Traceless chemical ligations from O-acyl serine sites

Article abstract of DOI:10.1039/c2ob07050b

Chemical ligation via O- to N-acyl transfer of O-acylated serine containing peptides affords serine containing native peptides via 8- and 11-membered cyclic transition states opening the door to a wide variety of potential applications to peptide elaborat

Full text of DOI:10.1039/c2ob07050b

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Cite this: Org. Biomol. Chem., 2012, 10, 4836
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COMMUNICATION
Traceless chemical ligations from O-acyl serine sites†
Mirna El Khatib,a Mohamed Elagawany,a,b,c Farukh Jabeen,a,d Ekaterina Todadze,a Oleg Bol’shakov,a
Alexander Oliferenko,a Levan Khelashvili,a Said A. El-Feky,b Abdullah Asirie and Alan R. Katritzky*a,e
Received 7th December 2011, Accepted 11th April 2012
DOI: 10.1039/c2ob07050b
remove.10–16 Another approach to overcome this limitation
Chemical ligation via O- to N-acyl transfer of O-acylated
involves the conversion of a cysteine residue into a serine
residue after NCL,10b,c however, this requires post-NCL modifi-
cations after NCL peptide synthesis.
serine containing peptides affords serine containing native
peptides via 8- and 11-membered cyclic transition states
opening the door to a wide variety of potential applications
to peptide elaboration. The feasibility of these traceless
chemical ligations is feasible as supported by computation.
To address these limitations, our group developed17–19 liga-
tions of S-acylated cysteine peptides to form native peptides
through expanded transition states with 11- and 14-membered
rings. The developed methodology required no auxiliary groups
and enabled the selective S-acylation of cysteine peptides by
N-acylbenzotriazoles in good yields and under mild conditions
followed by microwave-assisted chemical ligations of S-acyl
isopeptides. However, the challenge of ligation through 8-mem-
bered transition state and the low abundance of cysteine remained
an obstacle. Our current approach to this limitation is therefore
focused on serine which possesses the 1,2-hydroxylamine bifunc-
tionality7 (mimicking the SH/NH2 bifunctionality of cysteine) and
thus offers the possibility of chemoselective ligations by O- to
N-acyl transfer without the need of cysteine residues.
The total chemical synthesis of proteins has already greatly con-
tributed to knowledge of the relationship of protein structure to
their function in important biological processes.1–5 The develop-
ment of chemical ligation has facilitated the synthesis of large
peptides by linking the C-terminus of one unprotected peptide
with the N-terminus of another.1–3 Native chemical ligation
(NCL), first reported by Wieland et al.6 and later developed by
Kent,1,2 is a chemoselective and regioselective reaction of a
peptide-thioester and a terminal Cys-peptide that results in a
native amide bond at the ligation site through a rapid NCL S- to
N-acyl transfer via a cyclic transition state.1–3 The bifunctional
nature of the N-terminal cysteine 1,2-mercaptoamine moiety is
responsible for the observed chemoselectivity in NCL.7
While of great importance, NCL has limitations that include
the requirement of a N-terminal cysteine residue at the ligation
site to afford a peptide containing an internal cysteine. The low
abundance of cysteine in human proteins (1.7% of the residues)
also presents another limitation.8–10 In attempts to overcome the
limitation of low abundance of cysteine, considerable effort has
been devoted to developing thiol auxiliary groups, but such liga-
tions were found: (i) difficult to complete due to steric
hindrance10–16 and (ii) problematic since extraneous groups were
present in the ligated product which can be troublesome to
Initially, two problems existed for the acylation of the
hydroxyl group of serine: (i) difficulty in achieving O-acylation
without epimerization (especially in solid-phase synthesis)4,5 and
(ii) the facile hydrolysis of O-acyl serine ester linkages20 under
the aqueous conditions of classical NCL. These problems were
successfully overcome by our recently reported methodology for
the preparation of chirally pure O-acyl isopeptides in a single
step (74–91%) and under anhydrous conditions.20
Kiso et al.5 had demonstrated that O-acyl residues within a
backbone significantly altered the secondary native peptide struc-
ture. “O-Acyl isopeptides” are more hydrophilic and easier to
purify by HPLC5 than their corresponding native peptides.
N-Terminal serine isopeptides rapidly generate, by O→N intra-
molecular acyl migration, the corresponding native peptide via a
5-membered transition state (Fig. 1).21
aCenter for Heterocyclic Compounds, Department of Chemistry,
University of Florida, Gainesville, FL 32611-7200, USA.
E-mail: katritzky@chem.ufl.edu
We now demonstrate that this classic O- to N-acyl shift via a
5-membered transition state can be extended to eight-membered
bDepartment of Pharmaceutical Organic Chemistry, Faculty of
Pharmacy, Zagazig University, Zagazig-44519, Egypt
cDepartment of Organic Chemistry, College of Pharmacy, Misr
University for Science and Technology, Al-Motamayez District, 6th of
October, P.O. Box: 77, Egypt
dDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320,
Pakistan
eDepartment of Chemistry, King Abdulaziz University, Jeddah, 21589,
Saudi Arabia
†Electronic supplementary information (ESI) available: 1H and 13C
NMR for all compounds and HPLC/Mass-Spectrometry spectra for com-
pounds 4, 5, 7 and 8. See DOI: 10.1039/c2ob07050b
Fig. 1 O-Acyl isopeptide methodology.21
4836 | Org. Biomol. Chem., 2012, 10, 4836–4838
This journal is © The Royal Society of Chemistry 2012

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and eleven-membered transition states. Thus, “traceless” chemi-
cal ligation involving O- to N-acyl shift (at a Ser site) involving
neither cysteine nor an auxiliary group at the ligation site has
been achieved.
were coupled with Pg′′-Gly-Bt to give 6a–b which after depro-
tection of the protecting group Pg′′ provided O-acyl isotripep-
tides 7a–b (Scheme 3).
Intermediates 7a–b underwent ligation (Scheme 3) under
anhydrous conditions (piperidine 20 v/v% in DMF, MW 50 °C,
50 W, 1 h (for 7a) and 3 h (for 7b)). HPLC-MS showed the for-
mation of expected intramolecular ligated product 8a (99%,
retention time 21.67 min), hydrolysed form 9a (1%) and none of
the intermolecular by-product 10a. As for ligation on 7b,
HPLC-MS indicated the formation of the desired 8b (18%, reten-
tion time 17.86 min), hydrolysed form 9b (8%) and intermolecu-
lar by-product 10b (31%). The retention times and fragmentation
patterns of 7a and 7b were also studied by control experiments
(HPLC-MS of pure 7a and 7b). HPLC-MS, via (−)ESI-MS/MS,
demonstrated that products 7a and 8a, each of MW 466, pro-
duced different fragmentation patterns. In addition, product 10a
was isolated and its structure further confirmed by HRMS.
The ligation of 7a was also examined under aqueous con-
ditions (pH 7.6, 1 M buffer strength, MW 50 °C, 50 W, 1 h)
HPLC-MS of the aqueous product, which disclosed a small
amount corresponding to the ligated product 8a, together with a
major peak corresponding to a fragment having MW 366 that
corresponds to the removal of the Boc-group either from 7a or
from the ligated product 8a.
Unlike the sterically hindered and poorly organized for
binding eight-membered cyclic transition state in S-acyl tripep-
tides,18,19 the structurally similar O-acyl tripeptide 4a demon-
strated a preferential internal O- to N-acyl shift. This counter-
intuitive reactivity of O-acyl tripeptides is however rationalized
by the same computational protocol of virtual screening and
quantum chemical calculations. The effectiveness of confor-
mational preorganization was defined in terms of the b(N–C)
scoring function, i.e. the geometrical distance between the
nucleophilic amine nitrogen and the electrophilic ester carbon
atom. A full conformational search was performed using the
MMX force field (as implemented in PCModel v.9.3 software),
resulting in 572 conformations which were subsequently ranked
in descending order of the b(N–C) scoring function. The best
preorganized conformer shown in Fig. 2 had a value for b(N–C)
of 3.242 Å, significantly smaller than the value of 3.591 Å
found in the similar S-acyl structure (the GMMX routine of
Traceless chemical ligation by O- to N-acyl shift via an eight-
membered-TS at Ser site was demonstrated in 4a,c. Protected N-
(Pg-α-aminoacyl)benzotriazoles 1a–c were coupled with L-Ser-
OH using benzotriazole methodology22,23 giving intermediates
2a–c, which on O-acylation provided 3a–c. Deprotection of the
Cbz/Boc group of 3 by hydrogenation with Pd/C or by HCl–
dioxane afforded O-acyl isodipeptides 4a–c (Scheme 1).
Intermediates 4a,c underwent ligation under microwave
irradiation in piperidine–DMF 20 v/v%, 50 °C, 50 W, 1 h
(Scheme 2). Anhydrous conditions were chosen to avoid ester
hydrolysis.
Indeed, HPLC-MS indicated the formation of desired intra-
molecular ligated products 5a (57%, retention time 23.07 min)
and 5c (22%, retention time 39.50 min) and the presence of start-
ing materials 4a (43%, retention time, 19.61 min) and 4c (78%,
retention time, 38.65 min). The retention times and fragmenta-
tion patterns of 4a and 4c were also studied by control exper-
iments (HPLC-MS of pure 4a or 4c). HPLC-MS, via (−)
ESI-MS/MS, confirmed that compounds 4a and 5a with MW
409 have different fragmentation patterns. These data indeed
proved the formation of intramolecular ligated products 5a and
5c. Moreover, product 5a was isolated and the structure further
confirmed by HRMS.
Traceless chemical ligation by O- to N-acyl shift from a Ser
site via an 11-membered TS was achieved in isotripeptides 7a–
b. Amino-unprotected O-acyl isodipeptides 4a–b (Scheme 1)
Scheme 1 Preparation of O-acyl isodipeptides 4a–c.
Scheme 3 Preparation of O-acyl isotripeptides 7a–b and their ligation
to 8a–b.
Scheme 2 Chemical ligation of O-acyl isodipeptides 4a,c.
This journal is © The Royal Society of Chemistry 2012
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Conclusions
In conclusion, chemical ligation via O- to N-acyl transfer with 8-
and 11-membered transition states occurs successfully without
the use of either cysteine or an auxiliary group. The reactivity of
O-acyl peptides in traceless chemical ligation reactions is sup-
ported by theoretical and computational studies. Further ligation
investigations on other sized transition states as well as ligations
via threonine are ongoing in our laboratory.
Acknowledgements
We thank the University of Florida, The Kenan Foundation,
King Abdulaziz University, Jeddah, Saudi Arabia and the Higher
Education Commission, Pakistan for financial support. The
authors thank Kind Abdulaziz University for support of this
research by grant number 24-3-1432/HiCi. We thank
Dr C. D. Hall for helpful discussions, Dr J. Johnson for his help
in HLPC-MS analyses, Dr M. C. A. Dancel for her help with
HRMS analyses.
Fig. 2 Preorganised conformer of O-acyl peptide 4a.
Notes and references
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Fig. 3 Geometry optimized conformer of O-acyl peptide 4A.
PCModel and MMX force field were used for scanning all rota-
table bonds).19
As the O- to N-shift reaction occurs in the presence of piper-
idine, we believe general basic catalysis may be involved.
For calculation purposes this means that piperidine scavenges
one proton of the free amino group in structure 4a. We mimic
the catalysis conditions by removing the proximal hydrogen
atom from the N-terminus. To probe the reactivity, the preorga-
nized deprotonated structure 4A was subjected to geometry
optimization at the HF/6-31+G* level of theory.
The quantum chemical reaction energy Ereact is defined as
react = E2 − E1, where E1 and E2 are the energies of the starting
E
and final, geometry-optimised structures, respectively. Geometry
optimization resulted in Ereact = −61.74 kcal mol−1, which is
about 33 kcal mol−1 more favorable than the previously studied
S-acyl structure. The optimized structure is shown in Fig. 3.
Although the optimized structure does not show the ligation
completed, the cyclic transition state looks rather more organized
than in the corresponding S-acyl structure (Fig. 2b in ref. 19).
An apparent reason for that is the close hydrogen bond contact
made between the Phe donor nitrogen and the Gly donor NH,
with the NH⋯N distance of 1.91 Å. This explains the additional
stabilization of structure 4A as well as the higher yield of the
internal ligation product 5a.
23 A. R. Katritzky, A. A. Shestopalov and K. Suzuki, Arkivoc, 2005, vii, 36.
4838 | Org. Biomol. Chem., 2012, 10, 4836–4838
This journal is © The Royal Society of Chemistry 2012