the 13-membered cyclic transition state is confirmed
favorable in complete agreement with our previous study [31]. The
findings of this investigation hence offer prospects for a convergent
assembly of peptides and proteins with b-amino acid architecture.
Figure 1. (A) 12-Membered cyclic transition state proposed by Kemp
[23,24] for thiol capture ligations. (B) Cyclic transition state needed for
chemical ligation of isopeptide 23.
Demonstration of S ! N-Acyl Migration in S-Acyl
Monoisotetrapeptide 28 to Yield 29 via a 13-Membered
Cyclic Transition State
Computational Analysis
The varying regioselectivity demonstrated earlier in the S- to N-acyl
transfer reactions of isopeptides 7, 17, 23, and 28 is rationalized by
analyzing the cyclic transition states required to be formed by the
structures to undergo internal S ! N-acyl shift. It follows from
obvious steric considerations that larger rings are easier to form
Coupling of N-terminal amino-unprotected S-acyl monoisotripep-
tide 7 with Boc-b-Ala-Bt 2 gave 27. Deprotection of the Boc
group in 27 produced S-acyl monoisotetrapeptide 28 (Scheme 4),
which was subjected to microwave irradiation at 50 ꢀC for 4 h in
Figure 2. HPLC analysis of the disulfide dimer 26.
J. Pept. Sci. 2012; 18: 704–709 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

BOL’SHAKOV ET AL.
Scheme 4. Chemical ligation of S-acyl monoisotetrapeptide 28.
because the thermodynamic penalty for cyclization gradually
decreases as the ring size increases. On the basis of this basic
assumption, one would expect isopeptides 7, 17, 23, and 28
(forming 9-, 10-, 12-, and 13-membered transition states, respec-
tively) to be ordered as 7 < 17< 23< 28 with regard to their ability
to undergo internal S- to N-acyl transfer relative to intermolecular
transacylation. But the experimentally found order is somewhat
different: 7 < 23< 28ꢂ 17, inferring some other structural features
to influence the reactivity. To rationalize the observed reactivity, we
applied a computational protocol [32] previously designed by us
for the elucidation of similar S- to N-acyl transfer reactions [26,27].
This protocol includes a full conformational search and subse-
quently scoring of the generated conformers against a proximity
scoring function. The distance between the terminal amino group
and the thioester carbon atom denoted as b(N-C) is employed here
as such a function, because it readily measures the conformational
preorganization for the nucleophilic attack.
Full conformational search of isopeptide structures 7, 17, 23, and
28 was performed using the MMX force field, as implemented
in PCModel v.9.3 software [33]. As a result, 3–700 conformers
were generated for each isopeptide, which were subsequently
ranked in the ascending order of the b(N-C) proximity function.
The best b(N-C) scores for structures 7, 17, 23, and 28 are 3.32,
3.19, 3.18, and 3.43 , respectively. It is seen that the geometrical
proximity well reflects the better preorganization of the
10-membered ring forming g-amino acid terminus (17) over
the 9-membered ring forming b-amino acid terminus (7), in
accord with the steric arguments. But for structures 23 and 28,
the proximity function taken alone does not fully elucidate the
experimental reactivity. However, a closer inspection of the
structures of the best preorganized conformers reveals a possi-
ble explanation for that: NH–p interactions. The short contacts
(2.7–2.8 ) between the N–H bonds and the plane of the Z p-system
displayed in Figure 3 can be identified as a type of intermolecular
interaction, which frequently occurs in proteins.
Figure 3. Best preorganized conformers of 7, 17, 23, and 28 (upside
down) with NH–p contact visualized.
where one sees an amide NH–p contact capable to provide an
additional stabilization to the preorganized conformer. But in
the 9-, 12-, and 13-membered ring forming structures 7, 23,
and 28, respectively, the NH–p contacts are quite different: they
involve not the neighboring amide NH bond as in 17 but those
belonging to the terminal amino group. Such engagement some-
what locks the amino group bound to the Z phenyl ring and
makes it less available for the nucleophilic attack on the thioester
group. Thus, a combination of steric reasons and NH–p interac-
tions explains the observed reactivity of the title reaction.
In some situation, particularly if amide NH bonds are involved,
the NH–p interactions can be rather strong, up to 6 kcal/mol [34].
This is the case in the 10-membered ring forming structure 17,
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LONG-RANGE INTRAMOLECULAR S- TO N-ACYL TRANSFERS
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Conclusions
Stable, amino-unprotected S-acyl-monoisotri- and S-acyl-monoisotetra-
cysteine-peptides containing a-, b- and/or g-amino acid residues un-
dergo chemical ligations in which the cysteine S-acyl groups migrate
to the N-terminal amino acids via 9, 10-, 12-, and 13-membered cyclic
transition states to form the corresponding native tripeptide and tet-
rapeptide analogs. We have already demonstrated [32] by quantum
chemical calculations that cross-transition state H-bonding and tran-
sition state size can favor or disfavor the intramolecular reaction.
Now, we see that not only hydrogen bonding but also NH–p interac-
tions can affect regioselectivity of the S! N-acyl transfers. The
present work describing a range of novel long-range S- to N-acyl
migrations offers evidence that relates to the challenging problem
of successfully coupling large peptides and peptide analog fragments.
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Experimental
General Methods
Details of the syntheses and transformations with related analytical
data are reported in the Supplementary Information. Melting
points were determined on a capillary point apparatus equipped
with a digital thermometer. NMR spectra were recorded in CDCl3,
1
DMSO-d6 or CD3OD-d4 operating at 300 MHz for H and 75 MHz
for 13C with TMS as an internal standard. All microwave-assisted
reactions were carried out with a single mode cavity CEM Discover
microwave synthesizer. The reaction mixtures were transferred into
a 10-ml glass pressure microwave tube equipped with a magnetic
stirrer bar. The tube was closed with a silicon septum, and the
reaction mixture was subjected to microwave irradiation (Discover
mode; run time: 60 s; PowerMax-cooling mode).
Acknowledgements
We thank the University of Florida, the Kenan Foundation, and
King Abdulaziz University, Jeddah, Saudi Arabia for financial sup-
port. We thank Dr C. D. Hall and Dr E. Todadze for helpful discus-
sions and Dr J Johnson for help in HPLC-MS analyses.
27 Katritzky AR, Tala SR, Abo-Dya NE, Ibrahim TS, El-Feky SA, Gyanda K,
Pandya KM. Chemical ligation of S-acylated cysteine peptides to form
native peptides via 5-, 11-, and 14-membered cyclic transition states.
J. Org. Chem. 2011; 76: 85–96.
28 Katritzky AR, Angrish P, Todadze E. Chiral acylation with N-(protected
a-aminoacyl)benzotriazoles for advantageous syntheses of peptides
and peptide conjugates. Synlett 2009; 15: 2392–2411.
29 Hansen FK, Ha K, Todadze E, Lillicotch A, Frey A, Katritzky AR. Microwave-
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cysteine residues. Org. Biomol. Chem. 2011; 20: 7162–7167.
30 Nam N, Kim Y, You Y, Hong D, Kimb H, Ahn B. Water soluble pro-
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J. Pept. Sci. 2012; 18: 704–709 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci