Structure–Property Relationship Study of N‑(Hydroxy)Peptides for the Design of Self-Assembled Parallel β‑Sheets

Abstract

The design of novel and functional biomimetic foldamers remains a major challenge in creating mimics of native protein structures. Herein, we report the stabilization of a remarkably short β-sheet by incorporating N-(hydroxy)glycine (Hyg) residues into the backbone of peptides. These peptide–peptoid hybrids form unique parallel β-sheet structures by self-assembly upon hydrogenation. Our spectroscopic and crystallographic data suggest that the local conformational perturbations induced by N-(hydroxy)amides are outweighed by a network of strong interstrand hydrogen bonds.

Graphical Abstract

INTRODUCTION

In recent years, much interest has been devoted to the development of peptoids (N-substituted glycines)¹ due to their proteolytic stability and the ease to introduce various side chains without incorporating the asymmetric α-stereocenters typically found in peptides.² A major drawback in controlling these peptoid secondary structures is the lack of hydrogen-bonding motifs and the high backbone flexibility of the central glycine methylene. Interestingly, one subclass of peptoid blocks, namely, N-hydroxy-α-amino acids, can be found in numerous bioactive natural products, such as aurantimycins, polyoxypeptins, dentigerumycin, and penicisulfuranols A–F,³ and other bacterial siderophores.⁴ Despite an intriguing potential arising from the secondary hydroxamate functional group as a hydrogen-bond donor and acceptor, as well as a metal chelator,^5,6 the notorious lack of stability of this motif (pH-dependent decarboxylation and possible dismutation) has hampered most synthetic studies.⁷ Seemingly, only sparse examples of synthetic and structural studies have been reported on the propensity of N-(hydroxy)glycine (Hyg) to induce secondary interactions like β-turn⁸ and sheet-like structures (e.g., 1, Figure 1).⁹ Blackwell’s study demonstrated that Hyg residues positioned at hydrogen-bonded (inward) sites in peptoid sequences like 1 enabled the formation of antiparallel β-sheets. Therefore, we hypothesized that by intercalating α-amino acids in such peptoids, sturdy N-(hydroxy)peptoid–peptide β-sheet hybrids (e.g., 2, Figure 1) could potentially be generated. Given the important acidity of N-(hydroxyl)amides in comparison to typical amide groups,¹⁰ we became interested in evaluating their hydrogen-bond donor/acceptor abilities to form β-sheets. N-(hydroxy)⁹ and N-(alkoxy)peptoids¹¹ are known to preferentially adopt a trans-conformation, which is also appropriate to the design of novel β-sheet tertiary structures. Herein, we report the stabilizing effect of Hyg blocks inside a unique intra/intermolecular hydrogen-bond network for the self-assembly of particularly short β-sheets (Figure 1).¹² This work provides a foundation to understand and rationalize the role of N-(hydroxy)amides as hydrogen-bond donor/acceptor motifs in the backbone of peptide–peptoid hybrids.

Figure 1.

Proposed novel β-sheet scaffold built by intercalation of the Hyg blocks (left). Peptoid blocks synthesized in this study (right).

RESULTS AND DISCUSSION

Synthesis of Peptide–Peptoid Hybrids.

The syntheses of N-hydroxy-α-amino acids in both racemic¹³ and asymmetric¹⁴ manners have been previously reported; however, the insertion of these peptoid blocks into peptides has been fairly scarce.¹⁵ To develop a robust synthesis of N-(hydroxy)-peptoid–peptide hybrids, a series of N-(benzyloxy)glycine derivatives 3–6 were prepared with typical protecting groups for liquid- or solid-phase peptide synthesis (Figure 1). For the initial study of N-terminal elongation, dipeptide–peptoids 7a–c were synthesized starting from N-(OBn)-Gly-OEt 6a, which was coupled with three different Fmoc-protected amino acids (Scheme 1). As for N-alkyl peptoid blocks, N-alkoxy residues are known to have a low reactivity for amide-bond formation, but a cocktail of HATU/HOAt reagents was found suitable to the synthesis of dipeptide–peptoids 7a–c in 70–87% yields. Compounds 7a–c were then engaged in a sequence of deprotection/coupling toward the tripeptide–peptoids 9a–c. Unfortunately, under the large set of conditions tested,¹⁶ N-terminal free dipeptide 8 proved difficult to isolate and the coupling toward 9a–c failed. Instead, large amounts of degradation were observed along with the intramolecular cyclization into diketopiperazines (DKPs) 10a–c. Although Hyg favors trans amide bonds thermodynamically, the low transition-state energy barrier previously reported for the cis–trans isomerization of this motif (~16 kcal/mol)^7b can explain the ease of forming DKPs. While N-(benzyloxy)peptoid oligomers are known to be exclusively in a most stable trans amide conformation,¹¹ the fast kinetics of cis–trans isomerization do not allow the cis rotamer to be observed on the NMR time scale. To obtain a direct evidence of the genuine cis–trans isomerization, we thought to force the equilibrium toward the cis rotamer by exploiting the chelating nature of the Hyg residue. Dipeptide–peptoid 7c was therefore hydrogenated to the corresponding Fmoc-Ile-Hyg-OEt dipeptide, which was used as the chelating ligand to form a gallium(III) complex (3:1 ligand/metal ratio).¹⁷ In this complex, the strong nuclear Overhauser effect spectroscopy (NOESY) 1H–¹H cross-peak signal between the Ile H_α and the Hyg methylene detected by NMR supports the presence of a cis N-(hydroxy)amide rotamer. Taken together, this spectroscopic evidence of a cis rotamer and the facile DKP formation (8 → 10) further support that N-(hydroxy) and N-(benzyloxy)-amides undergo fast cis–trans isomerization.

Scheme 1. N-Terminal Elongation toward Tripeptide–Peptoids 9a–c.

^aReaction conditions: (a) Fmoc-AA-OH (4.0 equiv), HATU (4.0 equiv), HOAt (4.4 equiv), N,N-diisopropylethylamine (DIEA; 4.0 equiv) in CH₂Cl₂/DMF (3:1); (b) piperidine or NHEt₂; (c) 3 (4.0 equiv), HATU (4.0 equiv), HOAt (4.4 equiv), DIEA (4.0 equiv) in CH₂Cl₂.

To circumvent the formation of DKP 10, we turned our attention to a strategy of C-terminal elongation (Scheme 2). Dipeptides 11a–c were synthesized by coupling the acetyl Hyg 4 with three different t-butyl α-amino esters by activation with EDCi or HATU. When similar conditions were tested to couple Fmoc-Hyg 5 with Ile-OtBu, an unexpected cleavage of the benzyl protecting group occurred. The coupling was therefore optimized with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to deliver dipeptide 11d without epimerization.¹⁸ A smooth deprotection with trifluoroacetic acid (TFA) leads quantitatively to all of the corresponding free carboxylic acid of dipeptides 11a–d, which were directly coupled with 6a in the presence of HATU/HOAt to deliver tripeptide–peptoids 9a–d. Unfortunately, 9c was highly epimerized at the Ile α-stereocenter (2:3 dr). To improve the pivotal amide-bond formation, the coupling reaction was further evaluated with the unprotected block 6b. In this case, the epimerization of tripeptide–peptoids 9c′ could not be detected by ¹H NMR, but the racemization was later confirmed by the presence of a mixture of Ile/allo-Ile residues in the crystal structure of 12c (Figure 2). N-(hydroxy) and N-(benzyloxy)glycine blocks are less reactive than typical N-methylated amino acids, which are already known to be poor nucleophiles in amide-bond formation. Due to the oxygen electronegativity and the steric hindrance carried by the hydroxy- and benzyloxy-side chains during the approach to the activated carbonyl π* orbital, such peptoid ligation is particularly slow and difficult, leading ultimately to potential epimerization. To address this issue, several activating cocktails were evaluated for the difficult coupling of 6a. To our delight, tripeptide–peptoid 9c was prepared with ~17% of epimerization using EEDQ as the activating agent. Given the electron-withdrawing nature of the N-(benzyloxy) side chain, we hypothesized that the N-terminal acetyl of 11c might be unusually basic and participates into the Ile epimerization (see Figure SI-1). Thus, by switching to a Fmoc-protecting group in 11d, the EEDQ coupling was achieved successfully to produce 9d in 21% yield with minimal epimerization (~3%).^16,18 To circumvent the low coupling yield, further optimizations were required. A cocktail of pivaloyl chloride with lutidine (hindered base) provided tripeptide–peptoid hybrid 9d in 50% yield with low epimerization (<5%).¹⁶ Finally, benzyl groups from all synthetic tripeptide–peptoids 9a–d, 9c′ were removed under typical hydrogenation conditions to afford 12a–d in 79–99% yields.

Scheme 2. C-terminal Elongation toward Tripeptide–Peptoids 12a–d.

^aReaction conditions: (a) from 4 (1.0 equiv), DIPEA (1.5 equiv), for 11a,b: EDCi (1.2 equiv), HOBt (1.2 equiv) in CH₂Cl₂, for 11c: H AA-OtBu (2.0 equiv), HATU (1.5 equiv), HOAt (1.5 equiv) in dimethylformamide (DMF); for 11d: from 5 (1.0 equiv), EEDQ (1.2 equiv) in CH₂Cl₂; (b) TFA/CH₂Cl₂ (1:1); (c) for 9a–c′: 6a or 6b(2.0 equiv), HATU (2.0 equiv), HOAt (2.0 equiv), DIEA (2.0 equiv) in DMF, for 9d: PivCl (1.2 equiv), lutidine (1.2 equiv) in CH₂Cl₂; (d) Pd/C 10 mol %, H₂ in EtOH.

Figure 2.

NOESY data of 12c in the region of the N-OH protons (A) in CDCl₃, (B) in CD₃CN, and (C) in DMSO-d₆. (D) X-ray structure of 12c showing the solid-state conformation with interstrand hydrogen bonds represented by the dashed lines in the parallel β-sheet.

Structural Analysis of the Peptide–Peptoid Hybrids by Nuclear Magnetic Resonance (NMR) Spectroscopy.

The synthesis of “turnless” β-sheets inspired by short protein sequences or by peptidomimetics remains a major challenge.¹⁹ Indeed, motifs capable of enhancing nonbonded interstrand interactions to stabilize artificial short β-sheets are rare.²⁰ To assess the presence of secondary and potential tertiary structures in the synthetic peptide–peptoid hybrids 9a–d, 9c′, and 12a–d, a careful solution NMR spectral analysis at 18 °C was performed (Figure 2A–C).^16,21 A combination of ¹H, ¹H–¹H NOESY, and ¹H–¹³C heteronuclear multiple-quantum correlation (HMBC) NMRs in solvents of various polarity (CDCl₃, CD₃CN, DMSO-d₆) facilitated the conformational and structural analyses of these peptoid–peptide hybrids.¹⁶ The cis–trans equilibrium constants for both N-(benzyloxy) and N-(hydroxy)glycine residues in peptides 7a, 11a, and 9a were obtained with K_trans/cis values > 20 in each case, meaning that independently of the solvent, these amides have an exclusive thermodynamic preference for the trans geometry.^16,22 Interestingly, in all constructs 9, 11–12, the methylene protons (H_α/H_α′) of N-(hydroxy) or N-(benzyloxy)glycine blocks are always highly diastereotopic (doublets with a geminal coupling constant of ~17 Hz) with a Δδ(H_α/H_α′) of ~0.5 ppm, which typically characterize well-folded structures. Each hydroxyl proton OH¹ and OH² from Hyg(1) and Hyg(2) residues was easily assigned and differentiated based on distinct NOESY correlations as well as a clear HMBC correlation between OH² and the vicinal Ile carbonyl. Given the downfield chemical shifts of both OH¹ (δ 8.36–8.76 and 9.73–9.91 ppm) and OH² (δ 8.85–9.44 and 10.19–10.25 ppm) in CDCl₃ and DMSO-d₆, respectively, tripeptide–peptoids 9c′ and 12a–d, hydroxyl hydrogens were determined to participate in inter- or intramolecular hydrogen bonds. A deshielding of ~0.5 ppm between δ OH¹ and OH² suggests that the OH² hydroxyl is more strongly hydrogen bonded and likely to participate in the β-sheet stabilization. As shown in Figure 2C, all NOESY cross-peaks obtained for 12c in DMSO-d₆ can be attributed to interstrand interactions suggesting that no intermolecular assembly occurs in this solvent. In contrast, in CD₃CN and more markedly in CDCl₃ (Figure 2A,B), a stronger NOESY correlation between OH¹ on one strand and the Ile H_α on the second strand supports the formation of a β-sheet self-assembly. Indeed, in a pair of facing residues i,j on the opposite strands from a parallel β-sheet (e.g., Ile/allo-Ile in 12c,d) residue i is H-bonded to both residues j − 1 and j + 1, which in this case both are Hyg residues.²³ As established by Abraham, the difference in chemical shits of exchangeable protons or chemical shift deviation (CSD) due to solvent exposure Δδ = δ(DMSO-d₆) – δ(CDCl₃) can be used to assess the strength of intra- and intermolecular hydrogen bonds for the OH and NH groups.²⁴ The Δδ(H_N) of 0.53 and 0.57 ppm in constructs 12c,d suggest the presence of relatively strong hydrogen bonds likely arising from an intermolecular interaction between β-strands (see Table SI-8). Similarly, Δδ(OH²) of 0.80 and 0.77 ppm in 12c and 12d, respectively, support the existence of hydrogen bonds from the Hyg(2) hydroxyls, while the larger Δδ(OH¹) of 1.13 and 1.11 ppm firmly indicate that OH¹ hydroxyls are more solvent-exposed and not engaged in hydrogen bonding.¹⁶ Collectively, the downfield shifts observed for the exchangeable protons (OH/H_N) as well as the small CSD values for both Ile H_N and Hyg(2) OH in constructs 12c,d suggest that such hydrogens participate in strong interstrand hydrogen bonding. Furthermore, the self-assembly of 12c was characterized by monitoring the chemical shift deviations of OH/H_N protons as a function of concentration in CDCl₃ (Figures SI-12 and SI-13).^16,21 In the range of concentrations studied, changes in chemical shifts values fitted well to a dimerization isotherm equation representing the assembly of the strands into β-sheets.^20a These results also suggest that upon hydrogenation of the N-(benzyloxy)glycine residues, Hyg blocks play an important role in the self-assembly of constructs 12a–d into short β-sheets.

X-ray Analysis.

The structural features of β-sheets revealed by the NMR study were further confirmed in the crystal structure of construct 12c obtained by X-ray analysis (Figure 2D). In this crystal, the parallel β-sheets are packed in a head-to-head manner forming columns through a single and relatively weak hydrogen bond OH¹⋯O = C (N-terminal acetyl). Certainly, the low-lying conformation observed in the crystal lattice reflects closely the major conformer of 12c in solution (CDCl₃). Both H_N and OH² groups are shown to participate in intermolecular hydrogen bonding to stabilize a parallel β-sheet structure. Both Φ and ψ dihedral angles of Hyg(1/2) and Ile residues collected from the crystal structure are in agreement with an extended β-sheet arrangement (see the Ramachandran plot, Figure SI-14). A notable feature of the parallel β-sheet is the nitrogen pyramidalization in each N-(hydroxy)amide (ω(OH¹) 171.5° and ω(OH²) 170.9°). A similar pyramidalization was previously reported by Kirshenbaum¹¹ and others²⁵ for N-alkoxy peptoids. This phenomenon is likely responsible for decreasing the nitrogen lone pair resonance in the N-(hydroxy)amide, which in turn accentuates the basicity of the vicinal carbonyl. This stereoelectronic effect likely explains the unique hydrogen-bond acceptor nature of the N-(hydroxy)amide carbonyl responsible for the unusual parallel β-sheet alignment. In the original report from Blackwell, the central α-amino acid was instead a N-aryl peptoid block (i.e., construct 1 vs 2, Figure 1). In such constructs, the central carbonyls were also good hydrogen-bond acceptors, but these peptoid blocks could not participate in intermolecular H-bond, which could justify the preference for an antiparallel β-sheet direction. Our results on peptoid–peptide hybrids 12a–d complement well Blackwell’s study on peptoids in which Hyg residues were positioned at the H-bonded inward sites of the antiparallel β-sheets.⁹ Herein, we demonstrated that N-(hydroxy)amide groups can force intermolecular hydrogen bonds with Hyg at both H-bonded and non-H-bonded sites (inward and outward of strands) for stabilizing these novel β-sheets with a different parallel directionality.²⁶

β-Sheet Self-assembly and Thermal Stability.

Secondary and tertiary peptidyl structures have well-established signatures in far-UV circular dichroism (CD), but the assignment of the bands in the peptoids is more challenging due to their important flexibility. N-Alkoxy peptoid oligomers have been proposed by Kirshenbaum to adopt a polyproline II type (PPII) secondary structure that exhibit two maxima at 197 and 215 ± 5 nm.²⁷ The CD spectra obtained for tripeptide–peptoid hybrid 9d present similar bands at 195/216 nm in CH₃CN and 200/218 nm in HFIP, suggesting that a similar PPII secondary structure formed (Figure 3). As shown by the superimposition of the CD spectra in Figure 3, the hydrogenation of 9d into 12d triggers a dramatic change in the tripeptide–peptoid 3-dimensional structure. Typically, antiparallel β-sheets are characterized by two bands, a positive maximum at 193–195 nm and a negative exciton at 213–218 nm, profoundly resembling the two bands for parallel β-sheets at 200–203 and 214–221 nm.²⁸ Therefore, the combination of a positive exciton at 201 nm and a large CD band overlay at 213–219 nm obtained for construct 12d conclusively supports the formation of a parallel β-sheet. Furthermore, a positive band at 298 nm, characteristic of a π–π* transition, was also observed in the near-UV CD spectrum of 12d (Figure SI-15C).²⁹ This band arising from an interaction between the proximate fluorenyl π-systems of N-terminal Fmoc groups also supports the existence of a parallel arrangement between β-strands. Overall, the structural elucidation by CD from the Fmoc interaction at 298 nm and the exciton minima at 213/219 nm distinctively confirm a parallel β-sheet self-assembly of 12d in solution.

Figure 3.

CD spectra comparison of 9d and 12d showing the self-assembly process a CD spectra of 9d in acetonitrile (90 mM) and HFIP (103 mM) and of 12d in HFIP (116 mM) at 0 °C. Sample concentrations were accurately determined by UV-absorption of the fluorene (Fmoc) chromophore.

To test the stability of these novel parallel β-sheets, variable temperature NMR spectra (VT-NMR) were recorded between −60 and 110 °C (Figure 4). As expected, the spectra of tripeptide–peptoids 12c,d in DMSO-d₆ revealed no evidence of tertiary structures as shown by the small-temperature dependence coefficients (T_C, Δδ/T < −4.6 ppb/K) for H_N, while both N-(hydroxy)amide hydrogens (OH¹/OH²) might exist in a relatively strong intramolecular hydrogen bond (−4.6 < T_C < −4.0 ppb/K).^30,31 The T_C representing the slopes of the best-fitted linear regression of OH¹ and OH² chemical shift drifts as a function of temperature were invariably low for all of the constructs studied in DMSO-d₆ (Figure 4A and Table SI-7). It can be concluded that a strong intramolecular sixmembered ring hydrogen bond stabilizes each Hyg residue in DMSO-d₆ in single-stranded molecules (Figures SI-6 and SI-7).¹⁶ In contrast, the significantly larger T_C (OH¹) and T_C (OH²) values (−17.0 < T_C < −12.2 ppb/K) in CDCl₃ (Figure 4A) indicate that the hydrogen bonds are weaker in apolar solvents. These counterintuitive results could be explained by the competition between a strong interstrand H-bond and the innate intramolecular H-bond of the Hyg residues (as shown in DMSO-d₆). Competing intra- and intermolecular H-bonds result in a net weakening of the hydroxyls’ hydrogen-bond network. The results obtained for β-sheets 12c,d are in line with the interactions reported earlier by Blackwell for a peptoid tetramer β-sheet (e.g., 1, Figure 1). While examining the temperature dependency on the N-(hydroxy)amide chemical shifts in CDCl₃, it was found that both hydroxyl signals shifted in a nonlinear fashion (Figure 4B). The sigmoidal dependence of N-(hydroxy)amide proton (OH¹/OH²) chemical shifts as a function of temperature is distinctive of a β-sheet denaturation transition.³² The melting curves observed for β-sheets 12c,d indicate a rupture of folding at elevated temperatures. Thus, melting temperatures (T_m) corresponding to the hydrogen-bond network cleavage within the parallel β-sheets of 12c,d were estimated from the best-fitted curves of thermal transition to the Boltzmann equation.¹⁶ T_m(OH¹) of 23 and 14 ± 5 °C and T_m(OH²) of 46 and 29 ± 5 °C were calculated for the β-sheets of 12c and 12d, respectively (Figure 4B, Table SI-6). In both molecules, the significantly higher melting temperature (>15 °C) associated with the H-bond rupture of hydroxyl OH² is a direct evidence that the interstrand hydrogen bond from Hyg(2) stabilizes the entire structure.

Figure 4.

(A) Temperature dependence of the H_N and OH chemical shifts. (B) Plots of hydroxyl proton (OH¹/OH²) chemical shifts as a function of temperature in DMSO-d₆ and CDCl₃.

CONCLUSIONS

In conclusion, a coupling of the readily available N-(hydroxy)-glycine peptoid blocks (Hyg) has been developed with a low epimerization rate (3–5%) for C- and N-terminal insertion into peptide–peptoid hybrids. The synthetic constructs self-assembled in solution to form short parallel β-sheets, which were characterized by a combination of NMR and CD experiments and confirmed by the crystal structure of 12c. A detailed examination of the major differences in NOESY cross-strand correlations, CSDs, T_C, and T_m in various solvents clearly highlights the role of N-(hydroxy)amides from Hyg residues in the self-assembly and the stabilization of these tertiary structures.^8a Our results demonstrated that N-(hydroxy)amides not only favor exclusively trans amide conformers via intramolecular H-bonds (K_trans/cis > 20) but are also amenable to strong interstrand hydrogen bonds in solvents of low polarity. Recent studies from Del Valle have shown that the insertion of N-(amino)³³ and N-(hydroxy) α-amino acids^15f at specific non-hydrogen-bonded sites (outward positions) can stabilize the β-sheets within hairpins. In contrast, the present study establishes that a combination of N-(hydroxy)amides at hydrogen-bonded (inward) and non-hydrogen-bonded outward sites can be favorable due to a strong intramolecular and interstrand H-bond network. Given the typical difficulty to create stable β-sheets within short peptide constructs,^19,20 the ability of Hyg to improve the hydrogen-bond donor/acceptor properties of proteinogenic backbone amides will certainly find numerous applications in foldamer chemistry. Ongoing studies in our laboratory will leverage the unique stereoelectronic properties of Hyg and other N-hydroxy α-amino acids to study the assembly of the parallel and antiparallel β-hairpins in water.³⁴

EXPERIMENTAL SECTION

General Information.

All reagents used in the present paper were acquired from Alfa Aesar, Acros Organics, or Sigma Millipore. All bulk solvents were acquired from Fischer Scientific. Freshly distillated solvents were used in the reactions presented herein. Chloroform was dried over CaCl₂ overnight prior to distillation (BP 61 °C) and transferred under an argon atmosphere to a dark glass bottle with 3 Å molecular sieves for storage. Tetrahydrofuran was purified by refluxing with and distilling from sodium with benzophenone and transferred under an argon atmosphere to a dark glass bottle for storage. Dichloromethane was dried over CaCl₂ overnight prior to distillation (BP 40 °C) and transferred under an argon atmosphere to a dark glass bottle with 3 Å molecular sieves for storage. Toluene was dried over CaCl₂, CaH₂, or CaSO₄ and molecular sieves and transferred under an argon atmosphere to a dark glass bottle for storage. Full procedures can be found in Purification of Laboratory Chemicals by Armarego, W. L.F., and Chai C. L. L. editor (sixth edition). Reactions were performed in flame-dried glassware under a positive pressure of argon, unless the reaction occurs in water or aqueous solvent. Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise noted. Analytical thin-layer chromatography (TLC) was performed on 0.25 mm glass-backed 60 Å F-254 TLC plates. Flash chromatography was performed using 230–400 mesh silica gels (Silicycle, Inc.). The plates were visualized by exposure to UV light (254 nm) and developed by a solution of phosphomolybdic acid in ethanol, vanillin/sulfuric acid in ethanol, ninhydrin in ethanol, potassium permanganate in water/potassium carbonate/sodium hydroxide, or cerium-ammonium-molybdate in water/sulfuric acid and heat. Melting points were determined using an MPA 160 digital melting point apparatus. The matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra were performed on a Microflex LRF MALDI-TOF. The high-resolution mass spectra (HRMS) were obtained from the University of Florida using an Agilent 6230 TOF instrument, using electrospray ionization (ESI). The infrared spectra were recorded on a Nicolet iS10 FT-IR spectrophotometer (Thermo Scientific) with a SMART iTX ATR accessory. Optical rotation was measured on a JASCO P-2000 polarimeter.

The ¹H NMR spectra were recorded on a Varian Mercury400 (400 MHz) spectrometer using Vnmrj 4.2 software, Bruker AV-400 Ultrashield (400 MHz) spectrometer using Topspin 3.5 software, or Varian Mercury500 (500 MHz) spectrometer using Vnmrj 4.2 software and are reported in ppm using solvent as an internal standard (C₆D₆ at 7.16 ppm, CDCl₃ at 7.26 ppm, CD₃CN at 1.94 ppm, CD₃OD at 3.31 ppm, DMSO-d₆ at 2.50 ppm, and 1,4-dioxane-d₈ at 3.53 ppm). Data are reported as (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet; coupling constant(s) in hertz, integration). The 13C NMR spectra were recorded on a Bruker (100 MHz) spectrometer. Chemical shifts are reported in ppm, with solvent resonance employed as the internal standard (C₆D₆ at 128.1 ppm, CDCl₃ at 77.2 ppm, CD₃CN at 118.3 and 1.3 ppm, CD₃OD at 49.0 ppm, and DMSO-d₆ at 39.5 ppm). The NOESY spectra were recorded at 291 K, in the solvents mentioned above, at concentrations of 5–10 mM, with a 300 ms mixing time.

Variable temperature NMR were recorded on a Varian Mercury400 (400 MHz) spectrometer. The range of temperature studied was −60–70 °C in CDCl₃, −40–80 °C in CD₃CN, 0–100 °C in dioxane-d₈, and 20–110 °C in DMSO-d₆. The spectra were recorded with 16 scans every 10 °C, with a 5 min stabilization at each temperature before data acquisition. A final spectrum was recorded afterward at 20 °C to verify the absence of decomposition or transformation of the sample.

The CD spectra were recorded on a JASCO J-810 spectropolarimeter with a temperature controller module JASCO PFD-425S. The samples were prepared in a concentration range between 30 and 200 μM in CH₃CN or HFIP, and the sample concentrations were accurately determined by measuring the sample’s absorbance using a JASCO V-670 spectrophotometer based off the UV-absorption of the fluorenyl (Fmoc) group (ε₂₉₀ = 6089 M⁻¹ cm⁻¹) at 290 nm. The concentrations were obtained based on the Beer law: c = A/(ε·l), with c the sample concentration, A the measured absorptivity, ε the molar absorptivity, and l the path length of the cuvette. The raw CD data were recorded in mdeg with eight scans from 185 to 270 nm for the far-UV and 270–350 nm for the near-UV, every 0.1 nm at a speed of 100 nm per min. The CD spectra of the blank (pure solvent) were recorded and subtracted, while the baseline was set to 0 mdeg between 260 and 270 nm for the far-UV and 330–340 nm for the near-UV. The spectra were smoothed and baseline-corrected using the Spectragryph 1.2 software,³⁵ then converted into molar elipticity (deg cm² dmol⁻¹). The products of functionalization 3, 6a, 6b, SI-1, and SI-2 have been reported previously and characterized in refs 36d,c,b,e,a, respectively.

Synthesis of N-(benzyloxy)glycine (3).

O-Benzyl hydroxylamine hydrochloride (5.00 g, 31.1 mmol, 1.0 equiv) and glyoxylic acid monohydrate (4.33 g, 47.0 mmol, 1.5 equiv) were solubilized in AcOH/water (1:1 v/v, 100 mL) and stirred at RT during 30 min (completion monitored by TLC, oxime intermediate, R_f = 0.8, EtOAc/MeOH 1:1 v/v). The mixture was cool down to 0 °C with an ice bath and a solution of sodium cyanoborohydride (7.90 g, 125 mmol, 4.0 equiv) in AcOH/water (1:1 v/v, 50 mL) was added portion wise to avoid byproduct formation. The mixture was stirred for 5 h at RT. The reaction completion was monitored by TLC until the disappearance of the oxime intermediate. Water was then added and the crude reaction mixture was extracted with a mixture of iPrOH/CHCl₃ (5 × 20 mL). The combined organic layers were washed with saturated brine (3 × 20 mL), dried over Na₂SO₄, and filtrated, and the solvent was removed under reduced pressure (T < 35 °C) to afford 3 as a white solid (5.60 g, 30.9 mmol, 99% yield). R_f = 0.6 (100% EtOAc); HRMS-ESI (m/z): [M + H]⁺ calcd for C₉H₁₂NO₃, 182.0812; found, 182.0810 (−1.1 ppm). N-(Benzyloxy)-glycine 3 was pure enough to be engaged directly in the second step.

N-Acetyl-N-(benzyloxy)glycine (4).

Compound 3 (3.00 g, 16.6 mmol, 1.0 equiv) and sodium carbonate (1.76 g, 16.6 mmol, 1.0 equiv) were solubilized in water (17 mL). Acetic anhydride (1.86 g, 18.2 mmol, 1.1 equiv) was then added dropwise resulting in a precipitation and a yellow coloration. The mixture was stirred for an additional 30 min at RT. An initial wash using diethyl ether (2 × 20 mL) was achieved directly from the crude reaction mixture, and then, the aqueous layer was acidified with a solution of 2.0 M HCl to pH ~ 3. The aqueous layer was extracted with dichloromethane (3 × 20 mL), the combined organic layers were dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure to afford product 4 in a pure form as a yellowish powder (2.86 g, 12.8 mmol, 77% yield). R_f = 0.7 (EtOAc/MeOH 7:3 v/v); mp 109.0–115.0 (±0.6) °C; IR ν_max: 2928, 1706, 1596, 1456, 1379, 1260, 964, 911, 847, 749 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 9.97 (br, 1H), 7.37 (s, 5H), 4.87 (s, 2H), 4.31 (s, 2H), 2.15 (s, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 175.0, 172.5, 134.2, 129.5 (2C), 129.2, 128.8 (2C), 77.5, 49.4, 20.2; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₁H₁₃NO₄Na, 246.0737; found, 246.0728 (−3.7 ppm).

N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N-(benzyloxy)glycine (5).

Compound 3 (1.00 g, 5.52 mmol, 1.0 equiv) and sodium bicarbonate (928 mg, 11.0 mmol, 2.0 equiv) were solubilized in water (10 mL) by stirring 10 min at RT. The mixture was cooled down to 0 °C and (9H-fluoren-9-yl)methyl chloroformate (1.71 g, 6.63 mmol, 1.2 equiv) in dioxane (10 mL) was added to the mixture and stirred for 2 h at 0 °C. The dioxane was evaporated under reduced pressure, a solution of saturated sodium bicarbonate was added (pH ~ 9), and the aqueous phase was washed with hexanes (20 mL). The resulting aqueous layer was acidified with a solution of HCl (2.0 M) to pH ~ 4 allowing for the product precipitation. The product was extracted with dichloromethane (3 × 20 mL), the combined organic layers were dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure. The crude product was purified by precipitation into petroleum ether at RT and then filtrated to afford product 5 in a pure form as a white powder (1.73 g, 4.29 mmol, 77% yield). R_f = 0.7 (100% EtOAc); mp 132.0–137.0 (±0.6) °C; IR ν_max: 3039, 2954, 1757, 1737, 1666, 1427, 1350, 1248, 1184, 1116, 872, 760, 733 cm⁻¹; ¹H NMR (400 MHz, CD₃OD, δ): 7.77 (d, J = 7.5 Hz, 2H), 7.64 (d, J = 7.4 Hz, 2H), 7.37 (t, J = 7.3 Hz, 2H), 7.30 (m, 5H), 7.23 (m, 2H),4.64 (s, 2H), 4.58 (d, J = 6.0 Hz, 2H), 4.25 (t, J = 6.0 Hz, 1H), 4.02 (s, 2H); ¹³C{¹H} NMR (100 MHz, DMSO-d₆, δ): 169.9, 157.3, 144.1 (2C), 141.4 (2C), 135.5, 129.9 (2C), 128.9, 128.7 (2C), 128.2 (2C), 127.6 (2C), 125.5 (2C), 120.6 (2C), 76.5, 67.3, 51.6, 47.1; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₄H₂₂NO₅, 404.1492; found, 404.1485 (−1.7 ppm).

Substrates 6a and 6b were Prepared by the Following Procedures.

The appropriate alkoxyamine hydrochloride (1.0 mmol, 1.0 equiv), ptoluenesulfonic acid (0.1 mmol, 0.1 equiv), and a solution of ethyl glyoxylate (1.1 mmol, 1.1 equiv) in toluene (50% w/w) were mixed in EtOH (1.0 mL) and stirred at RT during 1 h until complete solubilization. The reaction was quenched with addition of a saturated NaHCO₃ solution (10 mL), and the product was extracted using dichloromethane (3 × 40 mL). The combined organic layers were washed with saturated brine (40 mL), dried over Na₂SO₄, filtrated, and the solvent was removed under reduced pressure to afford the corresponding ethyl glyoxylate oxime, which was pure enough to be engaged directly. Ethyl glyoxylate oxime (1.0 mmol, 1.0 equiv) was dissolved in EtOH (0.10 mL). BH₃.pyr (1.5 mmol, 1.5 equiv) was added to the mixture, which was cooled down to 0 °C. An ethanolic solution of HCl (6.0 M, 9.0 equiv, freshly prepared by addition of AcCl into EtOH at 0 °C) was added dropwise over a period of 3 h at 0 °C. After the addition of the acid, the mixture was stirred at RT for 30 min (the reaction completion was monitored by TLC until the disappearance of the ethyl glyoxylate oxime). Isolations of 6a and 6b were achieved as described below. Ethyl (benzyloxy)glycinate 6a: The reaction was quenched with addition of an aqueous solution of NaOH (2.0 M) at 0 °C and the crude mixture was concentrated under reduced pressure to remove EtOH. The resulting oil was taken into dichloromethane and washed with water (3 × 40 mL). Combined organic layers were washed with saturated brine (3 × 20 mL), dried over Na₂SO₄, and filtrated, and the solvent was removed under reduced pressure to afford product 6a as a yellowish oil (2.32 g, 11.1 mmol, 76% yield). R_f = 0.5 (hexanes/EtOAc 4:1 v/v); IR ν_max: 3068, 2923, 2854, 1685, 1451, 1231, 1121, 1002, 956, 758, 738 cm⁻¹; ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 171.1, 137.8, 128.4 (3C), 127.9 (2C), 76.1, 61.1, 53.5, 14.2. Ethyl hydroxyglycinate 6b: The crude mixture was concentrated under reduced pressure. The resulting oil was taken into dichloromethane and solid sodium carbonate was added to the solution at 0 °C. The mixture was then stirred overnight at room temperature. The solvent was filtrated and evaporated under reduced pressure to afford product 6b as a yellowish oil (2.43 g, 20.4 mmol, 72% yield). R_f = 0.4 (hexanes/EtOAc 3:2 v/v); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 171.1, 61.2, 54.9, 14.2.

General Procedure A, for N-O-benzyl α-Amino Ester Coupling With N-Fmoc α-Amino Acids.

Carboxylic acid 6a (4.0 mmol, 4.0 equiv) and the secondary amine Fmoc-AA-OH (1.0 mmol,1.0 equiv) were solubilized in CH₂Cl₂ and DMF (3:1; 4.0 mL). HOAt (4.4 mmol, 4.4 equiv) and HATU (4.0 mmol, 4.0 equiv) were added and the solution was stirred 10 min at RT. DIPEA (4.0 mmol, 4.0 equiv) was added to the mixture, which was stirred 4–15 h at RT. The reaction progress was monitored by TLC. CH₂Cl₂ (5.0 mL) was added and the reaction mixture was washed successively with a citric acid solution (10 mL, 5% w/w), saturated NaHCO₃ solution (10 mL), and saturated brine (10 mL). The organic layer was dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure to afford the crude product 7a–c, which was purified by chromatography.

Fmoc-N(Me)-Gly-N(OBn)-Ethyl-Glycinate (7a).

was prepared according to the general procedure A, using ethyl N-(benzyloxy)glycinate 6a (50 mg, 0.24 mmol, 1.0 equiv) and Fmoc-Sar-OH (149 mg, 0.48 mmol, 2.0 equiv). The crude product was purified by chromatography (hexanes/EtOAc 9:1 v/v) to afford 7a in a pure form as a white foam (104 mg, 0.21 mmol, 87% yield). R_f = 0.6 (hexanes/EtOAc 1:1 v/v); mp 42–45 °C; IR ν_max: 2944, 1747, 1702, 1450, 1399, 1372, 1204, 1146, 992, 758, 739, 700 cm⁻¹; Product 7a was characterized as a mixture of cis–trans rotamers in a ratio 2:3 by ¹H–¹³C HSQC: ¹H NMR (400 MHz, CDCl₃, δ) trans rotamer: 7.77 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 7.5 Hz, 2H), 7.40 (m, 5H), 7.34 (m, 4H), 4.93 (s, 2H), 4.39 (d, J = 7.0 Hz, 2H), 4.29 (s, 2H), 4.26 (s, 2H), 4.24 (t, J = 3.3 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.02 (s, 3H),1.28 (t, J = 6.7 Hz, 3H); cis rotamer: 7.72 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.5 Hz, 2H), 7.40 (m, 5H), 7.34 (m, 4H), 7.16 (m, 2H), 4.73 (s, 2H), 4.43 (d, J = 6.6 Hz, 2H), 4.29 (s, 2H), 4.24 (t, J = 3.3 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 4.07 (s, 2H), 2.91 (s, 2H), 1.25 (t, J =7.1 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ) trans rotamer: 167.7 (2C), 157.0, 144.1 (2C), 141.3 (2C), 134.3, 129.6 (2C),129.2, 128.8 (2C), 127.6 (2C), 127.1 (2C), 125.2 (2C), 120.0 (2C), 77.3, 67.9, 61.7, 50.6, 49.9, 47.2, 35.6, 14.1; cis rotamer: 167.6 (2C), 156.4, 144.2 (2C), 141.3 (2C), 134.1, 129.6 (2C), 129.2, 128.8 (2C), 127.6 (2C), 127.1 (2C), 125.1 (2C), 119.9 (2C), 77.5, 67.3, 61.7, 50.3, 50.1, 47.3, 36.0, 14.1; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₉H₃₁N₂O₆, 503.2177; found, 503.2161 (−3.2 ppm).

Fmoc-Gly-N(OBn)-Ethyl-Glycinate (7b).

was prepared according to the general procedure A, using ethyl N-(benzyloxy)glycinate 6a (70 mg, 0.33 mmol, 1.0 equiv) and Fmoc-Gly-OH (398 mg, 1.34 mmol, 4.0 equiv). The crude product was purified by chromatography (hexanes/EtOAc 70:30 v/v) to afford 7b in a pure form as a white foam (113 mg, 0.23 mmol, 70% yield). R_f = 0.2 (hexanes/EtOAc 70:30 v/v); mp 99–101 °C; IR ν_max: 3376, 2953, 1745, 1691, 1532, 1400, 1277, 1226, 992, 741, 730, 694 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 7.77 (d, J = 7.5 Hz, 2H), 7.61 (d, J =7.4 Hz, 2H), 7.40 (m, 7H), 7.32 (t, J = 7.3 Hz, 2H), 5.50 (br, 1H),4.91 (s, 2H), 4.39 (d, J = 7.2 Hz, 2H), 4.28 (s, 2H), 4.22 (m, 5H),1.27 (t, J = 7.1 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CD₃OD, δ): 173.1, 167.9, 157.7, 143.9 (2C), 141.2 (2C), 134.5, 129.4 (2C),128.8, 128.4 (2C), 127.4 (2C), 126.8 (2C), 124.9 (2C), 119.5 (2C), 76.9, 66.8, 61.3, 49.1, 47.1, 41.9, 13.0; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₂₉N₂O₆, 489.2020; found, 489.2013 (−1.4 ppm).

Fmoc-Ile-N(OBn)-Ethyl-Glycinate (7c).

was prepared according to the general procedure A, using ethyl N-(benzyloxy)glycinate 6a (50 mg, 0.24 mmol, 1.0 equiv) and Fmoc-Ile-OH (298 mg, 0.96 mmol, 4.0 equiv). The crude product was purified by chromatography (hexanes/EtOAc 80:20 v/v) to afford 7c in a pure form as a white foam (94 mg, 0.17 mmol, 72% yield). R_f = 0.4 (hexanes/EtOAc 70:30 v/v); mp 40–44 °C; IR ν_max: 3057, 2964, 1716, 1660, 1506, 1449, 1264, 1201, 1021, 732, 700 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 7.47 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 6.5 Hz, 2H), 7.41 (m, 7H), 7.32 (t, J = 7.4 Hz, 2H), 5.42 (d, J = 9.8 Hz, 1H),5.05 (dd, J = 32.3, 10.1 Hz, 2H), 4.86 (dd, J = 9.7, 6.0 Hz, 1H), 4.64 (d, J = 17.4 Hz, 1H), 4.40 (m, 2H), 4.25 (t, J = 7.2 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.99 (d, J = 17.5 Hz, 1H), 1.95 (m, 1H), 1.60 (m, 1H),1.26 (t, J = 7.1 Hz, 3H), 1.16 (m, 1H), 1.01 (d, J = 6.8 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 174.0, 167.3, 156.5, 144.0, 143.9, 141.3 (2C), 133.9, 129.6 (2C),129.2, 128.8 (2C), 127.7 (2C), 127.1 (2C), 125.2 (2C), 120.0 (2C), 77.9, 67.1, 61.6, 55.4, 48.6, 47.2, 37.3, 23.9, 15.7, 14.1, 11.2; HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₂H₃₇N₂O₆, 545.2646; found, 545.2631 (−2.8 ppm).

General Procedure B, for the Coupling of 4 With t-butyl α-Amino Ester Residues.

Carboxylic acid 4 (1.0 mmol, 1.0 equiv) and the H-AA-OtBu (1.2 mmol, 1.2 equiv) were solubilized in CH₂Cl₂ (5.0 mL) under an argon atmosphere and cooled down to 0 °C. EDCi (1.2 mmol, 1.2 equiv) and HOBt (1.2 mmol, 1.2 equiv) were added to the mixture. DIPEA (2.0 mmol, 2.0 equiv) was added to the mixture, which was stirred 15 h at RT. The reaction progress was monitored by TLC. CH₂Cl₂ (5.0 mL) was added and the mixture was washed successively with a citric acid solution (10 mL, 5% w/w), saturated NaHCO₃ solution (10 mL), and saturated brine (10 mL). The organic layer was dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure to afford the crude product 11a,b, which was purified by chromatography.

Ac-N(OBn)-Gly-N(Me)-tert-Butyl-Glycinate (11a).

was prepared according to the general procedure B, using N-acetyl-N-(benzyloxy)glycine 4 (100 mg, 0.45 mmol, 1.0 equiv) and H-Sar-OtBu (98 mg, 0.54 mmol, 1.2 equiv). The crude product was purified by chromatography (ether/EtOAc 1:1 v/v) to afford 11a in a pure form as an uncolored oil (106 mg, 0.30 mmol, 67% yield). R_f = 0.2 (hexanes/EtOAc 1:1 v/v); IR (dry film) ν_max: 2980, 2937, 2865, 1737, 1658, 1454, 1367, 1227, 1154, 1119, 1055, 1032, 1013, 846, 747, 700 cm⁻¹; Product 11a was characterized as a mixture of cis–trans rotamers in a ratio 2:5 by ¹H–¹³C HSQC: ¹H NMR (400 MHz, CDCl₃, δ) trans rotamer: 7.39 (m, 5H), 4.93 (s, 2H), 4.41 (s, 2H),4.04 (s, 2H), 2.99 (s, 3H), 2.16 (s, 3H), 1.45 (s, 9H); cis rotamer:7.39 (m, 5H), 4.93 (s, 2H), 4.33 (s, 2H), 3.91 (s, 2H), 2.98 (s, 3H),2.14 (s, 3H), 1.48 (s, 9H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ) trans rotamer: 175.1, 168.0, 167.2, 134.9, 129.5 (2C),128.9, 128.7 (2C), 82.0, 77.3, 50.3, 50.0, 35.6, 28.1, 20.4; cis rotamer: 175.1, 167.7, 167.4, 134.8, 129.5 (2C),128.9, 128.7 (2C), 82.9, 77.2, 52.1, 49.8, 35.4, 28.0 (3C), 20.4; HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₂₇N₂O₅, 351.1914; found, 351.1911 (−0.9 ppm).

Ac-N(OBn)-Gly-tert-Butyl-Glycinate (11b).

was prepared according to the general procedure B, using N-acetyl-N-(benzyloxy)glycine 4 (500 mg, 2.24 mmol, 1.0 equiv) and H-Gly-OtBu (376 mg, 2.69 mmol, 1.2 equiv). The crude product was purified by chromatography (hexanes/EtOAc 1:1 v/v) to afford 11b in a pure form as a yellowish oil (604 mg, 1.80 mmol, 67% yield). R_f = 0.3 (hexanes/EtOAc 1:1 v/v); IR (dry film) ν_max: 3288, 2980, 2941, 2865, 1736, 1685, 1620, 1545, 1454, 1404, 1364, 1223, 1154, 1055, 1033, 1016, 961, 853, 753, 697, 641 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 7.39 (s, 5H), 6.61 (br, 1H), 4.93 (s, 2H), 4.30 (s, 2H),3.93 (d, J = 5.1 Hz, 2H), 2.14 (s, 3H), 1.46 (s, 9H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 173.8, 168.5, 167.8, 134.1, 129.5 (2C),129.2, 128.8 (2C), 82.4, 77.4, 51.9, 42.0, 28.0 (3C), 20.3; HRMS-ESI (m/z): [M + H]⁺ Calcd for C₁₇H₂₅N₂O₅, 337.1758; found, 337.1754 (−1.2 ppm).

Ac-N(OBn)-Gly-tert-Butyl-Isoleucinate (11c).

N-acetyl-N-(benzyloxy)glycine 4 (700 mg, 3.14 mmol, 1.0 equiv), HATU (1.79 g, 4.71 mmol, 1.5 equiv), and HOAt (641 mg, 4.71 mmol, 1.5 equiv) were solubilized in DMF (8.0 mL) under an argon atmosphere. DIPEA (609 mg, 4.71 mmol, 1.5 equiv) was added to the mixture, which was preactivated 5 min at RT. H-Ile-OtBu (1.40 g, 6.28 mmol, 2.0 equiv) was then added and stirred 15 h at RT. The reaction progress was monitored by TLC. CH₂Cl₂ (10 mL) was added and the mixture was washed successively with a citric acid solution (15 mL, 5% w/w), saturated NaHCO₃ solution (15 mL), and saturated brine (15 mL). The organic layer was dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure to afford the crude product 11c in a pure form as a yellow oily solid (841.5 mg, 2.1 mmol, 68% yield). R_f = 0.4 (hexanes/EtOAc 1:1 v/v); ${[\alpha ]}_{\text{D}}^{18}-21.7$ (c 1.00, MeOH); IR (dry film) ν_max: 3358, 3276, 2962, 2932, 2873, 1735, 1720, 1689, 1650, 1556, 1536, 1448, 1412, 1391, 1365, 1287, 1257, 1151, 1133, 1079, 1028, 1009, 973, 948, 809, 737, 697 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 7.38 (s, 5H), 6.66 (d, J = 8.1 Hz, 1H),4.92 (s, 2H), 4.47 (dd, J = 8.5, 4.5 Hz, 1H), 4.34 (d, J = 16.5 Hz, 1H),4.23 (d, J = 16.5 Hz, 1H), 2.14 (s, 3H), 1.87 (m, 1H), 1.58 (m, 1H),1.45 (s, 9H), 1.15 (m, 1H), 0.92 (t, J = 7.4 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 173.8, 170.5, 167.4, 134.1, 129.4 (2C),129.1, 128.8 (2C), 82.1, 77.2, 56.9, 52.0, 38.1, 28.1 (3C), 25.3, 20.3, 15.3, 11.7; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₂₁H₃₂N₂O₅Na, 415.2203; found, 415.2204 (+0.2 ppm).

Fmoc-N(OBn)-Gly-tert-Butyl-Isoleucinate (11d).

N-Fmoc-N-(benzyloxy)glycine 5 (500 mg, 1.24 mmol, 1.0 equiv) and EEDQ (460 mg, 1.86 mmol, 1.5 equiv) were solubilized in CH₂Cl₂ (3.0 mL) under an argon atmosphere. The mixture was preactivated 5 min at RT. H-Ile-OtBu (832 mg, 3.72 mmol, 3.0 equiv) was then added and stirred 5 h at RT. The reaction progress was monitored by TLC. CH₂Cl₂ (3.0 mL) was added and the mixture was washed successively with a citric acid solution (10 mL, 5% w/w), saturated NaHCO₃ solution (10 mL), and saturated brine (10 mL). The organic layer was dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure to afford the crude product 11d in a pure form as a yellow oily solid (618 mg, 1.08 mmol, 87% yield). R_f = 0.3 (hexanes/EtOAc 4:1 v/v); ${[\alpha ]}_{\text{D}}^{18}-10.1$ (c 1.00, MeOH); IR (dry film) ν_max: 3343, 3067, 2965, 2936, 2880, 1725, 1685, 1523, 1451, 1414, 1392, 1368, 1340, 1249, 1216, 1143, 1092, 984, 912, 846, 757, 738, 698, 621 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 7.75 (d, J = 7.5 Hz, 1H),7.62 (m, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.32 (m, 7H), 6.55 (d, J = 8.4 Hz, 1H), 4.85 (s, 2H), 4.59 (dd, J = 6.6, 2.8 Hz, 2H), 4.48 (dd, J =8.5, 4.5 Hz, 1H), 4.28 (t, J = 6.6 Hz, 1H), 4.14 (d, J = 17.2 Hz, 1H),4.03 (d, J = 17.2 Hz, 1H), 1.84 (m, 1H), 1.42 (s, 9H), 1.40 (m, 1H),1.11 (m, 1H), 0.86 (m, 6H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 170.5, 167.4, 157.5, 143.5 (2C), 141.4 (2C) 134.9, 129.4 (2C),128.7, 128.5 (2C), 127.8 (2C), 127.2 (2C), 125.1 (2C), 120.0 (2C), 82.1, 77.2, 68.3, 56.8, 54.3, 47.1, 38.2, 28.0 (3C), 25.3, 15.3, 11.7; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₃₄H₄₀N₂O₆Na, 595.2779; found, 595.2763 (−2.7 ppm).

General Procedure C, for the Coupling of 6a or 6b With 11a–c.

t-Butyl protected dipeptide 11a–c (1.0 mmol, 1.0 equiv) was solubilized in dichloromethane (5.0 mL) and cooled down to 0 °C. Trifluoroacetic acid (5.0 mL) was added dropwise, the mixture was then stirred at 0 °C for 3–5 h. The reaction progress was monitored by TLC. The solvent and residual byproducts were evaporated under reduced pressure to afford the desired unprotected acid dimer. The resulting product and the ethyl (benzyloxy)glycinate 6a or ethyl hydroxyglycinate 6b (2.0 mmol, 2.0 equiv) were solubilized in CH₂Cl₂ or DMF (5.0 mL). Then, HOAt (2.0 mmol, 2.0 equiv) and HATU (2.0 mmol, 2.0 equiv) were added, and the solution was stirred 10 min at RT. DIPEA (2.0 mmol, 2.0 equiv) was added dropwise to the mixture, which was stirred 15 h at RT. The reaction progress was monitored by TLC. CH₂Cl₂ (5.0 mL) was added and the mixture was washed successively with a citric acid solution (10 mL, 5% w/w), saturated NaHCO₃ solution (10 mL), and saturated brine (10 mL). The organic layer was dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure to afford crude products 9a–d, which were further purified by chromatography.

Ac-N(OBn)-Gly-N(Me)-Gly-N(OBn)-Ethyl-Glycinate (9a).

was prepared according to the general procedure C, using N-(benzyloxy)glycinate 6a (740 mg, 3.54 mmol, 2.0 equiv) and peptide 11a (520 mg, 1.77 mmol, 1.0 equiv). The crude product was purified by chromatography (hexanes/EtOAc 7:3 v/v) to afford 9a as a white foam (277 mg, 0.57 mmol, 32% yield). R_f = 0.3 (hexanes/EtOAc 3:7 v/v); mp 47–49 °C; IR ν_max: 2931, 1743, 1658, 1454, 1373, 1204, 1021, 973, 844, 733, 700 cm⁻¹; Product 9a was characterized as a mixture of cis–trans rotamers in a ratio 1:2 by ¹H–¹³C HSQC: ¹H NMR (400 MHz, CDCl₃, δ) trans rotamer: 7.38 (m, 5H), 4.92 (s, 2H), 4.91 (s, 2H), 4.42 (s, 2H), 4.38 (s, 2H), 4.23 (s, 2H), 4.18 (m, 2H), 2.97 (s, 3H), 2.16 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H); cis rotamer:7.38 (m, 5H), 4.88 (s, 2H), 4.84 (s, 2H), 4.33 (s, 2H), 4.18 (m, 4H),3.98 (br, 2H), 2.80 (s, 3H), 2.10 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ) trans rotamer: 175.0, 172.3, 167.7, 167.5, 134.9, 134.2, 129.6 (2C), 129.5 (2C), 129.2, 129.0, 128.8 (2C), 128.7 (2C) 77.6 (2C), 61.6, 50.1 (2C), 49.6, 35.9, 20.4, 14.1; cis rotamer: 175.0, 173.3, 167.8, 167.4, 134.9, 134.1, 130.1 (2C), 129.6 (2C), 128.9, 128.8 (2C), 128.7, 128.5 (2C), 82.9, 77.0, 76.9, 61.9, 51.0, 50.1, 49.2, 35.4, 20.3, 14.1; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₃₂N₃O₇, 486.2235; found 486.2214 (−4.3 ppm).

Ac-N(OBn)-Gly-Gly-N(OBn)-Ethyl-Glycinate (9b).

was prepared according to the general procedure C, using N-(benzyloxy)glycinate 6a (242 mg, 1.16 mmol, 2.0 equiv) and peptide 11b (171 mg, 0.58 mmol, 1.0 equiv). The crude product was purified by precipitation into petroleum ether and filtration to afford 9b as a beige solid (127 mg, 0.27 mmol, 46% yield). R_f = 0.3 (hexanes/EtOAc 3:7 v/v); mp 91–96 °C; IR ν_max: 3239, 2979, 2953, 1745, 1695, 1674, 1644 1544, 1455, 1403, 1372, 1240, 1207, 1054, 1033, 1007, 972, 737 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 7.38 (s, 10H), 6.76 (br, 1H),4.91 (d, J = 10.7 Hz, 2H), 4.29 (s, 2H), 4.26 (d, J = 4.6 Hz, 2H), 4.24 (s, 2H), 4.18 (q, J = 7.2 Hz, 2H), 2.15 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 174.1, 172.5, 167.8, 167.3, 134.2, 133.9, 129.5 (4C),129.3, 129.1, 128.9 (2C), 128.8 (2C),77.8, 77.5, 61.8, 51.8, 50.0, 41.3, 20.4, 14.1; HRMS-ESI (m/z): [M +H]⁺ Calcd for C₂₄H₃₀N₃O₇, 472.2078; found, 472.2061 (−3.6 ppm).

Ac-N(OBn)-Gly-Ile-N(OBn)-Ethyl-Glycinate (9c).

was prepared according to the general procedure C, using N-(benzyloxy)glycinate 6a (363 mg, 1.74 mmol, 2.0 equiv) and peptide 11c (292 mg, 0.87 mmol, 1.0 equiv). The crude product was purified by precipitation into petroleum ether and filtration to afford 9c as an orange powder in a mixture of diastereoisomer (189 mg, 0.44 mmol, 41% yield, d.r. 3:2). R_f = 0.7 (hexanes/EtOAc 3:7 v/v); mp 117–120 °C; IR ν_max: 3279, 2964, 2933, 1751, 1637, 1544, 1454, 1390, 1375, 1212, 1019, 972, 840, 750, 699 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ) (S, S) isomer: 7.44 (m, 10H), 6.88 (d, J = 9.3 Hz, 1H), 5.10 (dd, J =9.3, 6.7 Hz, 1H), 5.00 (m, 2H), 4.93 (s, 2H), 4.58 (d, J = 8.3 Hz, 1H),4.33 (d, J = 3.5 Hz, 2H), 4.17 (m, 3H), 2.12 (s, 3H), 1.90 (m, 1H),1.55 (m, 1H), 1.24 (t, J = 7.1 Hz, 3H), 1.13 (m, 1H), 0.94 (d, J = 6.9 Hz, 3H), 0.86 (m, 3H); (R, S) isomer: 7.44 (m, 10H), 6.83 (d, J = 8.9 Hz, 1H), 5.19 (dd, J = 9.3, 4.3 Hz, 1H), 5.01 (s, 2H), 5.00 (m, 2H),4.63 (d, J = 8.4 Hz, 1H), 4.33 (d, J = 3.5 Hz, 2H), 4.17 (m, 3H), 2.12 (s, 3H), 1.97 (m, 1H), 1.39 (m, 1H), 1.24 (t, J = 7.1 Hz, 3H), 1.20 (m, 1H), 0.87 (m, 3H), 0.84 (m, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ) (S, S) isomer: 173.7, 172.9, 167.7, 167.4, 135.0, 134.4, 129.7 (2C),129.6 (2C), 128.8, 128.7 (2C), 128.6 (2C), 77.2, 76.5, 61.3, 53.0, 50.2, 48.1, 36.9, 23.9, 19.8, 15.0, 13.5, 10.4; (R, S) isomer: 173.7, 173.2, 167.7, 167.6, 135.0, 134.3, 129.7 (2C),129.6 (2C), 129.0, 128.7 (2C), 128.6 (2C), 77.1, 76.5, 61.3, 51.9, 50.2, 48.2, 36.3, 26.4, 19.8, 15.0, 13.5, 11.1; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₃₈N₃O₇, 528.2704; found, 528.2681 (−4.4 ppm).

Ac-N(OBn)-Gly-Ile-N(OH)-Ethyl-Glycinate (9c′).

was prepared according to the general procedure C, using N-hydroxyglycinate 6b (283 mg, 2.38 mmol, 2.0 equiv) and peptide 11c (400 mg, 1.19 mmol, 1.0 equiv). The crude product was purified by precipitation into petroleum ether and filtration to afford 9c′ as a light brown solid (231 mg, 0.53 mmol, 53% yield). R_f = 0.4 (hexanes/EtOAc 3:7 v/v); ${[\alpha ]}_{\text{D}}^{18}-14.1$ (c 1.00, MeOH); mp 118–120 °C; IR ν_max: 3280, 2968, 2934, 1753, 1655, 1637, 1541, 1372, 1245, 1201, 1018, 737, 696, 621 cm⁻¹; ¹H NMR (400 MHz, CD₃CN, δ): 8.38 (s, 1H), 7.43 (m, 5H), 6.88 (d, J = 8.9 Hz, 1H), 4.97 (dd, J = 9.1, 6.5 Hz, 1H), 4.92 (s, 2H), 4.44 (d, J = 17.5 Hz, 1H), 4.29 (d, J = 2.1 Hz, 2H),4.25 (d, J = 17.5 Hz, 1H), 4.17 (q, J = 7.0 Hz, 2H), 2.11 (s, 3H), 1.97 (m, 1H), 1.52 (m, 1H), 1.24 (t, J = 7.1 Hz, 3H), 1.11 (m, 1H), 0.94 (d, J = 6.9 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CD₃CN, δ): 173.8, 171.9, 167.9, 167.7, 134.9, 129.6 (2C),128.8, 128.6 (2C), 76.5, 61.2, 53.3, 50.1 (2C), 36.5, 23.8, 19.7, 15.1, 13.5, 10.6; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₁H₃₂N₃O₇, 438.2235; found, 438.2214 (−4.8 ppm).

Fmoc-N(OBn)-Gly-Ile-N(OBn)-Ethyl-Glycinate (9d).

Peptide 11d (190 mg, 0.37 mmol, 1.0 equiv), lutidine (0.03 mL, 0.44 mmol, 1.2 equiv), and 6a (308 mg, 1.47 mmol, 4.0 equiv) were solubilized in CH₂Cl₂ (1.0 mL) under an argon atmosphere. ClCOtBu (0.05 mL, 0.44 mmol, 1.2 equiv) was then added and stirred 15 h at RT. The reaction progress was monitored by TLC. CH₂Cl₂ (3.0 mL) was added and the mixture was washed successively with a citric acid solution (3 × 5.0 mL, 5% w/w), saturated NaHCO₃ solution (3 × 5.0 mL), and saturated brine (5.0 mL). The organic layer was dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure to afford the crude product 9d in a pure form as a yellowish oily gum (135 mg, 0.19 mmol, 51% yield). R_f = 0.65 (hexanes/EtOAc 6:4 v/v); ${[\alpha ]}_{\text{D}}^{18}-7.8$ (c 1.00, MeOH); IR ν(dry film) ν_max: 3320, 2961, 2934, 2876, 1743, 1712, 1655, 1526, 1451, 1419, 1389, 1344, 1200, 1094, 1022, 979, 911, 854, 739, 698 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ) 7.75 (d, J = 7.5 Hz, 2H), 7.64 (dd, J =7.3, 2.4 Hz, 2H), 7.37 (m, 14H), 6.59 (d, J = 9.4 Hz, 1H), 5.15 (dd, J = 9.4, 5.9 Hz, 1H), 5.03 (d, J = 10.1 Hz, 1H), 4.93 (d, J = 10.1 Hz, 1H), 4.86 (dd, J = 13.6, 10.1 Hz, 2H), 4.59 (m, 3H), 4.29 (m, 1H),4.16 (m, 3H), 4.06 (d, J = 17.1 Hz, 1H), 3.90 (d, J = 17.5 Hz, 1H),1.92 (m, 1H), 1.52 (m, 1H), 1.23 (t, J = 7.1 Hz, 3H), 1.03 (m, 1H), 0.95 (d, J = 6.8 Hz, 3H), 0.82 (t, J = 7.4 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ) 173.3, 167.8, 167.3, 157.7, 143.6 (2C), 141.4 (2C), 135.0, 133.9, 129.6 (2C), 129.4 (2C), 129.1, 128.8, 128.7, 128.5 (2C), 127.8 (2C), 127.2 (2C), 125.2 (2C), 120.0 (2C), 77.9, 77.5, 68.3, 61.6, 54.3, 53.3, 48.5, 47.1, 37.2, 23.9, 15.7, 14.1, 11.1; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₃₁H₄₅N₃O₈Na, 730.3099; found, 730.3087 (−1.6 ppm).

General Procedure D, for the Catalytic Hydrogenation of N-(benzyloxy)-Protecting Groups.

Peptides 9a–d (1.0 mmol, 1.0 equiv) were solubilized in EtOH (10 mL) and Pd/C (0.10 mmol,0.10 equiv, 10% w/w) was added to the solution. For 9d, the mixture was cooled down to 0 °C. The flask was placed under an atmosphere of a balloon of H_2,gas and the mixture was stirred for 1–18 h (reaction progress monitored by TLC). The reaction mixture was filtered through a pad of cleaned Celite with silica (1% w/w), and the solvent was evaporated to afford the crude product 12a–d, which were further purified by chromatography.

Ac-N(OH)-Gly-N(Me)-Gly-N(OH)-Ethyl-Glycinate (12a).

was prepared according to the general procedure D, using peptide 9a (63 mg, 0.13 mmol, 1.0 equiv). The crude product was purified by chromatography (gradient of hexanes/EtOAc 1:1 to EtOAc 100% and then EtOAc/MeOH 19:1) to afford 12a in a pure form as an uncolored oil (32 mg, 0.11 mmol, 81% yield). R_f = 0.10 (hexanes/EtOAc/AcOH 5:4:1); mp 118–121 °C; IR ν_max: 3433, 3193, 2924, 2852, 1741, 1636, 1460, 1401, 1208, 1124, 1023, 822, 562 cm⁻¹; product 12a was characterized as a mixture of cis–trans rotamers in a ratio 2:3 by ¹H–¹³C HSQC: ¹H NMR (400 MHz, DMSO-d₆, δ) trans rotamer: 10.18 (s, 1H), 9.76 (s, 1H), 4.42 (s, 2H), 4.29 (s, 2H), 4.28 (s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 2.93 (s, 3H), 2.02 (s, 3H), 1.20 (t,, J = 7.1 Hz, 3H); cis rotamer: 10.23 (s, 1H), 9.72 (s, 1H), 4.34 (s, 2H),4.33 (s, 2H), 4.24 (s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 2.81 (s, 3H), 2.02 (s, 3H), 1.20 (t,, J = 7.1 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ) trans rotamer: 172.8, 169.9, 169.6, 168.5, 61.9, 50.1, 49.5, 49.0, 36.4, 20.0, 14.1; cis rotamer: 172.8, 170.4, 169.4, 168.2, 61.9, 50.6, 50.5, 49.1, 35.4, 19.9, 14.1; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₁H₁₉N₃O₇Na, 328.1115; found, 328.1101 (−4.2 ppm).

Ac-N(OH)-Gly-Gly-N(OH)-Ethyl-Glycinate (12b).

was prepared according to the general procedure D, using peptide 9b (170 mg, 0.36 mmol, 1.0 equiv). The crude product was purified by chromatography (gradient of hexanes/EtOAc 1:1 to EtOAc 100% and then EtOAc/MeOH 19:1) to afford 12b in a pure form as a white solid (82 mg, 0.28 mmol, 79% yield). R_f = 0.05 (hexanes/EtOAc/AcOH 5:4:1); mp 140–142 °C; IR ν_max: 3299, 3097, 2911, 2778, 2680, 1743, 1678, 1646, 1593, 1564, 1464, 1411, 1388, 1376, 1257, 1218, 1189, 1027, 823, 786, 729, 615, 541 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, δ): 10.21 (s, 1H), 9.91 (s, 1H), 8.00 (br, 1H), 4.30 (s, 2H), 4.17 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 4.08 (d, J = 5.6 Hz, 2H),2.02 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H); ¹³C{¹H} NMR (100 MHz, DMSO-d₆, δ): 171.9, 170.9, 168.3, 168.0, 61.3, 51.4, 51.1, 40.4, 20.6, 14.5; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₀H₁₇N₃O₇Na, 314.0959; found, 314.0958 (+0.3 ppm).

Ac-N(OH)-Gly-Ile-N(OH)-Ethyl-Glycinate (12c).

was prepared according to the general procedure D, using peptide 9c′ (300 mg, 0.69 mmol, 1.0 equiv). The crude product was purified by chromatography (gradient of hexanes/EtOAc 1:1 to EtOAc 100% and then EtOAc/MeOH 19:1) to afford 12c in a pure form as a yellow solid (239 mg, 0.69 mmol, 99% yield). R_f = 0.54 (hexanes/EtOAc/AcOH 5:4:1); ${[\alpha ]}_{\text{D}}^{18}-14.2$ (c 1.00, MeOH); mp 97–99 °C; IR ν_max: 3276, 3210, 2965, 2–934, 1743, 1629, 1534, 1456, 1375, 1201, 1022, 634, 557 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, δ): 10.24 (s, 1H),9.89 (s, 1H), 7.82 (d, 1H), 5.02 (dd, J = 9.1, 6.0 Hz, 1H), 4.52 (d, J =17.3 Hz, 1H), 4.18 (s, 2H), 4.12 (q, J = 7.0 Hz, 2H), 4.08 (d, J = 17.4 Hz, 1H), 2.01 (s, 3H), 1.87 (m, 1H), 1.44 (m, 1H), 1.19 (t, J = 7.1 Hz, 3H), 1.05 (m, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (t, J = 7.3 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 173.5, 172.2, 169.8, 168.0, 61.8, 53.6, 51.4, 50.0, 36.0, 24.4, 20.1, 15.4, 14.1, 11.0; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₃₁N₂O₆, 348.1765; found, 348.1766 (+0.3 ppm).

Fmoc-N(OH)-Gly-Ile-N(OH)-Ethyl-Glycinate (12d).

was prepared according to the general procedure D, using peptide 9d (15 mg, 0.02 mmol, 1.0 equiv). The crude product was purified by chromatography (gradient of hexanes/EtOAc 4:1 to 2:6) to afford 12d in a pure form as a white powder (7 mg, 0.01 mmol, 62% yield). R_f = 0.38 (hexanes/EtOAc 2:3 v/v); ${[\alpha ]}_{\text{D}}^{18}-2.6$ (c 0.50, MeOH); mp 164–169 °C; IR ν_max: 3284, 2961, 2923, 2873, 1745, 1711, 1685, 1610, 1541, 1449, 1401, 1376, 1352, 1213, 1111, 1020, 974, 942, 738, 621 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, δ) 10.26 (s, 1H), 9.69 (s, 1H), 7.89 (d, J = 7.8 Hz, 3H), 7.70 (d, J = 7.5 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 5.06 (dd, J = 9.3, 5.9 Hz, 1H), 4.51 (d, J = 17.3 Hz, 1H), 4.26 (m, 3H), 4.13 (m, 5H), 1.88 (m, 1H), 1.45 (m, 1H), 1.18 (t, J = 7.1 Hz, 3H), 1.06 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.82 (t, J = 7.2 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ) 172.0, 169.9, 168.1, 158.1, 143.5 (2C), 141.3 (2C), 127.8 (2C),127.2 (2C), 125.2 (2C), 120.0 (2C), 68.9, 61.8, 54.4, 53.4, 49.9, 46.9, 35.8, 24.6, 15.4, 14.0, 10.9; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₂₇H₃₃N₃O₈Na, 550.2160; found, 550.2163 (+0.5 ppm).

N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N-benzylglycine (SI-1).

Benzylamine (200 mg, 1.87 mmol, 1.0 equiv) and glyoxylic acid monohydrate (344 mg, 3.73 mmol, 2.0 equiv) were solubilized in CH₂Cl₂ (9.0 mL) and stirred overnight at RT. The solvent was then evaporated and the N-benzyl-N-formylglycine intermediate was characterized by ¹H NMR (400 MHz, DMSO-d₆, δ): 8.31 (s, 1H),7.35 (m, 5H), 4.50 (s, 2H), and 3.83 (s, 2H). The crude N-benzyl-N-formylglycine was solubilized in a 1.0 M aqueous solution of HCl (9.0 mL) and stirred at reflux overnight. The water was then evaporated under reduced pressure to afford the desired N-benzylglycine hydrochloride (203 mg, 1.0 mmol, 54% yield) as a brown solid, which was used without any purification for the second step. R_f = 0.7 (CH₂Cl₂/MeOH/AcOH 16:3:1 v/v); N-benzylglycine hydrochloride (200 mg, 0.99 mmol, 1.0 equiv) was solubilized in dioxane (6.0 mL) and the solution was cooled down to 0 °C. A solution of sodium bicarbonate (200 mg, 2.44 mmol, 2.4 equiv) in water (2.0 mL) was then added and the mixture was stirred for 10 min at 0 °C. A solution of (9H-fluoren-9-yl)methyl chloroformate (471 mg, 1.82 mmol, 1.8 equiv) in dioxane (2.0 mL) was added to the mixture, which was slowly warm to RT and stirred overnight. Water was added and the aqueous layer (pH ~ 8) was washed with ethyl acetate (2 × 15 mL). The aqueous layer was acidified with a 5.0 M aqueous solution of HCl to pH ~ 3. The product was extracted with ethyl acetate (3 × 15 mL), the combined organic layers were dried over Na₂SO₄, filtrated, and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography (isocratic solvent mixture of hexanes/EtOAc, 4:1 v/v) to afford product SI-1 in a pure form as a white powder (107 mg, 0.28 mmol, 28% yield over 3 steps). R_f = 0.7 (100% EtOAc); mp 110.0–113.0 (±0.6) °C; IR ν_max: 3068, 2923, 2854, 1685, 1451, 1231, 1121, 1002, 956, 758, 738 cm⁻¹; Product SI-1 was characterized as a mixture of cis–trans rotamers in a 1:1 ratio by ¹H–¹³C HSQC: ¹H NMR (400 MHz, CD₃OD, δ) trans rotamer: 7.77 (d, J = 7.5 Hz, 2H), 7.50 (d, J = 7.5 Hz, 2H), 7.34 (t, J = 5.9 Hz, 2H), 7.23 (m, 5H), 6.96 (m, 2H), 4.53 (d, J = 5.8 Hz, 2H),4.32 (s, 2H), 4.22 (t, J = 5.8 Hz, 1H), 3.85 (s, 2H); cis rotamer: 7.73 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.37 (t, J = 5.8 Hz, 2H),7.29 (m, 5H), 7.16 (m, 2H), 4.49 (s, 2H), 4.47 (d, J = 6.3 Hz, 2H),4.22 (t, J = 5.8 Hz, 1H), 3.73 (s, 2H).

Ethyl Benzylglycinate (SI-2).

Benzylamine (1.00 g, 9.33 mmol, 1.0 equiv) and a solution of ethyl glyoxylate (1.05 g, 10.3 mmol, 1.1 equiv) in toluene (50% w/w) were solubilized in dry ethanol (19.0 mL) and stirred for 1 h at RT. A solution of sodium cyanoborohydride (1.18 g, 18.7 mmol, 2.0 equiv) in dry ethanol (3.0 mL) and acetic acid (0.05 mL, 0.93 mmol, 0.1 equiv) were added to the mixture, which was stirred overnight at RT. The reaction completion was monitored by TLC (R_f = 0.35, EtOAc/hexanes 1:1 v/v). The solvent was then evaporated and the crude was solubilized in dichloromethane (10 mL). The organic layer was washed with water (3 × 10 mL), dried over Na₂SO₄, and filtrated, and the solvent was evaporated under reduced pressure. The crude product was purified by chromatography (isocratic solvent mixture of hexanes/EtOAc, 7:3 v/v) to afford SI-2 in a pure form as a yellow oil (542 mg, 2.81 mmol, 30% yield). R_f = 0.3 (EtOAc/hexanes 1:1 v/v); IR ν_max: 3029, 2980, 2866, 1724, 1453, 1371, 1188, 1142, 1025, 736, 698 cm⁻¹.

Fmoc-Ile-N(OH)-Ethyl-Glycinate (SI-3).

was prepared according to the general procedure D, using peptide 7c (450 mg, 0.83 mmol, 1.0 equiv). The crude product was purified by chromatography (hexanes/EtOAc 7:3 v/v) to afford SI-3 in a pure form as a white solid (339 mg, 0.75 mmol, 90% yield). R_f = 0.3 (hexanes/EtOAc 7:3 v/v); ${[\alpha ]}_{\text{D}}^{18}-14.8$ (c 1.00, MeOH); mp 147–149 °C; IR ν_max: 3325, 3144, 2968, 1741, 1692, 1605, 1530, 1449, 1211, 1023, 758, 737, 646, 621 cm⁻¹; ¹H NMR (400 MHz, CDCl₃, δ): 8.35 (br, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.56 (dd, J = 7.3, 2.9 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 5.53 (d, J = 9.1 Hz, 1H),4.82 (t, J = 8.4 Hz, 1H), 4.45 (s, 2H), 4.37 (m, 2H), 4.20 (m, 3H),1.94 (m, 1H), 1.61 (m, 1H), 1.25 (t, J = 7.1 Hz, 3H), 1.20 (m, 1H),1.00 (d, J = 6.7 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 172.5, 168.4, 157.2, 143.8, 143.7, 141.3 (2C), 127.7 (2C), 127.1 (2C), 125.1 (2C), 120.0 (2C), 67.5, 61.8, 55.4, 49.4, 47.1, 36.4, 24.4, 15.6, 14.1, 11.1; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₃₁N₂O₆, 455.2177; found, 455.2157 (−4.4 ppm).

Ac-N(OH)-Gly-tert-Butyl-Isoleucinate (SI-4).

was prepared according to the general procedure D, using peptide 11c (450 g, 1.15 mmol, 1.0 equiv). The crude product was purified by chromatography (hexanes/EtOAc 6:4 v/v) to afford SI-4 in a pure form as an uncolored oil (344 g, 1.14 mmol, 99% yield). R_f = 0.2 (hexanes/EtOAc 6:4 v/v); ${[\alpha ]}_{\text{D}}^{18}-15.1$ (c 1.00, MeOH); IR (dry film) ν_max: 3268, 2967, 2934, 1732, 1659, 1637, 1536, 1458, 1393, 1367, 1248, 1142, 1038, 989, 846, 776, 582 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, δ): 9.87 (s, 1H), 6.02 (d, J = 8.4 Hz, 1H), 4.17 (m, 3H),2.01 (s, 3H), 1.74 (m, 1H), 1.41 (s, 3H), 1.37 (m, 1H), 1.18 (m, 1H),0.86 (m, 6H); ¹³C{¹H} NMR (100 MHz, DMSO-d₆, δ): 171.9, 170.9, 167.6, 81.2, 57.1, 51.2, 37.3, 28.1 (3C), 25.3, 20.6, 15.9, 11.8; HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₄H₂₆N₂O₅Na, 325.1734; found, 325.1732 (−0.6 ppm).

Ga(Fmoc-Ile-N(O)-Ethyl-Glycinate)₃ (SI-5).

Gallium sulfate (28 mg, 0.07 mmol, 3.5 equiv) was solubilized in DMF (0.30 mL). A solution of NH₃ in 1,4-dioxane (0.5 M, 0.20 mL) was added to the mixture. A solution of dipeptide SI-3 (10 mg, 0.02 mmol, 1.0 equiv) in DMF (0.30 mL) was added dropwise to the reaction mixture, which was stirred overnight at RT. The resulting crude product was precipitated into cold water, filtrated, and washed with water (3 × 1.0 mL). The solid was then solubilized in CH₂Cl₂, dried over Na₂SO₄, and filtrated, and the solvent was removed under reduced pressure to afford product SI-5 as a white powder (29 mg,0.02 mmol, 100% yield) mp 148–151 °C; IR ν_max: 3298, 2964, 2928, 1753, 1709, 1589, 1509, 1450, 1219, 1197, 1024, 758, 736 cm⁻¹; The broadness of the peaks in the ¹H NMR of product SI-5 suggested that several conformers or several ligand arrangements around the Ga center might co-exist; therefore, the following chemical shifts are representative of the major cis-conformation of the ligand SI-5: ¹H NMR (400 MHz, CD₃CN, δ): 7.82 (m, 2H), 7.64 (m, 2H), 7.41 (m, 2H), 7.33 (m, 2H), 6.02 (d, J = 8.1 Hz, 1H), 4.87 (d, J = 17.9 Hz, 1H), 4.21 (m, 7H), 1.79 (m, 1H), 1.52 (m, 1H), 1.21 (m, 3H), 1.08 (m, 1H), 0.88 (m, 6H); ¹³C{¹H} NMR (100 MHz, CDCl₃, δ): 170.3, 164.7, 156.3, 143.7, 143.6, 141.3 (2C), 127.8 (2C), 127.1 (2C), 125.1 (2C), 120.0 (2C), 67.2, 61.8, 54.0, 53.1, 47.2, 36.9, 24.7, 15.4, 13.9, 10.9; MALDI-TOF (m/z): [M + Na]⁺ calcd for C₇₅H₈₇N₆O₁₈GaNa, 1451.5225; found, 1453. 2538.