Direct/Reversible Amidation of Troponyl Alkylglycinates via Cationic Troponyl Lactones and Mechanistic Insights

Abstract

The conversion of troponyl alkylglycinate acid/ester/amide derivatives (Trag acid/ester/amide) into cationic troponyl lactones (CTLs) in the presence of trifluoroacetic acid and their amidation with amines is described. The reversible amidation of Trag amides, that is, the cleavage and reformation of the Trag amide bond via CTLs is demonstrated. The direct amidation of Trag esters with the amino group of amino acid esters/peptide esters via CTLs is achieved. The direct amidation of the amine group of hydroxyl amino acid esters is selective over esterification. The Trag amide bond is stable under basic ester hydrolysis and Fmoc removal conditions. Hence, the troponyl alkylglycinates could be applicable as protecting groups for amine functionality of amino acids and peptides. The reaction mechanism was investigated by using a deuterium probe and studied by NMR and electrospray ionisation mass spectrometry techniques. Deuterium incorporation at α-CH₂ strongly supported the formation of CTLs via ketene intermediates.

1. Introduction

Amide bonds are one of the most important and essential chemical bonds of biopolymers (proteins and peptides) and many bioactive molecules.¹ The synthesis of the amide bond is well-established from carboxylic acid and free amine in the presence of activating reagents.² Importantly, many methods are also available in the literature for the synthesis of the amide bond directly from carboxylic acid esters and amine in the presence of a metal catalyst and under constrained conditions such as high temperatures.³ The typical amide bonds are chemically robust and stable under physiological conditions. However, they can be cleavable under strong acidic or basic conditions at room/high temperatures.⁴ The stability of acyclic amide bonds is due to their resonance stabilization.⁴ On the other hand, strained cyclic lactams and nonplanar amide bonds have less amidic resonance stabilization because of nonplanarity of the amide bond.⁴ As a result, they are more reactive toward the nucleophilic addition reaction and are easily cleavable under mild conditions.⁴ In the literature, solvolysis of strained cyclic lactams and nonplanar amides is extensively studied.⁴ Further, the activation of amide bonds toward solvolysis by introducing electron-withdrawing groups at α-carbon is also demonstrated.^4d Overall, the stability of amide bonds mainly depends on the nature of the substituents at α-carbon of amide carbonyl and amide N-atom, which affect the steric and electronic environment of the respective amide bonds immensely.⁴ The cleavage of amide bonds with metal catalysts is also explored.⁵

Generally, the acyclic amide bonds derived from N,N-dialkyl glycine residues are quite stable, almost like other amide bonds. However, we have recently reported the cleavage of acyclic amide bonds derived from troponyl aminoethylglycine (Traeg) residues into esters under mild acidic conditions (Figure 1A).^6b Further, a plausible reaction mechanism for the cleavage of Traeg amide bonds via a reactive ketene intermediate is proposed based on the control experiments and literature reports.^6b The cleavage of Traeg amide bonds into “cationic troponyl lactone” was also observed during the mechanistical investigations. Previously reported control experiments revealed that the troponyl moiety and α-CH₂ of the glycinate unit of Traeg residues play a key role in the cleavage of the Traeg amide bond.^6b Such kind of organic transformations are unusual and interesting to explore. Hence, in-depth understanding with experimental evidences and exploring potential applications of this unusual amide cleavage is very important.

Figure 1.

(A) Previously reported methanolysis of Traeg amides. (B) Direct and reversible amidation of troponyl alkylglycinate (Trag) derivatives via CTLs (this report).

Herein, we report the syntheses and characterization of different cationic troponyl lactones (CTLs) from troponyl alkylglycinate amides and also from the Trag acid/esters derivatives. The reversible amidation: cleavage and reformation of the same Trag amide bond via CTLs is demonstrated (Figure 1B). The direct amidation of Trag esters/acid derivatives with amines via CTLs is described. The high probability of formation of CTLs via a reactive ketene intermediate is described by probing the reaction mechanism with deuterium labeling. The stability of Trag amide bonds under basic ester hydrolysis conditions and Fmoc removal conditions is demonstrated. Therefore, troponyl alkylglycinates could be potential candidates for the protection of amine groups of amino acids, peptides, and bioactive molecules through direct amidation.

2. Results and Discussion

2.1. Syntheses of Troponyl Alkylglycinate Derivatives

We began this study with the syntheses of troponyl alkylglycinate esters and amides by following previously reported procedures (Scheme 1A,B).⁶ Our previously reported troponyl aminoethylglycine amides contain N-Boc aminoethyl groups at glycinate nitrogen.^6bTraeg amides and Boc protecting groups are cleavable in acidic conditions. Hence, to avoid the interruption of the acid labile Boc protecting group in exploring the reactivity and potential applications of Traeg amide cleavage, the N-Boc aminoethyl group of Traeg is replaced with the alkyl chains.

Scheme 1. A) Synthesis of Troponyl Alkylglycinate Derivatives (Trag esters and amides); (B) Synthesis of N-Troponyl Amino Acid Esters; (C) Syntheses of N-troponyl proline amides.

Troponyl alkylglycinate esters (3a–d) are synthesized by following Scheme 1A. N-Troponyl amino acid esters such as N-troponyl proline ester (3e), N-troponyl glycine ester, and N-troponyl phenylalanine ester are synthesized by following Scheme 1B. Troponyl alkylglycinate amides (4a–d) and N-troponyl proline amides (4e/f) were synthesized through standard peptide coupling conditions.⁷N-Troponyl glycine (3aa) and N-troponyl phenylalanine (3ab) were synthesized to test the role of alkyl groups at glycinate nitrogen of Trag derivatives in formation of CTLs. All the synthesized new compounds are characterized by NMR and electrospray ionisation mass spectrometry (ESI-MS) and their spectral data are provided in the Supporting Information.

2.2. Reversible Amidation

In our pervious report, the formation of CTLs from troponyl aminoethylglycine amides was observed in the presence of 6.0 equiv of trifluoroacetic acid (TFA) in CD₃CN. Therefore, the troponyl octylglycinate amide 4b was treated with 6.0 equiv of TFA in CD₃CN to obtain CTL5b and which was studied by NMR (Scheme 2). The ¹³C NMR spectra clearly show the disappearance of troponyl carbonyl carbon and amide carbonyl carbon peaks after treatment with TFA. The appearance of new peaks representing the same carbons in CTL5b was noticed. The cationic carbon of CTL5b reportedly appeared between δ 150–156 and lactone carbonyl appeared most likely at δ 161 (Figure 2A).⁸

Scheme 2. Reversible Amidation of Troponyl Alkylglycinate Amides (4a–f) via CTLs.

Figure 2.

Comparative ¹³C NMR spectra of TFA-treated Trag amide 4b, Trag acid 3b-OH, and control monomer 3aa in CD₃CN, showing the formation of respective CTLs. (A) ¹³C NMR of 4b in CD₃CN obtained after 50 min of TFA treatment, showing the formation of 5b. (B) ¹³C NMR of 3b-OH in CD₃CN obtained after 2 h of TFA treatment, showing the formation of 5b. (C) ¹³C NMR of 3aa in CD₃CN obtained after 6 h of TFA treatment, showing the formation of 3aa-H⁺.

The mass spectra of the same sample exhibit a prominent mass peak at m/z 274.0 (M⁺) which is equivalent to the calculated molecular mass (m/z) of CTL5b. These NMR and ESI-MS studies strongly supported the formation of CTL5b from Trag amide 4b after TFA treatment within 30 min. Similar experiments were performed with other Trag amides 4a/c–e and characterized the formation of respective CTLs5a/c–e by NMR and ESI-MS (Scheme 2, conversion >95%; predicted from NMR, see SI for representative spectral data). All CTLs5a–e synthesized from 4a–e contain glycine ester and TFA peaks in their respective ¹H and ¹³C NMR spectra. The formation of CTLs5a–f from Trag amides 4a–f in the presence of 6.0 equiv of TFA in CD₃CN/CH₃CN is confirmed.

Next, the reactivity of CTLs5a–e was investigated. The CTL5b was converted into troponyl alkylglycinate ethyl ester as 3b in the presence of excess EtOH, which revealed that the hydroxyl nucleophile is reacting with lactone carbonyl and leads to lactone ring opening to give Trag ester 3b. On the basis of this, we envisaged that the neutralization of the reaction mixture containing CTL5b and the protonated glycine ester (H₃N⁺–CH₂–COOMe) leads to the formation of Trag amide 4b. The neutralization results in the generation of free amine nucleophile as amino acid ester (H₂N–CH₂–COOMe) which will react with lactone carbonyl carbon of CTL5b and forms the amide bond to give Trag amide 4b.

As expected, neutralization of the reaction mixture containing CTL5b and glycine ester salt with Et₃N at 0 °C produced the starting material 4b within 10 min (isolated yield 71%, Scheme 2). Similarly, other CTLs5a/c–e obtained from 4a/c–e were also subjected for the reversible amidation under similar conditions and isolated the starting materials 4a/c–e in good to moderated yields (Scheme 2). The amide cleavage and reformation was monitored by NMR in CD₃CN and ESI-MS. The obtained spectral data clearly revealed the cleavage and reformation of the Trag amide bond via CTL5b under given conditions. The representative ¹H/¹³C NMR spectral data are provided in the Supporting Information (Figures S1 and S2). The formation of undesired products was observed during the reversible amidation reaction. Hence, isolated yields of Trag amides 4 after reversible amidation were decreased. We noticed that the amidation of CTLs is faster than esterification even in the presence of a stoichiometric amount of amines.

Trag

The reversible amidation, cleavage (30 min) and reformation (10 min) of the amide bond 4 via CTLs5 within 45 min is confirmed. The cleavage and reformation of the same amide bond occurs via a CTL intermediate, and hence, this reaction can be called as reversible amidation. So far, no such reports for the reversible amidation reaction are available in the literature.

2.3. Direct Amidation

After confirming the formation of CTLs5a–e from Trag amides 4a–f, we were curious to explore the reactivity of Trag esters (3) in the presence of TFA in acetonitrile. At first, the Trag ester 3b was treated with TFA (6.0 equiv) in CD₃CN and studied the formation of CTL5b by NMR. The NMR studies revealed that the CTL5b is forming from Trag ester 3b at room temperature within 6–8 h (Scheme 3). However, the conversion is relatively slower than the respective amide 4b and also incomplete even after 8 h (>75%). This reaction was monitored by ¹H NMR, and 6.0 equiv of TFA was added to the Trag ester 3b in CD₃CN and characterized by ¹H NMR with 15 min time intervals for 3 h. These NMR results revealed the immediate protonation of troponyl carbonyl oxygen after the addition of TFA and formation of 3-H⁺ (Figures S5–S7). The slow reaction is most probably due to the reversible reaction between 3-H+ and 5, which is already discussed. The obtained spectral data are provided in the Supporting Information. Similarly, other Trag esters 3a/c–e were subjected to the formation of CTLs in the presence of TFA in CD₃CN. ¹H/¹³C NMR experiments confirmed the formation of respective CTLs5a/c–e. Importantly, in case of slow conversion (<75%), up to 8.0 equiv of TFA was used to enhance the conversion.

Scheme 3. Amidation of Trag Esters/Acids via CTLs5a–e with Benzyl Amine.

Next, Trag carboxylic acid derivatives 3b-OH/3c-OH were also subjected to CTLs syntheses in the presence of 6.0 equiv of TFA in CD₃CN and studied by NMR (Figure 2B). These studies revealed the formation of respective CTLs5b/c from 3b/c-OH with almost quantitatively (∼95.0%, Figures 2B and S3 and S4) within 3 h. In contrast to Trag esters 3a–e, the acid derivatives 3b/c-OH convert into respective CTLs more efficiently.

These freshly prepared CTLs5a–e in CD₃CN were then employed for the amidation with the amine group in Scheme 3. The CTLs obtained from 3a–e in the presence of TFA in CD₃CN were treated with benzylamine, followed by neutralization with Et₃N (6.5–8.5 equiv) at 0 °C to room temperature to obtain respective amides 6a–e. These amides were isolated in 57–70% yields. Trag monomers were also isolated (10–15%) along with these amides. The formation of CTLs from Trag ester/acid derivatives 3a–e and 3b/c-OH and their amidation are confirmed. Such kind of reactivity of carboxylic acid/esters under abovementioned conditions is not known in the literature. The formation of CTLs5a–e from troponyl alkylglycinate acid/ester derivatives (3/3-OH) is a major breakthrough of this work which facilitated the amidation of Trag acid/esters via a CTL intermediate.

Encouraged by the direct amidation of CTLs derived from Trag esters 3a–e, we sought to explore the potential applications of this unusual amide cleavage. For this purpose, amino acid esters and di/tripeptides as amines were employed for amidation with Trag esters 3b/c via respective CTLs5b/c in CH₃CN under optimized conditions (Scheme 4). As shown in Scheme 4, which demonstrates direct amidation of troponyl alkylglycinate esters and acids (3b/c and 3b/c-OH) with amino acid ester and peptide amines, we have employed various amino acid esters/peptide esters (including hydroxyl amino acid esters) for direct amidation with troponyl octylglycinate 3b under already optimized conditions. We have synthesized the Trag-containing peptides 4c/7–15 from respective amino acid/peptide esters through direct amidation (Scheme 4). The use of 1.0 equiv of 3b and 1.2 equiv of amines provided the isolated yields of amides 7–15 in 35–53%, and 3b was isolated in ∼15% (Scheme 4. Condition A). The decrease in isolated yields of 7–15 is due to incomplete conversion of 3b-H⁺ to reactive CTL5b. The formation of undesired products which could not be isolated and characterized was also observed (Figure S23).

Scheme 4. Amidation of Trag Ester 3b/c with Amino Acid/Peptide Esters.

We attempted to improve the reaction conditions to demonstrate the application of troponyl alkylglycinates as possible protecting groups for amine functionality. The amine starting material was considered as 1.0 equiv, and Trag was taken as 2.0 equiv (Scheme 4. Condition B). In this case, reasonable enhancement in isolated yields of 7–15 was observed for particular amides. For example, under reaction conditions A, 7 was isolated in 46%. While under reaction conditions B, 7 was isolated in 70% (Scheme 4A). The use of 2.0 equiv of Trog-OEt (3b) improved the yields of desired amides 7–15, and the Trog-OEt (3b) is isolated in 20–32%. We observed that the reaction worked more efficiently with freshly prepared amine trifluoroacetate salts [Boc-deprotected peptide esters with 30% TFA in dichloromethane (DCM)] when compare to amino acid ester hydrochlorides. In the case of amino acid ester hydrochlorides, the amino acid ester was first neutralized with 1.5 equiv of Et₃N in a minimum amount of CH₃CN before amidation reaction.

The direct amidation of troponyl octylglycinate/troponyl dodecylglycinate with amino acid/peptide esters was carried out in several ways. The amidation of CTLs is a very fast reaction. Hence, the generation of a free amine nucleophile on time to react with CTL during neutralization is important. The delay in generation of free amine nucleophile in the reaction mixture during neutralization causes the formation of more undesired products, and as a result, yields of desired Trag amides were decreased.

Importantly, only the amino group of hydroxyl amino acid esters was reacted with CTLs5 to give respective amides 12–15 (serine ethyl ester/threonine methyl ester/tyrosine methyl ester/4-hydroxy proline methyl ester, Scheme 4B). We could not observe hydroxyl-protected amino acid/peptide derivatives. As already mentioned, the most probable reason for this is that the amidation of CTLs is faster than esterification. Hence, CTLs can react selectively and rapidly with the amine functional group in the presence of the hydroxyl functional group to give Trag amides. Overall, the troponyl alkylglycinates would be attached to an amine functional group of amino acids esters and peptide esters without the use of peptide coupling reagents via CTL intermediates. Moreover, the substituent at the glycinate nitrogen could be changed as per requirements. Therefore, these molecules could be used in the design of prodrugs containing hydroxyl and amino functional groups.

2.4. Control Experiments

Next, the necessity of an alkyl substituent for the formation of CTLs from Trag derivatives is studied. Troponyl glycine 3aa and troponyl phenylalanine 3ab were treated with TFA (6.0 equiv) in CD₃CN and studied by NMR (Scheme 5). The analysis of obtained spectra revealed that no formation of CTLs was observed, and only protonation of troponyl carbonyl oxygen was characterized (Figure 2A). Even after 3 days, no change in spectra was observed. From these studies, it is confirmed that the alkyl group at glycinate nitrogen is necessary for the formation of CTLs.

Scheme 5. Role of the Alkyl Substituent at Glycinate Nitrogen in Formation of CTLs.

To demonstrate the troponyl alkylglycinates as orthogonal protecting groups, the stability of Trag-AA-OMe amide bonds was investigated under basic conditions such as ester hydrolysis conditions and Fmoc removal conditions.

Peptides 8/11 were treated with aq NaOH in MeOH/THF at room temperature (Scheme 6A). No amide hydrolysis of Trag-AA-OMe was observed under these conditions. Only selective ester hydrolyzed products 8/11-OH were obtained quantitatively (Page S114–117). Thus, Trag containing amide bonds are stable under ester hydrolysis conditions. The stability of Trag amides under Fmoc removal conditions was also demonstrated. Peptides 8/14 were treated with 20% piperidine in dimethylformamide (DMF) for 4 h and analyzed by ESI-MS. These experimental results revealed that Trag amides are stable under standard Fmoc removal conditions (Scheme 6B). The troponyl alkylglycinates would be attached at the amine group of amino acid and peptide esters via CTLs, and the Trag-AA-OMe amide bond is stable under basic ester hydrolysis and standard Fmoc removal conditions. Moreover, the Trag-(AA)_n-OMe amide bond is selectively cleavable under mild conditions (Scheme 6C; Figures S26–S28). Hence, troponyl alkylglycinates could be applicable as orthogonal protecting groups for amino functionality.

Scheme 6. Stability of the Trag Amide Bond under Ester Hydrolysis and Fmoc Removal Conditions and Regioselective Cleavage of the Trag Amide Bond.

2.5. Mechanism Investigation

2.5.1. Deuteration of Glycinate α-CH₂ with TFA-D

A plausible reaction mechanism for the cleavage of the troponyl aminoethylglycine amide bond via a reactive ketene intermediate is described in our previous report.^6b The reaction mechanism was proposed based on the control experiments and literature reports.^4d,9 These control experiments revealed the key role of α-CH₂ and the troponyl moiety in the cleavage of the amide bond. However, understanding the role of glycinate α-CH₂ in the formation of CTLs with experimental evidences is very important. Hence, we have attempted to probe the reaction mechanism using deuterium labeling.¹⁰ We envisaged that if the glycinate α-CH₂ involved in the reaction, deprotonation and reprotonation of α-CH₂ would occur. Hence, performing the reaction with deuterated TFA may lead to exchange of α-CH₂ protons with deuterium in the resultant CTLs and Trag amides.

At the outset, Trag amide 4b was treated with deuterated TFA (6.0 equiv of TFA-D) in CD₃CN to obtain respective CTLs and studied by NMR and ESI-MS (Scheme 7A). The obtained ¹H NMR spectrum exhibits a broad triplet along with a singlet at δ 4.61–4.60, and the ¹³C NMR spectra also exhibit a triplet along with a singlet at δ 52–50, unlike ¹H/¹³C NMR spectra of 5b obtained in the presence of TFA (comparative NMR spectral data are provided in Figure 3A–D). Further, the mass spectra of the same sample exhibit mass peaks at m/z 275 along with m/z 274 (M⁺) (Figure S20). NMR and MS results revealed the deuteration of α-CH₂ of the glycinate unit happens in the presence of TFA-D in CD₃CN. These triplet peaks in ¹H/¹³C NMR represent the proton and carbon of α-CHD (Figure 3A,C). Then, Trag amides 4a/c were also subjected to CTL formation in the presence of TFA-D in CD₃CN or CH₃CN and formation of respective α-deuterated CTLs5a′/c′ were consistently characterized by NMR and ESI-MS (Scheme 7A). The formation of α-dideuterated CTLs5a″–c″ was also observed in ESI-MS and NMR spectral data. Further, extending the reaction time in the presence of TFA-D from 30 min to 2 h to 12 h led to an increase in the formation of α-dideuterated CTLs (5a″–c″). Next, the α-deuterated CTLs obtained in the presence of TFA-D were then allowed to undergo reversible amidation. The obtained Trag amides contain undeuterated amides 4a–c, monodeuterated amides 4a′–c′, and also dideuterated amides 4a″–c″. The presence of these three compounds was characterized by NMR and ESI-MS. In ¹³C NMR, three representative peaks were observed for some of the characteristic carbons, and the mass analysis strongly supports NMR results (for representative spectral data, see Figures S17–S20).

Scheme 7. α-Deuteration Experiments.

Figure 3.

Expanded comparative spectral data showing the formation of α-deuterated CTL5b′ from 4b after treatment with TFA-D. (A,B) Expanded stacked ¹H NMR spectra of CTL5b/b′ synthesized in the presence of TFA-D/TFA in CD₃CN; triplet showing the α-deuteration as α-CHD (synthesized from 4b in CD₃CN); (C,D) expanded stacked ¹³C NMR spectra of CTL5b/b′ in CD₃CN synthesized in the presence of TFA-D/TFA (synthesized from 4b); triplet showing the α-deuteration as α-CHD.

The similar deuteration experiments were performed with Trag esters 3a–d and Trag acid 3c-OH in CD₃CN in the presence of TFA-D and studied by NMR. These studies confirmed the use of TFA-D results in the formation of respective α-deuterated CTLs (5a′–d′ and 5a″–d″) along with undeuterated CTLs5a–d with TFA-D (Scheme 7B). These CTLs were then allowed to undergo amidation with benzylamine. The mixture of deuterated (mono/dideuterated amides; 6a′–d′/6a″–d″) and undeuterated amides 6a–d were isolated after amidation (Scheme 7B). Most importantly, the formation of α-deuterated 3-H⁺ (Figure S25) strongly supported the slow conversion of 3-H⁺ to 5 which is due to occurrence of continuous reversible reaction between the 3-H⁺ and 5 (Scheme 7B, highlighted in the red box; for ¹H NMR, see Figure S25).

For control experiments, N-troponyl glycine (3aa) was also treated with TFA-D in CD₃CN and studied by NMR (Scheme 7C). These experimental results revealed that the protonation of troponyl carbonyl occurs, but the deuterium incorporation at α-CH₂ was not observed (Figure S24). These experiments suggest that the presence of an alkyl substituent at glycinate nitrogen is necessary to enhance the acidity of glycinate α-CH₂ after protonation of troponyl carbonyl for the formation of CTLs.

The formation of α-deuterated CTLs5a′–d′/5a″–d″ and α-deuterated Trag amides (4a′–c′/4a″–c″/6a′–d′/6a″–d″) was consistently characterized. The incorporation of deuterium clearly revealed the involvement of α-CH₂ in amide cleavage. However, it is very important to discuss the formation of dideuterated CTLs5a″–d″ and Trag amides 4a″–c″/6a″–d″. This can be explained by the continuous occurrence of reversible reaction between the CTLs5 and 3-H⁺, as presented in Scheme 7B (highlighted in the box). The cleavage of CTLs into Trag esters (3a–d) with excess EtOH nucleophile is already described. Hence, liberated EtOH during the formation of CTLs from Trag ethyl esters is further reacting with CTL carbonyl carbon to give protonated Trag esters (3-H⁺). Again, the protonated Trag ester 3-H⁺ converts into CTL5. The continuous occurrence of reversible reactions leads to the exchange of α-CH₂ protons with deuterium and results in formation of dideuterated products.

To confirm this, time-dependent direct/reversible amidation reactions were performed by using TFA-D. The Trag ester 3c was treated with 7.0 equiv of TFA-D in CH₃CN and maintained at room temperature for 6 h and performed the amidation reaction with benzylamine. The analysis of obtained Trag amides 6c/c′/c″ by ESI-MS revealed that monodeuterated Trag amide 6c′ is more. When the time increased to 16 h, the dideuterated amide 6c″ is more (Figures S21 and S22).

Then, the same experiments were also performed with Trag amide 4b. The Trag amide 4b was treated with TFA-D in CH₃CN and maintained at room temperature for 30 min. Then, the obtained CTLs were allowed to undergo reversible amidation reaction. NMR and MS analysis of isolated Trag amide revealed that only undeuterated and monodeuterated amides were formed (Figure S20). Next, when the time was increased to 2 h, the obtained Trag amides contained relatively more monodeuterated amide 4a′ than 4a/a″, whereas Trag amides obtained after 12 h of treatment with TFA-D contained relatively more dideuterated Trag amide 4a″ than 4a/4a′ (Figure S20). In the case of Trag amides, the reversible reaction occurs because of the presence of water in TFA-D and solvent. The conversion of Trag acid 3b-OH into CTL5b is already described, and reversible reaction between the CTL5b and 3-H⁺ was also observed using ¹H NMR (Figure S25).

The increase in time leads to increase in the formation of more α-dideuterated 6c″/4a″ amides. These experimental results strongly support the formation of dideuterated products which is due to continuous reversible reactions between the CTL5 and 3-H⁺, which causes exchange of α-protons with deuterium.

Further, we attempted to trap the ketene intermediate via [2 + 2] cycloaddition with imines. In the literature, the ketene and imine [2 + 2] cycloaddition reactions are reportedly known for the generation of β-lactam.^10d,12 Thus, we performed the Trag amide (10/6a/6c) cleavage reaction with TFA/without TFA in the presence of excess imine and studied their reaction mixture by ESI-MS. For control studies, we performed and studied a similar reaction without TFA. Their mass data are provided in the Supporting Information (Scheme S1; Page S118–122). In the case of Trag amide 10, a new mass peak at ∼469.28 was noticed in the presence of excess imine (N-benzylidene-1-phenylmethanamine) and TFA, while no such mass peak was appeared without TFA (Figures S178–S180). This mass peak is matched with calculated mass of respective β-lactam from proposed ketene and imine reactive species. The similar reactions were performed with the other Trag amides (6a/6c) and imine with TFA/without TFA. The formation of corresponding β-lactams from respective amides is noticed only in the presence of TFA (Figures S180–S184). However, the formation of CTL-charged species was more prominent as compared to that of β-lactam. Hence, these studies strongly supported the involvement of ketene as a reactive intermediate for the formation of CTLs.

The above mentioned experimental results strongly support the fact that the formation of CTLs5 from 3/3-OH/4 occurs via a ketene intermediate. The detailed mechanism is outlined in Figure 4.

Figure 4.

Plausible reaction mechanism for the formation of mono/dideuterated CTLs and troponyl alkylglycinate amides.

It is important to discuss the possibility of occurrence of CTL formation through an enolate intermediate without the formation of a ketene intermediate. The troponyl carbonyl oxygen is not a strong nucleophile to attack at the enol intermediate directly [the reported alkylation of troponyl carbonyl oxygen required a strong electrophile donor like triethyloxonium tetrafluoroborate (Meerwein’s reagent)].¹¹ In addition, troponyl carbonyl oxygen gets protonated immediately after the addition of TFA which further diminishes the nucleophilicity of troponyl carbonyl oxygen. Thus, formation of CTLs through the enolate intermediate is less probable. Therefore, the enol intermediate most probably is converted into a ketene intermediate, and the ketene intermediate rapidly reacts with troponyl carbonyl oxygen to give CTLs.

To summarize, the protonation of troponyl carbonyl is the very first step in initiating reaction, which results in generation of the 3/4-H⁺ intermediate. Then, the delocalization of the positive charge of the protonated amino tropylium cation to N-atom leads to a remarkable increase in the acidity of α-CH₂. As a result, the formation of the enol intermediate occurs, which most probably converts into a ketene intermediate. This reactive ketene intermediate instantly undergoes nucleophilic addition with troponyl carbonyl oxygen and produces CTL.

3. Conclusions

The troponyl alkylglycinate ester/amide derivatives and N-troponyl amino acid ester derivatives were synthesized. We have demonstrated the formation of CTLs5 from troponyl alkylglycinate acid/ester/amide derivatives (3-OH, 3, 4) in the presence of TFA/TFA-D in acetonitrile. The amidation of CTLs (5) with amine in the presence of a base (Et₃N) is established. The reversible amidation reaction, that is, the cleavage and reformation of the Trag-AA-OMe amide bond via CTLs are elucidated. The direct amidation of troponyl alkylglycinate acid/ester derivatives with amino acid/peptide esters via CTLs was successfully demonstrated. Importantly, the selective amidation of Trag ester with hydroxyl amino acid esters was observed.

The reaction mechanism was probed using deuterium to explore the possible reactive intermediates of the reaction. The formation of α-deuterated CTLs with TFA-D and the formation of α-deuterated Trag amides upon their amidation confirm the involvement of glycinate α-CH₂ in the formation of CTLs. Deuteration experimental results strongly supported the formation of CTLs via a ketene intermediate. The stability of the Trag-AA-OMe amide bond under ester hydrolysis conditions and standard Fmoc removal conditions is examined. The necessity of the alkyl substituent at glycinate nitrogen for the formation of CTLs is examined. The troponyl alkylglycinate amide bonds are stable under ester hydrolysis and Fmoc removal conditions. Further, Trag amides are selectively cleavable in the presence of other amide bonds. Thus, troponyl alkylglycinates are applicable as orthogonal protecting groups for amine functionality. To end, we have explored an unusual reactivity of troponyl alkylglycinate amide/ester/acid derivatives, which are activated by the protonation of troponyl carbonyl oxygen. To our knowledge, such kind of reactivity of carboxylic acid derivatives and troponyl carbonyl under these acidic conditions is not known in the literature.

4. Experimental Section

4.1. General

Materials and Instrumentation

All required chemicals and solvents were obtained from commercial suppliers and used without any further purification unless noted. Tropolone and TFA were purchased from Alfa Aesar, and TFA-D (98.5% D-enrichment) was purchased from Acros Organics. CD₃CN was obtained from Leonid chemicals with 99.80% D in a septum vial. Freshly distilled acetonitrile over calcium hydride was used. Reactions were monitored by thin layer chromatography (TLC) using Merck TLC silica gel 60 F₂₅₄ and visualized by UV and ninhydrin. Column chromatography was performed in 230–400 mesh silica. High-resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II spectrometer. ¹H NMR spectra were recorded on a Bruker AV-400 (400 MHz) instrument. Solvent residual peaks are used as internal reference standard (¹H NMR, CDCl₃ = 7.260 ppm; CD₃CN = 1.940 ppm; DMSO-d₆ = 2.50).¹¹H NMR chemical shifts were reported in ppm downfield from tetramethyl silane. Splitting patterns are abbreviated as follows: s: singlet, d: doublet, dd: doublet of doublet, t: triplet, q: quartet, dq: doublet of quartet, p: pentet, m: multiplet, and app: apparent. ¹³C NMR spectra were recorded on Bruker AV-400 (101 MHz) and Bruker Ascend-700 instruments, and solvent residual peaks are used as internal reference standard (CDCl₃ = 77.16 ppm; CD₃CN = 1.32 ppm; DMSO-d₆ = 39.52).¹³

A. General Procedure for the Syntheses of Troponyl Alkylglycinate Monomers (3a–d)

To a solution of 2-tosyloxy tropone (2, 2.4 gm, 8.677 mmol) in ethanol (50 mL) was added N-octylglycinate (1, 5.5 gm, 25.581 mmol) and refluxed for 42 h. Reaction was monitored by TLC. After completion of the reaction, the reaction mixture cooled to room temperature and was concentrated under vacuum on a rota evaporator. The reaction residue was redissolved in DCM (50 mL) and washed with water three times by using a separatory funnel. The organic layers were combined together and dried over Na₂SO₄ and then concentrated to dryness under vacuum. The concentrated residue was subjected to purification on silica gel by column chromatographic methods using ethyl acetate and hexanes as mobile phases. The desired product was obtained as yellow viscous liquid (2.6 gm, 93%) and characterized by ¹H/¹³C NMR and MS spectrometric methods.

B. General Procedure for the Syntheses of N-Troponyl Amino Acid Esters (3e, 3aa, 3ab)

2-tosyloxy tropone (500 mg, 1.81 mmol, 1.0 equiv) and 455 mg of glycine methyl ester hydrochloride (3.63 mmol, 2.0 equiv) were dissolved in ethanol, and to this, triethylamine (1.5 mL, 10.09 mmol, 6.0 equiv) was added and stirred at room temperature for 5 min. Then, the resultant reaction mixture is refluxed for 6–12 h. Completion of the reaction was judged by TLC. After completion of the reaction, the reaction mixture cooled to room temperature, and all volatiles were removed under reduced pressure to obtain the crude product. To the obtained crude product, water was added and extracted thrice with DCM. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product further purified through a silica gel column by using ethyl acetate and hexanes as mobile phases. The pure product (320 mg, yield = 84%) was obtained as a yellow solid.

C. General Procedure for the Ester Hydrolysis of Monomers (3-OH)

Ethyl ester of the monomers (3a–f) was dissolved in a 1:1 ratio of methanol and a tetrahydrofuran mixture. To this, 2.0 equiv of sodium hydroxide aqueous solution was added (sodium hydroxide was dissolved in a little amount of water). Then, the reaction mixture is stirred for 20 min. After completion of the reaction, all volatiles were evaporated under reduced pressure. To the crude product, 1.0 N HCl was added and extracted with DCM or ethyl acetate. The combined organic layers were evaporated in vacuo and dried completely. The obtained product was used for the peptide synthesis without any further purification and characterization. Note: 3b/c-OH was characterized by NMR and MS and then employed for CTLs5b/c syntheses.

Note: In the case of hydrolysis of troponyl proline (Trpro) monomer 3f, after completion of the reaction, all volatiles were evaporated. The crude residue was neutralized by using 1.0 N HCl to neutral pH (pH = 6.0–7.0). Then, water was evaporated. To the obtained crude product, methanol was added and filtered to remove NaCl salts. The filtrate was concentrated under vacuum and again redissolved in methanol and filtered. This process was done three times to remove NaCl salts. Then, the methanol filtrate was evaporated under reduced pressure, and the obtained product is allowed to dry and be used for peptide syntheses.

D. General Procedure for the Ester Hydrolysis of Trog Peptides (8-OH, 11-OH)

A troponyl octylglycine-phenylalanine peptide (90 mg, 0.199 mmol, 1.0 equiv) was dissolved in 1:1 methanol and tetrahydrofuran. To this solution, 16 mg of NaOH (0.398 mmol, 2.0 equiv) was added by dissolving it in a little amount of water. The resultant mixture was allowed to stir at room temperature for 10–20 min. The completion of reaction was judged by TLC. After completion of the reaction mixture, all volatiles were evaporated under reduced pressure to obtain the crude product. To the crude product, DCM was added, and excess acetic acid (400 μL) was added to neutralize sodium salts. After stirring for 2–3 min and transferring the solution into a separatory funnel, water was added and extracted. The combined organic layers were allowed to dry over sodium sulfate and concentrated under vacuum to obtain the pure product. If the presence of acetic acid was observed in the pure product, more water was added (relative to organic solvent) again and extracted with ethyl acetate. The obtained organic layer was dried over sodium sulfate and concentrated under reduced pressure. The obtained pure product (81 mg, yield 93%) was characterized by ¹H and ¹³C NMR in DMSO-d₆ and mass spectrometry.

E. General Procedure for Peptide Synthesis (4a/b/d/e/f)

Troponyl alkylglycine (3b-OH, 1.5 gm, 5.15 mmol) was dissolved in anhydrous DMF (8.0 mL) under nitrogen atmosphere and cooled to 0 °C. To this solution was added EDC·HCl or diisopropyl carbodiimide and allowed to stir for 10 min. Then, l-glycine methyl ester (970 mg, 1.5 equiv) was neutralized with Et₃N (3.0 equiv) in DMF (8.0 mL) and added to the cold reaction mixture. The reaction was stirred at 0 °C for 30 min. Then, it was stirred at room temperature or 48 °C (in cases of less conversion, reaction temperature was raised to 45–50 °C) overnight. Completion of the reaction was judged by TLC. After completion of the reaction, the mixture was concentrated under reduced pressure. A concentrated reaction residue was redissolved in DCM and then washed with water thrice followed by saturated sodium bicarbonate by following the extraction method. The washed organic layers were combined together and dried over Na₂SO₄. Then, concentrated under reduced pressure, the obtained crude product was purified through a silica gel column to obtain the pure product 4b (1.2 gm, 65%). The obtained product was characterized by ¹H/¹³C NMR and MS.

F. General Procedure for Syntheses of CTLs (5a–f) from Troponyl Alkylglycinate Amides (4a–f) and Reversible Amidation

Troponyl alkylglycinate amides 4d (30 mg) were dissolved in CD₃CN, and to this, 6.0 equiv of TFA-D was added at room temperature and allowed to stir. After 30 min, the formation of CTLs was confirmed by TLC and finally confirmed by ¹H/¹³C NMR and ESI-MS methods. The obtained CTLs contain TFA salts of amino acids esters. Then, to convert the CTL into starting material (respective CTL), the same reaction mixture was cooled to 0 °C and to this was added 6.5 equiv of triethylamine at 0 °C with moderate speed, and as a result, the reaction mixture turned to yellowish black. After the addition of the triethylamine, the resultant mixture brought to room temperature and allowed to stir at room temperature for 10 min. Then, all volatiles were evaporated under reduced pressure to obtain crude starting material. To this, crude product water was added and extracted with ethyl acetate. Organic layers were combined together and allowed to dry over sodium sulfate and then concentrated under reduced pressure to obtain the crude product. The obtained crude product was purified through silica gel column chromatography. The pure product (21 mg) was isolated (70%).

The use of TFA-D produces undeuterated CTLs5a–c and α-deuetrated CTLs5a′–c′ under same conditions in CD₃CN/CH₃CN. Further, reversible amidation of those CTLs produces undeuterated amides 4a–c and α-deuetrated amides 4a′–c′/4a″–c″.

Note: Importantly, the increase in the time gap between the amide cleavage and reversible amidation causes more dideuteration, which indicates that leaving the obtained CTLs and respective amine salts in TFA-D for a long time (2–12 h) causes dideuteration at the α-methylene group of the glycinate unit. This was studied by mass and NMR. The ¹³C NMR spectra of Trag amides obtained after reversible amidation after treatment with TFA-D have shown three sets of peaks (see the Supporting Information). For example, 4b peptide was subjected to CTLs5b/b′ formation in the presence of TFA-D. Then, the obtained CTLs (5b/b′) were employed for reversible amidation with different time intervals (30 min, 2 h, 12 h; see Figure S20). The increase in the formation of dideuterated amides 4a″–c″ was observed, and if the time is 12 h, the α-monodeuterated amides 4a′–c′ and α-dideuterated amides 4a″–c″ were observed almost in an equal ratio in their NMR and mass spectra (see Figures S17–S21). The α-dideuterated CTLs5a″–c″ were not observed in ¹H/¹³C NMR spectra, most probably because of overlapping of peaks.

G. General Procedure for the Syntheses of CTLs (5a–f) from Troponyl Alkylglycinate Esters (3a–f), Acids (3-OH), and Subsequent Amidation with Benzylamine (6a–E and 6a′–d′)

The troponyl alkylglycinate ester 3c (65 mg, 0.173 mmol) was dissolved in CD₃CN, and to this was added 7.0 equiv TFA or TFA-D. The resultant mixture was maintained at room temperature for 6–8 h to obtain respective CTLs (5) and the protonated form of starting material. After 6–8 h, to the same reaction mixture, benzylamine (0.208 mmol, 23 μL) was added, and the resultant mixture allowed to stir and cooled to 0 °C, and to this was added 6.5–8.5 equiv of triethylamine at 0 °C by using a syringe with moderate speed (for neutralization, equal amount of triethylamine with respect to TFA was added). After the addition of triethylamine, the reaction mixture turned to yellowish black. This reaction mixture is allowed to stir for 10 min at room temperature. Completion of reaction was judged by TLC. After completion of the reaction, all volatiles were evaporated under reduced pressure to obtain the crude product. To the crude product, water was added and extracted twice with ethyl acetate. The combined organic layers were allowed to dry over sodium sulfate and concentrated under reduced pressure. The obtained crude residue was subjected to purification through silica gel column chromatography by using ethyl acetate and hexanes as mobile phases. 6c (52 mg) as the pure product was obtained (70%).

To obtain the α-deuterated peptides 6a′–d′, the same procedure was followed and TFA-D was used instead of trifluoroacetic acid (TFA-H).

H. General Procedure for the Direct Amidation of Troponyl Alkylglycinates 3b/c with Amino Acid Ester Hydrochlorides and Peptides Trifluoroacetate Salts (4c/7–15)

Troponyl alkylglycinate ester 3b (290 mg, 0.927 mmol) was dissolved in 3.5 mL of anhydrous CH₃CN and to this was added 7.0 equiv TFA (6.489 mmol, 496 μL). The resultant mixture was left at room temperature for 6–8 h to obtain respective CTL5b. Then, the same reaction mixture was added to Boc-deprotected and fully dried diphenylalanine trifluoroacetate peptide salt (1.2 equiv H₃N⁺-Phe-Phe-OMe) in a round-bottom flask. The resultant mixture was allowed to stir at room temperature for complete dissolution of the peptide salt. The resultant mixture was cooled to 0 °C and to this was added 8.5 equiv (1.099 mL) of triethylamine at 0 °C by using a syringe with moderate speed under stirring. After the addition of triethylamine, the reaction mixture turned to yellowish black, and the reaction mixture was brought to room temperature and allowed to stir for 5–10 min at room temperature. Completion of reaction was judged by TLC. After completion of the reaction, all volatiles were evaporated under reduced pressure to obtain the crude product. To the crude product, water was added and extracted twice with ethyl acetate. The combined organic layers were allowed to dry over sodium sulfate and concentrated under reduced pressure. The obtained crude residue was subjected for purification through silica gel column chromatography by using ethyl acetate and hexanes as mobile phases. The pure product (265 mg, condition A, yield = 48%) was isolated as yellowish thick liquid. The rest of the reactions were performed in 0.32–0.34 mmol scale in 1.0–1.3 mL CH₃CN. Condition A: Trog-OEt = 1.0 equiv and amine = 1.2 equiv, and isolated yields are with respect to Trog-OEt. Condition B: Trog = 2.0 equiv and amine = 1.0 equiv, and isolated yields are with respect to amine.

Note: Reaction was performed in several possible ways such as the following: (1) solid amino acid/peptide ester was directly added to the freshly prepared respective CTLs5b solution in acetonitrile (containing TFA) and stirred well to ensure complete dissolution of amino acid or peptide ester salts. Then, it was cooled to 0 °C (2 min) and neutralized with an equal amount of triethylamine under stirring at 0 °C (the required Et₃N was calculated by considering the added TFA and amino acid ester salts); (2) when the amino acid ester hydrochlorides (valine methyl ester or phenylalanine methyl ester or tyrosine methyl ester or 4-hydroxyproline methyl ester) were used as amines, these amine salts were first dissolved in a minimum amount of acetonitrile and neutralized with 1.5 equiv of Et₃N. To the resultant solution, freshly prepared CTL5b solution in acetonitrile (containing TFA) was added and immediately cooled to 0 °C under vigorous stirring and neutralized with Et₃N with moderate speed to obtain Trog amides.

I. Reversible Amidation Reaction Monitoring by ¹H/¹³C NMR Experiments in CD₃CN

Troponyl alkylglycinate peptides (4a–f) were dissolved in CD₃CN and characterized by ¹H/¹³C NMR. To the same sample, 6.0 equiv of TFA was added and characterized by ¹H/¹³C NMR again. After the addition of TFA to the sample, as envisioned, both ¹H/¹³C NMR spectra are completely changed, and the characteristic tropone carbonyl peak at ∼δ 180–183.0 ppm was disappeared. These spectra contain TFA peaks. Then, again to the same sample in the NMR tube, 6.5 equiv of triethylamine was added at room temperature and stirred well. This sample was also characterized by ¹H/¹³C NMR spectra. Both ¹H/¹³C NMR spectra reappeared after neutralization (see Figure S1). Finally, the reaction mixture was evaporated under reduced pressure. To the crude product, water was added and extracted with ethyl acetate twice. The obtained organic layers were combined together and dried over sodium sulfate and concentrated under reduced pressure. The obtained crude product after extraction was purified through silica gel column chromatography.

J. Reaction Monitoring by NMR and Conversion of Troponyl Alkylglycinate Esters 3/3-OH into CTLs5

Troponyl alkylglycinate esters were dissolved in CD₃CN, and to this solution, 6.0–8.0 equiv of TFA (TFA-H/TFA-D) was added. After 6 h, ¹H and ¹³C NMR spectra were recorded.

Troponyl alkylglycinate acids (3/c-OH) were dissolved in CD₃CN, and to this solution, 6.0 equiv of TFA (TFA-H/TFA-D) was added. After 2 h, ¹H and ¹³C NMR spectra were recorded.

K. Stability of the Trag Amide Bond under Standard Fmoc Removal Conditions (8/14)

Trog-Hyp-OMe (14) peptide (62 mg, 0.14 mmol) was taken in a round-bottom flask and treated with 20.0% piperidine in DMF (1.0 mL) at room temperature for 4 h. Reaction was monitored by TLC. After 4 h, the reaction mixture was analyzed by ESI-MS. The TLC monitoring and ESI-MS experiments revealed that the Trag amide bond is stable under these conditions. The same reaction was performed with 36 mg of the Trag-Phe-Phe-OMe peptide (8) and analyzed by ESI-MS.

4.2. Analytical Data

• All CTLs were synthesized by following the general procedures F and G from respective troponyl alkylglycinate amides (4a–f) and acid derivatives 3b/c-OH in the presence of TFA/TFA-D. The NMR (¹H/¹³C/¹³CDEPT135) spectra of CTLs (5a/a′, 5c′, 5d, 5e) synthesized from amides 4a–e contain glycine methyl ester peaks and TFA peaks, whereas the NMR spectra of CTLs (5b/b′ and 5c) synthesized from 3-OH contain TFA peaks and protonated 3-OH peaks.

• The α-deuterated amides 4a′–c′ and 6a′–d′ contain three compounds such as undeuterated (4a), monodeuterated (4a′), and dideuterated (4a″) products which are characterized by NMR and MS. In ¹³C NMR, they have shown three sets of peaks. Hence, all observed chemical shift values are listed in ¹³C NMR data. This was further observed in their mass spectral data. ¹H NMR peaks appeared at the same chemical shifts for all three compounds, other than α-CH₂ and α-CHD.

Troponyl Hexylglycine Ethyl Ester (3a Trhg-OEt)

Synthesized by following the general procedure A. The pure product (2.0 gm, 85% yield) was isolated as yellow liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.06–6.90 (m, 2H), 6.83 (app d, J = 11.7 Hz, 1H), 6.52 (app dd, J = 18.5, 9.9 Hz, 2H), 4.31 (s, 2H), 4.18–4.05 (q, J = 7.1 Hz, 2H), 3.43–3.28 (m, 2H), 1.73–1.55 (m, 2H), 1.36–1.23 (m, 6H), 1.24–1.15 (m, 3H), 0.83 (t, J = 9.6, 4.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 181.31, 170.33, 156.72, 135.32, 133.79, 133.08, 123.77, 114.47, 60.79, 54.32, 53.25, 31.47, 26.64, 25.97, 22.51, 14.14, 13.93. HRMS (ESI-TOF) m/z: calcd for C₁₇H₂₅NO₃ [M + H]⁺, 292.1907; found, 292.1922.

Troponyl Octylglycine Ethyl Ester (3b Trog-OEt)

Synthesized by following the general procedure A. The pure product (2.6 gm, yield = 93%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.09–6.95 (m, 2H), 6.88 (app d, J = 11.7 Hz, 1H), 6.62–6.52 (m, 2H), 4.35 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.47–3.33 (m, 2H), 1.76–1.59 (m, 2H), 1.37–1.17 (m, 13H), 0.86 (t, J = 9.3, 4.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 181.49, 170.51, 156.87, 135.46, 133.91, 133.27, 123.92, 114.63, 60.96, 54.47, 53.37, 31.82, 29.39, 29.28, 27.12, 26.14, 22.68, 14.27, 14.14. HRMS (ESI-TOF) m/z: calcd for C₁₉H₂₉NO₃ [M + Na]⁺, 342.2040; found, 342.2064.

Troponyl Dodecylglycine Ethyl Ester (3c Trddg-OEt)

Synthesized by following the general procedure A. The pure product (450 mg, yield = 81%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.08–6.92 (m, 2H), 6.86 (app d, J = 11.7 Hz, 1H), 6.55 (app dd, J = 17.2, 9.8 Hz, 2H), 4.34 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H), 3.43–3.32 (m, 2H), 1.64 (m, J = 7.5 Hz, 2H), 1.35–1.16 (m, 21H), 0.84 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 181.41, 170.43, 156.81, 135.38, 133.84, 133.20, 123.84, 114.53, 60.88, 54.42, 53.32, 31.92, 29.64, 29.62, 29.57, 29.38, 29.34, 27.06, 26.09, 22.69, 14.22, 14.13. HRMS (ESI-TOF) m/z: calcd for C₂₃H₃₇NO₃ [M + Na]⁺, 398.2666; found, 398.2692.

Troponyl Phenethylglycine Ethyl Ester (3d Trpeg-OEt)

Synthesized by following the general procedure A. ) The pure product (2.5 gm, 80% yield) was obtained as a yellow semi solid. ¹H NMR (400 MHz, CD₃CN): δ 7.37–7.27 (m, 4H), 7.27–7.17 (m, 1H), 7.14–7.02 (m, 2H), 6.74 (app dd, J = 22.0, 11.2 Hz, 2H), 6.65–6.55 (m, 1H), 4.29 (s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.67 (dd, J = 8.8, 7.0 Hz, 2H), 2.98 (dd, J = 8.9, 6.9 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CD₃CN): δ 181.83, 170.92, 157.04, 140.09, 136.07, 134.82, 133.15, 129.86, 129.52, 127.37, 124.16, 114.24, 61.55, 56.33, 54.51, 33.17, 14.57. HRMS (ESI-TOF) m/z: calcd for C₁₉H₂₁NO₃ [M + H]⁺, 312.1594; found, 312.1610.

N-Troponyl Proline Methyl Ester (3e TrPro-OMe)

Proline methyl ester hydrochloride (602 mg, 2.0 equiv, 6.63 mmol) was dissolved in ethanol and neutralized with triethylamine (1.5 mL, 6.0 equiv, 10.9 mmol), and to this, 500 mg (1.0 equiv, 1.81 mmol) of 2-tosyloxy tropone was added and refluxed overnight. Completion of the reaction was monitored by TLC. After completion of the reaction, all volatiles were evaporated and washed with water. Combined organic layers were dried over sodium sulfate and concentrated to dryness. The obtained crude product was purified through silica gel column chromatography. The pure product (200 mg, 47% yield) was obtained as dark yellow gelatinous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.03 (app q, J = 10.7 Hz, 2H), 6.86 (app d, J = 11.7 Hz, 1H), 6.52 (t, J = 9.2 Hz, 1H), 6.42 (app d, J = 10.5 Hz, 1H), 5.21 (d, J = 8.4 Hz, 1H), 3.72 (s, 3H), 3.65–3.54 (m, 1H), 3.45 (dd, J = 16.1, 7.3 Hz, 1H), 2.19 (dd, J = 17.9, 11.1 Hz, 1H), 2.12–1.90 (m, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 180.69, 173.46, 155.35, 136.21, 134.86, 132.59, 122.77, 113.27, 62.78, 52.10, 51.57, 31.25, 22.72. HRMS (ESI-TOF) m/z: calcd for C₁₃H₁₅NO₃ [M + Na]⁺, 256.0944; found, 256.0951.

N-Troponyl Glycine Ethyl Ester (3aa TrGly-OEt)

Synthesized by following the general procedure B. The pure product (320 mg, yield = 84%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.60 (s, 1H), 7.32–7.23 (m, 1H), 7.20 (app t, J = 10.8 Hz, 2H), 6.71 (t, J = 9.4 Hz, 1H), 6.38 (app d, J = 10.3 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 4.09 (d, J = 5.6 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 177.09, 168.60, 154.66, 137.46, 136.03, 130.03, 123.23, 108.90, 61.89, 44.69, 14.22. HRMS (ESI-TOF) m/z: calcd for C₁₁H₁₃NO₃ [M + H]⁺, 208.0968; found, 208.0983.

N-Troponyl Phenylalanine Methyl Ester (3ab TrPhe-OMe)

Synthesized by following the general procedure B. The pure product (300 mg, yield = 58%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.56 (d, J = 7.7 Hz, 1H), 7.36–7.04 (m, 8H), 6.67 (t, J = 9.4 Hz, 1H), 6.38 (app d, J = 10.3 Hz, 1H), 4.47 (dd, J = 13.6, 7.4 Hz, 1H), 3.69 (s, 3H), 3.29 (dd, J = 13.7, 5.8 Hz, 1H), 3.20 (dd, J = 13.7, 7.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 176.99, 171.17, 154.08, 137.40, 135.94, 135.58, 130.07, 129.20, 128.83, 127.41, 123.39, 108.06, 57.09, 52.55, 38.41. HRMS (ESI-TOF) m/z: calcd for C₁₇H₁₇NO₃ [M + H]⁺, 284.1281; found, 284.1304.

Troponyl Octylglycine (3b-OH Trog-OH)

Synthesized by following the general procedure C. The pure product (120 mg, yield = 88%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CD₃CN): δ 7.21 (app dt, J = 18.5, 9.6 Hz, 2H), 6.97 (app d, J = 11.7 Hz, 1H), 6.87 (app d, J = 10.5 Hz, 1H), 6.76 (t, J = 9.3 Hz, 1H), 4.28–3.95 (m, 2H), 3.50–3.21 (m, 2H), 1.61 (s, 2H), 1.44–1.14 (m, 10H), 0.87 (t, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, CD₃CN): δ 182.74, 172.05, 158.58, 137.76, 135.76, 133.66, 126.49, 118.38, 55.61, 54.45, 32.51, 29.96, 29.94, 27.53, 26.55, 23.35, 14.42. HRMS (ESI-TOF) m/z: calcd for C₁₇H₂₅NO₃ [M + Na]⁺, 314.1727; found, 314.1741.

Troponyl Dodecylglycine (3c-OH Trddg-OH)

Synthesized by following the general procedure C. The pure product (75 mg, yield = 96%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.31–7.22 (m, 1H), 7.17 (app dd, J = 16.3, 11.1 Hz, 2H), 6.91–6.77 (m, 2H), 4.03 (s, 2H), 3.51–3.32 (m, 2H), 1.65 (d, J = 7.1 Hz, 2H), 1.38–1.13 (m, 21H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 182.48, 171.97, 158.27, 137.58, 135.00, 133.78, 126.95, 118.99, 55.48, 53.58, 31.92, 29.63, 29.56, 29.55, 29.35, 29.31, 26.97, 25.83, 22.70, 14.14. HRMS (ESI-TOF) m/z: calcd for C₂₁H₃₃NO₃ [M + H]⁺, 348.2533; found, 348.2543.

Trhg-Gly-OMe (4a)

Synthesized by following the general procedure E. The pure product (600 mg, 65% yield) was obtained as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.12 (s, 1H), 7.21–7.11 (m, 1H), 7.05 (app dd, J = 22.8, 11.2 Hz, 2H), 6.71 (app t, J = 9.7 Hz, 2H), 4.09 (d, J = 5.7 Hz, 2H), 3.93 (s, 2H), 3.72 (s, 3H), 3.43–3.22 (m, 2H), 1.75–1.53 (m, 2H), 1.38–1.19 (m, 6H), 0.86 (t, J = 6.6 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.20, 170.79, 170.24, 158.14, 136.36, 134.34, 134.04, 126.29, 118.14, 56.18, 52.50, 52.33, 41.27, 31.59, 26.82, 25.89, 22.68, 14.08. HRMS (ESI-TOF) m/z: calcd for C₁₈H₂₆N₂O₄ [M + H]⁺, 357.1785; found, 357.1778.

Trog-Gly-OMe (4b)

Synthesized by following the general procedure E. The pure product (1.2 gm, 65% yield) was obtained as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.13 (d, J = 5.3 Hz, 1H), 7.13 (app dd, J = 11.9, 8.3 Hz, 1H), 7.04 (app dd, J = 23.8, 11.1 Hz, 2H), 6.70 (app t, J = 10.0 Hz, 2H), 4.08 (d, J = 5.7 Hz, 2H), 3.92 (s, 2H), 3.71 (s, 3H), 3.38–3.25 (m, 2H), 1.71–1.51 (m, 2H), 1.26 (d, J = 15.0 Hz, 10H), 0.85 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.21, 170.73, 170.22, 158.11, 136.29, 134.35, 133.99, 126.23, 118.05, 56.16, 52.43, 52.31, 41.24, 31.84, 29.37, 29.30, 27.14, 25.88, 22.72, 14.18. HRMS (ESI-TOF) m/z: calcd for C₂₀H₃₀N₂O₄ [M + Na]⁺, 385.2098; found, 385.2087.

Trddg-Gly-OMe (4c)

Synthesized by following the general procedure H. The pure product (65 mg, yield = 53%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.09 (s, 1H), 7.16–7.08 (m, 1H), 7.01 (app dt, J = 14.8, 11.0 Hz, 2H), 6.67 (app t, J = 9.2 Hz, 2H), 4.06 (d, J = 5.7 Hz, 2H), 3.90 (s, 2H), 3.69 (s, 3H), 3.36–3.23 (m, 2H), 1.69–1.52 (m, 2H), 1.38–1.13 (m, 18H), 0.84 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.11, 170.63, 170.15, 158.04, 136.17, 134.26, 133.90, 126.10, 117.90, 56.09, 52.40, 52.22, 41.17, 31.94, 29.66, 29.65, 29.58, 29.36, 27.09, 25.85, 22.71, 14.16. HRMS (ESI-TOF) m/z: calcd for C₂₄H₃₈N₂O₄ [M + H]⁺, 419.2904; found, 419.2929.

Trpeg-Gly-OMe (4d)

Synthesized by following the general procedure E. The pure product (1.0 gm, 60% yield) was isolated as yellow thick liquid. FT-IR data (KBr plate) ν: 3307, 2911, 2851, 1747, 1661, 1541, 1446, 1213, 766, 697. ¹H NMR (400 MHz, CD₃CN): δ 7.41–7.26 (m, 3H), 7.27–7.18 (m, 3H), 7.17–7.05 (m, 2H), 6.82 (app dd, J = 32.9, 11.0 Hz, 2H), 6.69 (app dd, J = 10.0, 8.5 Hz, 1H), 4.01 (s, 2H), 3.86 (d, J = 5.9 Hz, 2H), 3.65 (s, 3H), 3.64–3.58 (m, 2H), 3.00–2.89 (m, 2H). ¹³C NMR (101 MHz, CD₃CN): δ 182.86, 171.23, 170.87, 157.87, 140.22, 136.41, 134.70, 134.23, 129.88, 129.47, 127.34, 125.81, 117.19, 56.36, 55.18, 52.59, 41.63, 33.27. HRMS (ESI-TOF) m/z: calcd for C₂₁H₂₂N₂O₄ [M + Na]⁺, 377.1472; found, 377.1464.

TrPro-Gly-OMe (4e)

Synthesized by following the general procedure E. The pure product (110 mg, 65% yield) was obtained as dark yellow gelatinous liquid. ¹H NMR (400 MHz, CD₃CN): δ 7.17–6.99 (m, 2H), 6.87 (s, 1H), 6.73 (app d, J = 11.8 Hz, 1H), 6.62–6.42 (m, 2H), 4.98 (dd, J = 8.2, 3.2 Hz, 1H), 3.83 (app qd, J = 17.5, 6.0 Hz, 2H), 3.67–3.61 (m, 3H), 3.45 (dt, J = 10.9, 7.1 Hz, 1H), 2.19 (tdd, J = 10.9, 10.5, 5.9 Hz, 1H), 2.03 (dt, J = 16.6, 6.4 Hz, 2H), 1.98–1.87 (m, 2H). ¹³C NMR (101 MHz, CD₃CN): δ 181.01, 174.04, 171.39, 156.60, 136.33, 135.34, 132.72, 123.00, 113.72, 64.89, 52.57, 52.54, 41.48, 32.41, 23.61. HRMS (ESI-TOF) m/z: calcd for C₁₅H₁₈N₂O₄ [M + Na]⁺, 313.1159; found, 313.1164.

TrPro-Phe-OMe (4f)

Synthesized by following the general procedure E. The pure product (240 mg, yield = 65%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CD₃CN): δ 7.36–7.26 (m, 2H), 7.26–7.15 (m, 3H), 7.14–6.95 (m, 2H), 6.74–6.61 (m, 2H), 6.60–6.46 (m, 1H), 6.41 (app d, J = 10.4 Hz, 1H), 5.01–4.87 (m, 1H), 4.61 (tt, J = 12.2, 6.1 Hz, 1H), 3.60 (s, 3H), 3.58–3.53 (m, 1H), 3.42 (dt, J = 10.8, 7.0 Hz, 1H), 3.08 (dt, J = 15.3, 7.7 Hz, 1H), 3.03–2.92 (m, 1H), 2.11 (dd, J = 10.7, 5.2 Hz, 1H), 1.91–1.72 (m, 3H). ¹³C NMR (101 MHz, CD₃CN): δ 180.98, 173.06, 172.75, 156.49, 138.06, 136.22, 135.24, 132.70, 130.33, 129.36, 127.71, 122.91, 113.60, 64.66, 54.29, 52.65, 52.50, 38.24, 32.16, 23.64. HRMS (ESI-TOF) m/z: calcd for C₂₂H₂₄N₂O₄ [M + Na]⁺, 403.1628; found, 403.1633.

α-Deuterated Trhg-Gly-OMe (4a/a′/a″)

Synthesized by following general procedure F, and 55 mg (starting 75 mg; yield = 73%) of the pure product was obtained as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.10 (s, 1H), 7.11 (app ddd, J = 11.8, 8.2, 1.1 Hz, 1H), 7.08–6.95 (m, 2H), 6.68 (app t, J = 9.5 Hz, 2H), 4.06 (d, J = 5.7 Hz, 2H), 3.91 (s, 1H), 3.89 (s, 1H), 3.70 (s, 3H), 3.37–3.23 (m, 2H), 1.59 (dd, J = 14.6, 7.3 Hz, 2H), 1.34–1.19 (m, 10H), 0.84 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.13, 183.11, 183.08, 170.66, 170.18, 158.04, 136.22, 134.29, 134.25, 134.22, 133.94, 126.16, 126.12, 126.08, 117.96, 117.88, 117.81, 56.11, 56.04, 56.82, 56.61, 52.41, 52.37, 52.33, 52.25, 41.19, 31.53, 26.76, 25.81, 23.54, 22.62, 14.03. HRMS (ESI-TOF) m/z: calcd for C₁₈H₂₅DN₂O₄ [M + H]⁺, 358.1848; found, 358.1857.

α-Deuterated Trog-Gly-OMe (4b/b′/b′)

Synthesized by following general procedure F, and 36 mg (starting 52 mg, yield = 69%) of the pure product was obtained as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.11 (s, 1H), 7.13 (app ddd, J = 11.8, 8.2, 1.2 Hz, 1H), 7.08–6.96 (m, 2H), 6.69 (t, J = 9.8 Hz, 2H), 4.08 (d, J = 5.7 Hz, 2H), 3.91 (s, 1H), 3.90 (s, 1H), 3.71 (s, 3H), 3.37–3.24 (m, 2H), 1.70–1.53 (m, 2H), 1.34–1.16 (m, 12H), 0.85 (t, J = 6.9 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃): δ 183.19, 183.16, 183.14, 170.69, 170.21, 158.08, 136.26, 134.33, 134.30, 134.26, 133.97, 126.20, 126.16, 126.12, 118.02, 117.94, 117.86, 56.15, 52.44, 52.40, 52.29, 42.17, 41.22, 31.83, 29.77, 29.35, 29.28, 27.12, 25.87, 23.58, 22.70, 14.16. HRMS (ESI-TOF) m/z: calcd for C₂₀H₂₉DN₂O₄ [M + Na]⁺, 386.2161; found, 386.2172.

α-Deuterated Trddg-Gly-OMe (4c/c′/c″)

Synthesized by following general procedure F, and 47 mg (starting 65 mg; yield = 72%) of the pure product was obtained as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.11 (s, 1H), 7.13 (app ddd, J = 11.8, 8.2, 1.2 Hz, 1H), 7.08–6.95 (m, 2H), 6.69 (t, J = 9.8 Hz, 2H), 4.08 (d, J = 5.7 Hz, 2H), 3.91 (s, 1H), 3.90 (s, 1H), 3.71 (s, 3H), 3.36–3.26 (m, 2H), 1.70–1.51 (m, 2H), 1.35–1.11 (m, 18H), 0.86 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.08, 183.06, 183.03, 170.59, 170.11, 157.98, 136.15, 134.23, 134.19, 134.16, 133.87, 126.09, 126.05, 126.01, 117.82, 117.75, 56.05, 55.56, 52.35, 52.30, 52.26, 52.19, 41.12, 31.89, 29.62, 29.60, 29.54, 29.32, 27.04, 25.79, 22.67, 14.10. HRMS (ESI-TOF) m/z: calcd for C₂₄H₃₇DN₂O₄ [M + Na]⁺, 442.2787; found, 442.2810.

CTL5a

Synthesized from 4a

¹H NMR (400 MHz, CD₃CN): δ 8.07 (app dd, J = 11.8, 8.6 Hz, 1H), 7.84 (app dd, J = 13.1, 8.6 Hz, 3H), 7.80–7.70 (m, 1H), 4.60 (s, 2H), 3.88–3.72 (m, 7H), 1.85–1.68 (m, 2H), 1.51–1.30 (m, 6H), 0.91 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CD₃CN): δ 160.05, 154.54, 152.81, 145.37, 140.46, 139.42, 132.26, 128.38, 54.84, 53.87, 51.13, 41.15, 32.12, 26.85, 25.52, 23.26, 14.26. HRMS (ESI-TOF) m/z: calcd for C₁₅H₂₀NO₂ [M]⁺, 246.1489; found, 246.1491.

CTL5b

Synthesized from 3b-OH

¹H NMR (400 MHz, CD₃CN): δ 8.14–8.03 (m, 1H), 7.90–7.80 (m, 3H), 7.76 (app tt, J = 11.5, 4.0 Hz, 1H), 4.61 (s, 2H), 3.84–3.72 (m, 2H), 1.83–1.67 (m, 2H), 1.50–1.24 (m, 12H), 0.89 (t, J = 6.8 Hz, 4H). ¹³C NMR (101 MHz, CD₃CN): δ 160.09, 154.62, 152.92, 145.44, 140.53, 139.49, 132.36, 128.46, 54.94, 51.19, 32.62, 29.99, 27.25, 25.65, 23.47, 14.44. HRMS (ESI-TOF) m/z: calcd for C₁₇H₂₄NO₂ [M]⁺, 274.1802; found, 274.1794.

CTL5c

Synthesized from 3c-OH

¹H NMR (400 MHz, CD₃CN): δ 8.07 (app dd, J = 12.1, 8.5 Hz, 1H), 7.84 (app dd, J = 14.8, 8.7 Hz, 3H), 7.75 (app dt, J = 11.4, 6.3 Hz, 1H), 4.61 (s, 2H), 3.84–3.70 (m, 2H), 1.77 (dt, J = 15.8, 7.9 Hz, 2H), 1.55–1.13 (m, 29H), 0.87 (t, J = 6.6 Hz, 5H). ¹³C NMR (101 MHz, CD₃CN): δ 160.05, 154.61, 152.91, 145.43, 140.54, 139.48, 132.35, 128.45, 54.94, 51.18, 32.83, 30.52, 30.41, 30.33, 30.25, 30.05, 27.26, 25.65, 23.55, 14.49. HRMS (ESI-TOF) m/z: calcd for C₂₁H₃₂NO₂ [M]⁺, 330.2428; found, 330.2406.

CTL5d

Synthesized from 4d

FT-IR data (KBr plate) ν: 3023, 2927, 2851, 1739, 1661, 1507, 1430, 1188, 1144, 714. ¹H NMR (400 MHz, CD₃CN): δ 8.04–7.93 (m, 1H), 7.88–7.78 (m, 3H), 7.74 (td, J = 8.2, 3.4 Hz, 1H), 7.34 (m, 4H), 7.32–7.24 (m, 1H), 4.56 (s, 2H), 4.13–4.03 (m, 2H), 3.81 (s, 2H), 3.79 (s, 3H), 3.17–3.06 (m, 2H). ¹³C NMR (101 MHz, CD₃CN): δ 160.19, 154.83, 152.61, 145.12, 140.61, 139.78, 137.69, 132.75, 130.16, 129.99, 128.38, 128.34, 55.80, 53.84, 51.26, 41.40, 31.78. HRMS (ESI-TOF) m/z: calcd for C₁₇H₁₆NO₂ [M]⁺, 266.1176; found, 266.1123.

CTL5e

Synthesized from 4e

¹H NMR (400 MHz, CD₃CN): δ 8.04 (app dd, J = 11.8, 8.6 Hz, 1H), 7.91–7.77 (m, 2H), 7.74 (app dd, J = 12.9, 5.4 Hz, 1H), 7.64 (app d, J = 11.8 Hz, 1H), 4.51 (t, J = 8.5 Hz, 1H), 4.09–3.97 (m, 1H), 3.91 (ddd, J = 13.2, 10.1, 4.8 Hz, 1H), 3.79 (d, J = 5.0 Hz, 5H), 2.70–2.53 (m, 1H), 2.31 (ddt, J = 14.2, 11.7, 7.0 Hz, 2H), 2.20 (ddd, J = 15.8, 8.9, 4.8 Hz, 1H). ¹³C NMR (101 MHz, CD₃CN): δ 162.83, 153.87, 152.79, 144.74, 139.89, 138.71, 131.44, 129.29, 60.41, 53.89, 52.42, 41.46, 30.43, 28.28, 23.22. HRMS (ESI-TOF) m/z: calcd for C₁₂H₁₂NO₂ [M]⁺, 202.0863; found, 202.0933.

CTL5a/a′

Synthesized from 4a

¹H NMR (400 MHz, CD₃CN): δ 8.08 (dd, J = 12.0, 8.4 Hz, 1H), 7.85 (dd, J = 19.9, 8.7 Hz, 3H), 7.76 (dt, J = 11.3, 6.1 Hz, 1H), 4.62 (s, 1H), 4.61 (d, J = 2.6 Hz, 1H), 3.84 (s, 2H), 3.81–3.73 (m, 5H), 1.85–1.71 (m, 2H), 1.53–1.38 (m, 2H), 1.35 (dd, J = 9.0, 5.0 Hz, 5H), 0.97–0.83 (m, 3H). ¹³C NMR (101 MHz, CD₃CN): δ 168.44, 160.18, 154.69, 152.96, 145.50, 140.57, 139.55, 132.41, 128.51, 54.99, 54.97, 53.94, 51.22, 50.96, 50.73, 41.39, 32.23, 26.96, 25.65, 23.36, 14.32. HRMS (ESI-TOF) m/z: calcd for C₁₅H₁₉DNO₂ [M]⁺, 247.1551; found, 247.1567.

CTL5b/b″b″

Synthesized from 3b-OH

¹H NMR (400 MHz, CD₃CN): δ 8.08 (app dd, J = 12.1, 8.5 Hz, 1H), 7.84 (app dd, J = 15.7, 8.6 Hz, 2H), 7.76 (app ddd, J = 11.5, 8.4, 6.0 Hz, 1H), 4.61 (s, 1H), 4.60 (d, J = 2.3 Hz, 1H), 3.85–3.72 (m, 2H), 1.77 (dt, J = 11.7, 7.9 Hz, 2H), 1.49–1.23 (m, 12H), 0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz, CD₃CN): δ 160.07, 154.62, 152.89, 145.43, 140.52, 139.48, 132.34, 128.44, 54.90, 54.87, 51.17, 51.15, 50.92, 50.69, 32.61, 30.52, 29.98, 27.24, 25.64, 23.46, 14.55, 14.44. HRMS (ESI-TOF) m/z: calcd for C₁₇H₂₃DNO₂ [M]⁺, 275. 1864; found, 275.1847.

CTL5c/c′/c″

Synthesized from 4c

¹H NMR (400 MHz, CD₃CN): δ 8.16–8.01 (m, 1H), 7.92–7.80 (m, 3H), 7.75 (ddd, J = 11.5, 8.4, 5.9 Hz, 1H), 4.61 (s, 2H), 4.60 (s, 1H), 3.86–3.71 (m, 7H), 1.76 (dt, J = 11.7, 8.0 Hz, 2H), 1.48–1.19 (m, 21H), 0.88 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CD₃CN): δ 168.32, 160.06, 154.56, 152.82, 145.36, 140.46, 139.42, 132.26, 128.39, 54.83, 53.84, 51.14, 51.11, 50.88, 50.65, 41.26, 32.75, 30.44, 30.33, 30.26, 30.17, 29.97, 27.18, 25.58, 23.49, 14.46. HRMS (ESI-TOF) m/z: calcd for C₂₁H₃₁DNO₂ [M]⁺, 331.2490; found, 331.2473.

Trhg-NHCH₂Ph (6a)

Synthesized by following the general procedure G. The pure product (25 mg, 62%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.99 (s, 1H), 7.33–7.26 (m, 4H), 7.23 (ddd, J = 9.0, 6.1, 3.3 Hz, 1H), 7.13 (app ddd, J = 11.9, 8.3, 1.3 Hz, 1H), 7.02 (app ddd, J = 17.0, 13.3, 6.3 Hz, 2H), 6.68 (app dd, J = 20.7, 9.8 Hz, 2H), 4.50 (d, J = 6.0 Hz, 2H), 3.92 (s, 2H), 3.38–3.21 (m, 2H), 1.65–1.45 (m, 2H), 1.35–1.15 (m, 8H), 0.86 (dd, J = 9.4, 4.1 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃): δ 183.20, 170.14, 158.06, 138.53, 136.24, 134.26, 133.91, 128.68, 127.69, 127.32, 126.20, 117.99, 56.68, 52.47, 43.44, 31.58, 26.83, 26.13, 22.65, 14.09. HRMS (ESI-TOF) m/z: calcd for C₂₂H₂₈N₂O₂ [M + Na]⁺, 375.2043; found, 375.2014.

Trog-NHCH₂Ph (6b)

Synthesized by following the general procedure G. The pure product (40 mg, yield = 57%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.22 (s, 1H), 7.50 (t, J = 5.0 Hz, 4H), 7.48–7.41 (m, 1H), 7.35 (app ddd, J = 11.9, 8.3, 1.2 Hz, 1H), 7.30–7.15 (m, 2H), 6.90 (app dd, J = 20.0, 9.8 Hz, 2H), 4.72 (d, J = 6.0 Hz, 2H), 4.15 (s, 2H), 3.58–3.45 (m, 2H), 1.76 (d, J = 6.9 Hz, 2H), 1.58–1.36 (m, 10H), 1.10 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.16, 170.10, 158.03, 138.51, 136.20, 134.23, 133.88, 128.65, 127.65, 127.29, 126.16, 117.96, 56.64, 52.44, 43.41, 31.83, 29.35, 29.26, 27.14, 26.14, 22.70, 14.17. HRMS (ESI-TOF) m/z: calcd for C₂₄H₃₂N₂O₂ [M + Na]⁺, 403.2356; found, 403.2364.

Trddg-NHCH₂Ph (6c)

Synthesized by following the general procedure G. The pure product (52 mg, yield = 70%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.23 (s, 1H), 7.51 (t, J = 4.9 Hz, 4H), 7.49–7.41 (m, 1H), 7.35 (app ddd, J = 11.9, 8.3, 1.3 Hz, 1H), 7.25 (app ddd, J = 16.9, 13.3, 6.2 Hz, 2H), 6.91 (app dd, J = 19.6, 9.8 Hz, 2H), 4.73 (d, J = 6.0 Hz, 2H), 4.16 (s, 2H), 3.62–3.46 (m, 2H), 1.77 (d, J = 6.8 Hz, 2H), 1.50 (m, 18H), 1.11 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.14, 170.09, 158.02, 138.51, 136.18, 134.22, 133.87, 128.64, 127.65, 127.28, 126.13, 117.93, 56.64, 52.44, 43.40, 31.99, 29.70, 29.63, 29.61, 29.42, 29.40, 27.14, 26.14, 22.77, 14.21. HRMS (ESI-TOF) m/z: calcd for C₂₈H₄₀N₂O₂ [M + Na]⁺, 459.2982; found, 459.3007.

Trpeg-NHCH₂Ph (6d)

Synthesized by following the general procedure G. The pure product (15 mg, 65%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.72 (s, 1H), 7.33–7.22 (m, 7H), 7.23–7.14 (m, 2H), 7.14–7.06 (m, 2H), 7.06–6.98 (m, 2H), 6.74 (app t, J = 9.9 Hz, 2H), 4.40 (d, J = 6.1 Hz, 2H), 3.92 (s, 2H), 3.67–3.53 (m, 2H), 2.95–2.77 (m, 2H). ¹³C NMR (101 MHz, CDCl₃): δ 183.26, 169.79, 157.64, 138.51, 138.48, 136.26, 134.84, 133.71, 128.87, 128.77, 128.69, 127.63, 127.32, 126.83, 126.79, 118.75, 56.96, 53.86, 43.40, 32.95. HRMS (ESI-TOF) m/z: calcd for C₂₄H₂₄N₂O₂ [M + Na]⁺, 395.1730; found, 395.1742.

TrPro-NHCH₂Ph (6e)

Synthesized by following the general procedure G. The pure product (32 mg, yield = 32%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.33–7.15 (m, 5H), 7.13–6.96 (m, 2H), 6.84 (app dd, J = 11.7, 3.8 Hz, 1H), 6.59 (app dd, J = 19.4, 10.0 Hz, 2H), 6.45 (app d, J = 10.5 Hz, 1H), 4.97 (dd, J = 7.9, 4.9 Hz, 1H), 4.47–4.30 (m, 2H), 3.82 (dt, J = 10.9, 6.7 Hz, 1H), 3.47–3.29 (m, 1H), 2.33–2.03 (m, 3H), 1.99–1.82 (m, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 180.75, 172.58, 155.92, 138.46, 136.01, 134.46, 132.72, 128.65, 127.67, 127.35, 123.59, 114.24, 64.15, 52.01, 43.36, 31.55, 31.43, 23.73. HRMS (ESI-TOF) m/z: calcd for C₁₉H₂₀N₂O₂ [M + Na]⁺, 331.1417; found, 331.1452.

α-Deuterated Trhg-NHCH₂Ph (6a/a′/a″)

Synthesized by following the general procedure G. The pure product (35 mg, 60%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.99 (s, 1H), 7.33–7.26 (m, 4H), 7.25–7.18 (m, 1H), 7.12 (app ddd, J = 11.9, 8.3, 1.2 Hz, 1H), 7.07–6.95 (m, 2H), 6.68 (app dd, J = 21.2, 9.8 Hz, 2H), 4.50 (d, J = 6.0 Hz, 2H), 3.92 (s, 1H), 3.91 (s, 1H), 3.37–3.21 (m, 2H), 1.64–1.44 (m, 2H), 1.33–1.13 (m, 6H), 0.86 (dd, J = 9.3, 4.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.19, 183.16, 183.13, 170.13, 158.04, 138.52, 136.24, 134.25, 134.21, 134.18, 133.92, 128.67, 127.68, 127.31, 126.20, 126.16, 126.12, 118.00, 117.92, 117.84, 56.67, 56.60, 56.37, 56.17, 52.47, 52.43, 52.39, 43.43, 31.57, 29.80, 26.82, 26.11, 22.65, 14.09. HRMS (ESI-TOF) m/z: calcd for C₂₂H₂₇DN₂O₂ [M + Na]⁺, 376.2106; found, 376.2080.

α-Deuterated Trog-NHCH₂Ph (6b/b′/b″)

Synthesized by following the general procedure G. The pure product (52 mg, yield = 57%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.00 (s, 1H), 7.31–7.25 (m, 4H), 7.22 (dq, J = 5.8, 4.0 Hz, 1H), 7.11 (app ddd, J = 11.9, 8.3, 1.2 Hz, 1H), 7.07–6.94 (m, 2H), 6.67 (app dd, J = 19.9, 9.8 Hz, 2H), 4.49 (d, J = 6.0 Hz, 2H), 3.92 (s, 1H), 3.90 (s, 1H), 3.34–3.23 (m, 2H), 1.53 (d, J = 6.9 Hz, 2H), 1.35–1.12 (m, 10H), 0.87 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.11, 170.08, 158.00, 138.50, 136.18, 134.17, 133.88, 128.63, 127.64, 127.27, 126.14, 126.10, 126.05, 117.94, 117.86, 117.78, 56.63, 56.52, 56.34, 56.12, 52.43, 52.39, 52.35, 43.38, 31.82, 29.34, 29.25, 27.12, 26.11, 22.68, 14.16. HRMS (ESI-TOF) m/z: calcd for C₂₄H₃₁DN₂O₂ [M + Na]⁺, 404.2419; found, 404.2409.

α-Deuterated Trddg-NHCH₂Ph (6c/c′/c″)

Synthesized by following the general procedure G. The pure product (73 mg, yield = 66%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.02 (s, 1H), 7.28 (t, J = 3.2 Hz, 4H), 7.23 (ddd, J = 8.8, 6.4, 2.8 Hz, 1H), 7.12 (app ddd, J = 11.9, 8.3, 1.2 Hz, 1H), 7.07–6.93 (m, 2H), 6.67 (app dd, J = 18.4, 9.7 Hz, 2H), 4.50 (d, J = 6.0 Hz, 2H), 3.93 (s, 1H), 3.91 (s, 1H), 3.36–3.24 (m, 2H), 1.65–1.46 (m, 2H), 1.39–1.16 (m, 18H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.05, 183.02, 170.04, 157.92, 138.48, 136.13, 134.16, 134.12, 134.09, 133.85, 128.59, 127.60, 127.22, 126.06, 126.02, 125.98, 117.87, 117.79, 117.71, 56.58, 56.29, 52.37, 52.33, 43.34, 31.95, 29.66, 29.59, 29.57, 29.38, 29.35, 27.10, 26.08, 26.06, 22.73, 14.17. HRMS (ESI-TOF) m/z: calcd for C₂₈H₃₉DN₂O₂ [M + Na]⁺, 460.3045; found, 460.3058.

α-Deuterated Trpeg-NHCH₂Ph (6d/d′/d″)

Synthesized by following the general procedure G. The pure product (27 mg, 65%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.74 (s, 1H), 7.35–7.22 (m, 7H), 7.23–7.14 (m, 2H), 7.10 (app dd, J = 13.4, 6.3 Hz, 2H), 7.02 (app t, J = 10.3 Hz, 2H), 6.74 (t, J = 9.5 Hz, 2H), 4.40 (d, J = 6.1 Hz, 2H), 3.91 (s, 1H), 3.90 (s, 1H), 3.66–3.54 (m, 2H), 2.94–2.79 (m, 2H). ¹³C NMR (101 MHz, CDCl₃): δ 183.19, 169.19, 157.59, 138.48, 138.44, 136.24, 134.79, 134.76, 133.71, 128.84, 128.74, 128.66, 127.60, 127.29, 126.80, 126.76, 118.74, 118.66, 118.58, 56.91, 53.84, 53.81, 43.36, 32.90. HRMS (ESI-TOF) m/z: calcd for C₂₄H₂₃DN₂O₂ [M + Na]⁺, 396.1793; found, 396.1800.

Trog-Pro-Leu-Phe-OMe (7)

Synthesized by following the general procedure H. Condition A: 110 mg (0.34 mmol scale, yield = 46%), condition B: 79 mg (0.156 mmol scale, yield = 70%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.01 (d, J = 9.1 Hz, 1H), 7.40 (d, J = 7.1 Hz, 2H), 7.26 (dd, J = 8.5, 6.3 Hz, 2H), 7.19 (d, J = 7.3 Hz, 1H), 7.12 (app ddd, J = 20.8, 7.6, 5.4 Hz, 3H), 6.93 (app d, J = 11.3 Hz, 1H), 6.74–6.55 (m, 2H), 4.78–4.63 (m, 2H), 4.48 (dd, J = 8.6, 2.9 Hz, 1H), 4.28 (q, J = 7.7 Hz, 1H), 3.64 (s, 3H), 3.63–3.56 (m, 2H), 3.56–3.43 (m, 3H), 3.41–3.29 (m, 1H), 2.95–2.83 (m, 1H), 2.13–1.90 (m, 2H), 1.90–1.61 (m, 4H), 1.62–1.50 (m, 1H), 1.47–1.15 (m, 10H), 0.97–0.80 (m, 3H), 0.69 (dd, J = 6.5, 3.7 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 180.81, 172.78, 171.64, 171.22, 170.24, 157.34, 138.88, 136.80, 135.27, 133.15, 129.33, 128.35, 126.47, 124.05, 114.35, 60.87, 56.27, 54.88, 52.08, 52.06, 51.16, 46.74, 40.32, 36.71, 31.84, 29.78, 29.43, 29.40, 29.32, 27.12, 25.36, 24.46, 23.95, 22.70, 22.61, 22.28, 14.17. HRMS (ESI-TOF) m/z: calcd for C₃₈H₅₄N₄O₆ [M + H]⁺, 663.4116; found, 663.4123.

Trog-Phe-Phe-OMe (8)

Synthesized by following the general procedure H. Condition A: 265 mg (0.927 mmol scale, yield = 48%), condition B: 61 mg (0.156 mmol scale, yield = 65%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.46 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.21–7.09 (m, 9H), 7.08–6.91 (m, 4H), 6.78–6.67 (m, 1H), 6.58 (d, J = 10.4 Hz, 1H), 4.79 (app tdd, J = 14.2, 8.2, 5.7 Hz, 2H), 3.90 (d, J = 16.7 Hz, 1H), 3.66 (s, 3H), 3.64–3.53 (m, 1H), 3.16 (app ddd, J = 25.7, 14.1, 5.6 Hz, 2H), 3.05–2.86 (m, 4H), 1.48–1.35 (m, 2H), 1.35–1.08 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 182.50, 171.70, 170.91, 170.35, 157.96, 137.05, 136.51, 136.32, 134.36, 133.97, 129.30, 129.26, 128.53, 128.40, 126.78, 126.15, 117.09, 55.87, 54.02, 53.57, 52.58, 52.26, 37.67, 37.27, 31.81, 29.30, 29.24, 27.05, 25.58, 22.68, 14.16. HRMS (ESI-TOF) m/z: calcd for C₃₆H₄₅N₃O₅ [M + Na]⁺, 622.3251; found, 622.3250.

Trog-Leu-Phe-OMe (9)

Synthesized by following the general procedure H. Condition A: 80 mg (yield = 0.344 mmol scale, 41%), condition B: 42 mg (0.156 mmol scale, yield = 48%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.46 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.25–7.09 (m, 5H), 7.09–6.97 (m, 2H), 6.91 (app d, J = 11.8 Hz, 1H), 6.76–6.65 (m, 1H), 6.61 (app d, J = 10.4 Hz, 1H), 4.81 (td, J = 8.6, 5.4 Hz, 1H), 4.50 (td, J = 8.3, 6.2 Hz, 1H), 3.94 (d, J = 16.9 Hz, 1H), 3.67 (d, J = 4.4 Hz, 3H), 3.57 (d, J = 16.9 Hz, 1H), 3.24 (dd, J = 14.2, 5.4 Hz, 1H), 3.16–3.01 (m, 2H), 3.00–2.85 (m, 1H), 1.66–1.54 (m, 2H), 1.52–1.37 (m, 3H), 1.37–1.11 (m, 10H), 0.93–0.83 (m, 6H), 0.80 (d, J = 6.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 182.42, 173.04, 171.18, 170.33, 157.98, 137.11, 136.29, 134.39, 133.98, 129.54, 129.27, 128.70, 128.48, 127.32, 126.77, 126.21, 117.09, 55.90, 54.01, 52.78, 52.20, 51.16, 40.53, 37.68, 37.23, 33.39, 31.79, 29.27, 29.24, 27.07, 25.26, 24.68, 22.90, 22.66, 21.69, 14.13. HRMS (ESI-TOF) m/z: calcd for C₃₃H₄₇N₃O₅ [M + Na]⁺, 588.3408; found, 588.3410.

Trog-Val-OMe (10)

Synthesized by following the general procedure H. Condition A: 60 mg (0.313 mmol scale, yield = 48%), condition B: 63 mg (0.235 mmol scale, yield = 67%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 8.01 (d, J = 8.7 Hz, 1H), 7.12 (app ddd, J = 11.9, 8.2, 1.2 Hz, 1H), 7.03 (dd, J = 16.6, 7.7 Hz, 2H), 6.77–6.61 (m, 2H), 4.52 (dd, J = 8.7, 5.0 Hz, 1H), 4.00 (d, J = 16.1 Hz, 1H), 3.83 (d, J = 16.1 Hz, 1H), 3.70 (s, 3H), 3.37–3.25 (m, 2H), 2.28–2.13 (m, 1H), 1.61 (d, J = 7.5 Hz, 2H), 1.28 (t, J = 15.6 Hz, 10H), 0.94 (dd, J = 19.0, 6.9 Hz, 6H), 0.86 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 183.21, 172.24, 170.20, 158.02, 136.10, 134.34, 133.81, 126.14, 117.86, 57.41, 56.32, 52.45, 52.10, 31.85, 31.14, 29.80, 29.41, 29.29, 27.16, 26.11, 22.72, 19.25, 17.86, 14.18. HRMS (ESI-TOF) m/z: calcd for C₂₃H₃₆N₂O₄ [M + Na]⁺, 427.2567; found, 427.2591.

Trog-Phe-OMe (11)

Synthesized by following the general procedure H. Condition A: 65 mg (0.34 mmol scale, yield = 42%), condition B: 54 mg (0.231 mmol scale, yield = 52%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.84 (d, J = 8.0 Hz, 1H), 7.23–7.07 (m, 6H), 7.06–6.93 (m, 2H), 6.67 (app dd, J = 9.9, 8.9 Hz, 1H), 6.56 (app d, J = 10.3 Hz, 1H), 4.84 (td, J = 7.6, 5.8 Hz, 1H), 3.89 (d, J = 3.7 Hz, 2H), 3.67 (s, 3H), 3.24–3.10 (m, 3H), 3.04 (dd, J = 13.9, 7.4 Hz, 1H), 1.55–1.38 (m, 2H), 1.36–1.06 (m, 13H), 0.86 (t, J = 6.9 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃): δ 182.79, 171.87, 169.87, 157.69, 136.25, 135.95, 134.11, 133.79, 129.31, 128.51, 126.97, 125.79, 117.30, 55.98, 53.40, 52.46, 52.27, 38.05, 31.82, 29.31, 29.26, 27.04, 25.90, 22.69, 14.16. HRMS (ESI-TOF) m/z: calcd for C₂₇H₃₆N₂O₄ [M + Na]⁺, 475.2567; found, 475.2590.

Trog-Thr-OMe (12)

Synthesized by following the general procedure H. Condition A: 57 mg (0.313 mmol scale, yield = 48%), condition B: 36 mg (0.156 mmol scale, yield = 56%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.95 (d, J = 9.2 Hz, 1H), 7.18–7.07 (m, 1H), 7.07–6.95 (m, 2H), 6.70 (app t, J = 9.9 Hz, 2H), 4.59 (dd, J = 9.3, 2.9 Hz, 1H), 4.30 (dd, J = 6.4, 2.9 Hz, 1H), 4.06 (d, J = 16.6 Hz, 1H), 3.83 (d, J = 16.6 Hz, 1H), 3.71 (s, 3H), 3.32 (dd, J = 14.8, 7.2 Hz, 3H), 1.71–1.55 (m, 2H), 1.26 (dt, J = 11.4, 8.0 Hz, 12H), 0.85 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 182.96, 171.29, 170.69, 158.20, 136.34, 134.43, 134.02, 126.33, 118.16, 68.58, 57.76, 56.21, 52.93, 52.44, 31.81, 29.75, 29.36, 29.25, 27.15, 25.93, 22.68, 20.26, 14.14. HRMS (ESI-TOF) m/z: calcd for C₂₂H₃₄N₂O₅ [M + Na]⁺, 429.2360; found, 429.2360.

Trog-Ser-OMe (13)

Synthesized by following the general procedure H. Condition A: 56 mg (0.313 mmol scale, yield = 44%), condition B: 47 mg (0.211 mmol scale, yield = 55%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.80 (d, J = 8.2 Hz, 1H), 7.20–7.03 (m, 2H), 6.98 (app d, J = 11.8 Hz, 1H), 6.72 (app t, J = 10.4 Hz, 2H), 4.65 (dt, J = 8.0, 3.1 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 4.10 (dd, J = 11.4, 3.3 Hz, 1H), 4.02–3.87 (m, 2H), 3.78 (d, J = 16.8 Hz, 1H), 3.34 (dd, J = 15.9, 6.7 Hz, 2H), 1.76–1.60 (m, 2H), 1.38–1.16 (m, 10H), 0.86 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 182.90, 170.81, 170.45, 158.31, 136.53, 134.54, 134.19, 126.37, 117.83, 62.77, 61.73, 56.54, 55.27, 53.39, 31.82, 29.35, 29.26, 27.18, 25.76, 22.69, 14.22, 14.16. HRMS (ESI-TOF) m/z: calcd for C₂₂H₃₄N₂O₅ [M + Na]⁺, 429.2360; found, 429.2374.

Trog-Hyp-OMe (14)

Synthesized by following the general procedure H. Condition A: 63 mg (0.34 mmol scale, yield = 44%), condition B: 65 mg (0.275 mmol scale, yield = 57%) of the pure product was isolated as yellow viscous liquid. Two sets of peaks were observed due to existence of trans and cis isomers around secondary amide in 8:2 ratio. Some of the peaks of both isomers in ¹H and ¹³C NMR are overlapped. Trans Isomer: ¹H NMR (400 MHz, CDCl₃): δ 7.10–6.98 (m, 2H), 6.83 (app dd, J = 11.4, 9.1 Hz, 1H), 6.66 (app dd, J = 10.7, 5.1 Hz, 1H), 6.63–6.52 (m, 1H), 4.82 (t, J = 10.5 Hz, 1H), 4.59 (t, J = 8.2 Hz, 1H), 4.47 (s, 1H), 4.07 (d, J = 16.6 Hz, 1H), 3.75–3.60 (m, 5H), 3.56–3.36 (m, 2H), 2.37–2.24 (m, 1H), 1.94 (ddd, J = 13.1, 8.4, 4.6 Hz, 1H), 1.75–1.57 (m, 2H), 1.41–1.12 (m, 10H), 0.86 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 180.98, 172.97, 168.43, 157.21, 136.02, 134.51, 132.69, 124.06, 115.58, 70.45, 57.70, 55.04, 54.71, 53.92, 52.30, 37.70, 31.83, 29.42, 29.31, 27.11, 26.16, 22.69, 14.15. Cis Isomer: ¹H NMR (400 MHz, CDCl₃): δ 7.10–6.98 (m, 2H), 6.83 (app dd, J = 11.4, 9.1 Hz, 1H), 6.66 (app dd, J = 10.7, 5.1 Hz, 1H), 6.63–6.52 (m, 1H), 4.76 (d, J = 6.2 Hz, 1H), 4.59 (t, J = 8.2 Hz, 1H), 4.35 (s, 1H), 3.97 (d, J = 12.4 Hz, 1H), 3.76 (s, J = 4.0 Hz, 3H), 3.74–3.61 (m, 5H). 3.56–3.36 (m, 2H), 2.59–2.43 (m, 1H), 2.24–2.11 (m, 1H). 1.75–1.57 (m, 2H), 1.41–1.12 (m, 10H), 0.86 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 180.96, 172.86, 169.46, 157.02, 136.11, 134.67, 132.52, 123.86, 115.02, 68.56, 57.58, 55.65, 54.79, 53.58, 52.74, 40.07, 31.83, 29.74, 29.31, 27.11, 25.91, 22.69, 14.15. HRMS (ESI-TOF) m/z: calcd for C₂₃H₃₄N₂O₅ [M + Na]⁺, 441.2360; found, 441.2375.

Trog-Tyr-OMe (15)

Synthesized by following the general procedure H. Condition A: 50 mg (0.313 mmol scale, yield = 35%), condition B: 42 mg (0.215 mmol scale, yield = 42%) of the pure product was isolated as yellow viscous liquid. ¹H NMR (400 MHz, CDCl₃): δ 7.85 (d, J = 8.3 Hz, 1H), 7.14 (app ddd, J = 11.9, 8.3, 1.2 Hz, 1H), 7.08–6.98 (m, 2H), 6.93 (d, J = 8.5 Hz, 2H), 6.71 (app dd, J = 9.9, 8.9 Hz, 1H), 6.67–6.57 (m, 3H), 4.82 (td, J = 8.0, 5.4 Hz, 1H), 4.02–3.86 (m, 2H), 3.67 (d, J = 5.1 Hz, 3H), 3.21 (td, J = 7.2, 3.6 Hz, 2H), 3.07 (dd, J = 14.0, 5.3 Hz, 1H), 2.91 (dd, J = 14.0, 7.9 Hz, 1H), 1.50 (dd, J = 13.8, 7.0 Hz, 2H), 1.34–1.08 (m, 10H), 0.85 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 182.78, 172.03, 170.15, 157.74, 155.73, 136.50, 134.19, 133.98, 130.31, 127.14, 126.31, 118.14, 115.63, 55.84, 53.69, 52.70, 52.38, 37.35, 31.84, 29.35, 29.28, 27.04, 26.12, 22.71, 14.18. HRMS (ESI-TOF) m/z: calcd for C₂₇H₃₆N₂O₅ [M + Na]⁺, 491.2516; found, 491.2535.

Trog-Phe-Phe-OMe (8-OH)

Synthesized by following the general procedure D. The pure product (66 mg, yield = 90%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, DMSO): δ 8.33 (d, J = 7.7 Hz, 1H), 8.09 (d, J = 8.7 Hz, 1H), 7.32–7.12 (m, 10H), 7.11–7.03 (m, 1H), 6.98 (app t, J = 10.3 Hz, 1H), 6.70 (app d, J = 11.7 Hz, 1H), 6.60–6.43 (m, 2H), 4.56 (td, J = 9.6, 4.1 Hz, 1H), 4.43 (td, J = 8.1, 5.6 Hz, 1H), 4.07 (app dd, J = 40.4, 16.9 Hz, 2H), 3.27–3.13 (m, 2H), 3.07 (td, J = 14.2, 4.7 Hz, 2H), 2.94 (dd, J = 13.8, 8.5 Hz, 1H), 2.73 (dd, J = 13.8, 10.1 Hz, 1H), 1.53–1.37 (m, 2H), 1.32–1.09 (m, 10H), 0.86 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, DMSO): δ 180.08, 172.75, 170.98, 168.39, 156.95, 137.84, 137.59, 134.93, 133.93, 131.53, 129.24, 129.12, 128.15, 127.95, 126.35, 126.14, 122.65, 113.58, 53.95, 53.80, 53.45, 52.60, 37.48, 36.73, 31.24, 28.73, 28.71, 26.38, 25.68, 22.10, 13.97. HRMS (ESI-TOF) m/z: calcd for C₃₅H₄₃N₃O₅ [M + Na]⁺, 608.3095; found, 608.3104.

Trog-Phe-OH (11-OH)

Synthesized by following the general procedure D. The pure product (81 mg, yield = 93%) was isolated as yellow viscous liquid. ¹H NMR (400 MHz, DMSO): δ 8.19 (d, J = 8.1 Hz, 1H), 7.32–7.13 (m, 5H), 7.12–7.02 (m, 1H), 6.98 (app t, J = 10.3 Hz, 1H), 6.70 (app d, J = 11.7 Hz, 1H), 6.61–6.46 (m, 2H), 4.45 (td, J = 8.6, 4.8 Hz, 1H), 4.18 (app dd, J = 38.8, 16.8 Hz, 2H), 3.25 (dt, J = 14.2, 7.1 Hz, 2H), 3.09 (dd, J = 13.7, 4.7 Hz, 1H), 2.89 (dd, J = 13.7, 9.0 Hz, 1H), 1.49 (dd, J = 13.8, 6.9 Hz, 2H), 1.34–1.10 (m, 10H), 0.85 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, DMSO): δ 180.12, 172.92, 168.47, 156.84, 137.65, 134.75, 133.77, 131.53, 129.20, 128.08, 126.30, 122.59, 113.56, 53.86, 53.54, 52.65, 37.01, 31.26, 28.78, 28.73, 26.41, 26.00, 22.12, 13.97. HRMS (ESI-TOF) m/z: calcd for C₂₆H₃₄N₂O₄ [M + H]⁺, 439.2591; found, 439.2605.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01540.

• Experimental procedures and analytical data, detailed NMR and mass spectral data for CTLs5 formation, direct amidation, reversible amidation, and α-deuteration, and copies of NMR and HRMS spectra (PDF)

The authors declare no competing financial interest.