Synthesis of thiophenylalanine-containing peptides via Cu(I)-mediated cross-coupling

Article abstract of DOI:10.1021/ol202947f

Aryl thiolates have unique reactive, redox, electronic, and spectroscopic properties. A practical approach to synthesize peptides containing thiophenylalanine has been developed via a novel Cu(I)-mediated cross-coupling reaction between thiolacetic acid a

Full text of DOI:10.1021/ol202947f

ORGANIC
LETTERS
2012
Vol. 14, No. 2
464–467
Synthesis of Thiophenylalanine-
containing Peptides via Cu(I)-mediated
Cross-Coupling
Christina R. Forbes and Neal J. Zondlo*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware
19716, United States
zondlo@udel.edu
Received November 1, 2011
ABSTRACT
Aryl thiolates have unique reactive, redox, electronic, and spectroscopic properties. A practical approach to synthesize peptides containing
thiophenylalanine has been developed via a novel Cu(I)-mediated cross-coupling reaction between thiolacetic acid and iodophenylalanine-
containing peptides in the solid phase. This approach is compatible with all canonical proteinogenic functional groups, providing general access
to aryl thiolates in peptides. Peptides containing thiophenylalanine (pKa 6.4) were readily elaborated to contain methyl, allyl, and nitrobenzyl
thioethers, disulfides, sulfoxides, sulfones, or sulfonates.
Cysteine residues play critical roles in protein folding,
structure, catalysis, response to oxidative stress, metal bind-
ing, modification, and cell signaling.1 These uses are depen-
dent on the redox properties, acidity, and nucleophilicity of
thiols and thiolates. The multiple oxidation states accessible
to thiols are critical in their versatility in function. Aryl thiols
may perform in similar roles but with reduced pKa and elec-
tronically tunable properties.2 Considering the special role of
tyrosine in biomolecular recognition, thiophenylalanine repre-
sents an intriguing hybrid of tyrosine and cysteine residues.3
Thiophenylalanine and related amino acids have di-
verse potential applications in fields including medicinal
chemistry, enzymology, protein design, materials science,
and nanotechnology. However, there are very few pub-
lished examples of thiophenylalanine-containing pep-
tides. The first such description, applied by Escher in
angiotensin II analogues, employed sulfonylation of phe-
nylalanine, followed by tin reduction, thiol protection,
and amine protection to generate the Boc amino acids.4
DeGrado extended this approach to prepare Fmoc-thio-
phenylalanine and employed its disulfide in a peptide as a
light-based switch of protein folding.5 In an alternative
synthetic approach, Still used diazonium chemistry to access
thiophenylalanine-containing peptides that were subse-
quently photocoupled to prepare thioether analogues of
diaryl ether antibiotics.6 Recently, Hecht incorporated
thiophenylalanine in place of an active site tyrosine of
(4) Escher, E.; Bernier, M.; Parent, P. Helv. Chim. Acta 1983, 66,
1355–65. Guillemette, G.; Bernier, M.; Parent, P.; Leduc, R.; Escher, E.
J. Med. Chem. 1984, 27, 315–20. Other syntheses of 4-ThioPhe: Johnson,
T. B.; Brautlecht, C. A. J. Biol. Chem. 1912, 12, 175–96. Elliott, D. F.;
Harrington, C. J. Chem. Soc. 1949, 1374–8. Colescott, R. L.; Herr, R. R.;
Dailey, J. P. J. Am. Chem. Soc. 1957, 79, 4232–5. Bergel, F.; Stock, J. A.
J. Chem. Soc. 1959, 90–7. Synthesis of S-t-butyl Boc-ThioPhe via Pd
cross-coupling: Rajagopalan, S.; Radke, G.; Evans, M.; Tomich, J. M.
Synth. Commun. 1996, 26, 1431–40.
(5) Lu, H. S. M.; Volk, M.; Kholodenko, Y.; Gooding, E.; Hochstrasser,
R. M.; DeGrado, W. F. J. Am. Chem. Soc. 1997, 119, 7173–80.
(6) Hobbs, D. W.; Still, W. C. Tetrahedron Lett. 1987, 28, 2805–8.
Hobbs, D. W.; Still, W. C. Tetrahedron Lett. 1989, 30, 5405–8. Related
approaches to aryl thioethers in peptides: West, C. W.; Rich, D. H. Org.
Lett. 1999, 1, 1819–22. Moreau, X.; Campagne, J.-M. J. Organomet.
Chem. 2003, 687, 322–6.
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Biophys. Biomol. Struct. 1997, 26, 357–71. Adams, S. R.; Campbell,
R. E.; Gross, L. A.; Martin, B. R.; Walkup, G. K.; Yao, Y.; Llopis, J.;
Tsien, R. Y. J. Am. Chem. Soc. 2002, 124, 6063–76. Chalker, J. M.;
Bernardes, G. J. L.; Lin, Y. A.; Davis, B. G. Chem. Asian J. 2009, 4,
630–40. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
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10.1021/ol202947f
Published on Web 01/06/2012
2012 American Chemical Society

topoisomerase I to examine the dependence of catalysis
on the nucleophilic phenolate.7
In all of these cases, significant effort was required to
synthesize protected thiophenylalanine. The development
of a practical synthetic approach to thiophenylalanine,
ideally employing commercially available amino acids with-
out separate, additional purification steps, could greatly
expand the applications of this amino acid. In view of the
recent advances in metal-catalyzed cross-coupling reactions
between aryl halides and thiols,8,9 we sought to develop an
alternative approach to protected thiophenylalanine em-
ploying commercially available 4-iodophenylalanine and
modification of a peptide via reaction on solid phase.
Copper catalysts have been most broadly employed for
cross-coupling reactions with thiols, typically using aryl
iodides. Therefore, we examined reaction conditions
on solid phase within the protected model peptide
Ac-T(4-I-Phe)PN-NH2, which we have previously used
to understand aromatic electronic effects in peptides.10
Table 1. Reaction Development
Figure 1. HPLC chromatograms of (a) Ac-T(4-I-Phe)PN-NH2
peptide starting material (1); (b) peptide after cross-coupling
reaction and peptide cleavage and deprotection, showing a
mixture of thioacetyl (2) and disulfide (3) products; and (c) the
result of treatment of the solution in (b) with DTT to effect
thiolysis and reduction of disulfide, showing clean overall con-
version to the 4-thiophenylalanine-containing product (4).
DIPEA as a base in toluene or t-amyl alcohol at 90À
110 °C, resulted in good conversion on solid phase to a
mixture of cross-coupled thioester and disulfide products.
This mixture could be cleanly converted to the thiophe-
nylalanine-containing peptide during standard cleavage/
deprotection/purification steps (Figure 1). At 110 °C in
toluene, iodophenylalanine underwent nearly complete
conversion to cross-coupled products in 16 h. If the
thioester product was desired, maximum yield was ob-
tained by running the reaction at 100 °C, which decreased
thiolysis and disulfide formation. The scope of this reac-
tion with different thiols was briefly examined (Table 2).
The reaction proceeded readily with thiophenol.6 Alkyl
thiols reacted poorly under these conditions.
To further examine the scope of this reaction, a series of
peptides, which included all canonical amino acid func-
tional groups, was synthesized and examined for cross-
coupling under the optimized reaction conditions (Figure 2).
The peptides included model peptides as well as the trp
cage miniprotein, with Tyr3 replaced by 4-I-Phe.12 Reac-
tion on the trp cage provides a critical test to confirm that
a All cross-coupled products, including overacetylated side products
in solution phase reactions, or disulfide products in solid phase reac-
tions. RN = Ac-Thr(tBu)- (solid) or Ac-Thr- (solution). RC = -Pro-
Asn(Trt)-NHRink (solid) or -Pro-Asn-NH2 (solution). Solid phase
reactions were followed by TFA cleavage/deprotection.
Initial efforts to employ t-BuSH proceeded without
success.11 We next examined cross-coupling with thiolacetic
acid (Table 1), which could be subjected to thiolysis or
hydrolysis in solution or on solid phase to yield the free
thiophenylalanine-containing peptide.9 The conditions of
Sawada et al., using 1,10-phenanthroline as a ligand and
(8) Kondo, T.; Mitsudo, T. Chem. Rev. 2000, 100, 3205–20. Ley,
S. V.; Thomas, A. W. Angew. Chem., Int. Ed. 2003, 42, 5400–49.
Beletskaya, I. P.; Ananikov, V. P. Chem. Rev. 2011, 111, 1596–636.
Kwong, F. Y.; Buchwald, S. L. Org. Lett. 2002, 4, 3517–20. Fernandez-
Rodriguez, M. A.; Shen, Q. L.; Hartwig, J. F. J. Am. Chem. Soc. 2006,
128, 2180–1. Correa, A.; Carril, M.; Bolm, C. Angew. Chem., Int. Ed.
2008, 47, 2880–3. Alvaro, E.; Hartwig, J. F. J. Am. Chem. Soc. 2009, 131,
7858–68. Bichler, P.; Love, J. A. Top. Organomet. Chem. 2010, 31, 39–64.
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6595–7. Pd catalyzed: Lai, C.; Backes, B. J. Tetrahedron Lett. 2007, 48,
3033–7. van den Hoogenband, A.; Lange, J. H. M.; Bronger, R. P. J;
Stoit, A. R.; Terpstra, J. W. Tetrahedron Lett. 2010, 51, 6877–81.
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Zondlo, N. J. Biopolymers 2006, 84, 192–204. Thomas, K. M.;
Naduthambi, D.; Zondlo, N. J. J. Am. Chem. Soc. 2006, 128, 2216–7.
(11) Pd-mediated cross-coupling with tBuSH: refs 4g and 8e.
(12) Neidigh, J. W.; Fesinmeyer, R. M.; Andersen, N. H. Nat. Struct.
Biol. 2002, 9, 425–30. Naduthambi, D.; Zondlo, N. J. J. Am. Chem. Soc.
2006, 128, 12430–1. Barua, B.; Lin, J. C.; Williams, V. D.; Kummler, P.;
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Org. Lett., Vol. 14, No. 2, 2012
465

Table 2. Thiol Scope of Reactions in the Solid Phase
a Includes disulfide products. b At 100 °C. RN = Ac-Thr(tBu)- (solid)
or Ac-Thr- (solution). RC = -Pro-Asn(Trt)-NHRink (solid) or -
Pro-Asn-NH2 (solution).
Figure 2. Crude HPLC chromatograms of cross-coupling reac-
tions with different peptides. SM = starting material peptide.13
the reaction conditions do not modify peptide structure in a
subtle manner that would not be identified by HPLC or MS.
Analysis by HPLC indicated clean reactions and high
conversions to cross-coupled product in all cases
(Figure 2). Trityl-protected cysteine residues exhibited
evidence of Cys deprotection and acetylation under reac-
tion conditions. After workup with DTT, both thiophe-
nylalanine and Cys were generated cleanly as free thiols.
Alternatively, to allow differentiation of Cys and thio-
phenylalanine, the peptide with t-butyl-protected Cys was
subjected to reaction conditions, cleanly generating the
peptide with free aryl thiol and protected Cys.
A photocleavable analogue of thiophenylalanine was also
prepared by alkylation with 2-nitrobenzyl bromide.14 In
addition the thioester could be deprotected and alkylation
effected on solid phase (Scheme 2).13
Scheme 1. Solution Alkylation Reactions on 4a
Analysis of the thiophenylalanine-containing trp cage
peptide by circular dichroism (Figure S31) indicated that
its structure and stability were similar to that of the native
tyrosine-containing peptide. In total, these data indicate
that the cross-coupling reaction may be employed to
modify complex peptides with diverse amino acid sub-
stitutions and implies generality in the application of this
reaction to synthesize peptides containing aryl thiols.
The UVÀvis spectra of T(4-SH-Phe)PN revealed a strong
dependence on pH, with a λmax of 249 nm as the thiol and an
intense λmax of 276 nm as the thiolate (ε276 = 17500) (Figure
S1). The pKa of thiophenylalanine was 6.4, significantly lower
than cysteine (pKa ∼8À8.5). Notably, thiophenylalanine is
primarily anionic at physiological pH. Thiophenylalanine
was weakly fluorescent as the thiolate (λmax = 405 nm).13
The reactivity of thiols provides the opportunity to
modify peptides to incorporate a range of functional groups,
which was examined in T(4-SH-Phe)PN peptides. While
alkyl thiols were poor substrates in cross-coupling reactions,
the aryl thiolate was readily alkylated with methyl iodide or
allyl iodide to generate the respective thioethers (Scheme 1).
a RN = Ac-Thr-, RC = -Pro-Asn-NH2.
Allyl thioethers have been shown by Davis to be superior
substrates to allyl ethers for cross-metathesis reactions.15
Aryl allyl thioethers thus have potential as reactive sub-
strates for bioorthogonal cross-metathesis reactions.15c
(14) Pelliccioli, A. P.; Wirz, J. Photochem. Photobiol. Sci. 2002, 1,
441–58.
(15) Lin, Y. A.; Chalker, J. M.; Floyd, N.; Bernardes, G. J. L.; Davis,
B. G. J. Am. Chem. Soc. 2008, 130, 9642–3. Lin, Y. A.; Chalker, J. M.;
Davis, B. G. J. Am. Chem. Soc. 2010, 132, 16805–11. Davis has
speculated on S-allyl-ThioPhe in cross-methathesis: Lin, Y. Y. A.;
Chalker, J. M.; Davis, B. G. ChemBioChem 2009, 10, 959–69.
(13) See the Supporting Information for details.
466
Org. Lett., Vol. 14, No. 2, 2012

Scheme 2. Solid Phase Alkylation of Ac-TXPN-NH2
Scheme 4. Solution Phase Peptide Oxidation Reactionsa
To explore this possibility, the S-allyl-thiophenylalanine-
containing peptide 6 was subjected to Davis’ cross-
metathesis conditions and found to react with allyl
alcohol at room temperature (Scheme 3).13
Scheme 3. Cross-metathesis on Ac-T(4-SAllyl-Phe)PN-NH2
Thiol oxidation state dramatically impacts protein
structure and function.1,16 Thiophenylalanine-containing
peptides were subjected to a variety of oxidation reac-
tions, including formation of the activated disulfide,
glutathionylated mixed disulfide, sulfoxide, sulfone, or
sulfonate (Scheme 4).17
a RN = Ac-Thr-, RC = Pro-Asn-NH2.
Aryl electronics can significantly affect protein struc-
ture and function.10,18 The derivatives described above
represent a continuum from strongly electron-donating
to electron-withdrawing groups. In the TXPN (X = aryl)
context, the strength of an aromatic-proline CH/π inter-
action is manifested in stabilization of a cis amide bond by
more electron-donating substituents. All model peptides
were examined by NMR and Ktrans/cis quantified to
identify the structural effects of aryl substitution (Table
3). The data indicate that thiophenylalanine and its
modified variants exhibit a range of electronic properties
that might be exploited for control of peptide structure.
We have described a practical approach to the synthesis
of thiophenylalanine-containing peptides that is com-
patible with all proteinogenic functional groups. The
method employs the commercially available amino
Table 3. Electronic Effects of Aryl Substution on Peptide
Structure (Ktrans/cis of the prolyl amide bond)
ΔGtrans/cis ΔΔGtrans/cis
Ac-TXPN-NH2, X =
Ktrans/cis kcal molÀ1 kcal molÀ1
4-SÀ-Phe (4, pH 8.5)
4-S-SPyridyl-Phe (20)
4-SAllyl-Phe (6)
2.2
2.9
3.3
3.5
3.5
3.6
3.7
4.3
4.4
4.4
4.7
5.9
3.2
2.7
À0.47
À0.63
À0.71
À0.74
À0.74
À0.76
À0.77
À0.86
À0.88
À0.88
À0.92
À1.05
À0.69
À0.59
0.22
0.06
À0.02
À0.05
À0.05
À0.07
À0.08
À0.17
À0.19
À0.19
À0.23
À0.36
0.00
4-SH-Phe (4, pH 4.0)
4-SCH3ÀPhe(17)
4-SPh-Phe(5)
4-S-SGlutathione-Phe (21)
4-SAc-Phe(2)
4-SO3À-Phe (24)
4-S(2-Nitrobenzyl)-Phe (18)
4-S(O)Me-Phe (22)
4-SO2Me-Phe (23)
Phe(F)10c
Tyr(Y)10c
0.10
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synthesized peptides, providing convenient access
to versatile thiol-modified aromatic amino acids.
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€
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Acknowledgment. We thank NSF (CAREER, CHE-
0547973) and NIH (RR17716) for funding this work.
Supporting Information Available. Experimental pro-
cedures, HPLC chromatograms, and spectral and char-
acterization data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Org. Lett., Vol. 14, No. 2, 2012
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