Enhanced Stereoselectivity of a Cu(II) Complex Chiral Auxiliary in the Synthesis of Fmoc-L-γ-carboxyglutamic Acid

Abstract

L-γ-Carboxyglutamic acid (Gla) is an uncommon amino acid that binds avidly to mineral surfaces and metal ions. Herein, we report the synthesis of N-α-Fmoc-L-γ-carboxyglutamic acid-γ,γ’-tert-butyl ester (Fmoc-Gla(O^tBu)₂-OH), a suitably protected analog for Fmoc-based solid phase peptide synthesis. The residue was synthesized using a novel chiral Cu(II) complex, whose structure-based design was inspired by the blue copper protein rusticyanin. The five-coordinate complex is formed by Shiff base formation between glycine and the novel ligand (S)-2-(N-(2-methylthio)-benzylprolyl)-aminobenzophenone in the presence of copper. Michael addition of di-tert-butyl methylenemalonate to the α-carbon of the glycine portion of the complex occurs in a diastereoselective fashion. The resulting (S,S) complex diastereomer can be easily purified by chromatography. Metal complex decomposition followed by Fmoc protection affords the enantiomerically pure amino acid. With the use of this novel chiral complex, the asymmetric synthesis of Fmoc-Gla(O^tBu)₂-OH was completed in nine steps from thiosalicylic acid in 14.5% overall yield.

Introduction

L-γ-Carboxyglutamic acid (Gla) occurs in natural proteins as a result of posttranslational modification of glutamic acid residues and serves both functional and structural roles.¹ For example, in the blood coagulation protein prothrombin, a host of Gla residues in the N-terminal domain of the protein bind calcium ions with high affinity resulting in a conformational change that enables phospholipid binding and ultimate coagulant activity.^2,3 The mineral-binding, Gla-containing domains of these proteins have been mimicked by integration of the Gla residue into synthetic peptides.^4,5 For example, we, as well as Murphy and coworkers, have shown that peptides containing Gla residues can undergo calcium-mediated binding to surfaces such as hydroxyapatite with high avidity^4,6,7 and can possibly be used as tools to better understand apatite remodeling⁸. This uncommon amino acid is typically incorporated into peptides synthetically, rather than enzymatically, using solid-phase peptide synthesis (SPPS). Syntheses are quite costly due to the commercial expense of the suitably protected analog of the Gla amino acid. This has served as motivation to develop synthetic approaches to this residue that are amenable to academic labs. Asymmetric routes^9,10 have greatly improved the yields of the amino acid in comparison to past racemic syntheses^11-13, however reducing the number of steps required to access the fully protected Gla analog (S)-1 necessary for Fmoc-based SPPS still poses a challenge, Figure 1.

Figure 1.

Fmoc-protected L-γ-carboxyglutamic acid.

One common approach for asymmetric amino acid synthesis is the use of chiral nucleophilic glycine equivalents. This method allows vast structural diversity at the amino acid side chain. Addition reactions to the Ni(II) complex of the Schiff base of glycine and (S)-BPB (Figure 2) generally give rise to product mixtures with a high excess of the diastereomer containing the (S)-amino acid.^14-16 Isolation of the major diastereomer through chromatography, followed by decomposition of the metal complex results in enantiomerically pure amino acid and recovery (>80%) of the chiral ligand.

Figure 2.

General route for preparation of enantiomerically pure Fmoc-amino acids starting from a chiral nucleophilic glycine equivalent.

These chiral templates have been used to gain access to a variety of glutamic acid^17-21 and proline^22,23 derivatives in a highly stereoselective fashion through Michael additions of activated olefins to the glycine enolate. An attractive benefit of using the BPB complexes for Michael additions is the mild conditions under which the reactions can be performed. Early transformations using excesses of sodium methoxide or triethylamine as a base provided moderate yields and diastereoselectivities^17,22. More recently developed conditions employing catalytic amounts (15 mol%) of DBU as a base could provide near quantitative yields of a single product diastereomer within minutes at ambient temperature^24-28. However, as will be shown, the previously reported four-coordinate, square planar Ni complex¹⁶ in Figure 2 proved to be ineffective for the synthesis of (S)-1. Using previously reported and optimized reaction conditions afforded a mixture of diastereomers that could not be separated by conventional normal-phase chromatography. Thus, both poor selectivity and poor chromatographic resolution resulted in the inefficient preparation of (S)-1 using this common chiral auxiliary.

Inspired by the axial binding interactions between methionine and copper in Blue Copper proteins, we set out to improve upon the results of others²⁹ by designing a novel chiral complex that contains an additional axial thioether ligand, which can better shield the top face of the coordination plane towards an incoming electrophile. This proported 5-coordinate, square pyramidal complex could effectively increase the ratio of diastereomers formed in the Michael addition reaction, and in addition, allow better resolution and subsequent separation of diastereomers. Herein, we synthesize the novel thioether-containing ligand (S)-7c (Figure 3a) and demonstrate the increased diastereoselectivity of the corresponding Cu(II)-containing glycine Schiff base (S)-9c (Figure 3b) using di-tert-butyl methylenemalonate as a Michael acceptor in our synthesis of γ-carboxyglutamic acid.

Figure 3.

(a) Novel thioether-containing chiral ligand (S)-7c. (b) Structure of the chiral BPB metal complexes containing an ortho-substituted benzyl group. (c) Highlighted residues bound to Cu(II) in the protein Rusticyanin (PDB: 1RCY), note the axial methionine ligation.

Results and Discussion

Our need for a more selective chiral glycine equivalent was realized during the optimization of reaction conditions for the Michael addition reaction employing the known Ni(II) complex of the Schiff base of glycine and (S)-2-(N-Benzylprolyl)-aminobenzophenone (BPB)^30,31 shown in Table 1. Initially, Michael additions of the glycinate (BPB-Ni-Gly) to di-tert-butyl methylenemalonate 14³² were optimized around conditions described in the literature.^21,22,33 The selectivity of the addition reaction was assessed by decomposing the resulting metal complex to free the amino acid, whose enantiomeric excess was measured using Marfey’s reagent (Table 1, note b). The diastereomeric ratio of the products favoring the (S,S) configuration was consistent with reports discussed earlier. A slightly higher yield was obtained by exchanging DMF with CH₃CN as the reaction solvent (Table 1, entries 1 and 2). Also, the reaction proceeded more slowly when DBU was exchanged for other amine and alkaline bases, and resulted in lower yields and selectivity (Table 1, entries 3-6). To our dismay, regardless of the reaction conditions, the product diastereomers were inseparable by chromatography on silica gel using typical solvent systems.^14,34 Owing to the fact that this method of amino acid synthesis relies on the stereodiscriminating ability of the complex³⁵ in the reaction and separation of products to obtain a high excess of enantiomerically pure amino acid, we began to examine methods to improve both of these features en-route to the synthesis of Gla residue (S)-1.

Table 1.

Solvent and Base Effect on the Addition of 14 to BPB-Ni-Gly.

Entry	Base	Solvent	Time(min)	Yield(%)a	ee(%)b
1	DBUc	DMF	10	70	80
2	DBUc	MeCN	10	75	82
3	NEt3d	1:1 MeCN:DMF	60	48	71
4	NH(iPr)2d	1:1 MeCN:DMF	60	41	58
5	NEt(iPr)2d	1:1 MeCN:DMF	60	25	70
6	KOHe	DCM	60	42	51

^acombined yield of major (S,S) and minor (S,R) diastereomers

^benantiomeric excess of the (S)-amino acid based on Marfey’s assay of the amino acid after decomposition of the metal complex diastereomers

^c0.15 equivalents of base

^d3.0 equivalents of base

^e10 equivalents of base.

Efforts by others have been made to improve the stereoselectivity of addition reactions through substitutions on the N-benzyl portion of the chiral complex.^36,37 Saghiyan and coworkers reported that 3,4-dimethyl-, 3,4-dichloro, and 2-chlorosubstitued benzyl analogs of the Ni(II) complex gave better selectivity due to increased shielding of the coordination plane.²⁹

Crystal structure analysis of the unsubstituted complex revealed that the benzyl group (Table 2, R = H, M = Ni) centers its aromatic ring directly above the metal in the coordination plane.³⁸ In comparison, when a chloro group is placed in the ortho position of the benzyl ring (Table 2, R = Cl, M = Ni), the aromatic ring moves off the metal atom and the Cl is positioned directly above the nickel.³⁹ Moreover, the sum of the Van-der-Waals radii of the Cl and Ni atoms is greater than the Ni-Cl atomic distance of 3.149 Å, implying a distinct axial interaction between the nickel and the chlorine.³⁹ This interaction was postulated to shield the top face of the complex towards an approaching electrophile, thus increasing the selectivity of the Michael addition. This crystallographic observation suggested to us that other bonafide axial ligand-metal interactions could be designed that enhance diastereoselectivity. Such axial interactions can be found in some Blue Copper proteins such as rustacyanin (Fig 3c), where sulfur coordination (from methionine) to a central Cu atom occurs above the coordination plane formed from a set of trigonal side-chain ligands at a Cu-S atomic distance of 2.885 Å.⁴⁰ We investigated the possibility of mimicking this naturally occurring complex by replacing the 2-chloro substituent on BPB with an methylthio-substituent and swapping the nickel with copper (Table 2, R = SMe, M = Cu) to form a purported 5-coordinate pyramidal complex. The axial sulfur donor should enhance the effective shielding of the coordination plane and increase the stereodiscriminating ability of the complex, and as a result, increase selectivity.

Table 2.

Michael Additions to Ni(II) complexes (S)-8a-c and Cu(II) complexes (S)-9a,c.

Entry	Complex	Product	Metal(M)	R	Yield(%)a	ee(%)b
1	(S)-8a	(S,S)-10a	Ni	H	75	82
2	(S)-9a	(S,S)-11a	Cu	H	64	49
3	(S)-8b	(S,S)-10b	Ni	Cl	94	85
4	(S)-8c	(S,S)-10c	Ni	SMe	81	90
5	(S)-9c	(S,S)-11c	Cu	SMe	70	93
*6	(S)-9c	(S,S)-11c	Cu	SMe	67	>99

^acombined yield of major (S,S) and minor (S,R) diastereomers

^benantiomeric excess of the (S)-amino acid based on Marfey’s assay of the amino acid after decomposition of the metal complex diastereomers

^*isolated yield of (S,S)-11c from product mixture (entry 5).

We began by preparing both the Ni(II) and Cu(II) complexes containing methylsulfide and chloro ortho-substituted benzyl groups to assess the ligating potential of the thioether and gauge the importance of swapping the nickel with copper. Based on the known propensity of thioethers to coordinate very weakly⁴¹ or not at all⁴² axially to Ni(II), we did not expect the thioether to coordinate to the Ni center, making it necessary to swap this metal with Cu. Lastly, the unsubstituted benzyl complexes were prepared to complete the comparative study.

Complexes of the Schiff base of glycine and 2-benzyl substituted ligands based on (S)-2-(N-Benzylprolyl)-aminobenzophenone (BPB) were synthesized according to Scheme 1 starting from the aryl chloride. N-benzylation of (S)-proline provided compounds (S)-6a-c, which were amidated with 2-aminobenzophenone to give chiral ligands (S)-7a-c. Metal complexes (S)-8a-c, (S)-9a, and (S)-9c were prepared using the corresponding ligands, metal salts, and glycine to form the Schiff base complex. Starting compound 5c was prepared by alkylating,^43,44 reducing,^45,46 and chlorinating⁴⁷ thiosalicylic acid (Scheme 2).

Scheme 1.

(a) (S)-Proline, NaOMe, MeOH, 45°C, 16 hrs; (b) N-MeIm, MsCl, 2-Aminobenzophenone, DCM, 48°C 16 hrs; (c) Glycine, Ni(NO₃)₂ · 6H₂O, NaOMe, MeOH, 55°C, 90 mins; (d) Glycine, CuSO₄·5H₂O, KOH, MeOH, RT, 1 hr.

Scheme 2.

(a) Iodomethane, K₂CO₃, Acetone, RT, 2 hrs; (b) LiAlH₄, THF, RT, 3 hrs; (c) Conc. HCl, Toluene, RT, 90 mins.

Evaluation of the chiral metal complexes was performed (Table 2) using the optimal conditions that were established earlier (Table 1, entry 2). As expected, the Cu(II) containing complex (S)-9a is less diastereoselective than the analogous Ni(II) containing complex (S)-8a due to the weaker ligand-metal coordination and distortion of the coordination plane in the Cu(II) complex versus the Ni(II) complex.³⁴ Improved selectivity and yield was observed using the 2-Cl-benzyl substituted Ni(II) complex (S)-8b, providing separable diastereomers in a combined yield of 94%. Further improvement in selectivity occurred using the 2-methylthio-substituted Ni(II) complex (S)-8c, albeit in lower yield than (S)-8b. This is somewhat surprising, since we did not expect the thioether to coordinate to the nickel. In fact, the crystal structure of (S)-8c in Figure 4 shows that this is indeed the case. Here, the benzylic moiety assumes a conformation that directs the thioether away from the metal center, however we cannot rule out any Ni-thioether interaction that may be occurring in solution. At this time, the reason for the slight increase in diastereoselectivity is not yet known.

Figure 4.

Crystal structure of Ni(II) complex (S)-8c showing the non-coordinated thioether. Second symmetry unique molecule and hydrogen atoms omitted for clarity. Atoms refined anisotropically but depicted with arbitrary radii. Crystal data: C₂₈H₂₇N₃NiO₃S, monoclinic, P2₁, a = 10.428(5) Å, b = 8.062(4) Å, c = 29.581 Å, β = 96.108(8), V = 2473(2) ų, Z= 4, D_calc = 1.462 g/cm³, 32913 reflections collected, 12271 unique, R_int = 0.0825, 651 parameters, R_f = 0.0674, wR2 = 0.1302, Flack parameter = −0.028(14), CCDC 795141.

Finally, the Cu(II) complex (S)-9c outperformed all others in terms of selectivity, resulting in an 11% increase in ee compared to the known Ni complex (S)-8a. Unlike the inseparable mixture of diastereomers resulting from the Michael addition to (S)-8a, the mixture of diastereomers from the reaction of (S)-9c were easily separated by chromatography (ΔR_f = 0.1, 3:1 hexanes:acetone) to allow isolation of the desired isomer (S,S)-11c in high stereochemical purity (Table 2, entry 6).

Although we were unable to obtain a crystal structure of (S)-9c, UV spectroscopic data (see supporting information), taken together with the increase in diastereoselectivity, supports axial coordination of the methyl sulfide similar to the coordination in complexes described by Belle and coworkers.⁴⁸ A UV difference spectrum was generated using the complexes (S)-9c and (S)-9a in CH₃CN and shows maxima at 309 (δε = −211) and 422 nm (Δε = 135), a shoulder at 335 nm (Δε = −761), and a broad band at 658 nm (Δε = 24). These features in the range of 300-450 nm and at 658 nm are consistent with sulfurcopper charge transfer bands and a copper d-d transition, respectively, and are consistent with assignments made for other compounds reported in the literature.⁴⁹

In addition, axial coordination of the thioether may also be responsible for the difference in the color of the solid compounds, changing from a brown color for (S)-9a to a green color for (S)-9c. Comparing the selectivity of complexes (S)-9a and (S)-9c, the ratio of diastereomeric products increases from 3:1 to >27:1, respectively. We attribute this large difference to a more effective shielding of the coordination plane via the addition of intramolecular axial ligand coordination to the metal.

With high diastereoselectivity and facile separation methods in hand, the synthesis of (S)-1 was finished by optimizing metal complex decomposition and Fmocprotection conditions. Typically, isolation of the amino acid from the metal complex is accomplished by decomposing the compound in a heated mixture of methanol and aqueous HCl.¹⁴ However for the Gla residue, milder conditions were sought to ensure that the acid-labile tert-butyl esters would remain intact. It was found that stirring any of the complexes ((S,S)-10a-c or (S,S)-11a,c) in THF with a minimal amount of dilute aqueous HCl decomposed the complex and preserved the t-butyl protecting groups. Concentrating the reaction mixture allowed for recovery of the ligands (S)-7a-c by filtration or phase separation. To complete the synthesis of (S)-1, (S,S)-11c was decomposed, Scheme 3. The filtrate was neutralized after ligand isolation and the soluble amino acid directly Fmoc-protected. From (S,S)-11c, the yield of (S)-1 was 68% over two steps.

Scheme 3.

(i) 1.5M aq. HCl, THF, RT, 7 hrs; (ii) EDTA, Fmoc-OSu, NaHCO₃, 1:1 MeCN:H₂O, 0°C to RT, 24 hrs.

Conclusion

We have successfully completed a facile chiral synthesis of Fmoc-Gla(O^tBu)₂-OH in nine steps from thiosalicylic acid in 14.5% overall yield. We have designed the novel chiral ligand (S)-7c, whose glycine Schiff base metal complexes show increased diastereoselectivity in the Michael addition reaction of di-tert-butyl methylenemalonate over previously reported ligands. Most notably, the Cu(II) complex (S)-9c allowed us to complete a highly stereoselective route to (S)-1 in three-steps with facile separation of diastereomers. The increase in selectivity is most likely due to an intramolecular axial coordination of a thioether to the Cu(II) center of a BPB system, a design inspired by the blue copper protein rusticyanin.

Experimental Section

Synthesis of (S)-1 from metal complexes 10a-c or 11a,c

In a typical procedure, 1.5M HCl (3.33 mL, 5 mmol) was added to a solution of the Ni(II) or Cu(II) complex of γ-carboxyglutamic acid (1 mmol) dissolved in THF (13 mL). The reaction was stirred for 7 hrs or until no more metal complex was present (TLC), and was then concentrated under vacuum to 1/6 the original volume. In the case of 10a or 11a, the precipitated ligand was removed from the aqueous portion by filtration and was washed with H₂O (2×1 mL). For 10b-c and 11c, the biphasic mixture was transferred to a separatory funnel and allowed to separate. The dense oily residue was removed and the aqueous portion was diluted with 2 mL of H₂O. In both cases, the aqueous portion was transferred to a clean flask and solid NaHCO₃ (336 mg, 4 mmol) was carefully added with stirring to neutralize the solution, followed by Na₂EDTA · H₂O (372 mg, 1 mmol), and was stirred for 5 minutes. Additional solid NaHCO₃ (336 mg, 4 mmol) was added, followed by a solution of Fmoc-OSu (337 mg, 1 mmol) in CH₃CN (5 mL). The reaction was stirred for 24 hrs under Ar, concentrated in vacuo to 1/2 its original volume, adjusted to pH 3 with 10% citric acid, and extracted with EtOAc (3×15mL). Combined organic extracts were washed with brine, dried with MgSO₄, concentrated, and purified on silica gel using an automated flash chromatography system employing a gradient of acetone in hexanes; product elutes at ~20% acetone to yield 357 mg of (S)-1 (0.68 mmol, 68% over 2 steps) as a waxy solid. [α]_D²⁰ = −8.6° (c 0.29, MeOH) [lit.⁵⁰ −8.6 (c 0.173, MeOH)]; ¹H NMR (400 MHz, CDCl₃): δ = 7.77 (m, 2H, ArH-4 and ArH-4′), 7.63 (m, 2H, ArH-1 and ArH-1′), 7.41 (m, 2H, ArH-3 and ArH-3′), 7.33 (m, 2H, ArH-2 and ArH-2′), 5.61 (NH), 4.42 (m, 2H, CHCH₂O), 4.33 (m, 1H, CHCH₂O), 4.24 (m, 1H, α-H), 3.38 (m, 1H, γ-H), 2.49 (m, 1H, β-H), 2.21 (m, 1H, β’-H), 1.48 (s, 18H, C(CH₃)₃). ¹³C NMR (100 MHz, CDCl₃): δ = 173.7 (C-1), 168.5 (C-5), 168.2 (C-5′), 156.2 (C-8), 144.0 (C-11), 143.9 (C-11′), 141.3 (C-12 and C12′) 127.7 (C-14 and C-14′), 127.1 (C-13 and C-13′), 125.3 (C-15), 125.2 (C-15′), 120.0 (C-16 and C-16′), 82.0 (C-6 and C-6′), 67.1 (C-9), 52.6 (C-2), 50.9 (C-4), 47.2 (C-10), 31.2 (C-3), 27.9 (C-7 and C-7′). ESI-MS: calcd for C₂₉H₃₄NO₈Na(M+Na⁺): 548.2; found 548.2.

2-(methylthio)benzoic acid (3c)

Thiosalicylic acid (1.54 g, 10 mmol) was dissolved in acetone (81 mL) and K₂CO₃ (4.14 g, 30 mmol) was added. Iodomethane (685 μL, 11 mmol) was added dropwise and stirred for 1 hr at room temperature. After which time, solvent was removed in vacuo and the residue dissolved in water (40 mL) and cooled to 0°C. With stirring, the pH of the solution was adjusted to ~1 with 2M HCl. The precipitated solid was filtered, washed with cold H₂O, and was allowed to air dry overnight to afford 1.61 g of the acid 3c (9.6 mmol, 96%) as a white solid (m.p. 168-170°C; lit.⁵¹ m.p. 169°C). ¹H NMR (400 MHz, CD₃OD): δ = 7.94 (dd, 1H, J = 7.8, 1.5 Hz, ArH-4), 7.46 (ddd, 1H, J = 8.1, 7.3, 1.6 Hz, ArH-2), 7.32 (d, 1H, J = 8.1 Hz, ArH-1), 7.13 (m, 1H, ArH-3), 2.38 (s, 3H, SCH₃). ¹³C NMR (100 MHz, CD₃OD): δ = 168.3 (C-7), 143.3 (C-1), 132.1 (C-5), 131.1 (C-3), 127.0 (C-2), 124.1 (C-6), 122.9 (C-4), 14.0 (C-8). ESI-MS: calcd for C₈H₈O₂S (MH⁻): 167.0; found 167.0.

2-(methylthio)benzyl alcohol (4c)

Acid 3c (16.64 g, 99 mmol) was dissolved in THF (225 mL) and added dropwise via addition funnel to a slurry of LiAlH₄ (7.14 g, 178 mmol) in THF (275 mL) under an Ar atmosphere. The mixture was stirred at room temperature for 3 hrs, after which 1M HCl (370 mL) was carefully added. The aqueous mixture was extracted 3×250 mL with ether. Combined organics were washed 2×100 mL with 1M NaOH, 1×100 mL H₂O, dried with MgSO₄ and concentrated to give 13.7 g of the alcohol 4c (89.1 mmol, 90%) as an oil. ¹H NMR (400 MHz, CDCl₃): δ = 7.42 (d, 1H, J = 7.2 Hz, ArH-4), 7.30-7.23 (m, 2H, ArH-2 and ArH-1), 7.18 (ddd, 1H, J = 7.5, 5.9, 2.8 Hz, ArH-3), 4.74 (s, 2H, CH₂OH), 2.48 (s, 3H, SCH₃). ¹³C NMR (100 MHz, CDCl₃): δ = 139.1 (C-2), 136.4 (C-1), 128.0 (C-5), 127.6 (C-3), 126.2 (C-6), 125.3 (C-4), 62.9 (C-7), 15.9 (C-8). APCI-MS: 4c (C₈H₁₀OS) readily ionized to a benzylic cation through loss of OH; calcd for C₈H₉S (M⁺): 137.0; found 137.0.

2-(methylthio)benzyl chloride (5c)

Alcohol 4c (1.23 g, 8 mmol) was dissolved in toluene (2.85 mL) and cooled to 0°C. Conc. HCl (1.73 mL, 20.8 mmol) was added dropwise at 0°C and the biphasic mixture was vigorously stirred to form an emulsion. Stirring was continued for 2 hrs at room temperature. The biphasic mixture was then diluted with 6.4 mL of toluene and transferred to a separatory funnel, allowed to separate, and the aqueous portion was removed. The organic portion was washed with 3 mL H₂O and stirred with solid NaHCO₃ (0.223 g, 2.66 mmol) for 15 minutes. The toluene solution was decanted and concentrated in vacuo to give 1.23 g of the chloride 5c (7.2 mmol, 90%) as an oil. ¹H NMR (400 MHz, CDCl₃): δ = 7.38 (d, 1H, J = 7.2 Hz, ArH-4), 7.32-7.27 (m, 2H, ArH-2 and ArH-1), 7.16 (ddd, 1H, J = 7.6, 5.7, 2.9 Hz, ArH-3), 4.74 (s, 2H, CH₂Cl), 2.50 (s, 3H, SCH₃). ¹³C NMR (100 MHz, CDCl₃): δ = 138.3 (C-2), 135.5 (C-1), 130.0 (C-5), 129.2 (C-3), 127.0 (C-6), 125.5 (C-4), 44.4 (C-7), 16.3 (C-8). APCI-MS: 5c (C₈H₉ClS) readily ionized to a benzylic cation through loss of Cl; calcd for C₈H₉S (M⁺): 137.0; found 137.0.

(S)-N-(2-(methylthio)benzyl)pyrrolidine-2-carboxylic acid (6c)

In a round-bottomed flask equipped with a reflux condenser, the benzyl chloride 5c (5.14 g, 30 mmol) was added dropwise via syringe through the condensor to a stirring solution of (S)-Proline (3.45 g, 30 mmol) and NaOMe (3.24 g, 60 mmol) in MeOH (30 mL) at 48°C. After stirring the mixture overnight at 48°C, the reaction was allowed to cool to room temperature and conc. HCl (30 mmol) was added via syringe to achieve a pH of ~5. CHCl₃ (30 mL) was added and stirred for 1 hr, after which the precipitate was filtered over celite and washed with CHCl₃. The filtrate was concentrated and the residue was purified on silica gel using an automated flash chromatography system employing a shallow gradient of methanol in CH₂Cl₂; product elutes at ~20% methanol to yield 5.57 g of the acid 6c (22.2 mmol, 74%) as a waxy solid. [α]_D²⁰ = −16.9° (c 1.0, MeOH); ¹H NMR (400 MHz, CD₃OD): δ = 7.47 (d, 1H, J = 7.9 Hz, ArH-1), 7.45-7.39 (m, 2H, ArH-3 and ArH-4), 7.23 (m, 1H, ArH-2), 4.48 (s, 2H, CH₂Ph), 3.95 (dd, 1H, J = 9.3, 5.0 Hz, α-H), 3.59 (m, 1H, δ-H), 3.22 (m, 1H, δ’-H), 2.54 (s, 3H, CH₃), 2.43 (m, 1H, β’-H), 2.21-2.04 (m, 2H, β-H and γ’-H), 1.90 (m, 1H, γ-H). ¹³C NMR (100 MHz, CD₃OD): δ = 171.6 (C-1), 139.2 (C-8), 131.6 (C-12), 130.5 (C-9), 128.9 (C-7), 127.9 (C-10), 125.8 (C-11), 68.6 (C-2), 55.6 (C-6), 54.4 (C-5), 28.6 (C-3), 23.1 (C-4), 15.4 (C-13). ESI-MS: calcd for C₁₃H₁₇NO₂S (MH⁻): 250.1; found 250.1.

(S)-N-(2-benzoylphenyl)-1-(2-(methylthio)benzyl)pyrrolidine-2-carboxamide (MeS-BPB) (7c)

N-methylimidazole (1.55 mL, 19.6 mmol) was added via syringe to a solution of benzyl proline 6c (2.24 g, 8.91 mmol) in CH₂Cl₂ (22 mL) under Ar atmosphere. The solution was cooled to 0°C and methanesulfonyl chloride (693 μL, 8.91 mmol) was added dropwise via syringe, after which the solution was warmed to room temperature and a solution of 2-aminobenzophenone (1.58 g, 8.02 mmol) in CH₂Cl₂ (8 mL) was added dropwise via addition funnel. The reaction flask was fitted with a reflux condenser and heated to 45°C and stirred overnight under Ar, allowed to cool to room temperature, then quenched with sat. aq. NH₄Cl (30 mL) and extracted with CH₂Cl₂ (3×50 mL). Organic extracts were concentrated and the residue was purified on silica gel using an automated flash chromatography system employing a gradient of ethyl acetate in hexanes; product elutes at ~40% ethyl acetate to yield 2.24 g of carboxamide 7c (5.21 mmol, 65% based on 2-aminobenzophenone) as a yellow oil. [α]_D²⁰ = −120.9° (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ = 10.99 (s, NH), 8.29 (d, 1H, J = 8.3 Hz, ArH-5), 7.75 (dd, 2H, J = 8.3, 1.2 Hz, ArH-9 and ArH-9’), 7.58 (m, 1H, ArH-11), 7.53-7.43 (m, 5H, ArH-6, ArH-2, ArH-4, ArH-10, and ArH-10′), 7.17-7.03 (m, 3H, ArH-8, ArH-3, and ArH-1), 6.96 (m, 1H, ArH-7), 3.84 (d, 1H, J = 13.5 Hz, CHHPh), 3.84 (d, 1H, J = 13.5 Hz, CHHPh), 3.34 (dd, 1H, J = 10.1, 4.6 Hz, α-H), 3.22 (m, 1H, δ-H), 2.45 (m, 1H, δ’-H), 2.31 (s, 3H, CH₃), 2.23 (m, 1H, β-H), 1.86 (m, 1H, β’-H), 1.78-1.67 (m, 2H, γ-H and γ’-H). ¹³C NMR (100 MHz, CDCl₃): δ = 197.3 (C-17), 174.1 (C-1), 138.3 (C-11), 138.2 (C-18), 137.6 (C-8), 135.9 (C-7), 132.7 (C-13), 132.5 (C-21), 131.8 (C-24), 129.9 (C-19), 129.6 (C-10), 128.2 (C-20), 127.6 (C-15), 126.5 (C-16), 125.1 (C-9), 124.6 (C-14), 122.5 (C-23), 121.8 (C-12), 68.3 (C-2), 57.2 (C-6), 54.1 (C-5), 30.8 (C-3), 24.0 (C-4), 15.5 (C-22). APCI-MS: calcd for C₂₆H₂₆N₂O₂S (MH⁺): 431.2; found 431.1.

Ni(II) complex of Schiff base of (S)-MeS-BPB and glycine (MeS-BPB-Ni-Gly) (8c)

In a round-bottomed flask fitted with an addition funnel, glycine (803 mg, 10.7 mmol) and Ni(NO₃)₂ · 6H₂O (1.87 g, 6.42 mmol) were added to a solution of benzophenone 7c (2.3 g, 5.35 mmol) in MeOH (16.5 mL) and heated to 55°C. A 2.5M solution of NaOMe (4.42 g, 81.85 mmol) in MeOH was added dropwise via the addition funnel at 55°C, after which the flask was fitted with a reflux condenser and stirred for 90 min. The reaction was allowed to cool to room temperature, then poured into 30 mL of 10% citric acid and extracted with CH₂Cl₂ (3×50 mL), concentrated, and purified on silica gel using an automated flash chromatography system employing a gradient of acetone in CH₂Cl₂; product elutes at ~80% acetone to yield 2.46 g of complex 8c (4.52 mmol, 84%) as a red solid. [α]_D²⁰ = +2474.0° (c 0.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ = 8.06 (d, 1H, J = 7.4 Hz, ArH-4), 7.95 (d, 1H, J = 8.6 Hz, ArH-5) 7.57-7.48 (m, 3H, ArH-11, ArH-10, and ArH-12), 7.28 (m, 1H, ArH-2), 7.26-7.20 (m, 2H, ArH-1 and ArH-3), 7.16 (ddd, 1H, J = 8.6, 7.1, 1.3 Hz, ArH-6), 7.1 (d, 1H, J = 7.1 Hz, ArH-13), 6.97 (m, 1H, ArH-9), 6.80 (dd, 1H, J = 8.2, 1.5 Hz, ArH-8), 6.70 (m, 1H, ArH-7), 4.50 (d, 1H, J = 13.0 Hz, CHHPh), 3.80 (d, 1H, J = 13.0 Hz, CHHPh), 3.80 (d, 1H, J = 20.1 Hz, Gly α-H), 3.67 (d, 1H, J = 20.1 Hz, Gly α’-H), 3.62-3.43 (m, 3H, Pro α-H, γ-H, and δ-H), 2.78 (m, 1H, β-H), 2.56 (m, 1H, β’-H), 2.50 (s, 3H, CH₃), 2.17-2.05 (m, 2H, γ’-H and δ’-H). ¹³C NMR (100 MHz, CDCl₃): δ = 180.3 (C-1), 177.1 (C-25), 171.0 (C-17), 142.4 (C-11), 140.0 (C-27), 134.5 (C-18), 133.1 (C-8), 132.8 (C-15), 131.8 (C-13), 131.3 (C-7), 129.8 (C-10), 129.7 (C-21), 129.5 (C-20), 129.2 (C-22), 126.9 (C-26), 126.2 (C-23), 125.6 (C-19), 125.5 (C-16), 125.3 (C-9), 124.5 (C-12), 120.8 (C-14), 70.2 (C-2), 61.2 (C-24), 60.4 (C-6), 57.8 (C-5), 30.4 (C-3), 23.8 (C-4), 16.5 (C-28). HRMS-MALDI-TOF: calcd for C²⁸H₂₈N₃O₃SNi (MH⁺): 544.120; found 544.117.

Cu(II) complex of Schiff base of (S)-MeS-BPB and glycine (MeS-BPB-Cu-Gly) (9c)

Glycine (2.5 g, 33.3 mmol) and CuSO₄ · 5H₂O (3.32 g, 13.3 mmol) were added to a solution of benzophenone 7c (2.87 g, 6.67 mmol) in MeOH (24 mL) at room temperature. A 4.6 M solution of KOH (2.61 g, 46.7 mmol) in MeOH was added to the mixture in one portion. The mixture was stirred for 1 hr at room temperature, then poured into 30 mL of 10% citric acid and extracted with CH₂Cl₂ (3×50 mL), concentrated, and purified on silica gel using an automated flash chromatography system employing a gradient of acetone in CH₂Cl₂; product elutes at ~80% acetone to yield 2.50 g of complex 9c (4.56 mmol, 68%) as green a green solid. [α]_D²⁰ = −639.4° (c 0.1, CHCl₃); HRMS-MALDI-TOF: calcd for C₂₈H₂₈N₃O₃SCu (MH⁺): 549.115; found 549.117.

Preparation of 10a-c and 11a,c

In a typical procedure, the metal complex (8a-c or 9a,c, 1 eq.) was dissolved in CH₃CN and DBU (0.15 eq.) was added at room temperature. Freshly prepared di-tert-butyl methylenemalonate (1 eq.) was added dropwise via syringe and stirred for 30 min. The reaction mixture was poured into 10% citric acid, extracted with CH₂Cl₂ (3x), concentrated, and purified on silica gel using an automated flash chromatography system employing a gradient of acetone in hexanes; product elutes at ~50% acetone to yield red (10a-c), brown (11a), and green (11c) solids. Combined yield of both diastereomers is shown for 10a-c, 11a, and 11c, however only spectral data for the major diastereomer (S,S) is listed.

Ni(II) complex of Schiff base of (S)-BPB and γ-carboxyglutamic acid (BPB-Ni-Gla) (10a)

Yield 75%. ¹H NMR (400 MHz, CDCl₃): δ = 8.19 (d, 1H, J = 8.1 Hz, ArH-4), 8.06 (d, 2H, J = 7.1 Hz, ArH-1 and ArH-1′), 7.52-7.47 (m, 3H, ArH-10, ArH-11, and ArH-9), 7.34 (m, 2H, ArH-2 and ArH-2′), 7.23-7.09 (m, 4H, ArH-12, ArH-3, ArH-8, and ArH-5), 6.67-6.59 (m, 2H, ArH-6 and ArH-7), 4.41 (d, 1H, J = 12.6 Hz, CHHPh), 3.90 (m, 1H, Gla γ-H), 3.84 (dd, 1H, J = 10.4, 5.2 Hz, Gla α-H), 3.74 (m, 1H, Pro γ-H), 3.61-3.52 (m, 2H, Pro δ-H and CHHPh), 3.46 (dd, 1H, J = 10.9, 5.8 Hz, Pro α-H), 2.97 (m, 1H, Gla β-H), 2.84 (m, 1H, Pro β-H), 2.53 (m, 1H, Pro β’-H), 2.24-2.05 (m, 3H, Pro γ’-H, Gla β’-H, and Pro δ’-H), 1.39 (s, 9H, C(CH₃)₃), 1.35 (s, 9H, C(CH₃)₃′). ¹³C NMR (100 MHz, CDCl₃): δ = 180.3 (C-1), 178.1 (C-25), 171.3 (C-17), 167.9 (C-28), 167.7 (C-28′), 142.4 (C-11), 133.5 (C-15), 133.4 (C-18), 133.3 (C-7), 132.1 (C-13), 131.5 (C-8), 129.6 (C-21), 129.1 (C-20), 128.8 (C-22), 128.7 (C-9), 128.6 (C-10), 127.8 (C-23), 126.9 (C-19), 126.1 (C-16), 123.5 (C-12), 120.5 (C-14), 81.8 (C-29), 81.5 (C-29′), 70.4 (C-2), 68.8 (C-24), 63.0 (C-6), 57.3 (C-5), 49.8 (C-27), 33.6 (C-26), 30.7 (C-3), 27.8 (C-30 and C-30′), 23.7 (C-4). HRMS-MALDI-TOF: calcd for C₃₉H₄₅N₃O₇NiNa (M+Na⁺): 748.251; found 748.257.

Ni(II) complex of Schiff base of (S)-Cl-BPB and γ-carboxyglutamic acid (Cl-BPB-Ni-Gla) (10b)

Yield 94%. ¹H NMR (400 MHz, CDCl₃): δ = 8.19 (dd, 1H, J = 7.6, 1.5 Hz, ArH-4), 8.07 (d, 1H, J = 8.7 Hz, ArH-5), 7.52-7.47 (m, 3H, ArH-11, ArH-12, and ArH-10), 7.34-7.25 (m, 2H, ArH-3 and ArH-1), 7.23 (m, 1H, ArH-13), 7.18-7.08 (m, 3H, ArH-2, ArH-9, and ArH-6), 6.68-6.61 (m, 2H, ArH-7 and ArH-8), 4.45 (d, 1H, J = 12.9 Hz, CHHPh), 3.93 (m, 1H, Gla γ-H), 3.85-3.78 (m, 2H, CHHPh and Gla α-H), 3.72 (m, 1H, Pro γ-H), 3.57-3.45 (m, 2H, Pro α-H and Pro δ-H), 3.07 (m, 1H, Pro β-H), 2.93 (m, 1H, Gla β-H), 2.63 (m, 1H, Pro β’-H), 2.24 (m, 1H, Pro γ’-H), 2.15-2.04 (m, 2H, Pro δ’-H and Gla β’-H), 1.39 (s, 9H, C(CH₃)₃), 1.34 (s, 9H, C(CH₃)₃′). ¹³C NMR (100 MHz, CDCl₃): δ = 179.4 (C-1), 178.0 (C-25), 171.3 (C-17), 167.9 (C-30), 167.6 (C-30′), 142.1 (C-11), 135.6 (C-27), 133.9 (C-8), 133.4 (C-15), 133.3 (C-18), 132.1 (C-13), 131.2 (C-7), 130.4 (C-10), 130.2 (C-26), 129.6 (C-21), 129.1 (C-20), 128.7 (C-22), 127.7 (C-19), 127.0 (C-9), 126.9 (C-23), 126.4 (C-16), 123.3 (C-12), 120.6 (C-14), 81.9 (C-31), 81.5 (C-31′), 70.8 (C-2), 68.7 (C-24), 59.8 (C-6), 57.3 (C-5), 49.7 (C-29), 33.6 (C-28), 30.3 (C-3), 27.7 (C-32 and C32′), 23.5 (C-4). HRMS-MALDI-TOF: calcd for C₃₉H₄₄ClN₃O₇NiNa (M+Na⁺): 782.212; found 782.217.

Ni(II) complex of Schiff base of (S)-MeS-BPB and γ-carboxyglutamic acid (MeS-BPB-Ni-Gla) (10c)

Yield 81%. ¹H NMR (400 MHz, CDCl₃): δ = 7.90 (d, 1H, J = 7.4 Hz, ArH-4), 7.84 (d, 1H, J = 8.6 Hz, ArH-5), 7.53-7.46 (m, 3H, ArH-11, ArH-10, and ArH-12), 7.24-7.04 (m, 6H, ArH-2, ArH-9, ArH-3, ArH-1, ArH-13, and ArH-6), 6.68-6.58 (m, 2H, ArH-7 and ArH-8), 4.34 (d, 1H, J = 13.0 Hz, CHHPh), 3.89 (dd, 1H, J = 7.1, 5.5 Hz, Gla γ-H), 3.85-3.71 (m, 2H, Gla α-H and Pro γ-H), 3.65 (d, 1H, J = 13.0 Hz, CHHPh), 3.54 (dd, 1H, J = 10.7, 6.8 Hz, Pro α-H), 3.45 (dd, 1H, J = 9.8, 7.0 Hz, Pro δ-H), 3.13 (m, 1H, Pro β-H), 3.01 (m, 1H, Gla β-H), 2.67 (m, 1H, Pro β’-H), 2.45 (s, 3H, CH₃), 2.27 (m, 1H, Pro γ’-H), 2.16-2.00 (m, 2H, Gla β’-H and Pro δ’-H), 1.40 (s, 9H, C(CH₃)₃), 1.33 (s, 9H, C(CH₃)₃′). ¹³C NMR (100 MHz, CDCl₃): δ = 179.2 (C-1), 177.8 (C-25), 170.6 (C-17), 167.9 (C-31), 167.5 (C-31′), 142.3 (C-11), 139.8 (C-27), 133.2 (C-18), 133.0 (C-15), 132.9 (C-8), 131.8 (C-13), 131.2 (C-7), 129.6 (C-10), 129.5 (C-21), 129.1 (C-20), 128.6 (C-22), 127.7 (C-23), 126.8 (C-19), 126.7 (C-16), 126.5 (C-26), 125.0 (C-9), 123.7 (C-12), 120.4 (C-14), 81.8 (C-32), 81.3 (C-32′), 71.1 (C-2), 68.6 (C-24), 60.8 (C-6), 57.7 (C-5), 49.7 (C-30), 33.6 (C-29), 30.3 (C-3), 27.7 (C-33 and C33′), 23.7 (C-4), 16.3 (C-28). HRMS-MALDI-TOF: calcd for C₄₀H₄₈N₃O₇SNi (MH⁺): 772.257; found 772.259.

Cu(II) complex of Schiff base of (S)-BPB and γ-carboxyglutamic acid (BPB-Cu-Gla) (11a)

Yield 64%. HRMS-MALDI-TOF: calcd for C₃₉H₄₅N₃O₇CuNa (M+Na₊): 753.245; found 753.245.

Cu(II) complex of Schiff base of (S)-MeS-BPB and γ-carboxyglutamic acid (MeS-BPB-Cu-Gla) (11c)

Yield 70%. [α]_D²⁰ = −1322.4° (c 0.1, CHCl₃, pure (S,S) diastereomer); HRMS-MALDI-TOF: calcd for C₄₀H₄₈N₃O₇SCu (MH⁺): 777.251; found 777.247.

Abbreviations

(BPB)

(S)-2-(N-Benzylprolyl)-aminobenzophenone

(MeCN)

acetonitrile

(DBU)

1,8-Diazabicyclo[5.4.0]undec-7-ene

(DMF)

dimethylformamide

(NEt₃)

triethylamine

[NH(iPr)₂]

diisopropylamine

[NEt(iPr)₂]

diisopropylethylamine

(DCM)

dichloromethane

(N-MeIm)

N-methylimidazole

(MsCl)

methanesulfonyl chloride

(THF)

tetrahydrofuran

(EDTA)

ethylenediaminetetraacetic acid

(Fmoc-OSu)

N-(9-fluorenylmethoxycarbonyloxy) succinimide

[Fmoc-Gla(OtBu)₂-OH]

N-α-(9-fluorenylmethyloxycarbonyl)-γ-carboxy-L-glutamic-acid-γ,γ’-di-tert-butyl ester