Construction of Five Tryptophan Isomers and Application of the Isomers to Solid-Phase Total Syntheses of Lysocin E Derivatives

Graphical Abstract

Five tryptophan (Trp) isomers were prepared in which the indole C3-substitution was changed to the C2/4/5/6/7-substitutions and were then applied to Fmoc-based solid-phase total syntheses of the analogues of antibiotic lysocin E. The biological evaluation revealed the importance of the precise three-dimensional shape of the original Trp residue.

Abstract

Tryptophan (Trp) plays a unique role in peptides and proteins as its indole ring possesses an electron-rich character and an N1−H hydrogen-bond donor. Because of its non-rotationally symmetric structure, synthetic alterations of the orientation of the indole ring would modulate the intrinsic structures and functions of peptides and proteins. Here we developed synthetic routes to the five Trp isomers in which the C3-substitution of the indole ring was changed to the C2/4/5/6/7-substitutions, and applied the five monomers to Fmoc-based solid-phase peptide synthesis. Specifically, the five monomers were prepared via Negishi cross-coupling reactions of C2/4/5/6/7-iodoindoles. To demonstrate the applicability of the monomers to the solid-phase synthesis, the five Trp isomers of macrocyclic antibiotic lysocin E were selected as target molecules and synthesized through peptide elongation, on-resin macrocyclization, and global deprotection. The Trp isomers displayed markedly weaker antibacterial activity than the parent natural product, revealing the biological importance of the precise three-dimensional shape of the original Trp residue of lysocin E. The present methods for the preparation and application of these five Trp isomers provide a new strategy for analyzing and modifying the specific functions of numerous Trp-containing peptides and proteins beyond this study.

Introduction

Tryptophan (Trp) is the sole proteinogenic amino acid containing two aromatic rings, fused benzene and pyrrole rings, in its side chain. The large C3-substituted indole moiety of Trp endows distinct physicochemical properties (Figure 1a).^{1, 2} The indole ring has an N1−H bond with hydrogen bonding ability³ and a permanent dipole moment pointing from N1 in the pyrrole ring to C4 in the benzene ring. Furthermore, because of its electron-rich character, the indole ring strongly interacts with cations and electron-deficient aromatic rings through cation-π⁴ and electron-donor acceptor (EDA) interactions,⁵ respectively. These factors play key roles in controlling the three-dimensional structures, dynamics, functions, and bioactivities of Trp-containing peptides and proteins.

Figure 1

(a) Physicochemical properties of the indole ring. The electrostatic potential surface and dipole moment were calculated by Spartan’18 with ωB97X-D/6-31G*. (b) A strategy for investigating the effect of indole orientation on bioactive peptides and proteins. (c) Structures of lysocin E (5 a), five Trp isomers 5 b–5 f, and menaquinone-4 (MK-4).

Noncovalent interactions of Trp are strongly influenced by the orientation of the indole ring because of its non-rotational symmetry. Therefore, alterations of the substitution patterns of the indole ring would provide valuable information for determining and modulating the original Trp functions in bioactive peptides and proteins. With this consideration in mind, we envisioned to devise a synthetic strategy to prepare five isomers 1 b–1 f of peptide/protein 1 a (Figure 1b), in which the original C3-substitution was changed to the C2-, C4-, C5-, C6-, and C7-substitutions. To realize an efficient and generic methodology for accessing these isomers, we decided to adopt Fmoc-based solid-phase peptide synthesis (SPPS)^{6, 7} because of its operational simplicity and applicability to the facile preparation of analogues without changing the overall procedure.^{8, 9} As a proof-of-concept study, we selected five isomers 5 b–5 f of lysocin E (5 a, Figure 1c)¹⁰ as the target molecules. Peptidic natural product 5 a has a 37-membered polyamide macrocycle and exerts potent antibacterial activity against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Our group achieved an Fmoc-based full solid-phase total synthesis of 5 a and performed structure-activity relationship (SAR) studies.^11-13 These studies revealed the essential role of the indole ring of residue-10 (d-Trp-10) upon EDA complexation with the electron-deficient naphthoquinone ring of menaquinone (MK) in the bacterial membrane.¹⁴ The binding of 5 a with MK disrupts the bacterial membrane integrity, eventually leading to bacteriolysis. Herein, we report the unified synthetic routes to five N_α-Fmoc-N_indole-Boc-protected Trp isomers 2 b–2 f, the incorporation of 2 b–2 f into the SPPS of lysocin E isomers 5 b–5 f, and the antibacterial activities of 5 b–5 f. The present study would serve as a new molecular editing strategy for proving the importance of the indole orientation of Trp of bioactive peptides and proteins.^{15, 16}

Results and Discussion

Efficient and unified syntheses of the N_α-Fmoc-N_indole-Boc-protected Trp isomers 2 b–2 f had not been reported (Figure 1b).^17-19 Thus, we designed short synthetic routes to the five isomers using Negishi cross-coupling reactions.^{20, 21} Compounds 2 b–2 f were retrosynthetically dissected into Cbz/Bn-protected chiral organozinc 3 and Boc-protected iodoindoles 4 b–4 f. In this strategy, the use of enantiomeric d-3 and l-3 would lead to d-2 b–d-2 f and l-2 b–l-2 f, respectively. Scheme 1 illustrates the construction of the d-series of the five isomers for the syntheses of lysocin E isomers. Upon performing the preliminary experiments, the C2/4/7-substituted iodoindoles 4 b/4 c/4 f were found to be less reactive for Negishi coupling than the C5/6-substituted iodoindoles 4 d/4 e, presumably due to the steric hindrance induced by the proximal C3-hydrogen atom or the N-Boc group. Accordingly, the conditions were judiciously optimized, ultimately establishing robust procedures. When 4 b–4 f were treated with 1.3 equiv of organozinc species d-3 under catalysis of Pd₂(dba)₃ (2.6 mol %) and Q-Phos (5.2 mol %) in DMF at 35 °C,^22-25 the requisite d-6 b, d-6 c, d-6 d, d-6 e, and d-6 f were obtained in 46 %, 62 %, 74 %, 75 %, and 51 % yields, respectively.

Scheme 1

Unified synthetic routes to five N_α-Fmoc-N_indole-Boc-protected Trp isomers. ^aBoc=tert-butoxycarbonyl, Bn=benzyl, Cbz=benzyloxycarbonyl, dba=dibenzylideneacetone, Fmoc=9-fluorenylmethoxycarbonyl.

Next, the highly stable N-Cbz and O-Bn protective groups of d-6 b–d-6 f required for the coupling reaction were simultaneously removed, and the free amines were capped with Fmoc groups to yield d-2 b–d-2 f. C2/4/5-substituted isomers d-6 b/d-6 c/d-6 d were subjected to Pd/C (6.6 mol %) under H₂ atmosphere in MeOH, leading to the corresponding amino acids. Subsequent Fmoc protection of the amine using FmocCl and NaHCO₃ in aqueous 1,4-dioxane successfully afforded d-2 b, d-2 c, and d-2 d in 77 %, 79 %, and 78 % yields, respectively, over two steps. Contrary to our expectation, the same conditions were not serviceable for d-6 e and d-6 f because of their different reactivities originating from the distinct indole substitutions. The hydrogenolysis reaction of C6-isomer d-6 e using Pd/C in MeOH caused an over-reduction, producing indoline d-7 e (30 %) along with desired d-2 e (33 %) after Fmoc protection. The higher reactivity of the C2=C3 double bond of d-6 e would be attributable to its lower shielding effect by the C6-attached amino acid moiety. To suppress the undesired reduction of the C2=C3 double bond, 150 equiv of 2-methyl-2-butene was added as a decoy substrate,^{26, 27} and EtOH was used as the solvent to decelerate the hydrogenolysis. The following Fmoc attachment gave rise to d-2 e in 58 % with a negligible amount of indoline d-7 e (3.3 %). On the other hand, Pd/C in MeOH was ineffective for the deprotection of C7-isomer d-6 f. In this case, the Cbz and Bn groups would be kinetically protected by the Boc-protected indole connected to the most hindered C7-position. Here, more reactive catalyst Pd(OH)₂/C (3.3 mol %) in the presence of MeOH and 2-methyl-2-butene (150 equiv) induced the requisite transformation. The ensuing treatment with FmocCl and NaHCO₃ produced the desired d-2 f in 53 % yield in two steps along with indoline d-7 f in 12 % yield. Hence, routes to all five isomers d-2 b–d-2 f were established.

Prior to the Fmoc-based SPPS using the thus-prepared monomers d-2 b–d-2 f, we performed a model study for the global deprotection of the side-chain protective groups. In general, the electron-rich aromatic rings often capture stable cations (e.g., Me₃C⁺ and Ph₃C⁺) that are generated upon the last removal of various acid-sensitive protective groups (e.g., t-Bu, Boc, and Tr), via Friedel-Crafts reactions. Compared with the original Trp residue, the five isomers would have higher reactivity toward these electrophiles due to the non-substituted C3-position of their indole rings. To investigate the reactivity of the C3-exposed indole, the Boc-protected d-8 and the Tr-protected d-9 were selected as model compounds, separately synthesized from C5-isomer d-2 d and a commercially available asparagine derivative, which were together subjected to acidic conditions (Table 1). When d-8 and d-9 were treated with CF₃CO₂H/H₂O (95/5) (entry 1), the C3-tritylated byproduct d-11 was produced in 88 % yield, and only a small amount of the desired d-10 (6.5 %) was obtained, corroborating the high nucleophilicity of the C3-position.²⁸ We therefore investigated an effective reductant of the tertiary cations. Application of the commonly used i-Pr₃SiH with CF₃CO₂H/H₂O was indeed effective, leading to the desired d-10 in 66 % yield (entry 2).²⁹ The indoline byproduct d-12, however, was formed in 18 % yield through C3-protonation and C2-hydride addition. To impede the hydride transfer, the bulkier t-Bu₂MeSiH was chosen instead of i-Pr₃SiH.^{30, 31} Although t-Bu₂MeSiH is rarely employed as a cation scavenger, the yield of d-10 was increased to 76 % and the yield of d-12 was decreased to 5.3 % (entry 3). Hence, the bulkiness of the two t-Bu groups allowed for suppressing the indole reduction while maintaining the reactivity against tertiary cations.

Table 1. Model study for the acid-promoted global deprotection.

entry	reagents	d-10	d-11	d-12
1	CF3CO2H/H2O (95/5)	6.5 %	88 %	–
2	CF3CO2H/H2O/i-Pr3SiH (95/3.5/1.5)	66 %	–^[a]	18 %
3	CF3CO2H/H2O/t-Bu2MeSiH (95/3.5/1.5)	76 %	–^[a]	5.3 %

• [a] A detectable amount of compound was not obtained.

Having established the deprotection conditions, we initiated Fmoc-based full solid-phase total syntheses of lysocin E (5 a) and five isomers 5 b–5 f (Scheme 2). The syntheses of the fully protected and resin-bound 15 a–15 f were conducted based on our previous synthesis of 15 a (Scheme 2).¹¹ Briefly, linear polyamide structures were constructed from the Wang-ChemMatrix resin-linked l-Glu-8 13 by combination of piperidine-mediated Fmoc removal and microwave-assisted condensation³² of the Fmoc-protected amino acids, including d-2 a–d-2 f. The amide linkages were formed using O-(7-aza-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt).³³ On the other hand, the ester linkage between the C_β−OH group of l-Thr-1 and l-Thr-12 was constructed using 14 by the action of N,N′-diisopropylcarbodiimide and N,N-dimethylaminopyridine (DMAP). After preparing the resin-linked linear intermediates, the Fmoc group of d-Gln-9 and the allyl group of l-Glu-8 were detached by using piperidine and Pd(PPh)₄/morpholine, respectively. Finally, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)³⁴ promoted the macrolactamization, giving rise to the 37-membered rings 15 a–15 f.

Scheme 2

Solid-phase total syntheses of lysocin E (5 a) and five isomers 5 b–5 f. DMAP=N,N-dimethylaminopyridine, HATU=O-(7-aza-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HOAt=1-hydroxy-7-azabenzotriazole, NMP=N-methyl-2-pyrrolidone, Pbf=2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, TBS=tert-butyldimethylsilyl, Tr=triphenylmethyl, t-Bu=tert-butyl.

Resin-bound macrocycles 15 a–15 f were submitted to the newly established deprotection conditions. Namely, CF₃CO₂H/H₂O/t-Bu₂MeSiH (95/3.5/1.5) simultaneously effected the removal of the two Pbf, two t-Bu, one Boc, one TBS, one Tr, and cleavage of the Wang-ChemMatrix resin. Remarkably, the undesired alkylated byproducts were not observed using this reagent mixture. After purification by high-performance liquid chromatography, 5 a, 5 b, 5 c, 5 d, 5 e, and 5 f were isolated in 17 %, 7.4 %, 3.4 %, 3.9 %, 7.0 %, and 0.4 %³⁵ yields, respectively, over 25 steps from 13. Thus, the five Trp isomers were successfully integrated into the Fmoc-based SPPS despite their distinct reactivities.

Having synthesized 5 a and five isomers 5 b–5 f, we evaluated their affinity with the target molecule, MK-4, and their activities against Gram-positive bacteria (Table 2). First, according to the previously developed method,¹⁴ complexation between 5 a–5 f and MK-4 in the DDM micelles was quantified as a dissociation constant (K_D) by isothermal titration calorimetry (ITC) experiments. The K_D value of lysocin E (5 a) and MK-4 was determined to be 0.093 μM. Although all the isomers 5 b–5 f possess the same electron-rich indole ring, the MK-affinities of 5 b–5 f were markedly lower. In fact, the binding heats of 5 b, 5 c, 5 e, and 5 f were too small to calculate the K_D values. Only the C5-isomer 5 d had a detectable affinity (K_D=0.237 μM), which was 2.5-fold weaker than that of the parent 5 a.³⁶ The data clarified that the orientation of the indole ring critically influenced the strong EDA complexation of the macrocyclic peptide with MK-4.

Table 2. MK-4 affinities and antibacterial activities of 5 a and five isomers 5 b–5 f.

• [a] The value is displayed as mean±SD from three independent experiments. [b] The value was determined by the microdilution method. [c] The value could not be determined due to the non-detectable binding heat. B. cereus=Bacillus cereus JCM20037, B. subtilis=Bacillus subtilis JCM2499, L. monocytogenes=Listeria monocytogenes 10403S, S. haemolyticus=Staphylococcus haemolyticus JCM2416. BCS=bovine calf serum, DDM=n-dodecyl-β-d-maltoside.

The antibacterial activities of five isomers 5 b–5 f were markedly weakened compared with that of lysocin E (5 a). The potencies were assessed as minimum inhibitory concentrations (MIC) against six Gram-positive bacterial strains, methicillin-susceptible S. aureus (MSSA1), MRSA4, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus haemolyticus. As reported previously,^{13, 14, 37} the parent 5 a was a highly effective antibacterial agent (MIC=1–4 μg/mL), and the activity toward MSSA1 was potentiated 64-fold (0.063 μg/mL) upon the addition of 10 % bovine calf serum (BCS). Five isomers 5 b–5 f all displayed weaker antibacterial activities (8–>64 μg/mL). Reflecting its strongest MK-4 affinity among the five isomers, the C5-isomer 5 d exhibited higher antibacterial activity than the other four isomers 5 b, 5 c, 5 e, and 5 f against all strains examined. Intriguingly, 10 % BCS lowered the MIC of 5 d from 16 μg/mL to 8 μg/mL, confirming its beneficial effect. For the first time, the ITC experiments and antibacterial assays together uncovered that subtle changes in the substitution positions of the indole ring were detrimental to the original MK-binding function and the MK-dependent antibacterial activity of 5 a.

To gain insight into the preliminary structural information of the differently substituted indole rings, we analyzed the hydrogen bonding of the indole N-proton by H/D exchange experiments. For this, lysocin E (5 a), C2-isomer 5 b, and C5-isomer 5 d were selected as representative peptides because of their disparate activities. The H/D exchange was initiated by the addition of 750 equivalents of D₂O to 5 a, 5 b, and 5 d in DMSO-d₆, and calculated by the change in the peak area of the proton of interest by measuring the ¹H NMR (Figure 2). The remaining ¹H peak area of the indole N-proton (10.0–11.0 ppm) was integrated to assess its involvement in the hydrogen bonding, and then compared with the remaining peak area of other N−H protons (7.50–9.00 ppm). The peak area of the indole proton (29 % for 5 a, 89 % for 5 b, 93 % for 5 d) was significantly larger than that of the other protons (16 % for 5 a, 8 % for 5 b, 3 % for 5 d), demonstrating that the indole protons participated in the hydrogen bonding.³⁸ Importantly, the slower H/D exchange of the indole protons of C2-isomer 5 b (89 %) and C5-isomer 5 d (93 %) than that of 5 a (29 %) suggested the existence of stable hydrogen bonds. Considering the negligible and weak activities of 5 b and 5 d, the indole rings of 5 b and 5 d are likely to be fixed to the unfavored locations for MK-complexation. Therefore, the three-dimensional alignment of the indole N−H of the parent 5 a was considered crucial for organizing the bioactive three-dimensional structure.

Figure 2

Relative remaining peak area of indole N−H and other N−H protons of 5 a, 5 b, and 5 d in H/D exchange experiments. The peak areas were calculated from the ¹H NMR spectra of 5 a, 5 b, and 5 d in (a) DMSO-d₆ without D₂O, (b) DMSO-d₆ with 750 equivalents of D₂O, respectively. The ¹H NMR spectra were immediately obtained after the addition of D₂O.

Conclusion

In summary, we devised a new general strategy for synthesizing five types of Trp isomer-containing peptides/proteins to explore the importance of their indole ring orientation. First, we prepared the C2-, C4-, C5-, C6-, and C7-isomers d-2 b–d-2 f of N_α-Fmoc-N_indole-Boc-protected Trp d-2 a by employing Negishi cross-coupling reactions with chiral organozinc d-3 and optimizing the protective group manipulations. Second, the doubly protected d-2 b–d-2 f were incorporated into the full solid-phase total syntheses of five isomers 5 b–5 f of macrocyclic lysocin E (5 a). Peptide elongation and macrocyclization from the resin-bound monomer gave rise to the fully protected 15 a–15 f. The last global deprotection necessitated new conditions because of the high nucleophilicity of the C3-unsubstituted indole rings of the Trp isomers. Specifically, t-Bu₂MeSiH was utilized with CF₃CO₂H and H₂O to realize the construction of five isomers of 5 a, 5 b–5 f. Third, the functional and biological evaluation showed that none of five isomers 5 b–5 f retained the strong MK-affinity and antibacterial activity of the parent 5 a. Thus, we revealed that the orientation of the original C3-subsituted indole plays a critical role in defining the three-dimensional shape required for stabilizing the EDA complex with MK and exerting MK-dependent potent antibacterial activity. These results also confirmed that the structures of natural products are precisely optimized for their dedicated functions through evolutionary selection. Because of the synthetic generality and robustness, the present strategy using these five isomers should be applicable to investigating the intrinsic functions of Trp in bioactive peptides and proteins other than lysocin E. Moreover, generation of the isomers would be useful for modulating and upgrading the original functions and biological activities of Trp-containing molecules.

Experimental Section

See the Supporting Information for details of the synthesis and characterization of compounds.

Supporting Information

Additional references cited within the Supporting Information.^39-46

Acknowledgments

This research was financially supported by Grants-in-Aid for Scientific Research (S) (JSPS, JP22H04970) to M.I., for Scientific Research (C) (JSPS, JP21K05286) to H.I., and for Scientific Research (B) (JSPS, JP23H02719) and the Institute for Fermentation, Osaka to H.H. A fellowship from MEXT (WISE Program) to J.F. is gratefully acknowledged. We thank Dr. Fumiaki Hayashi and Dr. Toshio Nagashima (RIKEN) for measurements of 800/200 and 600/150 MHz ¹H/¹³C NMR spectra.

Conflict of interest

The authors declare no conflict of interest.

Open Research

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.