Synthesis of β-Thiol Phenylalanine for Applications in One-Pot Ligation-Desulfurization Chemistry

Article abstract of DOI:10.1021/acs.orglett.5b00597

(Chemical Equation Presented) The efficient synthesis of a β-thiol phenylalanine derivative is described starting from Garner's aldehyde. The utility of this amino acid in peptide ligation-desulfurization chemistry is described, including the trifluoroethanethiol (TFET)-promoted one-pot assembly of the 62 residue peptide hormone augurin.

Full text of DOI:10.1021/acs.orglett.5b00597

Letter
pubs.acs.org/OrgLett
Synthesis of β‑Thiol Phenylalanine for Applications in One-Pot
Ligation−Desulfurization Chemistry
Lara R. Malins, Andrew M. Giltrap, Luke J. Dowman, and Richard J. Payne*
School of Chemistry, The University of Sydney, New South Wales, 2006, Australia
S
* Supporting Information
ABSTRACT: The efficient synthesis of a β-thiol phenylalanine derivative is described starting from Garner’s aldehyde. The
utility of this amino acid in peptide ligation−desulfurization chemistry is described, including the trifluoroethanethiol (TFET)-
promoted one-pot assembly of the 62 residue peptide hormone augurin.
significant research effort has focused on extending the scope of
native chemical ligation-based transformations to enable
ligation at residues other than Cys.3 This concept was catalyzed
by an initial report from Yan and Dawson,4 which
demonstrated that peptides and proteins produced via native
chemical ligation could be desulfurized to provide an alanine
(Ala) residue at the ligation junction. In the same report, the
authors proposed the concept of further expanding the
technology to other thiol-derivatized proteinogenic amino
acids at the N-terminus of peptide fragments through the use
of ligation−desulfurization chemistry (see Scheme 1B).5 Since
this early proposal, there has been a flourish of activity,
especially in the past decade, that has led to successful
syntheses of β-, γ-, and δ-thiol amino acids,6 including arginine
(Arg),7 aspartic acid (Asp),8 glutamic acid (Glu),9 glutamine
(Gln),10 phenylalanine (Phe),11 valine (Val),12 lysine (Lys),13
leucine (Leu),14 threonine (Thr),15 and proline (Pro)16
residues. These building blocks have also been successfully
employed in the synthesis of a small number of protein targets
to date.3,5b,17
wenty years following the original disclosure of the
1
Tconvergent assembly of unprotected peptide fragments,
native chemical ligation remains the most robust method for
the synthetic preparation of protein targets.2 The reaction,
which takes place in aqueous media at neutral pH, involves a
reversible trans-thioesterification step between a peptide
containing an N-terminal cysteine (Cys) and a peptide bearing
a C-terminal thioester functionality (Scheme 1A). This initial
capture step is followed by a rapid, intramolecular S- to N-acyl
shift to generate the native peptide bond. In recent years,
Scheme 1. (A) Mechanism of Native Chemical Ligation; (B)
Ligation−Desulfurization at Thiol-Derived Amino Acids
Although significant progress has been made to maximize the
scope of native chemical ligation, synthetic access to suitably
protected thiol-derived amino acid building blocks remains
challenging. With the exception of two commercially available
derivatives (penicillamine, a β-thiol surrogate of Val,12a and γ-
thiol Pro16a) and our recent disclosure of peptide ligations
promoted by the late-stage introduction of a 2-thiol tryptophan
(Trp) auxiliary onto unprotected peptides,18 most thiol-derived
amino acids require multiple synthetic steps. Indeed, a general
synthetic route to access a range of these important molecules
does not currently exist. Applications of ligation−desulfuriza-
tion technology at non-Cys junctions are therefore usually
Received: February 26, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b00597
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Letter
limited to the laboratories that developed the synthesis of a
given thiol-derived amino acid.17a
the hemiaminal moiety in 4 using p-toluenesulfonic acid in 1,4-
dioxane provided alcohol 5 in 79% yield. Oxidation of the
primary alcohol with pyridinium dichromate then afforded
carboxylic acid 6 in 62% yield. Finally, borohydride reduction of
the thiocyanate followed by trityl (Trt) protection using Trt−
OH in the presence of a Lewis acid afforded β-(trityl)thiol Phe
derivative 1 in a total of 7 steps and 10.8% overall yield (for the
oxidation−reduction pathway: 9 steps, 13.6% overall yield).
These synthetic routes therefore represent efficient alternatives
to the preparation of a β-thiol phenylalanine building block
previously described by Crich and Banerjee (9 total steps from
L-phenylalanine, 13.4% yield).11a,22
In seeking a robust and general synthetic strategy capable of
delivering a wide range of thiol-derived amino acids, we have
recently reported the use of Garner’s aldehyde19 as a common
chiral starting point for the synthesis of suitably protected β-
thiol Arg7 and β-selenol Phe20 building blocks. In principle,
Garner’s aldehyde can serve as a common starting point for the
incorporation of most of the side chains present in
proteinogenic amino acids as well as the incorporation of
thiol or selenol reaction auxiliaries to enable ligation−
desulfurization/deselenization chemistry3,5b at almost any
amino acid. Herein, we report the efficient preparation of a
suitably protected β-thiol Phe building block 1 from Garner’s
aldehyde (Scheme 2), with a view to further expanding the use
Model hexapeptide 7 was next prepared by Fmoc-strategy
SPPS starting from Rink amide resin, whereby β-thiol Phe
derivative 1 was incorporated at the N-terminus to afford resin-
bound peptide 8 (Scheme 3). Cleavage from the resin and
Scheme 2. Synthesis of Suitably Protected β-Thiol Phe (1)
from Garner’s Aldehyde (2)
Scheme 3. SPPS of Peptide 7 Bearing an N-Terminal β-Thiol
Phe Residue
purification by reversed-phase HPLC provided peptide 7 in
74% yield based on the original resin loading (see Supporting
Information for synthetic details). A variety of C-terminal
peptide thioesters (Ac-LYRANX-S(CH2)2CO2Et, X = Gly, Ala,
Met, Phe, Val) were also prepared to study the scope of the
ligation−desulfurization reactions with a range of amino acids
on the C-terminus of the acyl donor component.
With β-thiol peptide 7 and a variety of peptide thioesters
now in hand, we next performed ligation reactions under
standard native chemical ligation conditions [6 M guanidine
hydrochloride (Gn·HCl), 100 mM Na2HPO4] in the presence
of 50 mM TCEP and 2 vol % thiophenol at room temperature
and a final pH of 7.2−7.4 (Table 1). In all cases, ligations
proceeded in excellent yields (72−87%) and were complete
within 24 h, including with the more sterically demanding Val
thioester (entry 5). The ligation was also chemoselective in the
presence of the ε-amino group of lysine (see Supporting
Information for model ligation). Subsequent free-radical
desulfurization in the presence of TCEP, 2,2′-azobis-[2-(2-
imidazolin-2-yl)propane] dihydrochloride (VA-044)23 and
glutathione12a at 65 °C cleanly provided the native Phe residue
at the ligation junction affording native peptide products in 52−
87% yields (Table 1).
We were also interested in comparing the rate of ligation at
β-thiol Phe with that of native chemical ligation at Cys. To this
end, we prepared the model N-terminal peptide H-CSPGYS-
NH2 and evaluated the rate of ligation with Ac-LYRANG-
S(CH2)2CO2Et (see Supporting Information) compared with
the corresponding ligation of peptide 7. To our surprise,
ligation with β-thiol Phe-containing peptide 7 reached
completion in less than 30 min, proving to be only modestly
slower than ligation at the native Cys residue, despite the
additional steric bulk associated with the phenyl side chain (see
Supporting Information for kinetic data). In addition, we
conducted a competition experiment between peptide 7 and a
homologue bearing our N-terminal β-selenol Phe residue.20
This reaction was carried out under conditions commonly
employed for selenium-mediated ligations, namely in the
of this chiral molecule as a common starting point to access a
range of β-thiol amino acid derivatives. While β-thiol Phe has
already been demonstrated to be a competent Cys surrogate for
the ligation-based assembly of small peptides by Crich and
Banerjee,11a in this study we aimed to shorten the synthesis of
the amino acid and demonstrate the utility of the building block
in both stepwise ligation−desulfurization chemistry and in
efficient one-pot operations for the ligation-based assembly of
larger peptide and protein targets.
The synthesis of 1 began with a Grignard addition of
bromobenzene to Garner’s aldehyde 2 which gave alcohol 3 as
a 2:3 mixture of syn/anti diastereoisomers in excellent yield
(Scheme 2). Mesylation of diastereomeric 3 followed by
displacement with potassium thiocyanate then provided 4 in
38% yield over the two steps. Importantly, only the anti-
mesylate proved to be competent in the thiocyanate inversion
reaction and thus provided the syn-thiocyanate exclusively. The
corresponding syn-mesylate was recovered as unreacted starting
material. These results are consistent with an analogous
inversion performed with potassium selenocyanate for the
synthesis of β-selenol Phe.20 In order to improve the overall
yield of the inversion, oxidation of 3 could be performed under
Swern conditions followed by a DIBAL-H reduction to give
anti-enriched 3 (1:8 syn/anti).21 Mesylation and displacement
with potassium thiocyanate then provided 4, in 51% yield over
the 2 steps, as the syn-diastereoisomer. From here, cleavage of
B
DOI: 10.1021/acs.orglett.5b00597
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incubated for 6−7 h at 37 °C before purification by reversed-
phase HPLC. Gratifyingly, products from these one-pot
ligation−desulfurization reactions were isolated in 68−87%
yield over the two steps (average of 82−93% per step).
Table 1. β-Thiol Phe Ligation−Desulfurization Reactions
Having demonstrated the efficiency of the one-pot ligation-
desulfurization manifold at β-thiol Phe for model peptides, we
were next interested in using this methodology for the
construction of a more synthetically challenging target.
Specifically, we selected as a demonstrative example a 62-
amino acid fragment of the putative secreted peptide hormone
augurin 9, which is encoded by Esophageal Cancer Related
Gene-4 (Ecrg4) and expressed in endocrine tissue but has a
function that is as yet unknown.26 It was envisaged that this
Cys-free peptide target could be rapidly prepared using a
TFET-promoted one-pot ligation−desulfurization at β-thiol
Phe (Scheme 4). To this end, peptide thioester 10 (augurin 1−
thioester
(X =)
ligation
a
desulfurization
one-pot ligation−
a
a
yield
yield
desulfurization yield
Gly (G)
Ala (A)
Met (M)
Phe (F)
Val (V)
79%
86%
72%
83%
87%
87%
76%
52%
60%
67%
68%
79%
71%
87%
68%
a
Scheme 4. One-Pot Synthesis of Augurin 9
Isolated yields. Reaction conditions: Ligation: Thioester (1.1−1.3
equiv, 5.5−6.5 mM concentration), buffer (6 M Gn·HCl, 100 mM
Na2HPO4, 50 mM TCEP, 5 mM with respect to peptide 7), 2 vol %
thiophenol, 37 °C, pH 7.2−7.4, 24 h. Desulfurization: VA-044, buffer
(6 M Gn·HCl, 100 mM Na2HPO4, 500 mM TCEP), 40 mM
glutathione, 65 °C, 16 h. One-pot ligation−desulfurization:
Thioester (1.1−1.3 equiv, 5.5−6.5 mM concentration), buffer (6 M
Gn·HCl, 100 mM Na2HPO4, 50 mM TCEP, 5 mM final concentration
with respect to peptide 7), 2 vol % TFET, 30 °C, pH 7.0−7.4, 16 h;
then degas (Ar), dilute with buffer (6 M Gn·HCl, 100 mM Na2HPO4,
500 mM TCEP, pH adjusted to 6.0) to a final concentration of 2.5
mM with respect to peptide 7, addition of glutathione (40 mM), VA-
044 (20 mM), 37 °C, 6−7 h.
absence of TCEP (which is known to facilitate deseleniza-
tion)24 and in the presence of 4-mercaptophenylacetic acid
(MPAA) as both a thiol additive and mild reductant (see
Supporting Information). Interestingly, the competitive ligation
of peptide 7 (1.0 equiv) and the corresponding selenopeptide
dimer (S1, 1.0 equiv, see Supporting Information) with a
substoichiometric amount of peptide thioester occurred to
provide exclusively the thiol-Phe ligation product. As previously
suggested,20,25 it is postulated that the rate-determining step in
the selenol-mediated ligation, particularly in the absence of a
strongly reducing phosphine (e.g., TCEP), is the generation of
reactive selenol from the starting peptide, which exists in
oxidized form as the diselenide dimer (see Supporting
Information). Importantly, the observed rate differential
suggests that kinetically controlled ligation reactions employing
both thiol-Phe and selenol-Phe may be feasible for the iterative
assembly of target peptides and proteins from multiple
fragments.
We have recently reported the use of the alkyl thiol
trifluoroethanethiol (TFET) as an additive for one-pot native
chemical ligation−desulfurization reactions with Cys resi-
dues.17d Here, we were interested in employing TFET in
one-pot ligation−desulfurization reactions at β-thiol Phe in
order to streamline the methodology and reduce the number of
intermediary purification steps (Table 1). To this end, peptide
7 was reacted under modified ligation conditions [6 M Gn·HCl,
100 mM Na2HPO4, 50 mM TCEP] in the presence of 2 vol %
TFET at 30 °C and a final pH of 7.0−7.4. After 16 h, the
reaction was sparged with argon and diluted with degassed
buffer [6 M Gn·HCl, 100 mM Na2HPO4, 500 mM TCEP, pH
adjusted to 6.0] before the addition of VA-044 and reduced
glutathione to effect desulfurization of the β-thiol auxiliary in
the ligation products. The desulfurization reactions were
26), bearing a C-terminal Lys residue, and peptide 11 (augurin
27−62), bearing an N-terminal β-thiol Phe residue, were first
prepared using Fmoc-SPPS (see Supporting Information).
Following purification of the requisite fragments, the ligation
reaction was carried out in the presence of a slightly modified
buffer solution (6 M Gn·HCl, 0.2 M HEPES, 50 mM TCEP,
6 vol % TFET), most notably in the absence of phosphate to
minimize the potential for N-terminal pyroglutamate for-
mation27 at the terminal Gln residue of peptide thioester 10.
The pH of the reaction was also carefully controlled to
minimize base-catalyzed lactamization of the C-terminal Lys-
thioester moiety. After 16 h, the ligation was deemed to be
complete via HPLC-MS analysis. The crude ligation product 12
was subjected directly (without intermediary purification) to
the radical desulfurization conditions, cleanly affording the
target product bearing a native Phe residue at the ligation
junction. The efficiency of the one-pot protocol is reflected in
the analytical yield of the ligation−desulfurization pathway
(71% yield of product 9, with the corresponding ligation
product bearing an N-terminal pyroglutamate as a minor
byproduct in 20% yield; see Supporting Information). Although
the aggregation-prone nature of augurin28 hindered the facile
isolation of the target peptide, purified 9 was nonetheless
C
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2009, 48, 8090. (c) El Oualid, F.; Merkx, R.; Ekkebus, R.; Hameed, D.
S.; Smit, J. J.; de Jong, A.; Hilkmann, H.; Sixma, T. K.; Ovaa, H. Angew.
Chem., Int. Ed. 2010, 49, 10149.
(14) (a) Harpaz, Z.; Siman, P.; Kumar, K. S. A.; Brik, A.
ChemBioChem 2010, 11, 1232. (b) Tan, Z. P.; Shang, S. Y.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2010, 49, 9500.
(15) Chen, J.; Wang, P.; Zhu, J. L.; Wan, Q.; Danishefsky, S. J.
Tetrahedron 2010, 66, 2277.
(16) (a) Shang, S. Y.; Tan, Z. P.; Dong, S. W.; Danishefsky, S. J. J.
Am. Chem. Soc. 2011, 133, 10784. (b) Townsend, S. D.; Tan, Z.;
Dong, S.; Shang, S.; Brailsford, J. A.; Danishefsky, S. J. J. Am. Chem.
Soc. 2012, 134, 3912. (c) Ding, H.; Shigenaga, A.; Sato, K.; Morishita,
K.; Otaka, A. Org. Lett. 2011, 13, 5588.
(17) (a) Malins, L. R.; Payne, R. J. Aust. J. Chem. 2015, 68, 521−537.
(b) Shang, S.; Tan, Z.; Danishefsky, S. J. Proc. Natl. Acad. Sci. U.S.A.
2011, 108, 5986. (c) Kumar, K. S.; Bavikar, S. N.; Spasser, L.; Moyal,
T.; Ohayon, S.; Brik, A. Angew. Chem., Int. Ed. 2011, 50, 6137.
(d) Thompson, R. E.; Liu, X.; Alonso-García, N.; Pereira, P. J. B.;
Jolliffe, K. A.; Payne, R. J. J. Am. Chem. Soc. 2014, 136, 8161.
(18) Malins, L. R.; Cergol, K. M.; Payne, R. J. Chem. Sci. 2014, 5, 260.
(19) Garner, P. Tetrahedron Lett. 1984, 25, 5855.
obtained in 17% isolated yield. The rapid and efficient synthesis
of this difficult peptide target showcases the utility of one-pot
ligation−desulfurization reactions mediated by β-thiol Phe.
In summary, we have developed a novel synthetic route to β-
thiol Phe which highlights the generality of Garner’s aldehyde
as a common chiral precursor to both thiol- and selenol-derived
amino acids. We have expanded the scope of ligation reactions
at thiol Phe and explored the kinetics of the transformation for
the first time. Moreover, we have demonstrated that ligation
products can be desulfurized to provide native peptide products
through a streamlined one-pot ligation−desulfurization ap-
proach employing the thiol additive TFET. The utility of this
methodology was exemplified through the efficient, ligation-
based assembly of the 62-amino acid peptide hormone augurin.
Future work will focus on the use of β-thiol Phe in the synthesis
of other complex protein targets. Further studies will also focus
on the exploration of kinetically controlled and tandem ligation
reactions employing both β-thiol and β-selenol Phe derivatives.
ASSOCIATED CONTENT
* Supporting Information
(20) Malins, L. R.; Payne, R. J. Org. Lett. 2012, 14, 3142.
(21) Nishida, A.; Sorimachi, H.; Iwaida, M.; Matsumizu, M.; Kawate,
T.; Nakagawa, M. Synlett 1998, 389.
(22) Crich, D.; Banerjee, A. J. Org. Chem. 2006, 71, 7106.
(23) Wan, Q.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46,
9248.
■
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.
(24) Metanis, N.; Keinan, E.; Dawson, P. E. Angew. Chem., Int. Ed.
2010, 49, 7049.
(25) Gieselman, M. D.; Xie, L.; van Der Donk, W. A. Org. Lett. 2001,
3, 1331.
(26) Mirabeau, O.; Perlas, E.; Severini, C.; Audero, E.; Gascuel, O.;
Possenti, R.; Birney, E.; Rosenthal, N.; Gross, C. Genome Res. 2007,
17, 320.
(27) Khandke, K. M.; Fairwell, T.; Chait, B. T.; Manjula, B. N. Int. J.
Pept. Protein Res. 1989, 34, 118.
(28) Podvin, S.; Gonzalez, A.-M.; Miller, M. C.; Dang, X.; Botfield,
H.; Donahue, J. E.; Kurabi, A.; Boissaud-Cooke, M.; Rossi, R.;
Leadbeater, W. E.; Johanson, C. E.; Coimbra, R.; Stopa, E. G.; Eliceiri,
B. P.; Baird, A. PLoS One 2011, 6, e24609.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: richard.payne@sydney.edu.au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Australian Research Council
(ARC) through the award of an ARC Future Fellowship to
R.J.P. (FT30100150) and by the John Lamberton Research
Scholarship (L.R.M., A.M.G.) and the International Post-
graduate Research Scholarship Scheme (L.R.M.).
REFERENCES
■
(1) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776.
(2) Kent, S. B. Chem. Soc. Rev. 2009, 38, 338.
(3) Malins, L. R.; Payne, R. J. Curr. Opin. Chem. Biol. 2014, 22, 70.
(4) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526.
(5) (a) Rohde, H.; Seitz, O. Biopolymers 2010, 94, 551. (b) Dawson,
P. E. Isr. J. Chem. 2011, 51, 862. (c) Hackenberger, C. P. R.;
Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030.
(6) Wong, C. T. T.; Tung, C. L.; Li, X. Mol. Biosyst. 2013, 9, 826.
(7) Malins, L. R.; Cergol, K. M.; Payne, R. J. ChemBioChem. 2013, 14,
559.
(8) (a) Thompson, R. E.; Chan, B.; Radom, L.; Jolliffe, K. A.; Payne,
R. J. Angew. Chem., Int. Ed. 2013, 52, 9723. (b) Guan, X.; Drake, M. R.;
Tan, Z. Org. Lett. 2013, 15, 6128.
(9) Cergol, K. M.; Thompson, R. E.; Malins, L. R.; Turner, P.; Payne,
R. J. Org. Lett. 2014, 16, 290.
(10) Siman, P.; Karthikeyan, S. V.; Brik, A. Org. Lett. 2012, 14, 1520.
(11) (a) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064.
(b) Botti, P.; Tchertchian, S. 2006, WO/2006/133962.
(12) (a) Haase, C.; Rohde, H.; Seitz, O. Angew. Chem., Int. Ed. 2008,
47, 6807. (b) Chen, J.; Wan, Q.; Yuan, Y.; Zhu, J. L.; Danishefsky, S. J.
Angew. Chem., Int. Ed. 2008, 47, 8521.
(13) (a) Yang, R. L.; Pasunooti, K. K.; Li, F. P.; Liu, X. W.; Liu, C. F.
J. Am. Chem. Soc. 2009, 131, 13592. (b) Ajish Kumar, K. S.; Haj-Yahya,
M.; Olschewski, D.; Lashuel, H. A.; Brik, A. Angew. Chem., Int. Ed.
D
DOI: 10.1021/acs.orglett.5b00597
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