Tailored Approaches towards the Synthesis of l-S-(Trifluoromethyl)cysteine- and l-Trifluoromethionine-Containing Peptides

Article abstract of DOI:10.1002/ejoc.201601318

Among the fluorinated noncanonical amino acids, l-trifluoromethionine (TFM) and l-S-(trifluoromethyl)cysteine (TfmCys), fluorinated analogues of methionine and cysteine, are of particular interest because of their ability to locally increase the hydrophobicity of peptides. We report herein the synthesis of tert-butoxycarbonyl/benzyl-protected TFM and TfmCys by using a cheap and user-friendly radical trifluoromethylation approach. The benzyl protecting group of these fluorinated amino acids could be conveniently removed by hydrogenolysis, which circumvented troublesome saponification reactions. For the first time, TfmCys was inserted into a peptide sequence by liquid- or solid-phase peptide synthesis. Finally, a late trifluoromethylation strategy with the use of Togni's reagent on disulfide-bridged peptides was also efficient to incorporate TFM or TfmCys at both N-terminal and internal positions.

Full text of DOI:10.1002/ejoc.201601318

A Journal of
Accepted Article
Title: Tailored approaches towards the synthesis of L-S-
trifluoromethylcysteine and L-trifluoromethionine containing
peptides
Authors: Thierry Brigaud, Charlène Gadais, Nathalie Saraiva-Rosa,
Evelyne Chelain, and Julien Pytkowicz
This manuscript has been accepted after peer review and appears as an
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601318
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601318
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10.1002/ejoc.201601318
European Journal of Organic Chemistry
COMMUNICATION
Tailored approaches towards the synthesis of
L-S-
trifluoromethylcysteine and L-trifluoromethionine containing
peptides
Charlène Gadais,[a] Nathalie Saraiva-Rosa,[a] Evelyne Chelain,[a] Julien Pytkowicz,*[a] Thierry Brigaud*[a]
Abstract: Among the fluorinated noncanonical amino acids, L-
trifluoromethionine (TFM) and L-S-trifluoromethylcysteine (TfmCys),
fluorinated analogs of methionine and cysteine, are of particular
interest because of their ability to locally increase the hydrophobicity
of peptides. We report herein the synthesis of Boc/Benzyl protected
involved in electron transfer (Pseudomonas aeruginosa azurin:
Az) has been replaced by isostructural unnatural amino acid
analogues including a TFM one. A linear correlation was
established between the hydrophobicity of the side chains and
the diminution of protein reduction potential.[14] Recently, TFM
was used as lysine hydrophobic isostere in short peptides
inhibitors of histone methyltransferase PRC2 and was found to
inhibit the enzyme better than methionine containing peptides.[15]
TFM and TfmCys using
a cheap and user friendly radical
trifluoromethylation. The benzyl protecting group of these fluorinated
amino acids can be conveniently removed by hydrogenolysis
circumventing troublesome saponification reactions. For the first time, However, data concerning the hydrophobicity of methionine and
TfmCys was inserted in a peptide sequence by liquid or solid phase
peptide synthesis. Finally, a late trifluoromethylation strategy using
Togni’s reagent on disulfide bridged peptides was also efficient to
incorporate TFM or TfmCys at both N-terminal and internal positions.
its trifluoro analogue are scarce and most of them obtained from
fragment methods. Although several example of TFM containing
peptides or proteins can be found in the literature, the
incorporation of the S-trifluoromethyl analogue of cysteine has
been scarcely reported. Two octreotide analogues containing L-
S-trifluoromethylcysteine (TfmCys) were synthesized in 2008 by
Seebach, Togni and co-workers but unfortunately showed a
lower affinity towards the receptor than Octreotide itself.[16,17]
In the course of our ongoing studies dealing with the
measurement of the hydrophobic contribution of unnatural
trifluoromethylated amino acids (TfmAA’s) we are developing
their incorporation into short peptide sequences. Due to their
biological relevance, we turned our attention to TFM but also to
TfmCys that is, to our opinion, underused as cysteine fluorinated
analogue or NMR probe. Although several examples have been
reported in the literature for the bioincorporation[5-9] or the
solution phase[13] incorporation of TFM into peptides, its
integration by means of solid phase peptide synthesis (SPPS) is
very scarcely documented.[10] Because of the high leaving group
propensity of the SCF3 group, the direct incorporation of TfmCys
into peptides is much more challenging and, to our knowledge,
has never been reported so far neither by solution nor by solid
phase peptide synthesis. However its indirect incorporation into
peptides has been successfully achieved by late electrophilic S-
trifluoromethylation of sulfanyl group or cystine containing
peptides.[16] Today, an important panel of methods to obtain
SCF3 compounds is found in the literature.[18,19] In the particular
case of TfmCys and TFM syntheses, the majority of developed
strategies involves trifluoromethylation of available chiral thiols
(L-cysteine and L-homocysteine) or the dimeric disulfide
precursor (L-cystine and L-homocystine). Since the seminal work
of Taborsky et al[20] using very toxic reagents, more user friendly
syntheses have been developed. The CF3 moiety can be
delivered from trifluoromethyl iodide using anionic[21] or radical
chemistry under irradiation.[22,23] Langlois and co-workers
published an interesting alternative to generate the CF3 radical
from sodium trifluoromethanesulfinate and applied it to the
synthesis of TfmCys and TFM from L-cystinate and DL-
homocystinate methyl ester.[24] Alternatively, TfmCys derivatives
In the last decades, unnatural fluorinated amino acids have
proven to be efficient tools to advantageously modulate the
biophysical and biochemical properties of peptides or proteins.[1]
Moreover, due to its nearly total absence in biological media,
fluorine is an outstanding bioorthogonal atom useful as NMR
probe to explore conformational changes in peptides and
proteins and their interactions with other biological molecules.[2–4]
Among the fluorinated noncanonical amino acids successfully
designed for such purpose, L-trifluoromethionine (L-S-
trifluoromethylhomocysteine; TFM), the fluorinated analog of
methionine, was the subject of many relevant studies.[5] The
TFM was bioincorporated into bacteriophage lysozyme (LaL),[6,7]
DNA polymerase[8] or a green fluorescent protein (M2-EGFP)[9]
and used as a potential 19F reporter. More recently, the kinetic
and the presence of intermediate oligomeric species during the
formation of amyloid fibrils from IAPP or Aβ1-40 was
demonstrated by 19F NMR.[10,11] Moreover, various amide
derivatives of TFM showed antimicrobial properties[12] and a
small TFM containing peptides increased the chemotactic
activity of the tripeptide TFM-Leu-Phe.[13] Methionine is
frequently involved in hydrophobic interactions and the authors
often invoked the increases of the fluorinated peptide
hydrophobicity and the strengthening of the interaction with the
hydrophobic pocket of the receptor to explain their results. In
2006, the axial ligand methionine 121 from a blue copper protein
[a]
Dr. C. Gadais, N. Saraiva-Rosa, Dr. E. Chelain, Dr. J.Pytkowicz,
Prof. T. Brigaud
Laboratoire de Chimie Biologique (LCB), EA 4505
Université de Cergy-Pontoise
5 Mail Gay-Lussac, 95000 Cergy-Pontoise, France.
E-mail: julien.pytkowicz@u-cergy.fr
E-mail: thierry.brigaud@u-cergy.fr
https://www.u-cergy.fr/en/laboratories/cbl.html
could also be obtained from photolysis of an intermediate
Supporting information for this article is given via a link at the end of
the document
[25]
trifluorothioacetate.
However a mixture was obtained from
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10.1002/ejoc.201601318
European Journal of Organic Chemistry
COMMUNICATION
which the desired product was not easily separated from side
products. Recently, Togni and co-worker described a direct
cysteine trifluoromethylation using an electrophilic hypervalent
iodine(III) trifluoromethylated reagent. [26] These conditions were
successfully applied to synthesis of the Fmoc protected
trifluoromethionine[10] and more interestingly to the late stage
trifluoromethylation of dipeptides, β-dipeptides and even an
acyclic Octreotide analogue containing two cysteines.[17] In the
latter case however the authors obtained a mixture of mono, di
and tri-trifluoromethylated product since the reagent could also
attack the indol moiety of tryptophan.
and tert-butylhydroperoxide, these reagents are quite cheap.
The results are summarized in table 1. No conversion of the fully
deprotected cystine occurred presumably because of its low
solubility (entry 1). Even the N-Boc protected substrates in an
acetonitrile/methanol mixture proved to be unreactive (entries 2
and 3). As already noticed by Langlois et al., once the carboxylic
acid group is protected as an ester, the reaction proceeds in
pure acetonitrile to give the trifluoromethyl cysteine
hydrochloride 3a (entry 4). In the same conditions, the N-acetyl
cysteine methyl ester gave the expected trifluoromethyl cysteine
4a in 84% yield (entry 5).
In order to incorporate such SCF3 group containing amino
acids in a peptide chain, both amino and carboxylic protecting
groups should be selectively removed. We first try to perform the
saponification reaction of the cysteine methyl ester derivative 4a.
Unfortunately, even in mild basic conditions [27] the -elimination
reaction of the SCF3 group could not be avoided and a
significant amount of the elimination product 8 [28,17] was always
obtained beside the expected amino acid 7 (Scheme 1). In order
to obtain TfmCys and TFM ready to use for peptide synthesis,
we decided to prepare the orthogonally protected Boc/Bn
substrates which should provide the corresponding carboxylic
acid derivatives by hydrogenolysis. The S-trifluoromethylation of
the cystine and the DL-homocystine derivatives gave the
corresponding trifluoromethylthiolated amino acids in 33% and
42% yield respectively (Table 1, entries 6 and 7).
We report herein the synthesis of four model SCF3 group
containing tripeptides especially designed for probing the
consequences of the introduction of this fluorinated group on
peptides hydrophobicity. The fluorinated amino acids TfmCys
and TFM are positioned at the N-terminal position for peptides
1a and 1b and in the internal position for peptides 2a and 2b
(scheme 1). we developed three different approaches towards
the synthesis of these target peptides i) liquid synthesis from
conveniently protected TfmCys or TFM residues ii) solid phase
synthesis (SPPS) under microwave conditions iii) late stage
trifluoromethylation of dimeric cystine and homocystine
derivatives using Togni’s reagent.
Table 1. Radical trifluoromethylation of cystine and homocystine derivatives.
Entry
n
1
1
2
1
1
1
2
R1, R2
Conditions
A[b]
Product
Yield (%)[a]
1
H, H
-
-
2
Boc, H
Boc, H
HCl, Me
Ac, Me
Boc, Bn
Boc, Bn
B[c]
-
-
Figure 1. TfmCys and TFM target peptides.
3
B[c]
-
-
4
B[c]
3a
4a
5a
5b[g]
24
84
33
42
We first devoted our efforts to the synthesis of conveniently
protected TfmCys
and
TFM.
The electrophilic
S-
5
B[c]
trifluoromethylation using Togni’s reagent seemed very attractive
because of its compatibility with Fmoc and Boc protecting
groups.[10,26] In order to obtain the most versatile synthon for
liquid peptide coupling, we decided to start from Boc/Bn
protected cystine and homocystine. First, we tested the
electrophilic trifluoromethylation on the isolated cysteine
generated by reduction of the corresponding disulfides in
classical conditions. But unfortunately the reaction was unpaired
by a difficult separation of the desired product from the reagent
derived byproduct. Considering the moderate results obtained
via the electrophilic trifluoromethylation we decided to introduce
the trifluoromethyl group via a radical substitution reaction on the
disulfide group. Although the reaction developed by Langlois et
al. requires at least a twofold excess of trifluoromethylsulfinate
6
C[d]
7[e]
D[f]
[a] Isolated yields. [b] Conditions A : tBuOOH (3 equiv) / CF3SO2Na (3 equiv) /
H2O. [c] Conditions B : tBuOOH (4.5 equiv) / CF3SO2Na (3 equiv) / MeCN or
MeCN/MeOH. [d] Conditions C : tBuOOH (9.5 equiv) / CF3SO2Na (6 equiv) /
MeCN. [e] Reaction from Boc-DL-homocystine benzyl ester. [f] Conditions D :
tBuOOH (6 equiv) / CF3SO2Na (4.5 equiv) / MeCN. [g] Racemic mixture.
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10.1002/ejoc.201601318
European Journal of Organic Chemistry
COMMUNICATION
54% yield. To our knowledge this synthesis constitutes the first
introduction of the TfmCys in a peptide using the classical liquid
phase coupling methodology.
Scheme 1. Saponification reaction of the methyl ester 4a leading to the SCF3
-elimination as a side reaction.
The benzyl esters 5a and 5b were submitted to
hydrogenolysis in the presence of palladium to give the free
carboxylic acids 9a and 9b in excellent yields. The Boc
protection of TfmCys 5a could also be removed quantitatively in
classical acidic condition to give the benzyl ester 10 (scheme 2).
Scheme 3. Liquid phase synthesis of tripeptide 2a.
In other respect, the use of the Boc-TfmCys-OH amino acid
9a in Solid Phase Peptide Synthesis (SPPS) was investigated.
However because of the acidic lability of the Boc protection, the
Tfm-cysteine could only be inserted at the peptide N-terminus.
Thus the synthesis of the tripeptide 1a was investigated by
manual SPPS on a high loaded Wang Resin using HATU and
DIPEA as coupling mix. Unfortunately, in these conditions the
coupling reaction of the fluorinated amino acid occurred with a
very low yield. The microwave technology is known to increase
the yield of difficult peptide coupling and reduce the amount of
side-reactions often observed with unusual amino acids. Thus
the synthesis of 1a was investigated with an automated
microwave peptide synthesizer using DIC and oxyma pure as
Scheme 2. TfmCys and TFM target peptides.
Even if the electrophilic trifluoromethylation using Togni’s
reagent is known to proceed in better yields than the radical one,
the price of the reagents and the ease of purification made the
radical addition
a method of choice to prepare sufficient
quantities of Boc-TfmCys-OH (9a), Boc-TFM-OH (9b) and H-
TfmCys-OBn (10) appropriate for liquid or SPPS coupling.
coupling reagents,
a standard procedure for microwave
activation. In these conditions, the expected tripeptide 1a was
obtained in 30% yield from a single coupling of Boc-TfmCys-OH
9a at 50°C (scheme 4). To our knowledge, this result validates
for the first time, the incorporation of TfmCys in a peptide by
SPPS.
Since the benzyl ester protection is orthogonal to Boc and
tert-Butyl ester protections, the amino acid H-TfmCys-OBn (10)
is suitably designed for its incorporation into a short peptide
chain by liquid phase peptide synthesis. We chose the
hydrophobic H-Ala-TfmCys-Leu-OH peptide 2a as a model
(Scheme 3). For the liquid phase peptide coupling at the N-
terminal position of 10, we chose the mixed anhydride activation
because of the reduced basicity of N-methyl morpholine (NMM)
compared to Hunig’s base, typically used with other coupling
reagents. In these conditions, the dipeptide 11 resulting from the
coupling of 10 with the Boc-protected alanine, was obtained in
very good yield (91%) without any traces of SCF3 elimination
side products. The hydrogenolysis of the benzyl ester proceeded
smoothly to give the C-terminal unprotected dipeptide 12 that
was submitted to a coupling reaction in the same conditions with
the tert-butyl ester protected leucine to give the tripeptide 13 in
68% yield. To our great satisfaction, no SCF3 elimination side
product was detected at this step either. After a standard silica
gel purification, the protecting groups of 13 were removed using
TFA in dichloromethane (scheme 3) to yield the tripeptide 3 in
Scheme 4. Automated microwave activation SPPS of 1a.
In parallel to the direct introduction of TfmCys and TFM by
solution of solid phase peptide synthesis, we developed an
alternative strategy based on a late stage trifluoromethylation
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10.1002/ejoc.201601318
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COMMUNICATION
Scheme 5. Synthesis of H-TfmCys-Ala-Leu-OH (1a) and H-TFM-Ala-Leu-OH
(1b).
reaction using Togni’s reagent. This strategy inspired by the
pioneer works of Seebach, Togni et al.[17] allowed the synthesis
of tripeptides incorporating the trifluoromethylated amino acid in
N-terminal or central position. The strategy consists in the
introduction of the trifluoromethyl group at the end of synthesis
via the electrophilic trifluoromethylation reaction of dimeric
hexapeptides obtained from commercial cystine or homocystine.
As shown in the scheme 5, this method provided the
trifluoromethylated tripeptides 1a,b with TfmCys and TFM in N-
terminal position. After a classical Boc protection of the amine
functions of L-cystine or L-homocystine, the coupling reaction of
alanine methyl ester hydrochloride was performed through a
mixed anhydride activation of the two carboxylic acid functions
As presented in scheme 6, the synthesis of the tripeptide 2b
incorporating the trifluoromethionine in the central position has
also been achieved by electrophilic late trifluoromethylation of a
homocystine based hexapeptide. The first steps of this pathway
were initially tested from the DL-homocystine before being
carried out on enantiopure L-homocystine. The coupling reaction
between the Boc-protected L-alanine and homocystine methyl
ester hydrochloride was performed through the mixed anhydride
activation of alanine with isobutylchloroformate in the presence
of N-methylmorpholine to yield the corresponding di-Boc
protected dimethyl ester 18. After saponification of the ester
functions, the second coupling was carried out in the same
conditions with leucine tert-butyl ester hydrochloride to yield the
precursor of the trifluoromethylated tripeptide 20 in 57%. After
reduction of the disulfide, the trifluoromethylation reaction using
the electrophilic hypervalent iodine (III) trifluoromethylated
reagent leaded to the protected tripeptides 21 with a moderate
yield (21%). After removal of the protecting groups, the peptide
was purified by semi-preparative HPLC to provide the peptide
2b as its TFA salt in 47% yield.
with
isobutylchloroformate
in
the
presence
of
N-
methylmorpholine. The corresponding diBoc protected dimethyl
ester dipeptides 14a and 14b were obtained in 67% and 60%
respectively. After saponification of the methyl ester functions,
the diBoc protected diacids 15a and 15b were obtained in very
good yields. The two diacids were then engaged in a last
coupling with hydrochloride leucine tert-butylester using the
mixed anhydride activation, and the corresponding peptide
dimers 16a and 16b were obtained in quite good yields. The last
step before deprotection of the peptides is the electrophilic S-
trifluoromethylation using Togni’s reagent after reduction of the
disulfide bridges. The disulfide derivatives were cleaved by
treatment with tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) at room temperature. The addition of Togni’s reagent at
-78°C on the resulting thiols gave the trifluoromethylated N-
protected tert-butylester tripeptides 17a and 17b in moderate
40% and 11% yield respectively after purification by silica gel
chromatography. After simultaneous removal of the Boc and the
tert-butyl ester protecting groups with a large excess of TFA in
dichloromethane, the expected H-TfmCys-Ala-Leu-OH (1a) and
H-TFM-Ala-Leu-OH (1b) were quantitatively obtained as their
TFA salts.
Scheme 6. Synthesis of H-Ala-TFM-Leu-OH (2b)
In conclusion, we developed the synthesis of enantiopure L-
S-trifluoromethylcysteine and L-trifluoromethionine suitably
protected for peptide synthesis using
a
cheap radical
trifluoromethylation of disulfide precursors. Circumventing the
elimination of SCF3 moiety by hydrogenolysis of the benzyl ester
protected TfmCys, we were able to perform the liquid phase and
the solid phase synthesis of TfmCys containing peptides. We
also performed the late stage trifluoromethylation of dimeric
disulfide bridged hexapeptides derived from cystine or
homocystine. If this strategy was already described for TfmCys,
we demonstrated that it is also transposable for the synthesis of
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[10] Y. Suzuki, J. R. Brender, M. T. Soper, J. Krishnamoorthy, Y. Zhou, B. T.
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TFM containing peptides. These synthetic progresses will be of
major interest considering the relevant incorporation of these
two unnatural amino acids in peptides for biological issues. The
application of these short fluorinated peptides in terms of
hydrophobicity measurements will be reported in due course.
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Keywords: Fluorinated amino acids • (L)-S-trifluoromethyl
cysteine • (L)-trifluoromethionine • Fluorinated peptides • Solid
phase and solution phase peptide synthesis
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COMMUNICATION
COMMUNICATION
L-trifluoromethionine (TFM) and L-S-
trifluoromethylcysteine (TfmCys) are
synthesized using a cheap and user
friendly radical trifluoromethylation.
TfmCys is inserted for the first time in
a peptide sequence by liquid or solid
phase peptide synthesis. A late
trifluoromethylation strategy using
Togni’s reagent on disulfide bridged
peptides is also efficient for the
Charlène Gadais, Nathalie Saraiva-
Rosa, Evelyne Chelain, Julien
Pytkowicz,* Thierry Brigaud*
Page No. – Page No.
Tailored approaches towards the
synthesis of L-S-
trifluoromethylcysteine and L-
trifluoromethionine containing
peptides
incorporation of TFM or TfmCys at
both N-terminal and internal positions.
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