Trifluoromethylthiolation of Tryptophan and Tyrosine Derivatives: A Tool for Enhancing the Local Hydrophobicity of Peptides

The incorporation of fluorinated groups into peptides significantly affects their biophysical properties. We report herein the synthesis of Fmoc-protected trifluoromethylthiolated tyrosine (CF3S-Tyr) and tryptophan (CF3S-Trp) analogues on a gram scale (77–93% yield) and demonstrate their use as highly hydrophobic fluorinated building blocks for peptide chemistry. The developed methodology was successfully applied to the late-stage regioselective trifluoromethylthiolation of Trp residues in short peptides (66–80% yield) and the synthesis of various CF3S-analogues of biologically active monoamines. To prove the concept, Fmoc-(CF3S)Tyr and -Trp were incorporated into the endomorphin-1 chain (EM-1) and into model tripeptides by solid-phase peptide synthesis. A remarkable enhancement of the local hydrophobicity of the trifluoromethylthiolated peptides was quantified by the chromatographic hydrophobicity index determination method, demonstrating the high potential of CF3S-containing amino acids for the rational design of bioactive peptides.


■ INTRODUCTION
The introduction of fluorine atoms into biomolecules has become a well-established strategy in drug development, as they can be used to favorably improve or modulate their physicochemical and biological properties. 1 This approach is now widely exploited in the pharmaceutical industry, with 20− 25% of marketed drugs containing at least one fluorine atom. 2 Concurrently, peptides have emerged as a unique class of therapeutic agents in recent years.Over the past decade, their development has steadily increased, and therapeutic peptides now account for a significant portion of the pharmaceutical market. 3Moreover, tailor-made amino acids are becoming privileged scaffolds even in small-molecule drugs (which account for over 30% of pharmaceuticals). 4These paradigm shifts in medicinal chemistry call for further efforts to advance the synthesis of diverse fluorinated amino acids (F-AAs) for expanding the peptide design toolbox. 5n particular, the incorporation of F-AAs into peptides is a powerful tool for modulating various parameters, such as local hydrophobicity, 6 pK a values of proximal functionalities, 1b membrane permeability, 7 metabolic stability, 8 and inducing conformational constraints and self-assembly properties. 9urthermore, fluorine atoms are used as highly sensitive probes for 19 F NMR spectroscopy in biological media.9b,10 The most commonly investigated F-AAs contain either a fluorine atom or the CF 3 group. 11However, other fluorinated groups, especially motifs bearing chalcogen atoms, remain underexplored in the context of amino acid chemistry. 12Among them, the trifluoromethylthio group (CF 3 S) is of particular interest as it has one of the highest lipophilicity parameters (Hansch-Leo parameter; π = 1.44), 13 a strong electron-withdrawing effect (Hammett constant σ m = 0.40, σ p = 0.50), 14 and a favorable pharmacological profile. 15Therefore, the synthesis of trifluoromethylsulfanylated amino acids (CF 3 S-AAs) and their incorporation into peptides appears to be a promising strategy to improve their physicochemical properties.In particular, the local hydrophobicity and membrane permeability could be significantly increased, improving the drug profile of the peptides. 15,16ost of the CF 3 S-AAs reported so far involve the aliphatic AAs, in particular, the trifluoromethylcysteine (TfmCys) 16 and trifluoromethionine (TFM) analogues. 17They proved to be useful as sensitive 19 F NMR reporters to probe protein− protein interactions (PPIs), 10a as well as for their ability to enhance the local hydrophobicity of model peptides.6b A CF 3 Scysteine (CF 3 S-Cys) analogue was also prepared by direct trifluoromethylthiolation of the corresponding thiol. 18lthough there are numerous developed methods 15a, 16,19 and a plethora of shelf-stable and efficient reagents 20 for aromatic trifluoromethylthiolation, the synthesis of aromatic CF 3 S-containing amino acids (CF 3 S-AAs) is still in its infancy and has been very scarcely reported to date.A few years ago, Billard and co-workers demonstrated the reactivity of various substituted indoles toward electrophilic trifluoromethylthiolation using their first-and second-generation trifluoromethanesulfenamide reagents. 21While the reaction was effective in introducing the CF 3 S group at the C2-position of unprotected indole propionic acid and tryptamine, very limited conversion (ca.15%) was observed with Cbz-protected and unprotected Trp residues, and the desired products were not isolated (Scheme 1A).21a Nevertheless, the reaction conditions proved to be sensitive to the nature of the aromatic ring and are mainly limited to electron-rich aromatic compounds.Very recently, the synthesis of p-SCF 3 phenylalanine (Phe) through a photoredox-mediated Ni-catalyzed trifluoromethylthiolation pathway was reported. 22To the best of our knowledge, CF 3 S incorporation into tyrosine derivatives has not been reported so far.

The Journal of Organic Chemistry
As the demand for original fluorinated compounds continues to increase, the development of robust methods that allow efficient access to aromatic CF 3 S-AAs on a gram scale is of great importance.We have previously demonstrated that the p-chloro analogue 1 of the first-generation Billard's 21 reagent is more stable and promotes efficient electrophilic Friedel-Crafts trifluoromethylthiolation of diverse (hetero)arenes (Scheme 1B). 23Herein, we have investigated the Friedel-Crafts trifluoromethylthiolation with reagent 1 for the preparation of a series of aromatic CF 3 S-AAs (Scheme 1).We first report the synthesis of trifluoromethylthiolated tryptophan (CF 3 S-Trp) and tyrosine (CF 3 S-Tyr) amino acids, as well as their related monoamine analogues, such as trace amines and catecholamines.As an extension, the feasibility of the late-stage functionalization (LSF) was investigated on a series of Trpand/or Tyr-containing short peptides.In addition, solid-phase peptide synthesis (SPPS) synthesis of fluorinated analogues of the opioid agonist endomorphin-1 (EM-1) was performed using the ready-to-use Fmoc-protected CF 3 S-Trp and CF 3 S-Tyr building blocks.Finally, we report a significant enhancement of the local hydrophobicity and acidity originating from the CF 3 S group.

■ RESULTS AND DISCUSSION
The electrophilic trifluoromethylthiolation reaction was first studied on tryptophan derivatives 2a−c (Table 1).
The Fmoc-protecting group was selected to provide direct access to readily available building blocks for SPPS.In addition, the Fmoc group is known to tolerate the general acidic conditions used in our previous report. 23Trifluoromethylthiolation was first attempted on Fmoc-Trp-OEt 2a by applying the conditions of Billard et al. 21a with TsOH (2.5 equiv) as an activator in DCM at rt (conditions A) (Table 1, entry 1).Instead of the expected product 4a, the formation of CF 3 S-hexahydropyrrolo[2,3-b]indole 3a was observed in a low yield (29%) as a separable ca.1:1 mixture of endo-cis and exocis diastereomers (see the Supporting Information for structural elucidation). 24The formation of the intermediate compound 3a is consistent with the previously reported cascade trifluoromethylthiolation-cyclization of N-protected tryptamines. 25Similar to Qing et al., 25a we observed in the AAseries that 3a undergoes ring opening after a prolonged time in acidic media, yielding the expected CF 3 S-Trp 4a.To increase the activation of the electrophile and the rate of the ringopening process, we decided to replace TsOH with a stronger acid, namely, triflic acid (TfOH).The use of only 1.0 equiv of TfOH resulted in an inseparable 14:1 mixture of 3a and 4a in a 71% yield (Table 1, entry 2).Addition of TfOH in excess (2.5 equiv) increased both the selectivity for product 4a (3a/4a = ca.2:1) and the isolated yield of 3a and 4a (92%) (Table 1, entry 3).Increasing the reaction temperature to 50 °C in DCE (conditions B) allowed the exclusive formation of 4a in a 90% isolated yield in a shorter reaction time (Table 1, entry 4).The reaction also proved effective under Lewis acid activation.An initial experiment with BF 3 •OEt 2 under conditions A gave a mixture of 3a and 4a in a ratio of 2.1:1 (Table 1, entry 5).Application of conditions B resulted in an opposite 1:2.2 ratio of 3a/4a (Table 1, entry 6).Increasing the reaction time to 48 h improved the yield but had a limited effect on the ringopening step (Table 1, entry 7).Finally, a second addition of 2.5 equiv of BF 3 •OEt 2 after 24 h proved effective for complete conversion to 4a (Table 1, entry 8).It is noteworthy that trifluoromethylthiolation under Lewis acid activation gave a cleaner conversion compared to TfOH activation.To gain direct access to ready-to-use building blocks for SPPS, we decided to investigate the trifluoromethylthiolation of Fmoc-Trp-OH 2b.Compared to the ester series (Table 1, entry 7), the reaction on Fmoc-protected acid 2b using 2.5 equiv of BF 3 • OEt 2 under conditions B resulted in a complete conversion to the expected CF 3 S-Trp 4b after 24 h (Table 1, entry 9).With the optimized reaction conditions in hand, the trifluoromethylthiolation of 2b was carried out on a gram scale, yielding approximately 2 g of the desired product 4b (93% yield, Table 1, entry 10).The enantiopurity of 4b was analyzed by chiral HPLC, which showed no epimerization at the C α position (see the Supporting Information, Chapter 5).Next, we investigated the effect of the N-protection on the reaction outcome.Trifluoromethylthiolation starting from N-unprotected ethyl ester 2c under TfOH activation proceeded much faster (30 min) compared to 2a (Table 1, entry 3), and the corresponding CF 3 S-Trp 4c was isolated in a 96% yield (Table 1, entry 11).This result highlights the key role of the nature of the amino group in the ring-opening step.
We then decided to extend the scope of the reaction to biologically important tryptamine-based trace amines (Scheme 2).
The Journal of Organic Chemistry has been isolated in very low yield.21a Herein, the use of reagent 1 in the presence of TfOH allowed us to selectively obtain Fmoc-protected and N-methyl tryptamine analogues 4d and 4e (90 and 77% isolated yields, respectively), and to significantly improve the isolated yield (92%) of the unprotected CF 3 S-tryptamine 4f (Scheme 2).CF 3 S introduction was then attempted on serotonin and melatonin, two important biologically relevant representatives of the C5functionalized tryptamine derivatives.TfOH activation of Fmoc-protected serotonin 2g resulted in a ca.3:1 mixture of mono-SCF 3 (at the C2-indole position) and bis-SCF 3 (at the C2-and C4-indole positions) products.We assumed that the formation of the bis-SCF 3 compound arises due to the presence of the hydroxyl group at the C5-position, which causes a considerable increase in the electron density of the indole.Therefore, we decided to decrease the activation of the indole by protecting the free hydroxyl with a benzyl group (see Table S1 for optimization data).In addition, the BF 3 •OEt 2 activation was preferred to avoid the possible deprotection of the benzyl group by TfOH.Under these conditions, only the mono-SCF 3 serotonin derivative 4h was obtained in high yield (Scheme 2).When applied to melatonin 2i, which has a methoxy group at the C5-indole position, these conditions gave the expected mono-SCF 3 melatonin 4i in a 66% yield (Scheme 2).
We further explored the scope and limitations of our method with less electron-rich aromatic tyrosine (Tyr) derivatives (Table 2).
To access readily available building blocks for SPPS, Fmoc-Tyr-OH 5a was first subjected to the previously optimized conditions for Trp.The trifluoromethylthiolation of 5a using BF 3 •OEt 2 at rt (conditions A) and at 50 °C (conditions B) proceeded with a very low conversion (Table 2, entry 1).These results were attributed to the lower reactivity of the phenolic moiety compared to the indole.Replacing BF 3 •OEt 2 with TfOH significantly improved the reaction conversion (Table 2, entry 2).Moreover, increasing the amount of reagent 1 to 1.5 equiv resulted in complete conversion, and Fmocprotected CF 3 S-Tyr 6a was isolated in a 79% yield (Table 2, entry 3).A gram-scale trifluoromethylthiolation of 5a yielded ca.1.6 g of the enantiomerically pure Fmoc-protected building block 6a in 77% isolated yield (Table 2, entry 4).It is worthwhile to note that the phenol group of this amino acid should be orthogonally protected by a t-butyl group for incorporation into longer peptide sequences.In the case of the Fmoc-Tyr-OMe substrate 5b, harsher conditions (2.5 equiv of 1, 2 × 2.5 equiv of TfOH) were required to achieve quantitative CF 3 S-incorporation and the ester analogue 6b was isolated in an excellent yield of 93% (Table 2, entry 5).In contrast to the observation in the Trp series, the trifluoromethylthiolation of 5c without Fmoc protection of the Nterminus did not proceed (Table 2, entry 6).
Encouraged by the novel reactivity of tyrosine-based substrates, we investigated the CF 3 S-functionalization of tyramine, dopamine, and DOPA derivatives 5d−i (Scheme 3).
As with the Tyr series (see Table 2, entry 5), harsher conditions had to be applied to obtain Fmoc-protected and unprotected CF 3 S-tyramines 6d and 6e in good yields (Scheme 3).In the case of the dopamine and DOPA analogues, standard conditions (1.2 equiv of 1, 2.5 equiv of TfOH) are sufficient to achieve quantitative CF 3 S incorporation in the electron-rich catechol motif.However, a change in regioselectivity was observed because of the para-directing effect of the additional hydroxyl group (Scheme 3).This allowed straightforward synthesis of Fmoc-protected and unprotected CF 3 S-dopamines 6f and 6g as single regioisomers in 70 and 68% yields, respectively (Scheme 3).When applied to Fmoc-protected DOPA 5h, the reaction gave the corresponding 2−SCF 3 derivative 6h.Purification of 6h by flash chromatography showed that the product was not stable on silica after a prolonged time.Nevertheless, 6h could be isolated in satisfactory purity only by acidic workup.On the other hand, quantitative conversion of Fmoc-DOPA-OBn 5i could be achieved under BF 3 •OEt 2 activation (5.0 equiv, conditions B) to afford the corresponding 6i in a 77% yield (Scheme 3).The scope of the method was then tentatively extended to phenylalanine substrates (Phe), but no reaction occurred.The reactivity of the phenyl ring of Phe is low, and trifluoromethylthiolation occurs on the aniline ring of reagent 1.Such limitations were also demonstrated in our previous report on the trifluoromethylthiolation of arenes. 23SF is a powerful diversification tool for the synthesis of peptides featuring unnatural AAs with modified properties or biological activity.Because of its high reactivity, the Trp residue is often targeted for such modifications. 26We therefore investigated the feasibility of the method for LSF on a series of Trp-and/or Tyr-containing short peptides (Scheme 4).
Late-stage trifluoromethylthiolation was first investigated on a series of peptides with a Trp residue at the N-or C-terminal position (7a−b and 7c−d) and in the middle position of the peptide chain (7e).LSF of Fmoc-protected peptides was carried out using BF 3 •OEt 2 activation under the optimized conditions found for the Fmoc-Trp ester 2a (see Table 1, entry 8).CF 3 S-peptide 8a with the Fmoc-Trp residue at the Nterminal position was obtained in a 74% yield after 48 h (Scheme 4).LSF of the peptide 7b, with the free N-terminal amino group, was first performed under TfOH activation, which was the condition successfully used for the unprotected Trp ester 2c (see Table 1, Entry 11).However, the reaction a PG-Tyr-OR 5a−c (0.10 mmol, 1.0 equiv), ArNHSCF 3 1 (1.2 equiv), acid (2.5 equiv), conditions A (DCM; 1 mL, 0.1 M; rt).Conversion to 6a determined by 1 H NMR analysis (DMSO-d 6 ) of the crude reaction mixture.b n.d.: not determined.c Conditions B (DCE; 1 mL, 0.1 M; 50 °C), 24 h.d 1.5 equiv of ArNHSCF 3 1.e Reaction performed on a gram scale (4.0 mmol).f ArNHSCF 3 1 (2.5 equiv), TfOH (2 × 2.5 equiv, second addition after 18 h).

The Journal of Organic Chemistry
TfOH activation.Peptides 8d and 8e, having the Trp residue at the C-terminus and in the middle position of the peptide chain, were obtained in good yields (Scheme 4).Because of the difference in kinetics observed with peptides 8a and 8c, we decided to investigate the regioselectivity of the trifluoromethylthiolation on the Trp−Trp dipeptide 7f.CF 3 S incorporation under TfOH activation gave a mixture of mono-and bis-SCF 3 dipeptide 8f in a ratio of ca.2:1 (Scheme 4).Therefore, we have chosen the reaction conditions to selectively obtain the bis-SCF 3 dipeptide 8f.Under TfOH activation, the reaction gave a complex mixture, whereas, under BF 3 •OEt 2 activation, the dipeptide 8f was isolated in a 71% yield (Scheme 4).The chemoselectivity aspect of the late-stage SCF 3 incorporation, based on the difference in reactivity between Trp and Tyr residues, has also been investigated.Complete chemoselectivity in favor of the Trp residue was observed for the model dipeptide 7g.The reaction resulted in a quantitative conversion when 1.05 equiv of 1 was used and peptide 8g was obtained in a 78% yield (Scheme 4).A model Ala-Tyr dipeptide 7h was also tested for selective LSF of the Tyr residue.Unfortunately, the required harsher conditions (as in Table 2, entry 5) were not tolerated by the peptide substrate, resulting in a complex reaction mixture.To highlight the generalization of LS trifluoromethylthiolation to longer peptides of interest, we investigated the reaction on the analgesic peptide endomorphin-1 (EM-1).This substrate was selected as a relevant biologically active peptide target because it contains both Tyr and Trp residues.As expected, by applying the LSF conditions using a large excess of BF 3 •OEt 2 (20 equiv), only the formation of (CF 3 S)Trp-EM-1 was observed in 69% conversion, as determined by UPLC-MS and NMR analysis (Scheme 4).
As a complementary strategy to LSF, we decided to investigate the synthesis of the (CF 3 S)Trp-EM-1 analogue by SPPS.The (CF 3 S)Trp-EM-1 was obtained in a 26% yield using standard protocols and coupling reagents (HATU/ DIPEA) (Table 3).
Compared to endomorphin-1 (EM-1), the two fluorinated analogues were obtained in comparable yields and have similar cis/trans ratios of the peptidyl-prolyl amide bond.These results indicate that Fmoc-(CF 3 S)Trp-OH 4b and Fmoc-(CF 3 S)Tyr-OH 6a are suitable building blocks for SPPS and that their incorporation into EM-1 does not significantly affect the peptide conformation (Table 3).Interestingly, the retention times (tR) determined by reverse-phase HPLC were significantly longer for both CF 3 S-containing peptides compared to the parent EM-1 (ca. 3 min).This result supports the expected enhancement of local hydrophobicity.To achieve an accurate determination of the local hydrophobicity enhancement imparted by the CF 3 S group in a peptide context, we decided to measure the chromatographic hydrophobicity indexes of a series of four H 2 N-Ala-AA-Leu-OH tripeptides containing the respective Tyr, Trp, and (CF 3 S)Tyr or the (CF 3 S)Trp residue at the AA position (Figure 1).
The hydrophobicity index (HI) shift ΔHI (HI (CF3S)AA − HI AA ) is an assessment of the contribution of the CF 3 S substituent to the increase in local hydrophobicity.This method, developed by Valkóet al., 27 has already been successfully applied to measure the increased local hydrophobicity of peptides containing fluorinated AAs.6b,28 This short peptide sequence was chosen to prevent the formation of secondary structures that could affect hydrophobicity (see the Supporting Information, Chapter 6, for details).As shown in Figure 1, CF 3 S-containing peptides 11 and 12 have significantly higher HI compared to their non-fluorinated counterparts 9 and 10.These results corroborate the significant increase in RP-HPLC retention times measured with fluorinated EM-1 analogues.As observed in the nonfluorinated series (peptides 9 and 10), the (CF 3 S)Trpcontaining peptide 12 exhibits a higher HI than the (CF 3 S)Tyr peptide 11.It is noteworthy that the replacement of the Tyr residue with its CF 3 S-Tyr analogue provides a higher local enhancement of the hydrophobicity (ΔHI = HI (CF3S)Tyr − HI Tyr ca. 12 units) compared to the Trp series (ΔHI = HI (CF3S)Trp − HI Trp ca. 8 units).These results showed that (CF 3 S)Tyr and (CF 3 S)Trp are the most hydrophobic AAs determined so far by this method.6b Finally, we assessed the influence of the CF 3 S group on the acidity of the vicinal phenol function in a simplified tyramine model carrying an ortho CF 3 S.This functionality is highly important for peptide/protein active site interactions.The pK a values of the tyramine 5e and the CF 3 S-tyramine 6e triflate salts were determined by 1 H NMR spectroscopy in H 2 O/D 2 O 90:10 (see the Supporting Information, Chapter 7, for more details). 29It is noteworthy that in the case of CF 3 S-tyramine 6e, the pK a value could also be determined by 19 F NMR spectroscopy.As expected, the CF 3 S has a major impact on the pK a value with a 100-fold increase of the acidity of the vicinal hydroxyl group (pK a,(CF3S)Tyr = 8.1, pK a,Tyr = 9.9, Figure 1).The values of the pK a of CF 3 S-and CF 3 -phenols are comparable (8.25 30 and 8.42 31 for 2-CF 3 phenols in H 2 O), which is consistent with their similar electron-withdrawing nature (Hammet substituent constant: CF 3 : σ m = 0.43, σ p = 0.54; CF 3 S: σ m = 0.40, σ p = 0.50). 14

■ CONCLUSIONS
In this work, we have developed an efficient method for the aromatic trifluoromethylthiolation of tryptophan and tyrosine residues.Reactions were carried out using the electrophilic trifluoromethanesulfenamide reagent 1 in the presence of TfOH or BF 3 •OEt 2 as activating acids.Several trifluoromethylthiolated tryptophan analogues (CF 3 S-Trp) and their biologically active monoamine derivatives, protected or unprotected, were prepared in high yields following a thorough The Journal of Organic Chemistry methodological study.We also demonstrated the scope of our method by preparing the Fmoc-(CF 3 S)Trp-OH 4b and Fmoc-(CF 3 S)Tyr-OH 6a building blocks that are ready-to-use for SPPS on a gram scale.The late-stage CF 3 S-functionalization of short peptides showed total regioselectivity in favor of the Trp residue over other aromatic side chains and was successfully applied to the opioid agonist endomorphin-1 (EM-1).The synthesis of two trifluoromethylthiolated analogues of EM-1 was achieved by SPPS using standard protocols and Fmocprotected building blocks 4b and 6a.Finally, we investigated the effect of the incorporation of the CF 3 S group into aromatic Trp and Tyr residues on the physicochemical properties of the peptide framework.We demonstrated that the CF 3 Scontaining peptides exhibited a significant enhancement of the local hydrophobicity compared with their non-fluorinated counterparts.In this regard, the CF 3 S-Trp is more hydrophobic than the CF 3 S-Tyr.Moreover, the incorporation of a CF 3 S group adjacent to the phenol ring of tyramine significantly increases its acidity.Because of the key role played by Trp and Tyr residues in protein−protein interactions and the great biological importance of Trp-and Tyr-derived monoamines, the reported CF 3 S-containing analogues represent a new class of compounds endowed with unique properties. 32Their ability to dramatically increase local hydrophobicity and modulate the pK a of adjacent functional groups makes them very attractive for medicinal chemistry applications.The ease of implementation of the reported trifluoromethylthiolation should inspire further developments in the design of bioactive peptides.

Data Availability Statement
The data underlying this study are available in the published article and its online Supporting Information.
Experimental procedures, characterization data, copies of NMR spectra for all new compounds, HPLC traces of endomorphin-1 analogues, HPLC enantiopurity analysis of compounds 4b and 6a, HI determinations, and pKa determinations of compounds 5e and 6e (PDF) ■ AUTHOR INFORMATION The Journal of Organic Chemistry

Table 1 .
Scheme 1.Previous Reports on Direct Aromatic Trifluoromethylthiolation of Trp Derivatives or (Hetero)arenes Using the Trifluoromethanesulfenamide Reagents and Our Work Reported Herein Trifluoromethylthiolation of Tryptophan Derivatives 2a−c