One-pot peptide ligation-desulfurization at glutamate

Article abstract of DOI:10.1021/ol403288n

An efficient methodology for ligation at glutamate (Glu) is described. A γ-thiol-Glu building block was accessed in only three steps from protected glutamic acid and could be incorporated at the N-terminus of peptides. The application of these peptides in one-pot ligation-desulfurization chemistry is demonstrated with a range of peptide thioesters, and the utility of this methodology is highlighted through the synthesis of the osteoporosis peptide drug teriparatide (Forteo).

Full text of DOI:10.1021/ol403288n

Letter
pubs.acs.org/OrgLett
One-Pot Peptide Ligation−Desulfurization at Glutamate
Katie M. Cergol, Robert E. Thompson, Lara R. Malins, Peter Turner, and Richard J. Payne*
School of Chemistry, The University of Sydney, NSW 2006, Australia
S
* Supporting Information
ABSTRACT: An efficient methodology for ligation at
glutamate (Glu) is described. A γ-thiol-Glu building block
was accessed in only three steps from protected glutamic acid
and could be incorporated at the N-terminus of peptides. The
application of these peptides in one-pot ligation−desulfuriza-
tion chemistry is demonstrated with a range of peptide thioesters, and the utility of this methodology is highlighted through the
synthesis of the osteoporosis peptide drug teriparatide (Forteo).
Scheme 1. (A) Native Chemical Ligation−Desulfurization at
Cys/β-Thiol Amino Acids and (B) γ-Thiol-Mediated
Ligation−Desulfurization
ative chemical ligation is the most widely used method
Nfor the construction of peptides and proteins from
smaller peptide fragments.1−3 This methodology has greatly
expanded our understanding of protein structure and function
by facilitating access to a diverse range of proteins, including
post-translationally modified proteins that are difficult or
impossible to obtain through expression technologies.4−6
Mechanistically, the first step of the reaction involves a
reversible trans-thioesterification between a cysteine (Cys)
residue on the N-terminus of a peptide fragment with a
thioester moiety on the C-terminus of another peptide reaction
partner. A rapid S to N acyl shift, which proceeds through a
five-membered ring transition state, then facilitates the
formation of the native peptide bond in high yield and with
complete chemoselectivity. In addition to the rapid and high-
yielding nature of this reaction, a further strength of the
protocol is that ligations proceed in aqueous media and at
neutral pH in the presence of unprotected amino acid side
chains.
The low abundance of Cys residues within peptides and
proteins has prompted the development of post-ligation
desulfurization chemistry, whereby the Cys residue used to
facilitate the ligation can be desulfurized to provide an alanine
(Ala) residue,7 either through hydrogenation or via a radical-
mediated approach, for example, through the use of the popular
water-soluble radical initiator 2,2′-azobis[2-(2-imidazolin-2-
yl)propane]dihydrochloride (VA-044).8−10 To expand the
scope of ligation chemistry, extensive research efforts have
recently focused on the synthesis of β-, γ-, and δ-thiol-derived
amino acids as well as thiol-derived aromatic amino acids
(Scheme 1),11−25 which can be implemented in ligation−
desulfurization chemistry through a similar mechanism to
native chemical ligation.
homocysteine26 and other γ-thiol amino acids.8,27 Furthermore,
we report conditions for a one-pot ligation−desulfurization at
Glu whereby desulfurization can be carried out on the crude
ligation reaction, without the need for intermediate purification.
Synthesis of the initially proposed γ-thiol-Glu building block
1 proceeded from Boc-Glu(OtBu)-OAll (2) and began with
installation of a 2,4-dimethoxybenzyl (DMB)-protected thiol at
the γ-position (Scheme 2). This was facilitated by sulfenylating
reagent 3 following double deprotonation of 2 (see Supporting
Information for synthetic details).22,28−31 Gratifyingly, the
resulting DMB-protected γ-thiol-Glu 4 was isolated in good
yield (83%, Scheme 2) and as a single diastereoisomer (>99%
dr). Finally, 4 was subjected to Pd-catalyzed allyl ester
deprotection conditions to afford the desired γ-thiol-Glu
building block 1 in excellent yield. As it has recently been
shown that both diastereomers of β-thiol-Asp facilitate ligation
at comparable rates,22 we decided to focus only on the isolated
diastereomer for ligation studies.
Herein we report the first synthesis of a suitably protected γ-
thiol-Glu building block through a short and scalable route, as
well as its utility in peptide ligation−desulfurization chemistry.
Although this reaction would proceed through a six-membered
ring during the S to N acyl shift, owing to the γ-position of the
thiol (Scheme 1B), we envisaged that the ligation reaction
would still be facile based on prior ligation studies at
With suitably protected γ-thiol-Glu 1 prepared, this building
block was next incorporated at the N-terminus of model
Received: November 14, 2013
Published: December 2, 2013
© 2013 American Chemical Society
290
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Letter
thiol-Glu moiety in peptide 12 in ligation−desulfurization
chemistry using a variety of C-terminal model peptide
thioesters to probe the scope of these reactions (entries 1−5,
Table 1) (see Supporting Information for synthetic details).
Scheme 2. Synthesis of γ-Thiol-Glu Building Blocks 1 and 10
Table 1. Scope of γ-Thiol-Glu Ligation−Desulfurization
Chemistry
a
c
thioester
(X =)
ligation yield
(%)
desulfurization
a
one-pot yield
(%)
entry
yield (%)
b
1
2
3
4
5
Gly
Ala
72
77
83
80
68
89 (64)
73
67
72
74
56
b
Scheme 3. Synthesis of Model Peptide 12
91 (70)
b
Met
Phe
Val
98 (81)
b
84 (67)
b
98 (67)
a
Isolated yields after HPLC purification. Ligation: 5 mM 12 in buffer
(6 M Gn·HCl, 100 mM Na2HPO4, 50 mM TCEP), PhSH (2 vol %),
37 °C, pH 7.2−7.4, 16 h. Desulfurization: 500 mM TCEP in buffer (6
M Gn·HCl, 100 mM Na2HPO4), reduced glutathione (40 mM), VA-
b
044 (200 mM), pH 6.5−6.8, 65 °C, 16 h. Yield over two steps.
c
Desulfurization reactions were carried out at 37 °C in the one-pot
protocol.
peptide 5 following standard Fmoc strategy solid-phase peptide
synthesis (SPPS), starting from Rink amide resin, to afford
resin-bound peptide 6 (Scheme 3). Unfortunately, after
concomitant resin cleavage and deprotection (90:5:5 v/v/v
TFA/iPr3SiH/H2O), pentapeptide 7 and thiolactone 8 were
isolated, rather than the desired peptide displaying the γ-thiol-
Glu on the N-terminus. In this case, the acidic cleavage
conditions facilitated peptide splicing at the N-terminus,
resulting from attack by the nucleophilic thiol moiety, revealed
after deprotection of the acid-labile DMB protecting group,
onto the amide backbone via a favorable five-membered ring
transition state.26
To circumvent this unwanted pathway, we decided to
exchange the acid-labile DMB-thiol protecting group for an
acid-stable but reductively labile methyl disulfide protecting
group (Scheme 2). This transformation was achieved by
subjecting DMB-protected γ-thiol-Glu 1 to the reagent
dimethyl(methylthio)sulfonium tetrafluoroborate (9),32 which
facilitated this protecting group exchange in moderate yield
(55%, Scheme 2).33 Gratifyingly, incorporation of this modified
γ-thiol-Glu building block 10 into resin-bound peptide 11
employing (benzotriazol-1-yloxy)tripyrrolidinophosphonium-
hexafluorophosphate (PyBOP) and N-methylmorpholine
(NMM) afforded the desired model peptide 12 in good yield
after acidic deprotection, cleavage of the peptide from the resin
and HPLC purification (68%, Scheme 3), without any trace of
unwanted peptide splicing products.
Ligation reactions were carried out in ligation buffer (6 M
Gn·HCl, 100 mM Na2HPO4, 50 mM TCEP, 5 mM with
respect to 12) at 37 °C and pH 7.2−7.4 with the addition of
thiophenol as an aryl thiol catalyst. Pleasingly, each of the
ligation reactions proceeded to completion within 16 h and in
excellent yields after reverse-phase HPLC purification (68−
83%, Table 1). It should be noted that HPLC fractions
containing ligation product were immediately lyophilized to
avoid acid-mediated peptide splicing caused by the acidic
HPLC eluent.
Next we subjected the purified ligation products to radical-
based desulfurization10 using VA-044 as the radical initiator, in
the presence of TCEP and reduced glutathione.15 In all cases,
desulfurization reactions proceeded to completion within 16 h
at 65 °C, affording the native peptide products in excellent
yields after reverse-phase HPLC purification (Table 1, 84−98%
yield).
On the basis of prior reports of one-pot ligation−
desulfurization chemistry at Cys34 and β-thiol-Asp,22 we were
next interested in developing this concept to effect the one-pot
transformation at γ-thiol-Glu containing model peptides (Table
1, entries 1−5). We envisaged that this would not only
streamline the methodology by preventing additional purifica-
tion steps but also avoid peptide splicing facilitated by the γ-
thiol during purification. Specifically, each ligation reaction was
first carried out with careful monitoring and shown to proceed
to completion within 4 h, as determined by LC-MS analysis,
with the exception of the ligation reaction with model thioester
Ac-LYRANV-S(CH2)2CO2Et (entry 5) which required a
reaction time of 16 h to reach completion. After this time,
thiophenol was extracted from the reaction mixture by washing
with diethyl ether in order to prevent poisoning of the
desulfurization reaction by thiophenol.19,22 The ligation
reaction mixture was immediately treated with TCEP (500
mM), reduced glutathione (40 mM), and VA-044 (200 mM),
affording the desired peptide products in excellent yields over
At this stage, the stereochemical outcome of the sulfenylation
reaction was confirmed by single-crystal X-ray analysis of cyclic
derivative 13, which was synthesized following reductive
thiolactonization of 10, employing tris(2-carboxyethyl)-
phosphine (TCEP) followed by PyBOP and N,N-diisopropyl-
ethylamine (DIPEA) (Scheme 2). The stereochemistry of
thiolactone 13 was confirmed to be (2S,4S), indicating that the
(2S,4S) diastereoisomer of 4 was obtained following
sulfenylation.
With the desired model peptide 12 in hand, we next turned
our attention to investigating the utility of the N-terminal γ-
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Organic Letters
Letter
the two steps (Table 1, 56−74% yield). It should be noted that
the one-pot reaction was also attempted in the absence of VA-
044 to effect selective desulfurization in the presence of Cys
residues, as we have recently described at β-thiol-Asp.22
Unfortunately, due to peptide splicing which occurs at the
acidic pH required for the selective desulfurization protocol
(Scheme 3), a complex mixture of products resulted, and hence
we were unable to achieve the analogous selective desulfuriza-
tion reaction.
Scheme 4. Synthesis of Teriparatide (15)
For one-pot ligation−desulfurization reactions with peptide
thioesters bearing C-terminal Ala, Met, Phe, and Val residues,
peptide byproduct 14 was also observed, which may result from
reaction of the resulting VA-044 radical with the peptide radical
formed following H-abstraction from the γ-thiol (Figure 1).10
Information for synthetic details). Coupling of the γ-thiol-Glu
building block 10 and acidic cleavage and deprotection from
the resin then afforded the desired peptide fragment 17 in 18%
yield after reverse-phase HPLC purification (based on the
original resin loading) (Scheme 4). Ligation of peptide 17 with
Met-thioester 16 (see Supporting Information for synthetic
details) proceeded cleanly to completion over 16 h under the
ligation condition described previously. After HPLC purifica-
tion, the desired ligation product 19 was isolated in excellent
yield (82%). Desulfurization of 19 under the radical conditions
described above also proceeded smoothly to afford the native
teriparatide peptide (15) in excellent yield (95%) after
purification. Finally, we also synthesized teriparatide (15) by
employing our one-pot ligation−desulfurization strategy, which
gratifyingly afforded the desired peptide product in excellent
yield (71%, Scheme 4).
In conclusion, we have developed a concise and scalable
synthesis of a novel γ-thiol-Glu building block 10 which can be
readily incorporated into a variety of peptides to faciliate
ligation chemistry. The resulting γ-thiol-Glu peptides undergo
facile ligation reactions with a range of thioesters and can be
desulfurized to the native peptide products using radical-based
conditions. Furthermore, we have extended this methodology
to include a one-pot ligation−desulfurization cascade which
proved to be efficient and high-yielding, while reducing the
need for intermediate purification of the ligation products.
Moreover, this methodology has been successfully employed in
the high-yielding preparation of the FDA-approved osteopo-
rosis drug teriparatide (15), which suggests that this method-
ology will have wide utility in the synthesis of peptides and
proteins in the future. Further research in our laboratories will
focus on the expansion of this methodology to include the
synthesis of protein targets.
Figure 1. (a) Analytical HPLC trace (λ = 280 nm, 0−40% CH3CN
over 30 min) of crude reaction mixture for one-pot ligation−
desulfurization between 12 and Ac-LYRANG-S(CH2)2CO2Et without
the formation of byproduct 14. (b) Analytical HPLC trace (λ = 280
nm, 0−40% CH3CN over 30 min) of crude reaction mixture for one-
pot ligation−desulfurization between 12 and Ac-LYRANM-S-
(CH2)2CO2Et, indicating the formation of byproduct.
As this byproduct was not observed in the two-step ligation−
desulfurization procedures, it is likely that this pathway results
from trace amounts of aryl thiol remaining despite the
extraction protocol.34 Nonetheless, in all cases, this side
reaction did not significantly impact the isolated yield or the
overall efficiency of the reaction methodology (Figure 1).
Having demonstrated the scope and efficiency of the
ligation−desulfurization reactions at γ-thiol-Glu, including the
one-pot protocol, we next focused our attention on applying
the methodology to the synthesis of the peptide drug
teriparatide (15) (Forteo, Eli Lilly). Used in the treatment of
glucocorticoid-induced osteoporosis, this recombinant 34
amino acid parathyroid hormone is the only osteoporosis
drug on the market that stimulates bone growth.35 We
envisaged that this peptide could be easily accessed via a
ligation reaction between two fragments, namely, peptide
thioester 16, corresponding to teriparatide (1−18) and bearing
a C-terminal Met residue, and peptide 17 possessing an N-
terminal γ-thiol-Glu residue, representing teriparatide (19−38)
(Scheme 4). To this end, peptide 18 corresponding to
teriparatide (20−34) was first synthesized on trityl chloride
resin using standard Fmoc-SPPS procedures (see Supporting
292
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Letter
(29) Baldwin, J. E.; Moloney, M. G.; North, M. Tetrahedron 1989, 45,
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(30) Baldwin, J. E.; Moloney, M. G.; North, M. Tetrahedron 1989, 45,
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedures, analytical HPLC traces, and
characterization data. This material is available free of charge via
the Internet at http://pubs.acs.org.
6309.
(31) Baldwin, J. E.; North, M.; Flinn, A.; Moloney, M. G. J. Chem.
Soc., Chem. Commun. 1988, 828.
(32) Olsen, M. T.; Gray, D. L.; Rauchfuss, T. B.; De Gioia, L.;
Zampella, G. Chem. Commun. 2011, 47, 6554.
(33) Bishop, P.; Jones, C.; Chmielewski, J. Tetrahedron Lett. 1993, 34,
4469.
(34) Moyal, T.; Hemantha, H. P.; Siman, P.; Refua, M.; Brik, A.
Chem. Sci. 2013, 4, 2496.
(35) Saag, K. G.; Shane, E.; Boonen, S.; Marin, F.; Donley, D. W.;
Taylor, K. A.; Dalsky, G. P.; Marcus, R. N. Engl. J. Med. 2007, 357,
2028.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: richard.payne@sydney.edu.au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the ARC Future Fellowship scheme
(FT130100150) for research funding and the APA (RET)
and IPRS (LRM) schemes for PhD funding. We would also like
to thank Mr. James Montgomery (The University of Sydney)
for early contributions to this work.
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