Nanometer-Scale Water-Soluble Macrocycles from Nanometer-Sized Amino Acids

Abstract

This paper introduces the unnatural amino acids m-Abc^2K and o-Abc^2K as nanometersized building blocks for the creation of water-soluble macrocycles with well-defined shapes. m-Abc^2K and o-Abc^2K are homologues of the nanometer-sized amino acid Abc^2K, which we recently introduced for the synthesis of water-soluble molecular rods of precise length. [J. Am. Chem. Soc. 2007, 129, 7272]. Abc^2K is linear (180°), m-Abc^2K creates a 120° angle, and o-Abc^2K creates a 60° angle. m-Abc^2K and o-Abc^2K are derivatives of 3’-amino-[1,1’-biphenyl]-4-carboxylic acid and 2’-amino-[1,1’-biphenyl]-4-carboxylic acid, with two propyloxyammonium side chains for water solubility. m-Abc^2K and o-Abc^2K are prepared as Fmoc-protected derivatives Fmoc-m-Abc^2K(Boc)-OH (1a) and Fmoc-o-Abc^2K(Boc)-OH (1b). These derivatives can be used alone or in conjunction with Fmoc-Abc^2K(Boc)-OH (1c) as ordinary amino acids in Fmoc-based solid-phase peptide synthesis. Building blocks 1a–c were used to synthesize macrocyclic “triangles” 9a–c, “parallelograms” 10a,b, and hexagonal “rings” 11a–d. The macrocycles range from a trimer to a dodecamer, with ring sizes from 24 to 114 atoms, and are 1–4 nm in size. Molecular modeling studies suggest that all the macrocycles except 10b should have well-defined triangle, parallelogram, and ring shapes if all of the amide linkages are trans and the ortho-alkoxy substituents are intramolecularly hydrogen bonded to the amide NH groups. The macrocycles have good water solubility and are readily characterized by standard analytical techniques, such as RP-HPLC, ESI-MS, and NMR spectroscopy. ¹H and ¹³C NMR studies suggest that the macrocycles adopt conformations with all trans-amide linkages in CD₃OD, that the “triangles” and “parallelograms” maintain these conformations in D₂O, and that the “rings” collapse to form conformations with cis-amide linkages in D₂O.

Introduction

This paper introduces the unnatural amino acids m-Abc^2K and o-Abc^2K as nanometer-sized building blocks for the creation of water-soluble macrocycles with well-defined shapes. There is intense interest in nanometer-sized macrocycles with shapes such as hexagons,¹ triangles,² and parallelograms,^1c,3 because “shape-persistent” molecules are central to a bottom-up approach to functional well-defined nanometer-scale molecules and molecular assemblies (i.e., “nanotechnology”).^1–5 Most of these structures have been designed for solubility in organic solvents, and many are assembled from building blocks through carbon-carbon or metal-ligand bond formation. The creation of large (e.g., ≥ 2 nm) water-soluble macrocycles with well-defined shapes remains largely undeveloped. Such structures offer the promise of bridging the gap between the aqueous world of biology and the synthetic world of nanotechnology. If the structures are sufficiently easy to assemble from building blocks, they may even allow researchers from other disciplines to create molecular architectures for their own applications. Here, we describe our approach to large, water-soluble macrocycles.

We designed m-Abc^2K and o-Abc^2K as homologues of the nanometer-sized amino acid Abc^2K, which we recently introduced for the synthesis of water-soluble molecular rods of precise length.⁶ Abc^2K is a derivative of the nanometer-long amino acid 4’-amino-[1,1’-biphenyl]-4-carboxylic acid, with two propyloxyammonium side chains for water solubility. Abc^2K is linear (180°), m-Abc^2K creates a 120° angle, and o-Abc^2K creates a 60° angle.⁷ In this paper, we report the syntheses of Fmoc-protected derivatives Fmoc-m-Abc^2K(Boc)-OH (1a) and Fmoc-o-Abc^2K(Boc)-OH (1b) and the application of these derivatives to the synthesis of nanometer-scale water-soluble macrocycles.

Results and Discussion

The Fmoc-m-Abc^2K(Boc)-OH (1a) and Fmoc-o-Abc^2K(Boc)-OH (1b) building blocks were prepared in a fashion analogous to Fmoc-Abc^2K(Boc)-OH (1c), as shown in Scheme 1.⁶ Central to the synthesis of Abc^2K building blocks is the Suzuki cross-coupling reaction of 4-bromobenzoic acid derivative 6 with 3-aminophenylboronic acid or 2-aminophenylboronic acid. All of the reactions proceed in good yield, and the synthesis permits the preparation of gram-scale quantities of 1a and 1b. One noteworthy improvement to the synthesis involves the use of an unusual Grignard-based halogen-metal exchange reaction to generate 4-bromo-2,5-dimethoxybenzoic acid (3).⁸ This reaction, which was reported recently by Yokozawa and coworkers in a related system, proceeds in double the yield of the conventional Grignard reaction.^6,8

SCHEME 1.

Synthesis of Building Blocks 1a and 1b

To evaluate the utility of building blocks 1a–c for the preparation of macrocycles, we synthesized “triangles” 9a–c, “parallelograms” 10a,b, and hexagonal “rings” 11a–d (Figure 1). The triangles are composed of 3 o-Abc^2K units and 0, 3, or 6 Abc^2K units; the parallelograms are composed of 2 o-Abc^2K units, 2 m-Abc^2K units, and 0 or 4 Abc^2K units; and the rings are composed of 6 m-Abc^2K units and 0, 2, 4, or 6 Abc^2K units. The macrocycles range from a trimer to a dodecamer, with ring sizes from 24 to 114 atoms. The triangles are 1–3 nm on an edge, the parallelograms are 1–2 nm on an edge, and the rings are 2–4 nm across.

FIGURE 1.

Nanometer-scale macrocyclic peptides prepared from m-Abc^2K, o-Abc^2K, and Abc^2K (R = CH₂CH₂CH₂NH₃⁺ CF₃CO₂⁻).

The macrocycles were synthesized by Fmoc-based solid-phase synthesis of protected linear oligomers, followed by macrocyclization, deprotection, and RP-HPLC purification (Scheme 2). The linear oligomers were prepared on chlorotrityl resin with ca. 2 equiv of protected amino acid, HCTU coupling reagent, 2,4,6-collidine base, and 8–12 h coupling times. After final Fmoc-deprotection, the protected linear oligomers were cleaved from the resin and cyclized at tenth-millimolar concentrations with HCTU, 2,4,6-collidine, and 24 h reaction times. Analytical HPLC studies established that the purities of the unpurified cyclized peptides were generally high, with the exception of 11d, which proved difficult to cyclize. The cyclic peptides were generally easy to purify by RP-HPLC. Lyophilization afforded the trifluoroacetate salts as fluffy white solids. A typical synthesis starting with 0.05 mmol of chlorotrityl resin gave an initial resin loading of ca. 0.03 mmol and afforded 0.002–0.008 mmol of analytically pure macrocycle after a single purification.

SCHEME 2.

Synthesis of Cyclohexamer Triangle 9b from Building Blocks Fmoc-o-Abc^2K(Boc)-OH (1b) and Fmoc-Abc^2K(Boc)-OH (1c)

The macrocycles have good water solubility and are readily characterized by standard analytical techniques, such as RP-HPLC, ESI-MS, and NMR spectroscopy in D₂O or CD₃OD solution. Figure 2 shows characterization data for cyclododecamer 11d. The HPLC trace shows a single peak; the mass spectrum shows a series of multiply-charged molecular ions consistent with the molecular weight of the dodecamer, and the ¹H NMR spectrum is sharp and well resolved.

FIGURE 2.

Characterization data for cyclododecamer ring 11d.

¹H and ¹³C NMR studies suggest that the macrocycles adopt conformations with all trans-amide linkages in CD₃OD, that the “triangles” and “parallelograms” maintain these conformations in D₂O, and that the “rings” collapse to form conformations with cis-amide linkages in D₂O. The ¹H and ¹³C NMR spectra of ring 11a in CD₃OD are sharp and well resolved and clearly show sixfold symmetry (Figure 3a,b). The ¹H NMR spectrum shows six aromatic resonances and two sets of aliphatic resonances for each of the three methylene groups, consistent with a single type of m-Abc^2K unit. The ¹³C NMR spectrum shows one carbonyl resonance, twelve aromatic resonances, and six aliphatic resonances, also consistent with one type of m-Abc^2K unit and sixfold symmetry of ring 11a. All of the aromatic ¹H resonances are downfield (7–8 ppm), suggesting that all of the amide linkages are trans and that the ring adopts an open structure without significant interactions between the aromatic rings.

FIGURE 3.

NMR spectra of cyclohexamer ring 11a in CD₃OD and D₂O.

In D₂O, the ¹H and ¹³C NMR spectra of 11a are more complex (Figure 3c,d). Some of the aromatic ¹H resonances are shifted upfield (6–7 ppm), suggesting the presence of cis-amide linkages.⁹ The ¹³C NMR spectrum shows ca. four times as many resonances; while the ¹³C NMR spectrum in CD₃OD clearly shows two sets of propyloxyammonium groups, the ¹³C NMR spectrum in D₂O appears to show eight. Collectively, these data suggest the presence of two conformers in D₂O—a collapsed twofold-symmetrical cttctt-conformer, with two cis-amide linkages, in addition to the open ringlike tttttt-conformer, with all trans-amide linkages. We have previously documented the formation of both open (tttt) and collapsed (ctct) conformers in a related fourfold-symmetrical macrocycle and have prepared and studied control compounds that adopt cis-amide conformers.⁹ These related systems have also exhibited upfield shifting of the ¹H aromatic resonances, reflecting the proximity of the aromatic rings that is bought on by the cis-amide geometry.

Rings 11b–d show similar behavior. The ¹H NMR spectrum of 11d in CD₃OD shows one set of resonances associated with sixfold symmetry, and the aromatic resonances are downfield (7–8 ppm). The ¹H NMR spectrum in D₂O is more complex, and some of the aromatic resonances are shifted upfield (6–7 ppm), suggesting a collapsed conformer with cis-amide linkages. The ¹H NMR spectra of 11b and 11c in CD₃OD show downfield aromatic resonances and appear to be consistent with all trans-amide conformers with twofold symmetry. The spectra of 11b and 11c in D₂O are more complex, and some of the aromatic resonances are shifted upfield, suggesting cis-amide conformers.

In contrast to cyclohexamer ring 11a, cyclohexamer triangle 9b appears to adopt an open conformation with all trans-amide linkages in both D₂O and CD₃OD. The ¹³C NMR spectrum clearly shows threefold symmetry; the ¹H NMR spectrum, although slightly broadened, suggests threefold symmetry and shows downfield aromatic resonances (Figure 4). The cyclononamer triangle 9c also shows threefold symmetry and appears to adopt an open conformation with all trans-amide linkages in D₂O.

FIGURE 4.

NMR spectra of cyclohexamer triangle 9b in D₂O.

The cyclotrimer triangle 9a exhibits behavior atypical of the other macrocycles. It elutes from RP-HPLC as two peaks in a 2:1 ratio that re-equilibrate upon standing. The ¹H and ¹³C NMR spectra of 9a in D₂O and in CD₃OD show two conformers in a 2:1 ratio—a threefold-symmetrical conformer and an unsymmetrical conformer. The symmetrical conformer likely has all trans-amide linkages and must have threefold symmetry. The unsymmetrical conformer could either contain a cis-amide linkage or be an unsymmetrical atropisomer associated with slow rotation about the ortho-disubstituted biphenyl groups of the o-Abc^2K units (shown below). The small size of the macrocycle likely raises the energy barrier to conformational interconversion and slows equilibration of the two conformers.

Parallelogram 10b appears to adopt conformations with all trans-amide linkages in both D₂O and CD₃OD. The ¹³C NMR spectrum in D₂O shows a single twofold-symmetrical conformer. The ¹H NMR spectra show no significant upfield shifting of the aromatic resonances in either D₂O or CD₃OD, suggesting that the twofold-symmetrical conformer has all trans-amide linkages. Parallelogram 10a shows similar behavior at slightly elevated temperatures (e.g., 330 K). Some decoalescence of the ¹H NMR spectra in either D₂O or CD₃OD occurs at lower temperatures (e.g., 298 K), suggesting the occurrence of atropisomers or, less likely, cis-amide linkages.

Molecular modeling studies suggest that all the macrocycles except 10b should have well-defined triangle, parallelogram, and ring shapes if all the amide linkages are trans and the ortho-alkoxy substituents are intramolecularly hydrogen bonded to the amide NH groups. The macrocycles were modeled as simplified homologues in which the propyloxyammonium side chains (R = CH₂CH₂CH₂NH₃⁺) were replaced with methoxy groups (R = Me, Figure 5). Each molecule was modeled using Maestro/MacroModel v8.5 with the MMFFs implementation of the MMFF force field and MCMM conformational searching. The Ar–Ar and Ar–N bonds were rotated during the search procedure. Rotations about the Ar–CO and Ar–O bonds were not performed. Amide linkages were assumed to adopt trans conformations and were not allowed to adopt cis conformations (except as noted below). 1000 Monte-Carlo search steps were performed for each structure, and no effort was made to assure that all of the lowest-energy conformers or the global minimum were identified.^10,11

FIGURE 5.

Molecular models of low-energy conformers of macrocycles 9a–c, 10a,b, and 11a–d. The macrocycles were modeled as simplified homologues 9a'–c', 10a',b', and 11a'–d' in which the propyloxyammonium side chains (R = CH₂CH₂CH₂NH₃⁺) were replaced with methoxy groups (R = Me). Overlays represent conformers identified within the lowest 5.00 kJ/mol from 1000 Monte-Carlo conformational search steps.

Molecular modeling studies also suggest that introduction of cis-amide linkages permits the formation of collapsed structures. A simplified model of the cttctt-conformer of 11a (11a', R = Me) was generated and is shown with the tttttt-conformer in Figure 6. The conformer represents the global minimum among the cttctt-conformers and was identified from 1000 Monte-Carlo search steps starting with a cttctt-conformer.^12,13 In this conformer, the cis-amide linkages and adjacent aromatic rings create tight turn structures, and the other aromatic rings of the m-Abc^2K units stack.

FIGURE 6.

Molecular models of cttctt-and tttttt-conformers of macrocycle 11a. The macrocycle was modeled as simplified homologue 11a' in which the propyloxyammonium side chains (R = CH₂CH₂CH₂NH₃⁺) were replaced with methoxy groups (R = Me). Structures represent the lowest energy cttctt-and tttttt-conformers identified from 1000 Monte-Carlo conformational search steps.

Conclusion

In summary, we have now developed a family of nanometer-sized amino acids—Abc^2K, m-Abc^2K, and o-Abc^2K—that can easily be combined to create water-soluble nanometer-scale macrocycles with well-defined shapes. The macrocycles are easy to synthesize, purify, and characterize, because they are water-soluble cationic peptides. The macrocycles can adopt conformations with all trans-amide linkages and cavities as large as 4 nm or undergo hydrophobic collapse depending on their composition and on the solvent. The size, water solubility, and ease of synthesis of these compounds should help bridge the gap between biology and nanotechnology.

Experimental Section

General Procedures

Commercial solvents and reagents were used without further purification, unless otherwise stated. All solution-phase reactions were carried out under an atmosphere of nitrogen and were monitored by thin-layer chromatography (TLC) and carried out on 250 µm silica gel polyester plates on fluorescent silica gel with UV visualization. Column chromatography was performed on 40–63 µm silica gel (EMD Science) using flash chromatography. Solvents were removed by rotary evaporation, followed by further drying of residual solvents under vacuum (< 0.01 mmHg) or by air suction through a filter funnel. High resolution mass spectra were obtained by electrospray ionization (ESI) on a Waters Micromass LCT Premier (instrument variation σ < 5 ppm). NMR spectra were recorded using a 500 MHz Bruker AVANCE™ spectrometer. Chemical shifts are reported in parts per million (ppm) on the δ scale. ¹H NMR spectra in CD₃SOCD₃ were referenced with TMS (δ = 0.00 ppm); ¹H NMR spectra in D₂O were referenced to HDO (δ = 4.80 ppm); ¹H NMR spectra in CD₃OD were referenced to CHD₂OD (δ = 3.34). IR spectra were obtained using a Galaxy Series FTIR 5000. HPLC analysis was performed on an analytical RP-HPLC instrument, using a C₁₈ column (Alltech, Platinum Rocket, 3µm packing, 7 mm × 53 mm). Preparative RP-HPLC was performed using an Agilent C₁₈ column (1 inch diameter).

4-Bromo-2,5-dimethoxybenzoic acid (3).⁸

A flame dried 3-necked 100-mL round bottomed flask equipped with a magnetic stirring bar, rubber septum, ground-glass stopper, and a nitrogen inlet adapter, was charged with 1,4-dibromo-2,5-dimethoxybenzene (2) (5.0 g, 16.9 mmol). THF (ca. 25 mL) was added by syringe until a clear solution was formed. A 2.0 M solution of i-PrMgCl in THF (8.7 mL) was then added by syringe in a single portion at 25 °C to a stirring solution of 2, and was then stirred under an atmosphere of nitrogen for 20 h. Dry ice (ca. 20 g) was added in small portions resulting in a color change of the reaction mixture from light yellow to bluish-green. The solution returned to its original light yellow color after being allowed to mix for an additional 90 min. The reaction mixture was acidified with concd HCl (aq) (ca. 5 mL) and then extracted into ether (3 × 100 mL) with water (75 mL). The organic layer was washed with water (ca. 100 mL) and then extracted into aq 1 M KOH (3 × ca. 30 mL). The alkaline solution was acidified with concd HCl (aq), and the resultant white precipitate was washed with water to provide 4-bromo-2,5-dimethoxybenzoic acid (3) as a white powder (3.7 g, 85%).¹⁵

H-m-Abc^2K(Boc)-OCH₂COPh (7a)

Diether 6⁶ (5.63 g, 8.86 mmol) was dissolved in THF (150 mL) by heating to ca. 60 °C in an oil bath. To the warmed solution was added 3-aminophenylboronic acid (1.16 g, 8.86 mmol), PdCl₂(dppf)•CH₂Cl₂ (0.345 g, 0.423 mmol), K₂CO₃ (5.86 g, 42.3 mmol), water (30 mL), and the mixture was stirred at 60 °C for 24 h under an atmosphere of nitrogen. The solution was then concentrated by rotary evaporation and the resultant oil was dissolved in CH₂Cl₂ (ca. 100 mL). The organic layer was washed with water (ca. 100 mL), dried (MgSO₄), and filtered. The filtrate was concentrated by rotary evaporation to afford a brown oil. Purification using column chromatography (silica gel, ethyl acetate–hexanes, 2/1, v/v) provided H-m-Abc^2K(Boc)-OCH₂COPh (7a) as an off-white solid (3.60 g, 63%): mp 118–120 °C; IR (KBr) 3430, 3361, 1729, 1681 cm⁻¹; ¹H NMR, (500 MHz, CD₃SOCD₃, 298 K) δ 8.03 (d, J = 7.2 Hz, 2 H), 7.72 (t, J = 7.4 Hz, 1 H), 7.59 (t, J = 7.8 Hz, 2 H), 7.45 (s, 1 H), 7.08 (t, J = 7.8 Hz, 1 H), 7.03 (s, 1 H), 6.90 (t, J = 5.4 Hz, 1 H), 6.85 (t, J = 5.5 Hz, 1 H), 6.80 (s, 1 H), 6.72 (d, J = 7.7 Hz, 1 H), 6.59 (d, J = 7.9 Hz, 1 H), 5.72 (s, 2 H), 5.15 (br s, 2 H), 4.05 (t, J = 6.1 Hz, 2 H), 3.93 (t, J = 6.2 Hz, 2 H), 3.12 (q, J = 6.5 Hz, 2 H), 3.06 (q, J = 6.4 Hz, 2 H), 1.81 (quintet, J = 6.4 Hz, 2 H), 1.77 (quintet, J = 6.5 Hz, 2 H), 1.37 (s, 9 H), 1.32 (s, 9 H); ¹³C NMR (125 MHz, CD₃SOCD₃, 298 K) δ 192.8, 164.3, 155.6, 155.5, 152.6, 148.8, 148.16, 137.5, 136.7, 133.9, 133.9, 128.8, 128.4, 127.7, 117.7, 116.9, 116.6, 115.2, 114.8, 113.3, 77.4, 77.3, 67.2, 66.8, 66.2, 37.0, 36.7, 29.3, 29.0, 28.14, 28.09; HRMS (ESIMS) m/z for C₃₇H₄₇N₃O₉Na [M+Na]⁺ calcd 700.3210, found 700.3194.

Fmoc-m-Abc^2K(Boc)-OCH₂COPh (8a)

A solution of H-m-Abc^2K(Boc)-OCH₂COPh (7a) (3.60 g, 5.31 mmol), pyridine (0.52 mL, 6.4 mmol), and CH₂Cl₂ (100 mL) was cooled to 0 °C in an ice-bath. Fmoc-Cl (1.51 g, 5.84 mmol) in CH₂Cl₂ (50 mL) was added in drops over 5 min. After 30 min, the solution was allowed to warm to 25 °C and stirred for an additional 30 min. The mixture was then washed with water (ca. 100 mL), dried (MgSO₄), filtered, and the filtrate was concentrated by rotary evaporation to give a yellow oil. The yellow oil was dissolved in CH₂Cl₂ (ca. 15 mL), precipitated using ethyl acetate (ca. 50 mL) and hexanes (ca. 100 mL), and filtered to provide Fmoc-m-Abc^2K(Boc)-OCH₂COPh (8a) as a white solid (4.36 g, 91%): mp 118–120 °C; IR (KBr) 3369, 1733, 1697 cm⁻¹; ¹H NMR, (500 MHz, CD₃SOCD₃, 298 K) δ 9.63 (s, 1 H), 8.01 (d, J = 8.5 Hz, 2 H), 7.89 (d, J = 7.5 Hz, 2 H), 7.75 (d, J = 7.5 Hz, 2 H), 7.70 (t, J = 7.4 Hz, 1 H), 7.65 (br s, 1 H), 7.58 (t, J = 7.7 Hz, 2 H), 7.51-7.44 (m, 2 H), 7.42 (t, J = 7.5 Hz, 2 H), 7.37-7.30 (m, 3 H), 7.21 (d, J = 7.7 Hz, 1 H), 7.05 (s, 1 H), 6.66 (br s, 2 H), 5.68 (s, 2 H), 4.49 (d, J = 6.7 Hz, 2 H), 4.31 (t, J = 6.7 Hz, 2 H), 4.05 (t, J = 6.1 Hz, 2 H), 3.94 (t, J = 6.3 Hz, 2 H), 3.12 (q, J = 6.6 Hz, 2 H), 3.02 (q, J = 6.2 Hz, 2 H), 1.82 (quintet, J = 6.5 Hz, 2 H), 1.76 (quintet, J = 6.5 Hz, 2 H), 1.34 (s, 9 H), 1.32 (s, 9 H); ¹³C NMR (125 MHz, CD₃SOCD₃, 298 K) δ 192.7, 164.3, 155.54, 155.50, 153.4, 152.5, 148.8, 143.7, 140.7, 138.8, 137.4, 135.6, 133.9, 128.8, 128.3, 127.7, 127.6, 127.0, 125.0, 123.5, 120.1, 119.2, 118.3, 117.6, 116.8, 115.2, 77.4, 77.3, 67.3, 66.8, 66.3, 65.5, 46.5, 37.0, 36.6, 29.3, 29.1, 28.10, 28.08; HRMS (ESIMS) m/z for C₅₂H₅₇N₃O₁₁Na [M+Na]⁺ calcd 922.3891, found 922.3863.

Fmoc-m-Abc^2K(Boc)-OH (1a)

Zn dust (8.0 g, 122 mmol) was added to a solution of Fmoc-m-Abc^2K(Boc)-OCH₂COPh (8a) (4.36 g, 4.84 mmol), AcOH (90 mL), and H₂O (10 mL) and was stirred at 25 °C for ca. 18 h. The suspension was then diluted with CH₂Cl₂ (75 mL) and stirred for an additional ca. 1 h, concentrated to approximately one-half of its original volume, and partitioned between CH₂Cl₂ (75 mL) and aq 0.2 N HCl (ca. 75 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 75 mL), and the combined organic layers were washed with H₂O (ca. 100 mL), saturated aq sodium chloride (ca. 100 mL), and dried (MgSO₄). Filtration and concentration by rotary evaporation afforded a yellow oil. The oil was dissolved in ether (ca. 20 mL) and CH₂Cl₂ (ca. 10 mL) and the resulting solution was added in drops to hexanes (ca. 150 mL) over ca. 2 min. The resulting white suspension was filtered to afford Fmoc-m-Abc^2K(Boc)-OH (1a) as a white solid (3.78 g, 94%): mp 98–100 °C; IR (KBr) 3361, 1702 cm⁻¹; ¹H NMR, (500 MHz, CD₃SOCD₃, 298 K) δ 12.58 (br s, 1 H), 9.77 (br s, 1 H), 7.91 (d, J = 7.5 Hz, 2 H), 7.76 (d, J = 7.4 Hz, 2 H), 7.64 (br s, 1 H), 7.49 (br s, 1 H), 7.43 (t, J = 7.4 Hz, 2 H), 7.39-7.26 (m, 4 H), 7.18 (d, J = 7.6 Hz, 1 H), 6.99 (s, 1 H), 6.87 (t, J = 5.6 Hz, 1 H), 6.84 (t, J = 5.7 Hz, 1 H), 4.49 (d, J = 6.5 Hz, 2 H), 4.31 (t, J = 6.7 Hz, 1 H), 4.01 (t, J = 6.0 Hz, 2 H), 3.92 (t, J = 6.2 Hz, 2 H), 3.10 (q, J = 6.5 Hz, 2 H), 3.01 (q, J = 6.2 Hz, 2 H), 1.81 (quintet, J = 6.4 Hz, 2 H), 1.74 (quintet, J = 6.4 Hz, 2 H), 1.34 (s, 18 H); ¹³C NMR (125 MHz, CD₃SOCD₃, 298 K) δ 166.8, 155.5, 153.3, 151.7, 148.8, 143.7, 140.7, 138.7, 137.6, 134.3, 128.3, 127.6, 127.0, 125.0, 123.5, 120.9, 120.1, 119.1, 117.4, 116.7, 115.0, 77.38, 77.34, 67.2, 66.2, 65.5, 46.5, 36.9, 36.6, 29.2, 29.1, 28.1; HRMS (ESIMS) m/z for C₄₄H₅₁N₃O₁₀ [M+Na]⁺ calcd 804.3472, found 804.3470.

H-o-Abc^2K(Boc)-OCH₂COPh (7b)

Diether 6⁶ (2.31 g, 3.47 mmol) was dissolved in THF (80 mL) by heating to ca. 60 °C in an oil bath. To the warmed solution was added 2-aminophenylboronic acid (0.874 g, 3.99 mmol), K₂CO₃ (2.40 g, 17.4 mmol), water (20 mL), PdCl₂(dppf)•CH₂Cl₂ (0.142 g, 0.174 mmol), and the mixture was stirred at 60 °C for 24 h under an atmosphere of nitrogen. The solution was then concentrated by rotary evaporation to an oily slurry and was partitioned between water (150 mL) and CH₂Cl₂ (75 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 75 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (ca. 100 mL) and dried (MgSO₄). The suspension was then filtered and concentrated by rotary evaporation to afford a brown oil. The oil was purified using column chromatography (silica gel, ethyl acetate–hexanes, 2/1, v/v) to provide H₂N-o-Abc^2K(Boc)-OCH₂COPh (7a) as an off-white solid (1.61 g, 69%): mp 54–56 °C; IR (KBr) 3367, 1701 cm⁻¹; ¹H NMR, (500 MHz, CD₃SOCD₃, 298 K) δ 8.03 (d, J = 7.0 Hz, 2 H), 7.72 (t, J = 7.5 Hz, 1 H), 7.59 (t, J = 7.7 Hz, 2 H), 7.46 (s, 1 H), 7.06 (t, J = 7.0 Hz, 1 H), 6.98 (J = 7.5 Hz, 1 H), 6.95 (s, 1 H), 6.82 (t, J = 5.5 Hz, 2 H), 6.61 (t, J = 7.3 Hz, 1 H), 5.72 (s, 2 H), 4.72 (br s, 2 H), 4.02 (t, J = 6.0 Hz, 2 H), 3.93 (t, J = 6.5 Hz, 2 H), 3.11 (q, J = 6.5 Hz, 2 H), 3.00 (q, J = 6.0 Hz, 2 H), 1.80 (quintet, J = 6.5 Hz, 2 H), 1.68 (quintet, J = 6.25 Hz, 2 H), 1.37 (s, 9 H), 1.32 (s, 9 H); ¹³C NMR (125 M, Hz, CD₃SOCD₃, 298 K) δ 192.8, 164.5, 155.50, 155.48, 152.5, 149.2, 145.6, 134.6, 133.89, 133.85, 130.5, 128.8, 128.43, 127.7, 122.1, 118.1, 117.6, 115.9, 114.9, 114.8, 77.34, 77.29, 67.1, 66.8, 65.8, 37.0, 36.3, 29.3, 29.2, 28.13, 28.09;. HRMS (ESIMS) m/z for C₃₇H₄₈N₃O₉ [M+H]⁺ calcd 678.3391, found 678.3391.

Fmoc-o-Abc^2K(Boc)-OCH₂COPh (8b)

A solution of H-o-Abc^2K(Boc)-OCH₂COPh (7b) (1.51 g, 2.23 mmol), pyridine (0.220 mL, 2.68 mmol), and CH₂Cl₂ (75 mL) was cooled to 0 °C in an ice-bath. Fmoc-Cl (1.51 g, 3.70 mmol) in CH₂Cl₂ (20 mL) was added in drops over 10 min. After 20 min, the solution was allowed to warm to 25 °C and stirred for an additional 30 min. The mixture was then washed with water (150 mL), dried (MgSO₄), filtered, and the filtrate was concentrated by rotary evaporation to afford a yellow oil. Purification using column chromatography (silica gel, ethyl acetate–hexanes, 2/1, 5% CH₂Cl₂, v/v) provided Fmoc-o-Abc^2K(Boc)-OCH₂COPh (8b) as a white solid (2.0 g, 100%): mp 68–70 °C; IR (KBr) 3373, 1732, 1695 cm⁻¹; ¹H NMR, (500 MHz, CD₃SOCD₃, 298 K) δ 8.51 (br s, 1 H), 8.05 (d, J = 8.1 Hz, 2 H), 7.88 (d, J = 7.9 Hz, 2 H), 7.72 (t, J = 7.7 Hz, 1 H), 7.65-7.55 (m, 4 H), 7.48 (s, 2 H), 7.44-7.34 (m, 3 H), 7.34-7.27 (m, 3 H), 7.24 (t, J = 7.7 Hz, 1 H), 6.96 (s, 1 H), 6.80 (t, J = 5.5 Hz, 1 H), 6.73 (t, J = 5.5 Hz, 1 H), 5.75 (s, 2 H), 4.30 (d, J = 7.0 Hz, 2 H), 4.22 (t, J = 7.0 Hz, 1 H), 4.01-3.92 (m, 2 H), 3.85 (t, J = 6.3 Hz, 2 H), 3.08 (q, J = 6.4 Hz, 2 H), 2.86 (q, J = 6.2 Hz, 2 H), 1.78 (quintet, J = 6.5 Hz, 2 H), 1.60 (quintet, J = 6.5 Hz, 2 H), 1.32 (s, 9 H), 1.31 (s, 9 H); ¹³C NMR (125 MHz, CD₃SOCD₃, 298 K) δ 192.7, 164.5, 155.5, 155.4, 154.1, 152.3, 149.0, 143.6, 140.6, 135.5, 133.89, 133.87, 133.5, 131.6, 130.6, 128.8, 128.2, 127.8, 127.6, 127.0, 125.1, 124.6, 120.0, 118.7, 117.7, 114.9, 77.4, 77.3, 67.1, 66.9, 66.3, 65.9, 46.4, 37.1, 36.3, 29.2, 29.0, 28.1; HRMS (ESIMS) m/z for C₅₂H₅₇N₃O₁₁Na [M+Na]⁺ calcd 922.3891, found 922.3895.

Fmoc-o-Abc^2K(Boc)-OH (1b)

Zn dust (3.2 g, 49 mmol) was added to a solution of Fmoc-o-Abc^2K(Boc)-OCH₂COPh (8b) (1.85 g, 2.06 mmol), AcOH (90 mL), and H₂O (10 mL) and was stirred at 25 °C for 18 h. The suspension was then diluted with CH₂Cl₂ (100 mL) and stirred for an additional 3 h, washed with 0.2 N HCl (100 mL), H₂O (100 mL), and saturated aqueous sodium chloride (100 mL). The organic layer was dried over MgSO₄ and concentrated by rotary evaporation to give a yellow oil. The oil was dissolved in ether (ca. 20 mL) and the resultant solution was added in drops to hexanes (ca. 80 mL) over ca. 2 min. The resultant white suspension was filtered to provide Fmoc-o-Abc^2K(Boc)-OH (1b) as a white solid (1.54 g, 96%): mp 98–100 °C. An analytical sample of Fmoc-o-Abc^2K(Boc)-OH (1b) was prepared by purification of 50 mg by RP-HPLC (water–CH₃CN buffers with 0.1 % TFA), concentrating, dissolving the resultant oil in CH₂Cl₂, and passing the solution through a plug of silica gel (eluted with ethyl acetate): mp 98–100 °C; IR (KBr) 3500-3100, 1706 cm⁻¹; ¹H NMR, (500 MHz, CD₃SOCD₃, 320 K) δ 12.50 (br s, 1 H), 8.11 (br s, 1H), 7.85 (d, J = 7.6 Hz, 2 H), 7.56 (d, J = 7.4 Hz, 2 H), 7.49 (br d, J = 7.6 Hz, 1 H), 7.39 (t, J = 7.4 Hz, 2 H), 7.33 (td, J = 7.7, 1.2 Hz, 1 H), 7.31-7.23 (m, 4 H), 7.20 (td, J = 7.3, 0.9 Hz, 1 H), 6.84 (s, 1 H), 6.77 (br s, 1 H), 6.55 (br s, 1 H), 4.29 (d, J = 7.1 Hz, 2 H), 4.21 (t, J = 7.0 Hz, 1 H), 3.95 (t, J = 6.1 Hz, 2 H), 3.84 (t, J = 6.4 Hz, 2 H), 3.09 (q, J = 6.4 Hz, 2 H), 2.86 (q, J = 6.3 Hz, 2 H), 1.77 (quintet, J = 6.4 Hz, 2 H), 1.61 (quintet, J = 6.6 Hz, 2 H), 1.34 (s, 9 H), 1.32 (s, 9 H); ¹³C NMR (125 MHz, CD₃SOCD₃, 298 K) δ 166.9, 155.5, 155.4, 154.0, 151.5, 149.0, 143.6, 140.6, 135.4, 132.2, 131.8, 130.6, 128.0, 127.5, 126.9, 125.1, 124.9, 124.6, 121.2, 120.0, 117.6, 114.7, 67.1, 66.3, 65.9, 46.4, 37.0, 36.4, 29.1, 29.0, 28.11, 28.08; HRMS (ESIMS) m/z for C₄₄H₅₁N₃O₁₀Na [M+Na]⁺ calcd 804.3472, found 804.3463.

Representative Procedure for Macrocycle Synthesis: Synthesis of Cyclohexamer Triangle 9b

A Bio-Rad Poly-Prep column^® was charged with 2-chlorotrityl resin (73 mg, nominally 1.4 mmol/g, 0.10 mmol, Novabiochem). The resin was derivatized by gently agitating with a solution of Fmoc-o-Abc^2K(Boc)-OH (1b) (102 mg, 0.13 mmol) in 20% 2,4,6-collidine-CH₂Cl₂ (ca. 1 mL) for ca. 12 h. The solution was then drained using nitrogen pressure, and the resin was washed with CH₂Cl₂ (5 × ca. 5 mL, 1 min each). After the loading step, unreacted sites on the resin were capped using a solution of CH₂Cl₂-MeOH-DIPEA (17/2/1) for ca. 30 min. (Measurement of the weight gain in related experiments demonstrated the efficiency of the resin loading step to be ca. 50%.) The column was then drained, and the resin was washed with DMF (6 × ca. 5 mL, 1 min each) and then CH₂Cl₂ (6 × ca. 5 mL, 1 min each). The Fmoc group was removed by adding a solution of 20% piperidine–DMF (1 × ca. 5 mL for 1 min, 1 × ca. 5 mL for 20 min) to the resin followed by gentle agitation of the resultant suspension. The piperidine solution was drained, and the resin was washed with DMF (6 × ca. 5 mL, 1 min each) followed by CH₂Cl₂ (6 × ca. 5 mL, 1 min each).

Elongation of the protected linear Abc^2K oligomer was accomplished by pre-activating the appropriate Fmoc-protected Abc^2K building block (Abc^2K or o-Abc^2K for 9b; 78 mg, 0.10 mmol) with HCTU (0.10 mmol, 41 mg) in a ca. 1 mL solution of 20% 2,4,6-collidine–DMF. (These amounts correspond to ca. 2 equiv, based on 50% loading of the resin.) The coupling solution was then added to the resin and gently agitated for ca. 12 h. To determine if Abc^2K couplings were complete, a small amount of resin was treated in a new Bio-Rad column with a solution of CF₃COOH/water (9/1, v/v) and the column was vigorously agitated for ca. 2 h. The solution was then drained, concentrated by rotary evaporation, and the residue was dissolved in a mixture of water and CH₃CN and then injected into an analytical RP-HPLC instrument to determine if any unreacted amine was present. In all cases, the amount of uncoupled amine was not significant enough to warrant a second coupling. The coupling procedure was repeated until the desired length of the linear oligomer was obtained [H-(o-Abc^2K(Boc)-Abc^2K(Boc))₃-OH].

The protected linear Abc^2K(Boc) oligomer was cleaved from the resin using a solution of AcOH-TFE-CH₂Cl₂ (1/1/4) for ca. 12 h. The peptide solution was drained from the resin and concentrated to form a gel. From the gel, remaining acetic acid was azeotropically removed by rotary evaporating with hexanes (3×) and CH₂Cl₂ (3×) to provide a white a solid. (This step is particularly important to avoid acetylation of the aniline group by trace amounts of acetic acid during cyclization.) The protected linear oligomer was split into two equal portions, and the cyclization step was carried out using half the material. (The remaining half of the material was reserved for studies of the uncyclized linear peptide.)

Cyclization was accomplished by charging a 250-mL round bottomed flask with H-(o-Abc^2K(Boc)-Abc^2K(Boc))₃-OH (half of the material from above step) and 100 mL of 2% collidine-CH₂Cl₂. HCTU (0.1 mmol, dissolved in 1 mL of DMF) was added in drops over 2 min and the mixture was stirred for ca. 24 h. The reaction solution was then washed with ca. 100 mL of 0.2 N HCl (aq) to remove excess base, and the organic layer was concentrated to an oil. Global deprotection of the Boc groups was carried out in a small round bottomed flask equipped with a magnetic stirring bar, and mixed with 10–20 mL of CF₃COOH/CH₂Cl₂ (1/1, v/v) for ca. 4 h. The solution was then concentrated by rotary evaporation, and the resultant oil was purified by preparative RP-HPLC (water–CH₃CN with 0.1 % TFA). Pure fractions of the product (ca. 98% purity) were concentrated by rotary evaporation to remove most of the CH₃CN, frozen, and then lyophilized to afford a white powder (16 mg, 0.005 mmol, 9 % based on the nominal 1.4 mmol/g loading of the resin and use of half of the linear oligomer in the cyclization step). The remaining less pure HPLC fractions (< 98% purity) were also lyophilized (10 mg, 0.003 mmol, 6 %) and stored for future use. Macrocycles 9a,c, 10a,b, and 11a–d were prepared in a similar fashion.¹⁴

It should be noted that the percentage yields based on resin loading are significantly higher than the above percentages would indicate, because the actual resin loading was found to be roughly half (0.7–1.0 mmol/g) of the nominal resin loading (1.4 mmol/g). Treatment of 36 mg of 2-chlorotrityl resin (nominally 1.4 mmol/g, 0.050 mmol, Novabiochem,) with Fmoc-o-Abc^2K(Boc)-OH (1b) (51 mg, 0.065 mmol) in 20% 2,4,6-collidine-CH₂Cl₂ resulted in a weight gain of 19 mg, thus indicating 0.7 mmol/g actual loading. Treatment of 36 mg of 2-chlorotrityl resin (nominally 1.4 mmol/g, 0.050 mmol, Novabiochem,) with Fmoc-m-Abc^2K(Boc)-OH (1a) (51 mg, 0.065 mmol) in 20% 2,4,6-collidine-CH₂Cl₂ resulted in a weight gain of 27 mg, thus indicating 1.0 mmol/g actual loading. A control study in which 36 mg of 2-chlorotrityl resin (nominally 1.4 mmol/g, 0.050 mmol, Novabiochem) was treated with MeOH in 20% 2,4,6-collidine-CH₂Cl₂ resulted in no weight gain, thereby validating the use of gravimetric analysis to assess resin loading. Gravimetric analysis after elongation to the full-length linear peptides indicated a final resin loading of 0.3–0.6 mmol/g, suggesting that some loss of peptide from the labile 2-chlorotrityl resin occurs during the synthesis.