Silicon-Containing Dipeptidic Aspartame and Neotame Analogues

Article pubs.acs.org/Organometallics

Silicon-Containing Dipeptidic Aspartame and Neotame Analogues Steffen Dörrich,† Steffen Falgner,† Sarah Schweeberg,† Christian Burschka,† Peter Brodin,‡ Britt Marie Wissing,‡ Babro Basta,‡ Peter Schell,‡ Udo Bauer,‡ and Reinhold Tacke*,† †

Institut für Anorganische Chemie, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany AstraZeneca, R&D Mölndal, Pepparedsleden 1, S-43183 Mölndal, Sweden

‡

S Supporting Information *

ABSTRACT: A series of seven silicon-containing dipeptides ((S,R)-5, (S,S)-5, (S,R)-6, (S,R)-7, (S,S)-7, (S,R)-8, and (S,S)-8) was prepared in multistep syntheses, starting from the enantiopure silicon-containing α-methylated α-amino acids (R)- or (S)-α-[(trimethylsilyl)methyl]alanine ((R)-4 or (S)-4). For the preparation of (R)-4 and (S)-4, a robust synthesis on a multigram scale was developed, using an enzymecatalyzed stereoselective ester hydrolysis as the key step. Coupling of (R)-4 and (S)-4 with different S-configured aspartate residues yielded a series of dipeptides that can be best described as silicon analogues of the artificial sweeteners aspartame and neotame. The identity of these dipeptides was established by elemental analyses and NMR spectroscopic studies (1H, 13C, 15N, 29 Si). Some of the title compounds and some of their precursors were structurally characterized by single-crystal X-ray diffraction. The silicon-containing dipeptides were shown to display attractive ADME properties, including good solubility and plasma protein binding capabilities, no CYP inhibition, and no metabolic stability liabilities. Thus, the silicon-containing α-amino acids (R)-4 and (S)-4 proved to be promising building blocks for the design of biologically active peptides. Attempts to characterize the title compounds for their T1R2/R3 activating properties by testing their ability to induce GLP-1 secretion from the intestinal endocrine cell line STC-1 failed for unknown reasons. Sensory evaluation of the silicon-containing dipeptides demonstrated the aspartame analogue (S,R)-5 and the neotame analogue (S,R)-8 to be very potent artificial sweeteners, whereas the corresponding diastereomers tasted bitter. The silicon compounds (S,R)-5 and (S,R)-8 are approximately 50 and 600 times, respectively, as sweet as sucrose on a weight basis.

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INTRODUCTION In light of our systematic investigations on silicon-containing αamino acids1 and their application in the synthesis of siliconcontaining peptidic drugs,1b,c and inspired by Weidmann’s finding that phenylalanine (1) and β-(trimethylsilyl)alanine (2) can behave as bioisosteric building blocks in biologically active peptides,2 we have been interested in the design of siliconbased dipeptidic sweeteners (Scheme 1) (for recent reviews dealing with silicon-containing α-amino acids and peptides, see ref 3). Previous work, in which α-methyl-phenylalanine (3) was used for the development of artificial sweeteners,4 inspired us to incorporate the α-methylated α-amino acids (R)- and (S)-α[(trimethylsilyl)methyl]alanine ((R)-4 and (S)-4)1h,5 into several dipeptides (5−8). Keeping in mind that the commercial artificial dipeptidic sweeteners aspartame ((S,S)-9) and neotame ((S,S)-10) and the related sweetener (S,S)-11 (Scheme 1) are built up from (S)-aspartic acid and (S)-phenylalanine (or (S)-α-methyl-phenylalanine), the analogous silicon-containing dipeptides (S,R)-5, (S,R)-6, (S,R)-7, and (S,R)-8 were regarded as potential sweeteners.5 Linear biologically active peptides that are built up from natural α-amino acids show a high conformational flexibility and therefore adopt a multitude of different conformations in solution, although only a few of these represent a bioactive conformation. However, incorporation of nonproteinogenic α-methylated α-amino acids leads to conformationally restricted © 2012 American Chemical Society

peptides, which can serve as valuable tools in the search for both the preferred bioactive conformation and the active site of the receptor.6 This approach has also been used for the development of the theory of sweet taste.7 With these points in mind, the incorporation of the silicon-containing α-methylated α-amino acids (R)-4 and (S)-4 into the dipeptides 5−8 offered great scope to further our understanding of taste mechanisms. The design of the target dipeptides presented herein takes into account that the absolute configuration of the aspartate residue (S configuration) is important for the sweet taste of dipeptidic sweeteners.4d Thus, the aspartame analogues 5 and 6 and the neotame analogues 7 and 8 synthesized in this study contain S-configured aspartate residues. We report here on the synthesis of the silicon-based dipeptides (S,R)-5, (S,S)-5, (S,R)6, (S,R)-7, (S,S)-7, (S,R)-8, and (S,S)-8 and the evaluation of their ADME profiles. In addition, we report on our (failed) attempts to evaluate the sweet-tasting properties of these dipeptides by testing their ability to induce GLP-1 secretion from the mouse intestinal endocrine cell line STC-1. Furthermore, the results of sensory evaluations of compounds (S,R)-5, (S,S)-5, (S,R)-7, (S,S)-7, (S,R)-8, and (S,S)-8 are described. The studies Special Issue: Organometallics in Biology and Medicine Received: May 22, 2012 Published: July 25, 2012 5903

dx.doi.org/10.1021/om300442e | Organometallics 2012, 31, 5903−5917

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Scheme 1. Structural Formulas of the α-Amino Acids 1−4, the Target Dipeptides 5−8, and the Artificial Sweeteners Aspartame (9), Neotame (10), and 11

Scheme 2. Modified Syntheses of the Enantiomerically Enriched Intermediates (R)-13 and (S)-14 and the Enantiopure Silicon-Containing α-Amino Acids (R)-4 and (S)-4

reported in this paper represent an extension of our ongoing investigations on silicon-containing drugs8,9 and odorants.10,11

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RESULTS AND DISCUSSION Syntheses. The silicon-based aspartame and neotame analogues (S,R)-5, (S,S)-5, (S,R)-6, (S,R)-7, (S,S)-7, (S,R)-8, and (S,S)-8 were synthesized, starting from the siliconcontaining α-amino acids (R)-4 and (S)-4. The enantioselective syntheses of (R)-4 and (S)-4 have already been reported elsewhere;1h however, the scale-up of these syntheses required several modifications (Scheme 2). The enzyme-catalyzed preparation of the key intermediate in the synthesis of (R)-4 and (S)-4, (R)-3-ethoxy-2-methyl-3-oxo2-[(trimethylsilyl)methyl]propanoic acid ((R)-13), was performed on the 100 g scale to give (R)-13 in 97% yield. For reasons not yet fully understood, the enantiomeric excess of (R)-13 was only 75% ee (compared to 85% ee reported in ref 1h); however, this problem could be overcome by appropriate modifications to the subsequent transformations (R)-13 → (R)-4 and (R)-13 → (S)-14 → (S)-4 (Scheme 2). The enantiomeric excess of (R)-4 was increased stepwise from 75% ee to 90% ee and finally to >99% ee through 2-fold crystallization of (R)-4 from methanol/water (16% yield). As the synthesis of (S)-14 and its subsequent crystallization from n-hexane led to an increase of the enantiomeric excess from 75% to 95% ee (30% yield), a much higher yield (56% (relative to (S)-14) or 17% (relative to (R)-13)) was obtained in the subsequent transformations of (S)-14 into (S)-4 (compared to (R)-13 → (R)-4; for details, see the Experimental Section). Both silicon-containing α-amino acids were obtained with an enantiomeric excess of >99% ee. The silicon-based aspartame analogues (S,R)-5, (S,S)-5, and (S,R)-6 were synthesized according to Schemes 3 and 4 and were isolated as the corresponding ammonium trifluoroacetates (S,R)-5·TFA, (S,S)-5·TFA, and (S,R)-6·TFA (TFA = trifluoroacetic acid). In the first step, compounds (R)-4 and (S)-4 were treated with thionyl chloride in methanol (or ethanol) to give

Scheme 3. Syntheses of the Silicon-Based Aspartame Analogues (S,R)-5·TFA and (S,R)-6·TFA

the hydrochlorides of the corresponding methyl (or ethyl) esters (R)-15, (S)-15, and (R)-16 (yields: (R)-15·HCl, 99%; (S)-15·HCl, 99%; (R)-16·HCl, 99%). Coupling of (R)-15·HCl, (S)-15·HCl, and (R)-16·HCl with N-tert-butoxycarbonyl-4-tertbutoxy-(S)-aspartic acid ((S)-17) using N-methylmorpholine as the auxiliary base and O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate (HCTU, 18) as the coupling reagent yielded the protected dipeptidic intermediates (S,R)-19 (86% yield), (S,S)-19 (98%), and (S,R)-20 (98%), respectively. Removal of the tert-butoxycarbonyl (Boc) and tert-butyl protecting groups by treatment with an excess of trifluoroacetic 5904


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the tert-butoxycarbonyl (Boc) protecting group was accomplished by using a mixture of chlorotrimethylsilane and phenol,12 without affecting the benzyl ester group, to furnish the N-deprotected dipeptides (S,R)-23 (70% yield) and (S,S)23 (90%). Reaction of (S,R)-23 and (S,S)-23 with 2,2dimethylpropanal (24), in the presence of magnesium sulfate, yielded the N-2,2-dimethylpropylidene-substituted derivatives (S,R)-25 and (S,S)-25, respectively. The corresponding N-3,3butylidene analogues (S,R)-26 and (S,S)-26 were synthesized analogously by treating (S,R)-23 or (S,S)-23 with 3,3dimethylbutanal (27). Compounds (S,R)-25, (S,S)-25, (S,R)26, and (S,S)-26 were isolated as crude products, followed by removal of the benzyl (Bn) protecting group and hydrogenation of the imine moiety in a single step by reaction with hydrogen in the presence of palladium/carbon as the catalyst to give the target compounds (S,R)-7 (95% yield), (S,S)-7 (89%), (S,R)-8 (92%), and (S,S)-8 (93%) (the yields are reported relative to the respective intermediates (S,R)-23 and (S,S)-23). The identities of the silicon-based aspartame and neotame analogues (S,R)-5·TFA, (S,S)-5·TFA, (S,R)-6·TFA, (S,R)-7, (S,S)-7, (S,R)-8, and (S,S)-8 and the intermediates (R)-15·HCl, (S)-15·HCl, (R)-16·HCl, (S,R)-19, (S,S)-19, (S,R)-20, (S,R)22, (S,S)-22, (S,R)-23, and (S,S)-23 were established by NMR spectroscopic studies ( 1 H, 13 C, 15 N, 19 F, 29 Si), mass spectrometric investigations, and elemental analyses (C, H, N) or high-resolution mass spectrometry. In addition, compounds (S,R)-5·2H2O, (S,R)-5·TFA, (S,R)-7·H2O, (S,R)-8·2.5H2O, (R)-15·HCl, (S,R)-19·0.22H2O, (S,R)-20, (S,R)-22, and (S,S)-22 were characterized by crystal structure analyses. Determination of the Enantiomeric and Diastereomeric Excesses. The enantiomeric excess of (R)-4 and (S)-4

Scheme 4. Synthesis of the Silicon-Based Aspartame Analogue (S,S)-5·TFA

acid afforded the target compounds (S,R)-5·TFA (54% yield), (S,S)-5·TFA (89%), and (S,R)-6·TFA (50%). The silicon-based neotame analogues (S,R)-7, (S,S)-7, (S,R)8, and (S,S)-8 were synthesized according to Schemes 5 and 6, starting from (R)-15·HCl or (S)-15·HCl. Coupling of (R)15·HCl and (S)-15·HCl with N-tert-butoxycarbonyl-4-benzyloxy-(S)-aspartic acid ((S)-21) using N-methylmorpholine as the auxiliary base and 18 as the coupling reagent yielded the protected dipeptidic intermediates (S,R)-22 (97% yield) and (S,S)-22 (98%), respectively. Subsequent selective removal of

Scheme 5. Syntheses of the Silicon-Based Neotame Analogues (S,R)-7 and (S,R)-8

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Scheme 6. Syntheses of the Silicon-Based Neotame Analogues (S,S)-7 and (S,S)-8

excess of the silicon-containing α-amino acids (R)-4 and (S)-4 was then accomplished by their derivatization according to Scheme 7 and subsequent GC-MS studies of the analytical samples obtained, using the 1/1 mixture of (R,R)-29 and (R,S)29 as the reference sample (for further details, see the Experimental Section and the Supporting Information). According to these studies, compounds (R)-4 and (S)-4 were obtained with an enantiomeric excess of >99% ee. As the purchased aspartic acid was enantiomerically pure and all steps in the synthesis of the target dipeptides should not affect the absolute configuration of the stereogenic centers, an enantiomeric excess of >99% ee and a diastereomeric excess of >99% de of the target compounds and their intermediates can be assumed. This assumption is further supported by the NMR spectra of the diastereomeric compounds, which did not indicate any epimerization at the stereogenic centers. Crystal Structure Analyses. Compounds (S,R)-5·2H2O, (S,R)-5·TFA, (S,R)-7·H2O, (S,R)-8·2.5H2O, (R)-15·HCl, (S,R)-19·0.22H2O, (S,R)-20, (S,R)-22, and (S,S)-22 were structurally characterized by single-crystal X-ray diffraction. The crystal data and the experimental parameters used for the crystal structure analyses are given in the Supporting Information. The molecular (zwitterionic) structures of (S,R)5, (S,R)-7, and (S,R)-8 in the crystal of the respective hydrates and the structure of the cation of (S,R)-5·TFA are depicted in Figures 1−4. All the bond lengths and angles of the compounds studied are in the expected ranges and do not need any further discussion, except for the Si−C−C angles (119.75(7)−123.81(14)°), which differ significantly from the ideal tetrahedral angle. The same phenomenon has also been observed for the crystal

was determined, after Mosher-type derivatization (Scheme 7),13 by GC-MS studies and NMR spectroscopic measurements. For Scheme 7. Syntheses of the Diastereomers (R,R)-29 and (R,S)-29, Starting from rac-4 (→(R,R)-29/(R,S)-29, 1/1 Mixture), (R)-4 (→(R,R)-29), and (S)-4 (→(R,S)-29)

this purpose, rac-15·HCl (obtained from rac-4 analogously to the synthesis of (R)-15·HCl and (S)-15·HCl) was treated with (S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanonyl chloride ((S)-28) to afford a 1:1 mixture of the two reference compounds (R,R)-29 and (R,S)-29 (yield 84%). Their identities were established by elemental analyses (C, H, N), NMR spectroscopic studies (1H, 13C, 15N, 19F, 29Si), and mass spectrometric investigations (GC/EI-MS). The determination of the enantiomeric 5906


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Figure 4. Molecular (zwitterionic) structure of (S,R)-8 in the crystal of (S,R)-8·2.5H2O.

Figure 1. Comparison of the molecular (zwitterionic) structure of (S,R)-5 in the crystal of (S,R)-5·2H2O (left) with that of (S,S)-1117 in the crystal of (S,S)-11·2H2O (right).

the Si−C−C bond angle appears to be an intrinsic structural feature of silicon-containing amino acids of this particular formula type. As can be seen from Figures 1, 2, and 4, the dipeptides (S,R)5, (S,R)-7, and (S,R)-8 adopt a zwitterionic structure in the crystal. In these structures, the charged ammonium and carboxylate groups of the zwitterions are close together and form a “zwitterionic ring” in the aspartate residue, with intramolecular N−H···O hydrogen bonds.14 The N···O distances observed for (S,R)-5 (2.938(4) Å), (S,R)-7 (2.5674(11) Å), and (S,R)-8 (2.6619(8) Å) are similar to those found in the crystal structures of aspartame ((S,S)-9; 2.987 Å),15 its α,α-dialkylated analogue (S,S)-11 (3.056 Å),4b and neotame ((S,S)-10; 3.187 Å).16 Also, the C terminus of the silicon-containing dipeptides shows structural similarities with their corresponding carbon analogues (SiMe3/Ph exchange) in the solid state. This is shown for the structures of (S,R)-5 and (S,S)-11 in Figure 1.17 Biological Studies. The silicon-containing aspartame and neotame analogues were profiled for their ADME properties using neotame as a reference compound. All compounds were investigated for their solubility in HBSS buffer (pH 7.4) and their octananol/water (pH 7.4) distribution coefficient (log D value). Furthermore, they were studied for their human plasma protein binding (hPPB) capability and their cytochrome P450 (CYP) inhibition using the two isoforms CYP3A4 and CYP2D6. To determine the rate of decomposition in vitro, the intrinsic clearance (CLint) and the half-life (t1/2) of the test compounds were measured in human liver microsomes. As shown in Table 1, in reference to neotame no significant impact on the ADME properties of the test compounds was observed. Overall, the silicon-containing dipepetidic aspartame and neotame analogues studied showed an attractive property profile, including good solubility and plasma protein binding capabilities, no CYP inhibition, and no metabolic stability liabilities. Thus, the silicon-containing α-amino acids (R)-4 and (S)-4 proved to be promising building blocks for the design and development of biologically active peptides. To get some preliminary information about their T1R2/R3 activating and thereby their sweet-tasting properties, the title compounds as well as aspartame and neotame were tested for their ability to induce GLP-1 secretion from the mouse intestinal endocrine cell line STC-1.18 This cell line can be activated19,20

Figure 2. Molecular structure of the cation in the crystal of (S,R)5·TFA.

Figure 3. Molecular (zwitterionic) structure of (S,R)-7 in the crystal of (S,R)-7·H2O.

structures of rac-β-(trimethylsilyl)alanine (2)1g and (R)α-[(trimethylsilyl)methyl]alanine (4).1h Thus, the extension of 5907


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Table 1. ADME Properties of (S,R)-5, (S,S)-5, (S,R)-6, (S,R)-7, (S,S)-7, (S,R)-8, (S,S)-8, and Neotame IC50 (μM) compd a

(S,R)-5 (S,S)-5b (S,R)-6 (S,R)-7 (S,S)-7 (S,R)-8 (S,S)-8 neotame a

solubility (μM)

log D

hPPB (% free)

CYP3A4

CYP2D6

CLint (μL min−1 mg−1)

t1/2 (min)

36 61 77 96 97 89 89 91

0.3 0.3 0.8 2.4 2.3 2.8 2.7 1.8

nd nd 72 20 16 31 27 17

>20 >20 >20 >20 >20 >20 >20 >20

>20 >20 >20 >20 >20 >20 >20 >20

139

Studied as (S,R)-5·TFA. bStudied as (S,S)-5·TFA.

and (S,R)-8 showed lower sweetness intensities than their respective analogues (S,S)-9 (aspartame), (S,S)-10 (neotame), and (S,S)-11, they are very potent artificial sweeteners.

and release GLP-1 after treatment with sweet- and bitter-tasting compounds. The artificial sweetener sucralose and phenylthiocarbamide (PTC), a bitter taste receptor (T2R) agonist, were used as positive controls and resulted in GLP-1 release as measured by ELISA. However, treatment with the other test compounds, including neotame and aspartame, at concentrations up to 2 mM did not result in any significant increase in GLP-1 production. A possible reason for this is species variation in the T1R2/R3 sweet taste receptors. STC-1 is a cell line of murine origin, and it is known that neither aspartame nor neotame are active at the mouse sweet taste receptor.21 Further attempts using a human entero endocrine cell line such as NCI-H716, which is known to secrete GLP-1, failed to show GLP-1 secretion properties upon compound exposure for unknown reasons.22 Sensory Evaluation. The silicon-containing dipeptides (S,R)-5, (S,S)-5, (S,R)-8, and (S,S)-8 were taste-checked by two volunteers (for details, see the Experimental Section). Compounds (S,R)-5 (aspartame analogue) and (S,R)-8 (neotame analogue) were found to be approximately 50 and 600 times, respectively, as sweet as sucrose on a weight basis, whereas the corresponding diastereomers (S,S)-5 and (S,S)-8 tasted bitter (Table 2). This is in good agreement with the

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CONCLUSION In this study, we have succeeded in scaling up the synthesis of the enantiopure silicon-containing α-methylated α-amino acids (R)- and (S)-α-[(trimethyl)silyl)methyl]alanine ((R)-4 and (S)-4). Coupling of (R)-4 and (S)-4 with different S-configured aspartate residues yielded a series of silicon-containing dipeptidic aspartame and neotame analogues. These dipeptides showed attractive ADME property profiles, including good solubility and plasma protein binding capabilities, no CYP inhibition, and no metabolic stability liabilities. Thus, the silicon-containing α-amino acids proved to be promising building blocks for the design of biologically active peptides. Attempts to characterize the title compounds for their T1R2/ R3 activating properties by testing their ability to induce GLP-1 secretion from the mouse intestinal endocrine cell line STC-1 failed for unknown reasons (no significant increase in GLP-1 secretion up to a concentration of 2 mM). Sensory evaluation of the silicon-containing dipeptides (S,R)-5, (S,S)-5, (S,R)-8, and (S,S)-8 demonstrated the aspartame analogue (S,R)-5 and the neotame analogue (S,R)-8 to be very potent artificial sweeteners, whereas the corresponding diastereomers (S,S)-5 and (S,S)-8 tasted bitter. The silicon compounds (S,R)-5 and (S,R)-8 are approximately 50 and 600 times, respectively, as sweet as sucrose on a weight basis.

Table 2. Taste and Sweetness Intensities of (S,R)-5, (S,S)-5, (S,R)-8, (S,S)-8, (S,S)-9, (S,R)-9, (S,S)-10, (S,R)-10, (S,S)11, and (S,R)-11 compd

tastea

(S,R)-5b (S,S)-5c (S,R)-8 (S,S)-8 (S,S)-9 (S,R)-9 (S,S)-10 (S,R)-10 (S,S)-11 (S,R)-11

sweet (50) bitter sweet (600) bitter sweet (100−200)d bitterd sweet (8000−10000)e not sweetf sweet (200)g bitterg

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EXPERIMENTAL SECTION

Chemistry: General. All syntheses in organic solvents were carried out under dry argon. The organic solvents used were dried and purified according to standard procedures and stored under dry argon. Starting materials and reagents were purchased from Sigma-Aldrich (Schnelldorf, Germany), ABCR (Karlsruhe, Germany), or Acros (Geel, Belgium) and were used without further purification. The enzyme (porcine liver esterase, 165 U mg−1, EC 3.1.1.1) was purchased from Fluka (Buchs, Switzerland). Reversed-phase medium-pressure liquid chromatography (RP-MPLC) was performed as follows: pressure, 16 bar; column, 50 × 2.5 cm; RP-18 silica gel, YMC ODS-A, 15 μm; detector, Knauer Variable Wavelength Monitor. Bulb-to-bulb distillations were accomplished by using a Büchi GKR-50 Glass Oven apparatus. Lyophilization was carried out with a Christ alpha 1-2 apparatus. Enzymatic reactions were performed by using a Schott TitroLine alpha titrator with pH-stat function. Melting points were determined with a Büchi Melting Point B-540 apparatus using samples in open glass capillaries or by differential scanning calorimetry using a Mettler Toledo DSC 823e apparatus. The 1H, 13C, 15N, 19F, and 29Si NMR spectra were recorded at 23 °C, unless otherwise stated, on a Bruker Avance 400 (1H, 400.1 MHz; 19F, 376.5 MHz) or a Bruker

a

Sweetness intensities (in parentheses) refer to sucrose as 1. bStudied as (S,R)-5·TFA. cStudied as (S,S)-5·TFA. dReference 23. eReference 24. fReference 24b. gReference 4b.

known impact of the absolute configuration on the taste properties of the related dipeptides (S,S)-9, (S,R)-9, (S,S)-10, (S,R)-10, (S,S)-11, and (S,R)-11 (Table 2). Thus, the amino acids (R)-4 and (S)-phenylalanine as well as (S)-4 and (R)phenylalanine behave as bioisosteric building blocks in these dipeptides. Although the silicon-containing dipeptides (S,R)-5 5908


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was dissolved in hydrochloric acid (6 M, 700 mL) at 20 °C, and the resulting mixture was heated under reflux for 1 h. The solvent was removed under reduced pressure, the solid residue was dissolved in water (40 mL), and the resulting solution was freeze-dried to furnish a colorless powder ((R)-4·HCl; 5.08 g; crude product, not further purified), which was dissolved in a mixture of methanol (95 mL) and an aqueous sodium hydroxide solution (1 M, 22.5 mL; 22.5 mmol of NaOH).26 The solution was kept undisturbed at 20 °C for 4 days, and the resulting precipitate was separated by suction filtration and washed with diethyl ether (2 × 5 mL) to furnish (R)-4 (2.93 g, 16.7 mmol; 16% yield; enantiomeric excess, >99% ee) as a colorless crystalline solid. The NMR data of the product (solvent, D2O) were identical with those reported in ref 1h. Synthesis of (S)-2-Amino-2-methyl-3-(trimethylsilyl)propanoic Acid ((S)-α-[(Trimethylsilyl)methyl]alanine, (S)-4). This compound was synthesized according to ref 1h. A solution of thionyl chloride (2.74 g, 23.0 mmol) in dichloromethane (25 mL) was added dropwise at 0 °C within 15 min to a stirred solution of (S)-14 (5.00 g, 19.2 mmol; enantiomeric excess, 95% ee) in a mixture of dichloromethane (25 mL) and N,N-dimethylformamide (50 μL). The resulting mixture was warmed to 20 °C and was then stirred at this temperature for 15 h. The solvent and the excess thionyl chloride were removed under reduced pressure (20 °C, 0.02 mbar), the residue was dissolved in acetone (20 mL), and the resulting solution was added dropwise at 0 °C within 10 min to a stirred suspension of sodium azide (1.37 g, 21.1 mmol) in acetone (30 mL). The mixture was warmed to 20 °C and was then stirred at this temperature for 3 h. The resulting precipitate was separated by suction filtration, washed with acetone (3 × 40 mL), and discarded. The filtrate (including the wash solutions) was heated under reflux for 16 h, the mixture was cooled to 20 °C, and the solvent was removed under reduced pressure. Subsequently, hydrochloric acid (6 M, 300 mL) was added to the residue in a single portion at 20 °C, and the resulting mixture was heated under reflux for 16 h. The solvent was removed under reduced pressure, the solid residue was dissolved in water (400 mL), and the resulting aqueous solution was extracted continuously with diethyl ether (500 mL) over a period of 1 day using a perforator, and the extract was discarded. The remaining aqueous phase was freeze-dried to furnish a colorless powder ((S)-4·HCl; 3.30 g; crude product, not further purified), which was dissolved in a mixture of methanol (36 mL) and an aqueous sodium hydroxide solution (1 M, 14.6 mL; 14.6 mmol of NaOH).26 The solution was kept undisturbed at 10 °C for 1 day, and the resulting precipitate was separated by suction filtration and washed with diethyl ether (2 × 5 mL) to furnish (S)-4 (1.89 g, 10.8 mmol; 56% yield; enantiomeric excess, >99% ee) as a colorless crystalline solid. The NMR data of the product (solvent, D2O) were identical with those reported in ref 1h. Synthesis of (S)-Aspart-1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Methyl Ester Hydrotrifluoroacetate ((S,R)-5·TFA). Trifluoroacetic acid (4.45 g, 39.0 mmol) was added in a single portion at 20 °C to (S,R)-19 (242 mg, 525 μmol), and the resulting solution was stirred at 20 °C for 20 min. Subsequently, the volatile components were removed under reduced pressure, and water (150 mL) was added to the residue. The aqueous phase was extracted continuously with diethyl ether (400 mL) over a period of 8 h using a perforator, and the extract was discarded. The remaining aqueous solution was freed of water under reduced pressure, the solid residue was crystallized from water (500 μL; cooling of a boiling solution to 20 °C and crystallization over a period of 2 days), and the resulting crystals were washed with n-heptane (−20 °C, 500 μL). Recrystallization from water/methanol (10/1 v/v, 500 μL; cooling of a boiling solution to 20 °C and crystallization over a period of 2 days) and washing of the resulting crystals with n-heptane (−20 °C, 500 μL) furnished (S,R)-5·TFA (119 mg, 284 μmol; 54% yield) as a colorless crystalline solid. Mp: 219 °C dec. 1H NMR (400.1 MHz, [D6]DMSO): δ 0.00 (s, 9 H; Si(CH3)3), 1.18 (δA) and 1.20 (δB) (AB system, 2J(A,B) = 13.7 Hz, 2 H; SiCHAHB), 1.44 (s, 3 H; CCH3), 2.41 (δA), 2.61 (δB), and 3.79 (δX) (ABX system, 2J(A,B) = 17.0 Hz, 3 J(A,X) = 9.1 Hz, 3J(B,X) = 4.1 Hz, 3 H; CHAHBCHX), 3.55 (s, 3 H; OCH3), 8.6 ppm (br s, 1 H; C(O)NH), NH3 and C(O)OH not

Avance 500 NMR spectrometer (1H, 500.1 MHz; 13C, 125.8 MHz; 15 N, 50.7 MHz; 29Si, 99.4 MHz) using [D6]DMSO, C6D6, CD3OH, CD3OD, or CD2Cl2 as the solvent. Chemical shifts (ppm) were determined relative to internal [D5]DMSO (1H, δ 2.49 ppm; [D6]DMSO), internal C6HD5 (1H, δ 7.28 ppm; C6D6), internal CHD2OH (1H, δ 3.30 ppm; CD3OH), internal CHD2OD (1H, δ 3.30 ppm; CD3OD), internal CHDCl2 (1H, δ 5.32 ppm; CD2Cl2), internal [D6]DMSO (13C, δ 39.5 ppm; [D6]DMSO), internal C6D6 (13C, δ 128.0 ppm; C6D6), internal CD3OH (13C, δ 49.0 ppm; CD3OH), internal CD3OD (13C, δ 49.0 ppm; CD3OD), internal CD2Cl2 (13C, δ 53.8 ppm; CD2Cl2), external H2NC(O)H (90% in [D6]DMSO) (15N, δ −268.0 ppm; [D6]DMSO, C6D6, CD3OH, CD3OD, CD2Cl2), external CFCl3 (19F, δ 0 ppm; [D6]DMSO, C6D6, CD3OD), or external TMS (29Si, δ 0 ppm; [D6]DMSO, C6D6, CD3OH, CD3OD, CD2Cl2). Analysis and assignment of the 1H and 13C NMR spectroscopic data was supported by 1H,1H gradient selected COSY along with 13C,1H gradient selected HMQC and HMBC experiments. Assignment of the 13C NMR spectroscopic data was additionally supported by DEPT 135 experiments. The 15N NMR spectra were obtained by using inverse correlation 15N,1H HMQC and HMBC experiments. The spin systems were analyzed by using the WINDAISY software package (version 4.05, Bruker).25 Coupling constants are given as their absolute values. The assignment of the NMR spectroscopic data does not take into account H/D exchange processes when using CD3OD as the solvent. In addition, the assignment of the NMR spectroscopic data does not take into account that compounds (S,R)-7, (S,S)-7, (S,R)-8, and (S,S)-8 can adopt a zwitterionic structure in solution. EI-MS spectra (70 eV) and CI-MS spectra (reactant gas, methane) were recorded on a Varian 320-MS SQ mass spectrometer. HRMS spectra were recorded on a Bruker MicrOTOF mass spectrometer using solutions in methanol (ESI). Elemental analyses were performed by using a VarioMicro apparatus (Elementar Analysensysteme GmbH) or a EURO EA Elemental Analyzer (EuroVector). Synthesis of rac-2-Amino-2-methyl-3-(trimethylsilyl)propanoic Acid (rac-α-[(Trimethylsilyl)methyl]alanine, rac-4). This compound was synthesized according to ref 1h. Synthesis of (R)-2-Amino-2-methyl-3-(trimethylsilyl)propanoic Acid ((R)-α-[(Trimethylsilyl)methyl]alanine, (R)-4). This compound was synthesized according to ref 1h. A solution of thionyl chloride (14.6 g, 123 mmol) in dichloromethane (50 mL) was added dropwise at 0 °C within 15 min to a stirred solution of (R)-13 (23.7 g, 102 mmol; enantiomeric excess, 75% ee) in a mixture of dichloromethane (150 mL) and N,N-dimethylformamide (200 μL). The resulting mixture was warmed to 20 °C and was then stirred at this temperature for 18 h. The solvent and the excess thionyl chloride were removed under reduced pressure (20 °C, 0.02 mbar), the residue was dissolved in acetone (200 mL), and the resulting solution was added dropwise at 0 °C within 10 min to a stirred suspension of sodium azide (7.28 g, 112 mmol) in acetone (150 mL). The mixture was warmed to 20 °C and was then stirred at this temperature for 3 h. The resulting precipitate was separated by suction filtration, washed with acetone (3 × 40 mL), and discarded. The filtrate (including the wash solutions) was heated under reflux for 16 h, the mixture was cooled to 20 °C, and the solvent was removed under reduced pressure. Subsequently, hydrochloric acid (6 M, 700 mL) was added to the residue in a single portion at 20 °C, and the resulting mixture was heated under reflux for 6 h. The solvent was removed under reduced pressure, the solid residue was dissolved in water (500 mL), and the resulting aqueous solution was extracted with diethyl ether (3 × 100 mL) and then freed of water under reduced pressure. The solid residue was dissolved in water (250 mL), and the resulting solution was freezedried to furnish a colorless powder ((R)-4·HCl; 17.3 g; crude product, not further purified), which was dissolved in a mixture of methanol (324 mL) and an aqueous sodium hydroxide solution (1 M, 76.6 mL; 76.6 mmol of NaOH).26 The solution was kept undisturbed at 20 °C for 7 days, and the resulting precipitate was separated by suction filtration and washed with diethyl ether (3 × 20 mL) to furnish (R)-4 (3.96 g, 22.6 mmol; 22% yield; enantiomeric excess, 90% ee) as a colorless crystalline solid. To increase the enantiomeric excess, compound (R)-4 (3.96 g, 22.6 mmol; enantiomeric excess, 90% ee) 5909


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detected. 13C NMR (125.8 MHz, CD3OH): δ −0.4 (Si(CH3)3), 25.1 (CCH3), 29.2 (SiCH2), 36.3 (CH2CH), 51.1 (CH2CH), 52.7 (OCH3), 60.0 (CCH3), 168.3 (C(O)NH), 173.1 (C(O)OH), 175.5 ppm (C(O)OCH3), CF3 and CF3C not detected. 15N NMR (50.7 MHz, CD3OH): δ −346.8 (NH3), −245.7 ppm (C(O)NH). 19F NMR (376.5 MHz, [D6]DMSO): δ −73.4 ppm. 29Si NMR (99.4 MHz, CD3OH): δ −1.0 ppm. Anal. Calcd for C14H25F3N2O7Si: C, 40.19; H, 6.02; N, 6.69. Found: C, 40.17; H, 6.28; N, 6.79. Synthesis of (S)-Aspart-1-yl-(S)-2-[(trimethylsilyl)methyl]alanine Methyl Ester Hydrotrifluoroacetate ((S,S)-5·TFA). Trifluoroacetic acid (4.45 g, 39.0 mmol) was added in a single portion at 20 °C to (S,S)-19 (250 mg, 543 μmol), and the resulting solution was stirred at 20 °C for 20 min. Subsequently, the volatile components were removed under reduced pressure, and water (40 mL) was added to the residue. The aqueous phase was extracted with diethyl ether (3 × 15 mL), the extracts were discarded, and the remaining aqueous solution was freed of water under reduced pressure. The solid residue was dissolved in methanol/water (7/3 v/ v, 1 mL) and was further purified by RP-MPLC (eluent, methanol/ water (7/3 v/v); flow rate, 15 mL min−1; detector wavelength, 210 nm). The resulting product was dissolved in water (12 mL), and the solution was freeze-dried to furnish (S,S)-5·TFA (202 mg, 483 mmol; 89% yield) as a colorless solid. Mp: 250 °C dec. 1H NMR (500.1 MHz, [D6]DMSO): δ 0.01 (s, 9 H; Si(CH3)3), 1.20 (δA) and 1.28 (δB) (AB system, 2J(A,B) = 14.5 Hz, 2 H; SiCHAHB), 1.39 (s, 3 H; CCH3), 2.64 (δA), 2.74 (δB), and 3.91 (δX) (ABX system, 2J(A,B) = 17.5 Hz, 3 J(A,X) = 8.5 Hz, 3J(B,X) = 3.8 Hz, 3 H; CHAHBCHX), 3.56 (s, 3 H; OCH3), 8.6 ppm (br s, 1 H; C(O)NH), NH3 and C(O)OH not detected. 13C NMR (125.8 MHz, CD3OH): δ −0.1 (Si(CH3)3), 25.1 (CCH3), 28.5 (SiCH2), 37.1 (CH2CH), 51.3 (CH2CH), 52.9 (OCH3), 60.0 (CCH3), 168.5 (C(O)NH), 174.1 (C(O)OH), 176.1 ppm (C(O)OCH3), CF3 and CF3C not detected. 15N NMR (50.7 MHz, CD3OH): δ −348.1 (NH3), −247.5 ppm (C(O)NH). 19F NMR (376.5 MHz, [D6]DMSO): δ −73.5 ppm. 29Si NMR (99.4 MHz, CD3OH): δ −0.9 ppm. Anal. Calcd for C14H25F3N2O7Si: C, 40.19; H, 6.02; N, 6.69. Found: C, 39.97; H, 6.36; N, 6.75. Synthesis of (S)-Aspart-1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Methyl Ester Hydrochloride ((S,R)-5·HCl). A 2 M solution of hydrogen chloride in diethyl ether (50 mL, 100 mmol of HCl) was added in a single portion at 20 °C to (S,R)-19 (502 mg, 1.09 mmol), and the resulting solution was stirred at 20 °C for 41 h. Subsequently, the volatile components were removed under reduced pressure, and water (60 mL) was added to the residue. The aqueous phase was extracted with diethyl ether (3 × 25 mL), and the extracts were discarded. The remaining aqueous solution was freed of water under reduced pressure, and the solid residue was dissolved in methanol/ water (3/2 v/v, 700 μL) and was further purified by RP-MPLC (eluent, methanol/water (3/2 v/v); flow rate, 14 mL min−1; detector wavelength, 210 nm). The resulting product was dissolved in water (15 mL), and the solution was freeze-dried to furnish (S,R)-5·HCl (308 mg, 904 μmol; 83% yield) as a colorless solid. Mp: 234 °C dec. 1 H NMR (500.1 MHz, CD3OH): δ 0.04 (s, 9 H; Si(CH3)3), 1.22 (δA) and 1.29 (δB) (AB system, 2J(A,B) = 14.5 Hz, 2 H; SiCHAHB), 1.54 (s, 3 H; CCH3), 2.50 (δA), 2.70 (δB), and 3.98 (δX) (ABX system, 2J(A,B) = 17.1 Hz, 3J(A,X) = 5.3 Hz, 3J(B,X) = 9.0 Hz, 3 H; CHAHBCHX), 3.65 (s, 3 H; OCH3), 8.6 ppm (br s, 1 H; C(O)NH), NH3 and C(O)OH not detected. 13C NMR (125.8 MHz, CD3OH): δ −0.4 (Si(CH3)3), 25.2 (CCH3), 27.7 (SiCH2), 38.3 (CH2CH), 52.3 (CH2CH), 52.6 (OCH3), 59.8 (CCH3), 169.3 (C(O)NH), 175.7 (C(O)OCH3), 176.1 ppm (C(O)OH). 15N NMR (50.7 MHz, CD3OH): δ −347.5 (NH3), −246.0 ppm (C(O)NH). 29Si NMR (99.4 MHz, CD3OH): δ −1.0 ppm. Anal. Calcd for C12H25ClN2O5Si: C, 42.28; H, 7.39; N, 8.22. Found: C, 42.31; H, 7.49; N, 7.99. Synthesis of (S)-Aspart-1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Ethyl Ester Hydrotrifluoroacetate ((S,R)-6·TFA). Trifluoroacetic acid (4.45 g, 39.0 mmol) was added in a single portion at 20 °C to (S,R)-20 (100 mg, 211 μmol), and the resulting solution was stirred at 20 °C for 20 min. Subsequently, the volatile components were removed under reduced pressure, and water (150 mL) was added to the residue. The aqueous phase was extracted continuously with

diethyl ether (400 mL) over a period of 8 h using a perforator, and the extract was discarded. The remaining aqueous solution was freed of water under reduced pressure, and the solid residue was dissolved in methanol/water (80/20 v/v, 500 μL) and was further purified by RPMPLC (eluent, methanol/water (80/20 v/v); flow rate, 15 mL min−1; detector wavelength, 210 nm). The resulting product was dissolved in water (8 mL), and the solution was freeze-dried to furnish (S,R)6·TFA (46.0 mg, 106 μmol; 50% yield) as a colorless solid. Mp: 235 °C dec. 1H NMR (500.1 MHz, CD3OD): δ 0.05 (s, 9 H; Si(CH3)3), 1.22 (δA) and 1.29 (δB) (AB system, 2J(A,B) = 14.5 Hz, 2 H; SiCHACHB), 1.26 (δX), 4.10 (δA), and 4.13 (δB) (ABX3 system, 2 J(A,B) = 10.8 Hz, 3J(A,X) = 7.1 Hz, 3J(B,X) = 7.2 Hz, 5 H; OCHAHBC(HX)3), 1.55 (s, 3 H; CCH3), 2.64 (δA), 2.83 (δB), and 4.05 ppm (δX) (ABX system, 2J(A,B) = 17.6 Hz, 3J(A,X) = 8.8 Hz, 3J(B,X) = 4.8 Hz, 3 H; CHAHBCHX), NH3, C(O)NH, and C(O)OH not detected. 13C NMR (125.8 MHz, CD3OD): δ −0.2 (Si(CH3)3), 14.4 (OCH2CH3), 25.2 (CCH3), 29.3 (SiCH2), 37.4 (CH2CH), 51.6 (CH2CH), 59.9 (CCH3), 62.5 (OCH2CH3), 168.6 (C(O)NH), 174.6 (C(O)OH), 175.3 ppm (C(O)OCH2CH3), CF3C and CF3C not detected. 15N NMR (50.7 MHz, CD3OD): δ −348.3 (NH3), −246.7 ppm (C(O)NH). 19F NMR (CD3OD, 376.5 MHz): δ −76.9 ppm. 29Si NMR (99.4 MHz, CD3OD): δ −0.9 ppm. Anal. Calcd for C15H27F3N2O7Si: C, 41.66; H, 6.29; N, 6.48. Found: C, 41.35; H, 6.32; N, 6.19. Synthesis of N-(2,2-Dimethylpropyl)-(S)-aspart-1-yl-(R)-2[(trimethylsilyl)methyl]alanine Methyl Ester ((S,R)-7). Compound 24 (127 mg, 1.47 mmol) and anhydrous magnesium sulfate (354 mg, 2.94 mmol) were added at 20 °C to a stirred solution of (S,R)-23 (580 mg, 1.47 mmol) in dichloromethane (25 mL), and the resulting mixture was stirred at this temperature for 24 h. The magnesium sulfate (including its hydrate) was filtered off and discarded, and the solvent of the filtrate was removed under reduced pressure to furnish N-(2,2-dimethylpropylidene)-4-benzyloxy-(S)aspart-1-yl-(R)-2-[(trimethylsilyl)methyl]alanine methyl ester ((S,R)25; 596 mg; crude product, not further purified) as a colorless oily liquid (EI-MS: m/z (%) 447 (1) [M+ − CH3], 91 (100) [C7H7+]). At 20 °C, palladium/carbon (200 mg, 10 wt %, 188 μmol of Pd) was added to a stirred solution of (S,R)-25 (596 mg; crude product) in methanol (25 mL), and the resulting suspension was stirred at this temperature under an atmosphere of hydrogen for 20 min. The insoluble components were filtered off, washed with methanol (2 × 10 mL), and discarded. The filtrate and the wash solutions were combined, and the solvent was removed under reduced pressure. The residue was dissolved in methanol (1.5 mL) and further purified by RP-MPLC (eluent, methanol/water (7/3 v/v); flow rate, 15 mL min−1; detector wavelength, 210 nm) to furnish (S,R)-7 (521 mg, 1.39 mmol; 95% yield) as a colorless solid. Mp: 140 °C dec. 1H NMR (500.1 MHz, [D6]DMSO): δ −0.00 (s, 9 H; Si(CH3)3), 0.87 (s, 9 H; C(CH3)3), 1.227 (δA) and 1.230 (δB) (AB system, 2J(A,B) = 3.0 Hz, 2 H; SiCHAHB), 1.42 (s, 3 H; CCH3), 2.22 (δA) and 2.28 (δB) (AB system, 2J(A,B) = 11.2 Hz, 2 H; CHAHBNH), 2.30 (δA), 2.40 (δB), and 3.35 (δX) (ABX system, 2J(A,B) = 15.9 Hz, 3J(A,X) = 10.1 Hz, 3J(B,X) = 4.3 Hz, 3 H; CHAHBCHX), 3.54 (s, 3 H; OCH3), 8.2 ppm (br s, 1 H; C(O)NH), CH2NH, and C(O)OH not detected. 13C NMR (125.8 MHz, CD3OD): δ −0.1 (Si(CH3)3), 25.6 (CCH3), 27.4 (C(CH3)3), 28.5 (SiCH2), 31.5 (C(CH3)3), 36.1 (CH2CH), 52.9 (OCH3), 59.4 (CH2NH), 60.2 (CCH3), 60.7 (CH2CH), 168.0 (C(O)NH), 175.7 (C(O)OCH3), 176.6 ppm (C(O)OH). 15N NMR (50.7 MHz, CD3OD): δ −340.5 (CH2NH), −244.4 ppm (C(O)NH). 29Si NMR (99.4 MHz, CD3OD): δ −1.0 ppm. EI-MS: m/z (%) 374 (1) [M+], 172 (100). Anal. Calcd for C17H34N2O5Si: C, 54.51; H, 9.15; N, 7.48. Found: C, 54.14; H, 8.87; N, 7.47. Synthesis of N-(2,2-Dimethylpropyl)-(S)-aspart-1-yl-(S)-2[(trimethylsilyl)methyl]alanine Methyl Ester ((S,S)-7). Compound 24 (49.1 mg, 570 μmol) and anhydrous magnesium sulfate (137 mg, 1.14 mmol) were added at 20 °C to a stirred solution of (S,S)-23 (225 mg, 570 μmol) in dichloromethane (25 mL), and the resulting mixture was stirred at this temperature for 24 h. The magnesium sulfate (including its hydrate) was filtered off and discarded, and the solvent of the filtrate was removed under reduced 5910


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Article

Synthesis of N-(3,3-Dimethylbutyl)-(S)-aspart-1-yl-(S)-2[(trimethylsilyl)methyl]alanine Methyl Ester ((S,S)-8). Compound 27 (45.2 mg, 451 μmol) and anhydrous magnesium sulfate (109 mg, 906 μmol) were added at 20 °C to a stirred solution of (S,S)23 (178 mg, 451 μmol) in dichloromethane (10 mL), and the resulting mixture was stirred at this temperature for 16 h. The magnesium sulfate (including its hydrate) was filtered off and discarded, and the solvent of the filtrate was removed under reduced pressure to furnish N-(3,3-dimethylbutylidene)-4-benzyloxy-(S)-aspart-1-yl-(S)-2-[(trimethylsilyl)methyl]alanine methyl ester ((S,S)26; 199 mg; crude product, not further purified) as a colorless oily liquid (EI-MS: m/z (%) 461 (2) [M+ − CH3], 91 (100) [C7H7+]). At 20 °C, palladium/carbon (75 mg, 10 wt %, 70.5 μmol of Pd) was added to a stirred solution of (S,S)-26 (199 mg; crude product) in methanol (10 mL), and the resulting suspension was stirred at this temperature under an atmosphere of hydrogen for 20 min. The insoluble components were filtered off, washed with methanol (2 × 10 mL), and discarded. The filtrate and the wash solutions were combined, and the solvent was removed under reduced pressure. The residue was dissolved in methanol (1 mL) and further purified by RP-MPLC (eluent, methanol/water (7/3 v/v); flow rate, 13 mL min−1; detector wavelength, 210 nm) to furnish (S,S)-8 (163 mg, 419 μmol; 93% yield) as a colorless solid. Mp: 149 °C dec. 1H NMR (500.1 MHz, CD3OD): δ 0.06 (s, 9 H; Si(CH3)3), 0.97 (s, 9 H; C(CH3)3), 1.28 (δA) and 1.32 (δB) (AB system, 2J(A,B) = 14.6 Hz, 2 H; SiCHAHB), 1.51 (s, 3 H; CCH3), 1.60 (δA), 1.64 (δB), 2.95 (δC), and 2.98 (δD) (ABCD system, 2J(A,B) = 12.9 Hz, 3J(A,C) = 12.5 Hz, 3 J(A,D) = 4.6 Hz, 3J(B,C) = 4.9 Hz, 3J(B,D) = 12.6 Hz, 2J(C,D) = 11.8 Hz, 4 H; CHAHBCHCHDNH), 2.52 (δA), 2.62 (δB), and 3.98 (δX) (ABX system, 2J(A,B) = 17.0 Hz, 3J(A,X) = 10.1 Hz, 3J(B,X) = 4.5 Hz, 3 H; CHAHBCHX), 3.67 ppm (s, 3 H; OCH3), C(O)NH, CH2NH, and C(O)OH not detected. 13C NMR (125.8 MHz, CD3OD): δ −0.1 (Si(CH3)3), 25.0 (CCH3), 28.9 (SiCH2), 29.5 (C(CH3)3), 30.6 (C(CH3)3), 37.5 (CH2CH), 40.8 (CH2CH2NH), 44.6 (CH2CH2NH), 52.9 (OCH3), 59.5 (CH2CH), 60.0 (CCH3), 168.3 (C(O)NH), 175.7 (C(O)OCH3), 175.9 ppm (C(O)OH). 15N NMR (50.7 MHz, CD3OD): δ −333.8 (CH2NH), −243.6 ppm (C(O)NH). 29Si NMR (99.4 MHz, CD3OD): δ −0.9 ppm. EI-MS: m/z (%) 388 (1) [M+], 186 (100). Anal. Calcd for C18H36N2O5Si: C, 55.64; H, 9.34; N, 7.21. Found: C, 55.51; H, 9.46; N, 7.14. Synthesis of Diethyl Methyl[(trimethylsilyl)methyl]malonate (12). This compound was synthesized according to ref 1h. Synthesis of (R)-3-Ethoxy-2-methyl-3-oxo-2-[(trimethylsilyl)methyl]propanoic Acid ((R)-13; Enantiomeric Excess, 75% ee). This compound was synthesized according to ref 1h. Porcine liver esterase (60 mg, 165 U mg−1, EC 3.1.1.1) was added in a single portion at 38 °C to a stirred emulsion of 12 (100 g, 384 mmol) in water (3 L). The mixture was stirred at 38 °C for 94 h while the pH value was adjusted to 7.60 with a 1 M aqueous sodium hydroxide solution by using a pH-stat titrator. The enzyme was separated by suction filtration (Celite), washed with water (3 × 150 mL), and discarded. The aqueous phase was extracted continuously with diethyl ether (1.5 L) over a period of 4 days using a perforator, and the extract was discarded. The remaining aqueous solution was acidified with concentrated hydrochloric acid (40 mL) and extracted continuously with diethyl ether (1.5 L) over a period of 18 h using a perforator. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by bulb-to-bulb distillation (110 °C, 0.02 mbar) to furnish (R)-13 (86.9 g, 374 mmol; 97% yield; enantiomeric excess, 75% ee) as a colorless liquid. The NMR data of the product (solvent, [D6]DMSO) were identical with those reported in ref 1h. Synthesis of [(S)-3-tert-Butoxy-2-methyl-3-oxo-2[(trimethylsilyl)methyl]propanoic Acid ((S)-14; Enantiomeric Excess, 95% ee). This compound was synthesized according to ref 1h. At −20 °C, 2-methylpropene (83.0 g, 1.48 mol) was passed through a stirred mixture of (R)-13 (40.0 g, 172 mmol; enantiomeric excess, 75% ee), dichloromethane (400 mL), and concentrated sulfuric acid (4.00 mL) over a period of 1 h. The mixture was warmed to 20 °C and was then stirred at this temperature for 20 h. Subsequently,

pressure to furnish N-(2,2-dimethylpropylidene)-4-benzyloxy-(S)aspart-1-yl-(S)-2-[(trimethylsilyl)methyl]alanine methyl ester ((S,S)25; 200 mg; crude product, not further purified) as a colorless oily liquid (EI-MS: m/z (%) 447 (2) [M+ − CH3], 91 (100) [C7H7+]). At 20 °C, palladium/carbon (150 mg, 10 wt %, 141 μmol of Pd) was added to a stirred solution of (S,S)-25 (200 mg; crude product) in methanol (20 mL), and the resulting suspension was stirred at this temperature under an atmosphere of hydrogen for 20 min. The insoluble components were filtered off, washed with methanol (2 × 10 mL), and discarded. The filtrate and the wash solutions were combined, and the solvent was removed under reduced pressure. The residue was dissolved in methanol (1 mL) and further purified by RP-MPLC (eluent, methanol/water (4/1 v/v); flow rate, 15 mL min−1; detector wavelength, 210 nm) to furnish (S,S)-7 (191 mg, 510 μmol; 89% yield) as a colorless solid. Mp: 121 °C dec. 1H NMR (500.1 MHz, [D6]DMSO): δ −0.01 (s, 9 H; Si(CH3)3), 0.87 (s, 9 H; C(CH3)3), 1.23 (δA) and 1.28 (δB) (AB system, 2J(A,B) = 14.5 Hz, 2 H; SiCHAHB), 1.40 (s, 3 H; CCH3), 2.22 (δA) and 2.27 (δB) (AB system, 2J(A,B) = 10.9 Hz, 2 H; CHAHBNH), 2.30 (δA), 2.39 (δB), and 3.33 (δX) (ABX system, 2J(A,B) = 15.9 Hz, 3J(A,X) = 9.5 Hz, 3J(B,X) = 4.6 Hz, 3 H; CHAHBCHX), 3.55 (s, 3 H; OCH3), 8.3 ppm (br s, 1 H; C(O)NH), CH2NH and C(O)OH not detected. 13C NMR (125.8 MHz, CD3OD): δ −0.1 (Si(CH3)3), 25.1 (CCH3), 27.4 (C(CH3)3), 28.8 (SiCH2), 31.6 (C(CH3)3), 36.3 (CH2CH), 52.9 (OCH3), 59.6 (CH2NH), 60.0 (CCH3), 60.7 (CH2CH), 168.3 (C(O)NH), 175.8 (C(O)OCH3), 176.7 ppm (C(O)OH). 15N NMR (50.7 MHz, CD3OD): δ −340.5 (CH2NH), −244.4 ppm (C(O)NH). 29Si NMR (99.4 MHz, CD3OD): δ −0.9 ppm. EI-MS: m/z (%) 374 (1) [M+], 172 (100). Anal. Calcd for C17H34N2O5Si: C, 54.51; H, 9.15; N, 7.48. Found: C, 54.25; H, 8.93; N, 7.45. Synthesis of N-(3,3-Dimethylbutyl)-(S)-aspart-1-yl-(R)-2[(trimethylsilyl)methyl]alanine Methyl Ester ((S,R)-8). Compound 27 (53.3 mg, 532 μmol) and anhydrous magnesium sulfate (129 mg, 1.07 mmol) were added at 20 °C to a stirred solution of (S,R)-23 (210 mg, 532 μmol) in dichloromethane (10 mL), and the resulting mixture was stirred at this temperature for 2.5 h. The magnesium sulfate (including its hydrate) was filtered off and discarded, and the solvent of the filtrate was removed under reduced pressure to furnish N-(3,3-dimethylbutylidene)-4-benzyloxy-(S)-aspart-1-yl-(R)-2-[(trimethylsilyl)methyl]alanine methyl ester ((S,R)26; 285 mg; crude product, not further purified) as a colorless oily liquid (EI-MS: m/z (%) 461 (2) [M+ − CH3], 91 (100) [C7H7+]). At 20 °C, palladium/carbon (110 mg, 10 wt %, 103 μmol of Pd) was added to a stirred solution of (S,R)-26 (285 mg; crude product) in methanol (10 mL), and the resulting suspension was stirred at this temperature under an atmosphere of hydrogen for 20 min. The insoluble components were filtered off, washed with methanol (2 × 10 mL), and discarded. The filtrate and the wash solutions were combined, and the solvent was removed under reduced pressure. The residue was dissolved in methanol (1 mL) and further purified by RP-MPLC (eluent, methanol/water (7/3 v/v); flow rate, 13 mL min−1; detector wavelength, 210 nm) to furnish (S,R)-8 (191 mg, 492 μmol; 92% yield) as a colorless solid. Mp: 114 °C dec. 1H NMR (500.1 MHz, CD3OD): δ −0.05 (s, 9 H; Si(CH3)3), 0.95 (s, 9 H; C(CH3)3), 1.31 (δA) and 1.34 (δB) (AB system, 2J(A,B) = 14.5 Hz, 2 H; SiCHAHB), 1.54 (s, 3 H; CCH3), 1.59 (δA), 1.61 (δB), 2.95 (δC), and 2.96 (δD) (ABCD system, 2J(A,B) = 13.0 Hz, 3J(A,C) = 12.2 Hz, 3 J(A,D) = 4.9 Hz, 3J(B,C) = 4.9 Hz, 3J(B,D) = 12.4 Hz, 2J(C,D) = 12.1 Hz, 4 H; CHAHBCHCHDNH), 2.52 (δA), 2.66 (δB), and 3.96 (δX) (ABX system, 2J(A,B) = 17.0 Hz, 3J(A,X) = 9.9 Hz, 3J(B,X) = 4.5 Hz, 3 H; CHAHBCHX), 3.67 ppm (s, 3 H; OCH3), C(O)NH, CH2NH, and C(O)OH not detected. 13C NMR (125.8 MHz, CD3OD): δ −0.1 (Si(CH3)3), 25.6 (CCH3), 28.6 (SiCH2), 29.5 (C(CH3)3), 30.6 (C(CH3)3), 37.6 (CH2CH), 41.0 (CH2CH2NH), 44.6 (CH2CH2NH), 52.9 (OCH3), 59.5 (CH2CH), 60.1 (CCH3), 168.7 (C(O)NH), 175.7 (C(O)OCH3), 176.2 ppm (C(O)OH). 15N NMR (50.7 MHz, CD3OD): δ −334.3 (CH2NH), −244.4 ppm (C(O)NH). 29Si NMR (99.4 MHz, CD3OD): δ −1.0 ppm. EI-MS: m/z (%) 388 (1) [M+], 186 (100). Anal. Calcd for C18H36N2O5Si: C, 55.64; H, 9.34; N, 7.21. Found: C, 55.46; H, 9.27; N, 7.07. 5911


Organometallics

Article

ethanol (400 mL) and potassium hydroxide (55.0 g, 980 mmol) were added one after another in single portions, and the resulting mixture was heated under reflux for 54 h and then cooled to 20 °C. The solvent was removed under reduced pressure, water (3 L) was added, the resulting mixture was extracted continuously with diethyl ether (1.5 L) over a period of 2 days using a perforator, and the extract was discarded. The remaining aqueous phase was acidified with concentrated hydrochloric acid (80 mL) and then extracted continuously with diethyl ether (1.5 L) over a period of 45 h using a perforator. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was dried in vacuo (20 °C, 0.02 mbar, 2 h) and then crystallized from n-hexane (200 mL) at −20 °C over a period of 1 day to furnish (S)-14 (30.5 g, 117 mmol; 68% yield; enantiomeric excess, 75% ee) as a colorless crystalline solid. Further product fractions of (S)-14 (in total 11.2 g, 43.0 mmol; 25% yield; enantiomeric excess, 75% ee) were obtained by applying the crystallization procedure described above to the mother liquor (crystallization from 50 mL, 12 mL, and 2 mL n-hexane, respectively) to give (S)-14 in a total yield of 93% as a crystalline solid (enantiomeric excess, 75% ee). Enhancement of the enantiomeric excess was accomplished by recrystallizing (S)-14 (19.7 g, 75.7 mmol; enantiomeric excess, 75% ee) from n-hexane (129 mL) at −20 °C over a period of 1 day to furnish (S)-14 (5.86 g, 22.5 mmol; 30% yield (relative to 19.7 g of (S)-14); enantiomeric excess, 95% ee) as a colorless crystalline solid. The NMR data of the product (solvent, C6D6) were identical with those reported in ref 1h. Synthesis of rac-2-Amino-2-methyl-3-(trimethylsilyl)propionic Acid Methyl Ester Hydrochloride (rac-α[(Trimethylsilyl)methyl]alanine Methyl Ester Hydrochloride, rac-15·HCl). Thionyl chloride (6.79 g, 57.1 mmol) was added at −20 °C to a stirred suspension of rac-4 (1.00 g, 5.70 mmol) in methanol (50 mL). After the mixture had been warmed to 20 °C, it was stirred at this temperature for 5 min and then heated under reflux for 2 h. The solvent and excess thionyl chloride were removed under reduced pressure, and the resulting residue was washed with diethyl ether (2 × 10 mL) and then dissolved in water (150 mL). The resulting solution was freeze-dried to furnish rac-15·HCl (1.28 g, 5.67 mmol; 99% yield) as a colorless solid. Mp: 253 °C dec. 1H NMR (500.1 MHz, [D6]DMSO, 27 °C): δ 0.01 (s, 9 H; Si(CH3)3), 1.26 (δA) and 1.30 (δB) (AB system, 2J(A,B) = 14.4 Hz, 2 H; SiCHAHB), 1.51 (s, 3 H; CCH3), 3.72 (s, 3 H; OCH3), 8.7 ppm (br s, 3 H; NH3). 13 C NMR (125.8 MHz), [D6]DMSO, 27 °C): δ −0.7 (Si(CH3)3), 23.3 (CCH3), 27.3 (SiCH2), 52.8 (OCH3), 58.4 (CCH3), 171.6 ppm (C(O)OCH3). 15N NMR (50.7 MHz, [D6]DMSO, 27 °C): δ −319.5 ppm. 29Si NMR (99.4 MHz, [D6]DMSO, 27 °C): δ −1.0 ppm. EIMS:27 m/z (%) 174 (4) [M+ − CH3], 73 (100) [Si(CH3)3+]. HRMS (ESI): m/z calcd for [M − HCl + Na]+, 212.1077; found, 212.1077. Synthesis of (R)-2-Amino-2-methyl-3-(trimethylsilyl)propionic Acid Methyl Ester Hydrochloride ((R)-α[(Trimethylsilyl)methyl]alanine Methyl Ester Hydrochloride, (R)-15·HCl). Thionyl chloride (508 mg, 4.27 mmol) was added at −20 °C to a stirred suspension of (R)-4 (75.0 mg, 428 μmol) in methanol (3 mL). After the mixture had been warmed to 20 °C, it was stirred at this temperature for 5 min and then heated under reflux for 2 h. The solvent and excess thionyl chloride were removed under reduced pressure, and the resulting residue was washed with diethyl ether (2 × 3 mL) and then dissolved in water (3 mL). The resulting solution was freeze-dried to furnish (R)-15·HCl (96.0 mg, 425 μmol; 99% yield) as a colorless solid. Mp: 271 °C dec. The NMR data of the product (solvent, [D6]DMSO) were identical with those obtained for rac-15·HCl. EI-MS:27 m/z (%) 174 (5) [M+ − CH3], 73 (100) [Si(CH3)3+]. HRMS (ESI): m/z calcd for [M − HCl + Na]+, 212.1077; found, 212.1078. Synthesis of (S)-2-Amino-2-methyl-3-(trimethylsilyl)propionic Acid Methyl Ester Hydrochloride ((S)-α[(Trimethylsilyl)methyl]alanine Methyl Ester Hydrochloride, (S)-15·HCl). Thionyl chloride (678 mg, 5.70 mmol) was added at −20 °C to a stirred suspension of (S)-4 (100 mg, 570 μmol) in methanol (3 mL). After the mixture had been warmed to 20 °C, it was stirred at this temperature for 5 min and then heated under reflux for 2 h.

The solvent and excess thionyl chloride were removed under reduced pressure, and the resulting residue was washed with diethyl ether (2 × 2 mL) and then dissolved in water (3 mL). The resulting solution was freeze-dried to furnish (S)-15·HCl (128 mg, 567 μmol; 99% yield) as a colorless solid. Mp: 258 °C dec. The NMR data of the product (solvent, [D6]DMSO) were identical with those obtained for rac-15·HCl. EI-MS:27 m/z (%) 174 (6) [M+ − CH3], 73 (100) [Si(CH3)3+]. HRMS (ESI): m/z calcd for [M − HCl + Na]+, 212.1077; found, 212.1077. Synthesis of (R)-2-Amino-2-methyl-3-(trimethylsilyl)propionic Acid Ethyl Ester Hydrochloride ((R)-α[(Trimethylsilyl)methyl]alanine Ethyl Ester Hydrochloride (R)16·HCl). Thionyl chloride (6.81 g, 57.2 mmol) was added at −20 °C to a stirred suspension of (R)-4 (1.00 g, 5.70 mmol) in ethanol (100 mL). After the mixture had been warmed to 20 °C, it was stirred at this temperature for 5 min and then heated under reflux for 16 h. The solvent and excess thionyl chloride were removed under reduced pressure, and the resulting residue was washed with diethyl ether (3 × 30 mL) and then dissolved in water (50 mL). The resulting solution was freeze-dried to furnish (R)-16·HCl (1.35 g, 5.63 mmol; 99% yield) as a colorless solid. Mp: 263 °C dec. 1H NMR (500.1 MHz, [D6]DMSO): δ 0.02 (s, 9 H; Si(CH3)3), 1.25 (δX), 4.13 (δA), and 4.20 (δB) (ABX3 system, 2J(A,B) = 10.9 Hz, 3J(A,X) = 3J(B,X) = 7.1 Hz, 5 H; OCHAHBC(HX)3), 1.24 (δA) and 1.26 (δB) (AB system, 2J(A,B) = 13.1 Hz, 2 H; SiCHAHB), 1.49 (s, 3 H; CCH3), 8.5 ppm (br s, 3 H; NH3). 13C NMR (125.8 MHz, [D6]DMSO): δ −0.5 (Si(CH3)3), 13.8 (OCH2CH3), 23.6 (CCH3), 27.3 (SiCH2), 58.4 (CCH3), 62.0 (OCH2CH3), 171.4 ppm (C(O)OC2H5). 15N NMR (50.7 MHz, [D6]DMSO): δ −319.5 ppm. 29Si NMR (99.4 MHz, [D6]DMSO): δ −1.0 ppm. EI-MS:27 m/z (%) 188 (5) [M+ − CH3], 130 (100) [M+ − Si(CH3)3]. HRMS (ESI): m/z calcd for [M − HCl + Na]+, 226.1234; found, 226.1233. N-tert-Butoxycarbonyl-4-tert-butoxy-(S)-aspartic Acid ((S)17). This compound was commercially available (Sigma-Aldrich). O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium Hexafluorophosphate (18). This compound was commercially available (Sigma-Aldrich). Synthesis of N-tert-Butoxycarbonyl-4-tert-butoxy-(S)-aspart1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,R)19). (S)-17 (987 mg, 3.41 mmol), 18 (1.54 g, 3.72 mmol), and Nmethylmorpholine (941 mg, 9.30 mmol) were added one after another in single portions at 20 °C to a stirred suspension of (R)-15·HCl (700 mg, 3.10 mmol) in dichloromethane (30 mL). The resulting mixture was stirred at this temperature for 30 min and then extracted with water (2 × 20 mL). The combined aqueous solutions were extracted with dichloromethane (3 × 20 mL) and discarded, the solvent of the combined organic solutions was removed under reduced pressure, and the solid residue was crystallized from methanol/water (58/42 v/v, 76 mL; slow cooling of a boiling solution to 20 °C and crystallization over a period of 1 day). The precipitate was washed with methanol (−20 °C, 10 mL) and dried under vacuum (0.05 mbar, 20 °C, 4 h) to furnish (S,R)-19 (1.23 g, 2.67 mmol; 86% yield) as a colorless crystalline solid. Mp: 239 °C. 1H NMR (500.1 MHz, C6D6): δ 0.14 (s, 9 H; Si(CH3)3), 1.26 (δA) and 1.73 (δB) (AB system, 2J(A,B) = 14.6 Hz, 2 H; SiCHAHB), 1.43 (s, 9 H; C(CH3)3; Boc or tBu), 1.54 (s, 9 H; C(CH3)3; Boc or tBu), 1.83 (s, 3 H; CCH3), 2.58 (δA), 2.92 (δB), 4.80 (δM), and 6.0 (br, δx) (ABMX system, 2J(A,B) = 16.8 Hz, 3J(A,M) = 7.1 Hz, 3J(B,M) = 4.5 Hz, 3J(M,X) = 8.8 Hz, 4 H; CHAHBCHMNHX), 3.47 (s, 3 H; OCH3), 7.4 ppm (br s, 1 H; C(O)NH). 13C NMR (125.8 MHz, C6D6): δ −0.4 (Si(CH3)3), 25.8 (CCH3), 27.7 (SiCH2), 27.9 (C(CH3)3; Boc or tBu), 28.3 (C(CH3)3; Boc or tBu), 37.2 (CH2CH), 51.4 (CH2CH), 51.9 (OCH3), 59.1 (CCH3), 79.7 (C(CH3)3; Boc or tBu), 81.0 (C(CH3)3; Boc or tBu), 155.8 (OC(O)NH), 170.2 (CC(O)NH), 171.7 (C(O)OC(CH3)3), 174.9 ppm (C(O)OCH3). 15N NMR (50.7 MHz, C6D6): δ −293.8 (OC(O)NH), −252.2 ppm (CC(O)NH). 29Si NMR (99.4 MHz, C6D6): δ −1.1 ppm. CI-MS: m/z (%) 489 (1) [M + C2H5]+, 461 (5) [M + H]+, 349 (100). Anal. Calcd for C21H40N2O7Si: C, 54.76; H, 8.75; N, 6.08. Found: C, 54.38; H, 8.69; N, 6.13. Synthesis of N-tert-Butoxycarbonyl-4-tert-butoxy-(S)-aspart1-yl-(S)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,S)19). (S)-17 (894 mg, 3.09 mmol), 18 (1.39 g, 3.36 mmol), and N-methylmorpholine (852 mg, 8.42 mmol) were added one after 5912


Organometallics

Article

another in single portions at 20 °C to a stirred suspension of (S)15·HCl (634 mg, 2.81 mmol) in dichloromethane (30 mL). The resulting mixture was stirred at this temperature for 30 min and then extracted with water (2 × 20 mL). The combined aqueous solutions were extracted with dichloromethane (3 × 20 mL) and discarded, the solvent of the combined organic solutions was removed under reduced pressure, and the remaining residue was dissolved in methanol/water (4/1 v/v, 4 mL) and then further purified by RP-MPLC (eluent, methanol/water (4/1 v/v); flow rate, 9 mL min−1; detector wavelength, 210 nm) to furnish (S,S)-19 (1.27 g, 2.76 mmol; 98% yield) as a colorless solid. Mp: 244 °C. 1H NMR (500.1 MHz, C6D6): δ 0.16 (s, 9 H; Si(CH3)3), 1.35 (δA) and 1.98 (δB) (AB system, 2J(A,B) = 14.7 Hz, 2 H; SiCHAHB), 1.42 (s, 9 H; C(CH3)3; Boc or tBu), 1.56 (s, 9 H; C(CH3)3; Boc or tBu), 1.85 (s, 3 H; CCH3), 2.54 (δA), 2.98 (δB), 4.76 (δM), and 6.0 (br, δx) (ABMX system, 2J(A,B) = 16.7 Hz, 3 J(A,M) = 5.9 Hz, 3J(B,M) = 4.6 Hz, 3J(M,X) = 8.8 Hz, 4 H; CHAHBCHMNHX), 3.46 (s, 3 H; OCH3), 7.6 ppm (br s, 1 H; C(O)NH). 13C NMR (125.8 MHz, C6D6): δ −0.5 (Si(CH3)3), 26.2 (CCH3), 26.9 (SiCH2), 27.9 (C(CH3)3; Boc or tBu), 28.3 (C(CH3)3; Boc or tBu), 36.7 (CH2CH), 51.7 (CH2CH), 52.0 (OCH3), 59.1 (CCH3), 79.8 (C(CH3)3; Boc or tBu), 80.9 (C(CH3)3; Boc or tBu), 155.8 (OC(O)NH), 170.1 (CC(O)NH), 171.7 (C(O)OC(CH3)3), 175.3 ppm (C(O)OCH3). 15N NMR (50.7 MHz, C6D6): δ −293.6 (OC(O)NH), −255.5 ppm (CC(O)NH). 29Si NMR (99.4 MHz, C6D6): δ −1.1 ppm. EI-MS: m/z (%) 445 (3) [M+ − CH3], 57 (100) [C(CH3)3+]. Anal. Calcd for C21H40N2O7Si: C, 54.76; H, 8.75; N, 6.08. Found: C, 54.76; H, 8.40; N, 5.93. Synthesis of N-tert-Butoxycarbonyl-4-tert-butoxy-(S)-aspart1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Ethyl Ester ((S,R)-20). (S)-17 (1.81 g, 6.26 mmol), 18 (2.83 g, 6.84 mmol), and Nmethylmorpholine (1.74 g, 17.2 mmol) were added one after another in single portions at 20 °C to a stirred suspension of (R)-16·HCl (1.35 g, 5.63 mmol) in dichloromethane (150 mL). The resulting mixture was stirred at this temperature for 11 h and then extracted with water (3 × 50 mL). The combined aqueous solutions were extracted with dichloromethane (3 × 50 mL) and discarded, the solvent of the combined organic solutions was removed under reduced pressure, and the solid residue was then crystallized from methanol/ water (73/27 v/v, 190 mL; slow cooling of a boiling solution to 20 °C and crystallization over a period of 1 day) to furnish (S,R)-20 (2.62 g, 5.52 mmol; 98% yield) as a colorless crystalline solid. Mp: 242 °C dec. 1 H NMR (500.1 MHz, CD2Cl2): δ 0.03 (s, 9 H; Si(CH3)3), 1.21 (δA) and 1.46 (δB) (AB system, 2J(A,B) = 14.6 Hz, 2 H; SiCHAHB), 1.24 (δX), 4.10 (δA), and 4.12 (δB) (ABX3 system, 2J(A,B) = 10.9 Hz, 3 J(A,X) = 7.1 Hz, 3J(B,X) = 7.2 Hz, 5 H; OCHAHBC(HX)3), 1.437 (s, 9 H; C(CH3)3; Boc or tBu), 1.440 (s, 9 H; C(CH3)3; Boc or tBu), 1.53 (s, 3 H; CCH3), 2.55 (δA), 2.73 (δB), 4.37 (δM), and 5.6 (br, δX) (ABMX system, 2J(A,B) = 16.9 Hz, 3J(A,M) = 8.0 Hz, 3J(B,M) = 5.1 Hz, 3J(M,X) = 9.9 Hz, 4 H; OCHAHBCHMNHX), 7.0 ppm (br s, 1 H; C(O)NH). 13C NMR (125.8 MHz, CD2Cl2): δ 0.0 (Si(CH3)3), 14.5 (OCH2CH3), 25.8 (CCH3), 28.1 (SiCH2), 28.4 (C(CH3)3; Boc or tBu), 28.7 (C(CH3)3; Boc or tBu), 38.2 (CH2CH), 51.4 (CH2CH), 59.3 (CCH3), 61.9 (OCH2CH3), 80.5 (C(CH3)3; Boc or tBu), 82.1 (C(CH3)3; Boc or tBu), 155.9 (OC(O)NH), 170.4 (CC(O)NH), 171.9 (C(O)OC(CH3)3), 175.0 ppm (C(O)OCH2CH3). 15N NMR (50.7 MHz, CD2Cl2): δ −294.3 (OC(O)NH), −251.6 ppm (CC(O)NH). 29Si NMR (99.4 MHz, CD2Cl2): δ 0.8 ppm. EI-MS: m/z (%) 459 (4) [M+ − CH3], 57 (100) [C(CH3)3+]. Anal. Calcd for C22H42N2O7Si: C, 55.67; H, 8.92; N, 5.90. Found: C, 55.65; H, 8.79; N, 6.13. N-tert-Butoxycarbonyl-4-benzyloxy-(S)-aspartic Acid ((S)21). This compound was commercially available (Sigma-Aldrich). Synthesis of N-tert-Butoxycarbonyl-4-benzyloxy-(S)-aspart1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,R)22). (S)-21 (2.77 g, 8.57 mmol), 18 (3.87 g, 9.35 mmol), and Nmethylmorpholine (2.37 g, 23.4 mmol) were added one after another in single portions at 20 °C to a stirred suspension of (R)-15·HCl (1.76 g, 7.79 mmol) in dichloromethane (60 mL). The resulting mixture was stirred at this temperature for 30 min and then extracted with water (2 × 30 mL). The combined aqueous solutions were

extracted with dichloromethane (3 × 20 mL) and discarded, the solvent of the combined organic solutions was removed under reduced pressure, and the remaining residue was dissolved in methanol (1.6 mL) and then further purified by RP-MPLC (eluent, methanol/water (4/1 v/v); flow rate, 19 mL min−1; detector wavelength, 225 nm) to furnish (S,R)-22 (3.73 g, 7.54 mmol; 97% yield) as a colorless solid. Mp: 250 °C. 1H NMR (500.1 MHz, C6D6): δ 0.14 (s, 9 H; Si(CH3)3), 1.26 (δA) and 1.72 (δB) (AB system, 2J(A,B) = 14.6 Hz, 2 H; SiCHAHB), 1.54 (s, 9 H; C(CH3)3), 1.81 (s, 3 H; CCH3), 2.61 (δA), 2.93 (δB), 4.80 (δM), and 6.0 (br, δx) (ABMX system, 2J(A,B) = 16.9 Hz, 3J(A,M) = 7.0 Hz, 3J(B,M) = 4.5 Hz, 3J(M,X) = 8.8 Hz, 4 H; CHAHBCHMNHX), 3.46 (s, 3 H; OCH3), 4.99 (δA) and 5.06 (δB) (AB system, 2J(A,B) = 12.4 Hz, 2 H; OCHAHB), 7.13−7.17 (m, 1 H; p-CH, C6H5), 7.18−7.22 (m, 2 H; m-CH, C6H5), 7.23−7.26 (m, 2 H; o-CH, C6H5), 7.4 ppm (br s, 1 H; C(O)NH). 13C NMR (125.8 MHz, C6D6): δ −0.4 (Si(CH3)3), 25.9 (CCH3), 27.6 (SiCH2), 28.3 (C(CH3)3), 35.9 (CH2CH), 51.3 (CH2CH), 52.0 (OCH3), 59.1 (CCH3), 66.6 (OCH2), 79.9 (C(CH3)3), 128.2 (p-C, C6H5), 128.4 (o-C, C6H5), 128.6 (m-C, C6H5), 136.2 (i-C, C6H5), 155.8 (OC(O)NH), 169.9 (CC(O)NH), 172.0 (C(O)OCH2C6H5), 174.8 ppm (C(O)OCH3). 15N NMR (50.7 MHz, C6D6): δ −293.9 (OC(O)NH), −252.7 ppm (CC(O)NH). 29Si NMR (99.4 MHz, C6D6): δ −1.1 ppm. EI-MS: m/z (%) 479 (10) [M+ − CH3], 91 (100) [C7H7+]. Anal. Calcd for C24H38N2O7Si: C, 58.28; H, 7.74; N, 5.66. Found: C, 58.14; H, 7.70; N, 5.70. Synthesis of N-tert-Butoxycarbonyl-4-benzyloxy-(S)-aspart1-yl-(S)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,S)22). (S)-21 (1.13 g, 3.49 mmol), 18 (1.58 g, 3.82 mmol), and Nmethylmorpholine (970 mg, 9.59 mmol) were added one after another in single portions at 20 °C to a stirred suspension of (S)-15·HCl (720 mg, 3.19 mmol) in dichloromethane (40 mL). The resulting mixture was stirred at this temperature for 30 min and then extracted with water (2 × 20 mL). The combined aqueous solutions were extracted with dichloromethane (3 × 20 mL) and discarded, the solvent of the combined organic solutions was removed under reduced pressure, and the remaining residue was dissolved in methanol (1 mL) and then further purified by RP-MPLC (eluent, methanol/water (75/ 25 v/v); flow rate, 18 mL min−1; detector wavelength, 225 nm) to furnish (S,S)-22 (1.55 g, 3.13 mmol; 98% yield) as a colorless oily liquid. 1H NMR (500.1 MHz, C6D6): δ 0.16 (s, 9 H; Si(CH3)3), 1.30 (δA) and 1.96 (δB) (AB system, 2J(A,B) = 14.7 Hz, 2 H; SiCHAHB), 1.56 (s, 9 H; C(CH3)3), 1.84 (s, 3 H; CCH3), 2.52 (δA), 3.02 (δB), 4.78 (δM), and 5.9 (br, δx) (ABMX system, 2J(A,B) = 17.1 Hz, 3J(A,M) = 6.0 Hz, 3 J(B,M) = 4.7 Hz, 3 J(M,X) = 9.3 Hz, 4 H; CHAHBCHMNHX), 3.44 (s, 3 H; OCH3), 4.99 (δA), and 5.01 (δB) (AB system, 2J(A,B) = 12.3 Hz, 2 H; OCHAHB), 7.13−7.17 (m, 1 H; p-CH, C6H5), 7.18−7.21 (m, 2 H; m-CH, C6H5), 7.22−7.26 (m, 2 H; o-CH, C6H5), 7.6 ppm (br s, 1 H; C(O)NH). 13C NMR (125.8 MHz, C6D6): δ −0.5 (Si(CH3)3), 26.2 (CCH3), 27.0 (SiCH2), 28.3 (C(CH3)3), 35.4 (CH2CH), 51.5 (CH2CH), 52.0 (OCH3), 59.1 (CCH3), 66.5 (OCH2), 80.0 (C(CH3)3), 128.2 (p-C, C6H5), 128.4 (oC, C6H5), 128.6 (m-C, C6H5), 136.2 (i-C, C6H5), 155.8 (OC(O)NH), 170.0 (CC(O)NH), 171.9 (C(O)OCH2C6H5), 175.3 ppm (C(O)OCH3). 15N NMR (50.7 MHz, C6D6): δ −293.7 (OC(O)NH), −255.3 ppm (CC(O)NH). 29Si NMR (99.4 MHz, C6D6): δ −1.1 ppm. EI-MS: m/z (%): 479 (10) [M+ − CH3], 91 (100) [C7H7+]. Anal. Calcd for C24H38N2O7Si: C, 58.28; H, 7.74; N, 5.66. Found: C, 58.00; H, 7.78; N, 5.61. Synthesis of 4-Benzyloxy-(S)-aspart-1-yl-(R)-2[(trimethylsilyl)methyl]alanine Methyl Ester ((S,R)-23). A mixture of chlorotrimethylsilane (543 mg, 5.00 mmol), phenol (1.41 g, 15.0 mmol), and dichloromethane (5 mL) was added at 20 °C in a single portion to (S,R)-22 (534 mg, 1.08 mmol), and the resulting mixture was stirred at this temperature for 20 min. Subsequently, dichloromethane (30 mL) and an aqueous sodium hydroxide solution (1 M, 10 mL) were added. The organic phase was separated and washed with an aqueous sodium hydroxide solution (1 M, 3 × 10 mL). The combined aqueous layers were extracted with dichloromethane (3 × 20 mL), and the combined organic extracts were washed sequentially with water (10 mL) and a saturated aqueous sodium chloride solution (2 × 10 mL). The organic solution was dried over 5913


Organometallics

Article

(S)-3,3,3-Trifluoro-2-methoxy-2-phenylpropanonyl Chloride ((S)-28). This compound was commercially available (Sigma-Aldrich; enantiomeric excess, >99% ee). Synthesis of N-[(R)-3,3,3-Trifluoro-2-methoxy-2-phenylpropanonyl]-(R)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((R,R)-29) and N-[(R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanonyl]-(S)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((R,S)29). Propylene oxide (124 mg, 2.13 mmol) and (S)-28 (134 mg, 530 μmol) were added one after another in single portions to a stirred suspension of rac-15·HCl (120 mg, 531 μmol) in tetrahydrofuran (5 mL) at 20 °C, and the resulting mixture was heated under reflux for 2 h. Subsequently, the volatile components were removed under reduced pressure, and the residue was dissolved in diethyl ether (30 mL). The resulting solution was washed successively with a saturated aqueous sodium carbonate solution (3 × 15 mL), hydrochloric acid (2 M, 3 × 15 mL), and a saturated aqueous sodium chloride solution (2 × 10 mL). The organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was dissolved in methanol (800 μL) and then further purified by RP-MPLC (eluent, methanol/water (4/1 v/v); flow rate, 19 mL min−1; detector wavelength, 210 nm) to furnish a 1/1 mixture of the two diastereomers (R,R)-29 and (R,S)-29 (181 mg, 446 μmol; 84% yield) as a colorless liquid. 1H NMR (500.1 MHz, C6D6; data for two diastereomers (molar ratio, 1/1); the resonance signals of (R,S)-29 are marked with asterisks):28 δ −0.01*/0.07 (s, 9 H; Si(CH3)3), 0.92*/ 1.02 (δA) and 1.35/1.38* (δB) (AB system, 2J(A,B) = 14.6*/14.6 Hz; 2 H; SiCHAHB), 1.78*/1.80 (s, 3 H; CCH3), 3.27/3.49* (q, 5J(H,F) = 1.5/1.8 Hz*, 3 H; COCH3), 3.45/3.47* (s, 3 H; C(O)OCH3), 7.1*/7.4 (br s, 1 H; NH), 7.14−7.19 (m, 1 H; p-CH, C6H5), 7.20−7.26 (m, 2 H; m-CH, C6H5), 7.86−7.90 ppm (m, 2 H; o-CH, C6H5). 13C NMR (125.8 MHz, C6D6; data for two diastereomers (molar ratio, 1/1); the resonance signals of (R,S)-29 are marked with asterisks):28 δ −0.6*/− 0.5 (Si(CH3)3), 24.8*/25.1 (CCH3), 28.0*/28.4 (SiCH2), 51.9/52.0* (C(O)OCH3), 54.7/55.1* (q, 4J(C,F) = 1.9/1.9* Hz; COCH3), 58.88*/58.91 (CCH3), 84.3*/84.4 (q, 2J(C,F) = 26.0/26.0* Hz; CCF3), 124.5*/124.7 (q, 1J(C,F) = 290.0/290.0* Hz; CCF3), 127.9*/ 128.5 (q, 4J(C,F) = 1.5/1.5* Hz; o-CH, C6H5), 128.57*/128.58 (m-CH, C6H5), 129.47*/129.50 (p-CH, C6H5), 133.1/133.7* (i-C, C6H5), 165.0/165.2* (C(O)NH), 174.1/174.3* ppm (C(O)OCH3). 15N NMR (50.7 MHz, C6D6; data for two diastereomers (molar ratio, 1/1); the resonance signal of (R,S)-29 is marked with an asterisk):28 δ −252.5*/− 252.1 ppm; 19F NMR (376.5 MHz, C6D6; data for two diastereomers (molar ratio, (1/1); the resonance signal of (R,S)-29 is marked with an asterisk):28 δ −68.27*/−68.32 ppm. 29Si NMR (99.4 MHz, C6D6; data for two diastereomers (molar ratio, 1/1); the resonance signal of (R,S)29 is marked with an asterisk):28 δ −1.23*/−1.20 ppm. GC/EI-MS (data for two diastereomers (molar ratio, 1:1); the signal of (R,S)-29 is marked with an asterisk):29 tR = 10.6*/10.7 min; m/z (%) 390 (40) [M+ − CH3], 346 (100) [M+ − C(O)OCH3]. Anal. Calcd for C18H26F3NO4Si: C, 53.32; H, 6.46; N, 3.45. Found: C, 53.25; H, 6.62; N, 3.54. Determination of the Enantiomeric Excess of (R)-4 and (S)-4 by NMR Spectroscopy. The enantiomeric excesses of (R)-4 and (S)4 were determined by 1H, 13C, 15N, 19F, and 29Si NMR experiments after Mosher-type derivatization13 (rac-4 → rac-15·HCl → (R,R)-29/ (R,S)-29; (R)-4 → (R)-15·HCl → (R,R)-29; (S)-4 → (S)-15·HCl → (R,S)-29) (see section above). The NMR spectra were recorded at 23 °C on a Bruker Avance 400 (19F, 376.5 MHz) or Bruker Avance 500 NMR spectrometer (1H, 500.1 MHz; 13C, 125.8 MHz; 15N, 50.7 MHz; 29Si, 99.4 MHz). The composition of the samples used for the NMR experiments was as follows: sample substance (29), 18 mg; C6D6, 750 μL. Determination of the Enantiomeric Excess of (R)-4 and (S)-4 by Gas Chromatography. The enantiomeric excesses of (R)-4 and (S)-4 were determined by capillary gas chromatography after Moshertype derivatization13 (see section below). The experimental conditions were as follows: gas chromatograph, Varian 450-GC; column, Varian FactorFour VF-5 ms capillary column (length, 30 m; internal diameter, 0.25 mm; film thickness, 0.25 μm); carrier gas, helium; constant column flow, 1 mL min−1; temperature program, 80 °C (2 min) to 280

anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The remaining residue was dissolved in methanol (1.2 mL) and then further purified by RP-MPLC (eluent, methanol/water (7/3 v/v); flow rate, 16 mL min−1; detector wavelength, 210 nm) to furnish (S,R)-23 (297 mg, 753 μmol; 70% yield) as a colorless liquid. 1H NMR (400.1 MHz, CD2Cl2): δ 0.02 (s, 9 H; Si(CH3)3), 1.24 (δA) and 1.48 (δB) (AB system, 2J(A,B) = 14.6 Hz, 2 H; SiCHAHB), 1.54 (s, 3 H; CCH3), 1.8 (br s, 2 H; NH2), 2.59 (δA), 2.94 (δB), and 3.63 (δX) (ABX system, 2J(A,B) = 16.8 Hz, 3J(A,X) = 8.4 Hz, 3J(B,X) = 3.9 Hz, 3 H; CHAHBCHX), 3.66 (s, 3 H; OCH3), 5.13 (δA) and 5.14 (δB) (AB system, 2J(A,B) = 12.4 Hz, 2 H; OCHAHB), 7.29−7.39 (m, 5 H; C6H5), 7.9 ppm (br s, 1 H; C(O)NH). 13C NMR (125.8 MHz, CD2Cl2): δ −0.3 (Si(CH3)3), 25.7 (CCH3), 27.8 (SiCH2), 39.7 (CH2CH), 52.4 (CH2CH), 52.5 (OCH3), 58.5 (CCH3), 66.8 (OCH2), 128.5 (o-C, C6H5), 128.6 (p-C, C6H5), 128.9 (m-C, C6H5), 136.3 (i-C, C6H5), 172.1 (C(O)OCH2C6H5), 172.3 (CC(O)NH), 175.4 ppm (C(O)OCH3). 15N NMR (50.7 MHz, CD2Cl2): δ −352.6 (NH2), −253.0 ppm (C(O)NH). 29Si NMR (99.4 MHz, CD2Cl2): δ −0.8 ppm. EI-MS: m/z (%) 379 (21) [M+ − CH3], 91 (100) [C7H7+]. HRMS (ESI): m/z calcd for [M + Na]+, 417.1816; found, 417.1815. Synthesis of 4-Benzyloxy-(S)-aspart-1-yl-(S)-2[(trimethylsilyl)methyl]alanine Methyl Ester ((S,S)-23). A mixture of chlorotrimethylsilane (737 mg, 6.78 mmol), phenol (1.98 g, 21.0 mmol), and dichloromethane (10 mL) was added at 20 °C in a single portion to (S,S)-22 (747 mg, 1.51 mmol), and the resulting mixture was stirred at this temperature for 20 min. Subsequently, dichloromethane (60 mL) and an aqueous sodium hydroxide solution (1 M, 60 mL) were added. The organic phase was separated and washed with an aqueous sodium hydroxide solution (1 M, 3 × 20 mL). The combined aqueous layers were extracted with dichloromethane (3 × 30 mL), and the combined organic extracts were washed sequentially with water (10 mL) and a saturated sodium chloride solution (2 × 10 mL). The organic solution was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The remaining residue was dissolved in methanol (1 mL) and then further purified by RP-MPLC (eluent, methanol/water (7/3 v/v); flow rate, 15 mL min−1; detector wavelength, 220 nm) to furnish (S,R)-23 (537 mg, 1.36 mmol; 90% yield) as a colorless liquid. 1H NMR (500.1 MHz, CD2Cl2): δ 0.01 (s, 9 H; Si(CH3)3), 1.22 (δA) and 1.59 (δB) (AB system, 2J(A,B) = 14.7 Hz, 2 H; SiCHAHB), 1.56 (s, 3 H; CCH3), 1.8 (br s, 2 H; NH2), 2.62 (δA), 2.92 (δB), and 3.61 (δX) (ABX system, 2 J(A,B) = 16.7 Hz, 3J(A,X) = 8.4 Hz, 3J(B,X) = 3.8 Hz, 3 H; CHAHBCHX), 3.67 (s, 3 H; OCH3), 5.127 (δA) and 5.134 (δB) (AB system, 2J(A,B) = 14.4 Hz, 2 H; OCHAHB), 7.30−7.39 (m, 5 H; C6H5), 8.0 ppm (br s, 1 H; C(O)NH). 13C NMR (125.8 MHz, C6D6): δ −0.5 (Si(CH3)3), 26.4 (CCH3), 27.0 (SiCH2), 39.2 (CH2CH), 51.9 (OCH3), 52.6 (CH2CH), 58.6 (CCH3), 66.2 (OCH2), 128.2 (p-C, C6H5), 128.5 (o-C, C6H5), 128.7 (m-C, C6H5), 136.5 (i-C, C6H5), 171.7 (C(O)OCH2C6H5), 172.0 (CC(O)NH), 175.4 ppm (C(O)OCH3). 15N NMR (50.7 MHz, CD2Cl2): δ −352.6 (NH2), −253.9 ppm (C(O)NH). 29Si NMR (99.4 MHz, C6D6): δ −1.1 ppm. EI-MS: m/z (%): 379 (1) [M+ − CH3], 91 (100) [C7H7+]. HRMS (ESI): m/z calcd for [M + Na]+, 417.1816; found, 417.1814. 2,2-Dimethylpropanal (24). This compound was commercially available (Sigma-Aldrich). Synthesis of N-(2,2-Dimethylpropylidene)-4-benzyloxy-(S)aspart-1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,R)-25). See synthesis of (S,R)-7. Synthesis of N-(2,2-Dimethylpropylidene)-4-benzyloxy-(S)aspart-1-yl-(S)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,S)-25). See synthesis of (S,S)-7. Synthesis of N-(3,3-Dimethylbutylidene)-4-benzyloxy-(S)aspart-1-yl-(R)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,R)-26). See synthesis of (S,R)-8. Synthesis of N-(3,3-Dimethylbutylidene)-4-benzyloxy-(S)aspart-1-yl-(S)-2-[(trimethylsilyl)methyl]alanine Methyl Ester ((S,S)-26). See synthesis of (S,S)-8. 3,3-Dimethylbutanal (27). This compound was commercially available (Sigma-Aldrich). 5914


Organometallics

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°C (13 min) with 20 °C min−1; injector temperature, 220 °C; split, 1/100; detector, Varian 320-MS SQ (EI-MS, 70 eV); reconstructed ion current (RIC), mass range m/z 200−410. The retention times of the derivatives of (R)-4 ((R,R)-29) and (S)-4 ((R,S)-29) were as follows: (R,R)-29, 10.7 min; (R,S)-29, 10.6 min. Derivatization of (R)-4 and (S)-4 for the Determination of the Enantiomeric Excess by Gas Chromatography. A 1.25 M solution of hydrogen chloride in methanol (4 mL, 5.00 mmol of HCl) was added at 20 °C to 4 (1.00 mg, 5.70 μmol), and the resulting solution was heated under reflux for 15 min. The solvent and excess hydrogen chloride were removed under reduced pressure to furnish 15·HCl (crude product, not further purified). 15·HCl was dissolved in tetrahydrofuran (3 mL), followed by sequential addition of propylene oxide (1.32 mg, 22.7 μmol) and (S)-28 (1.44 mg, 5.70 μmol) at 20 °C. The resulting mixture was heated under reflux for 30 min, and the volatile components were removed under reduced pressure. The residue was dissolved in diethyl ether (700 μL), the resulting solution was extracted with water (300 μL), and a 2 μL sample of the organic phase was injected into the gas chromatograph. Crystal Structure Analyses. A 1 M solution of sodium hydroxide (48 μL, 48 μmol of NaOH) was added at 20 °C to a solution of (S,R)5·HCl (16.4 mg, 48 μmol) in methanol (500 μL), and the resulting mixture was kept undisturbed at 10 °C for 35 days to give suitable single crystals of (S,R)-5·2H2O. Suitable single crystals of (S,R)-22 were obtained by undisturbed storage of the oily product obtained by the synthesis of (S,R)-22 as described above. Suitable single crystals of the other compounds were obtained by crystallization from water at 20 °C ((S,R)-5·TFA)], from methanol/water (83/17 v/v) at 10 °C ((S,R)7·H2O), from methanol/water (88/12 v/v) at 10 °C ((S,R)-8·2.5H2O), from dichloromethane/n-hexane (1/1 v/v) at 10 °C ((R)-15·HCl), from methanol/water (58/42 v/v) at 20 °C ((S,R)-19·0.22H2O), from methanol/water (68/32 v/v) at 20 °C ((S,R)-20), and from nhexane at 20 °C (cooling of a boiling solution to 20 °C; (S,S)-22). The crystals were mounted in inert oil (perfluoropolyalkyl ether, ABCR) on a glass fiber and then transferred to the cold nitrogen gas stream of the diffractometer ((S,R)-5·2H2O, (R)-15·HCl, and (S,R)22, Stoe IPDS, graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å); (S,R)-5·TFA, (S,R)-7·H2O, (S,R)-8·2.5H2O, (S,R)-19·0.22H2O, (S,R)-20, and (S,S)-22, Bruker Nonius KAPPA APEX II, Montel mirror, MoKα radiation (λ = 0.710 73 Å)). The structures were solved by direct methods.30 All non-hydrogen atoms were refined anisotropically.30 A riding model was employed in the refinement of the CH hydrogen atoms. CCDC-877521 ((S,R)-5·2H2O), CCDC-877522 ((S,R)-5·TFA), CCDC-877523 ((S,R)-7·H2O), CCDC877524 ((S,R)-8·2.5H2O), CCDC-877525 ((R)-15·HCl), CCDC877526 ((S,R)-19·0.22H2O), CCDC-877527 ((S,R)-20), CCDC-877528 ((S,R)-22), and CCDC-877529 ((S,S)-22) contains supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/data_request/cif. Determination of Physicochemical Properties. (a). Materials. Hank's balanced saline solution (HBSS) and 2-[4-(2-hydroxyethyl)piperazino]ethanesulfonic acid (HEPES) were purchased from GIBCO (Carlsbad, CA). The solvents used for the experiments were of analytical grade, and the water used was obtained from a water purification system (Elgastat Maxima, ELGA, Lane End, U.K.). The HBSS buffer (pH 7.4) was prepared as follows: an aqueous solution of HEPES (12.5 mL, 1 M) was added to HBSS (500 mL), and the pH was titrated to 7.4 with an aqueous sodium hydroxide solution (10 mM). (b). Solubility in HBSS Buffer (pH 7.4). The liquid handling for the solubility assay was automated in a 96-well format using a Multiprobe II HT EX robot (Packard, Meriden, CT). The test compounds (10 μL, 10 mM in DMSO) were diluted in HBSS buffer (990 μL, pH 7.4) and shaken at room temperature on a flatbed shaker. After 16 h, samples of 400 μL were filtered through a 8 × 12 Whatman GF/B well filter (assisted by vacuum). The filtered samples were analyzed on an Agilent 1100 HPLC system (Waldbronn, Germany) with a diode array detector and coupled to a Micromass LC-TOF mass spectrometer (Waters, Wythenshawe, U.K.) equipped with an electrospray interface.

The software used for evaluation of the data was QuanLynx (Waters). As standards for the concentration estimations, samples with the same degree of dilution were prepared, but with acetonitrile instead of the buffer. (c). log D Values. The log D values were obtained by determining the capacity factor k′. Thus, an Agilent 1100 HPLC system was used to inject samples onto a reversed-phase LC column (Waters XTerra C18, 3.5 μm, 100 × 2.1 mm). The outlet from the LC column was connected to a Micromass LC-TOF mass spectrometer (Waters) equipped with an electrospray interface. The pumps were programmed to deliver the following gradient at a flow rate of 300 μL min−1 (mobile phase A, 5% acetonitrile/95% aqueous ammonium acetate solution (10 mM, pH 7.4); mobile phase B, 95% acetonitrile/5% aqueous ammonium acetate solution (10 mM, pH 7.4)): 100% A (2 min), linear gradient from 100% A to 100% B (2−17 min), 100% B (3 min). Standardized k′́ data were obtained by calibrating against a set of compounds with precisely determined k′́ values (warfarin, testosterone, metoprolol, propranolol, felodipine). The log D values in octanol/water are also known for these compounds, which allowed mapping of the results onto a log D scale. Determination of hPPB Values. (a). Materials. The potassium phosphate buffer (122 μM, pH 7.4) was of analytical grade. The water used in the experiments was obtained from a water purification system (Elgastat Maxima). (b). Assays. Equilibrium dialysis was carried out using a laboratorymade dialysis plate, made of polyether ether ketone (PEEK), consisting of two symmetric halves, and a semipermeable membrane with a molecular cutoff of 6−8 KDa (Spectra/Por, Spectrum Laboratories Inc., CA). The membrane was first soaked in water for 30 min, before being placed between the dialysis plate halves. A phosphate buffer (122 μM, 190 mL, pH 7.4) was added into one side of the chamber, and an equivalent volume of plasma with spiked test compounds (10 mM of each compound) was added to the other side of the membrane. The test compounds were dialyzed for 18 h at 37 ± 1 °C in an air incubator on an orbital shaker. A 50 mL volume from the buffer side (representing the unbound concentration) and an equivalent sample volume from the plasma side (representing the total concentration) were transferred from the dialysis cells to a 96-deep-well plate for LC-MS analysis. Determination of CYP Inhibition. (a). Materials. Nicotinamide adenine dinucleotide phosphate (NADPH, reduced form, tetrasodium salt, 98%) and Tris base were purchased from Sigma-Aldrich (St. Louis, MO). The potassium phosphate buffer (pH 7.4) and acetonitrile were of analytical grade. The water used in the experiments was obtained from a water purification system (Elgastat Maxima). The recombinant human enzymes used were prepared in house,31 except for CYP2D6, which was purchased from CYPEX (Dundee, U.K.). The following coumarin substrates, biotransformed into fluorescent metabolites, were used as probes for each individual CYP: CYP3A4, 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC; Gentest, Woburn, MA); CYP2D6, 7-methoxy-4-(aminomethyl)coumarin (MAMC; Gentest). A fluorescence plate reader (SpectraMax GeminiXS, Molecular Devices, Sunnyvale, CA) was used to measure the levels of metabolites formed. (b). Assays. A fluorescence-based method according to ref 32 in 96well format was used to determine the inhibition of two different CYPs (3A4 and 2D6). Dilution series of the test compounds were prepared at eight different concentrations (for CYP3A4, 50.0, 16.7, 5.56, 1.85, 0.617, 0.206, 0.0686, and 0.0229 μM; for CYP2D6, 20.0, 6.67, 2.22, 0.741, 0.247, 0.0823, 0.0274, and 0.00914 μM). For each CYP, a mixture of the enzyme, the corresponding coumarin substrate, potassium phosphate buffer (pH 7.4), and water (concentrations and volumes were CYP dependent) were added to each well in a black 96-well plate. The test compounds at different concentrations were added. After 10 min of preincubation, the cofactor NADPH was added to initiate the reaction. After 20−50 min (CYP and substrate dependent), the reaction was terminated by addition of 0.1 M Tris base in acetonitrile/water (4/1 v/v). The plates were transferred to the fluorescence plate reader, where the wavelengths were set individually for the different coumarin substrates and their respective fluorescent 5915


Organometallics

Article

and (S,S)-23, and tables and CIF files giving the crystallographic data for (S,R)-5·2H2O, (S,R)-5·TFA, (S,R)-7·H2O, (S,R)-8·2.5H2O, (R)-15·HCl, (S,R)-19·0.22H2O, (S,R)-20, (S,R)-22, and (S,S)-22. This material is available free of charge via the Internet at http://pubs.acs.org.

metabolites (CYP3A4, 7-hydroxy-4-(trifluoromethyl)coumarin (HFC); CYP2D6, 7-hydroxy-4-(aminomethyl)coumarin (HAMC)). The responses were exported to Excel, with which the IC50 curves (XLfit) were plotted (percent inhibition versus concentration) and IC50 values calculated for each test compound and enzyme. Determination of Intrinsic Clearance and Half-Lives in Liver Microsomes. (a). Materials. Human liver microsomes were prepared according to ref 33 and stored as granulates at −70 °C. Nicotinamide adenine dinucleotide phosphate (NADPH, reduced form, tetrasodium salt, 98%) was purchased from Sigma-Aldrich. The potassium phosphate buffer (93 mM, pH 7.4) and acetonitrile were of analytical grade. The water used in the experiments was obtained from a water purification system (Elgastat Maxima). (b). Assays. The metabolic stability in human liver microsomes was tested by a substrate depletion method.34 The liquid handling was automated in a 96-well format using a Tecan Genesis Workstation 200 equipped with a Tecan GenMate 96 pipetting robot (Männerdorf, Switzerland). A mixture of microsomes (0.5 mL−1), potassium phosphate buffer (93 mM, pH 7.4), and the test substrate (1 μM) was prepared in the 96-well plates and incubated at 37 °C. The reaction was initiated by addition of the cofactor NADPH (1 mM). Aliquots were withdrawn from the incubation mixture at 0, 3, 7, 15, and 30 min, and the reaction was terminated by precipitation of the proteins with 3.5 parts of cold acetonitrile. After centrifugation (20 min, 2900g), the supernatant was diluted with an equal amount of water and then analyzed by LC-MS. The LC system consisted of an Agilent 1100 pump and a CTC HTS PAL injector (Zwingen, Switzerland). A Waters Quattro Ultima triple-quadrupole mass spectrometer (Wythenshawe, U.K.), equipped with an electrospray ion source, was used for quantification by selected reaction monitoring. The peaks in the chromatograms were integrated by the QuanLynx software, and the peak areas were exported to Excel, with which log [area] versus time (min) was plotted (XLfit). The rate of compound disappearance over time was calculated from the slope of the line (rate constant k). Both clearance values (CLint) and half-lives (t1/2) were calculated from k. Determination of T1R2/R3 Activating Properties. (a). Materials. STC-1 cells (Origin CSH; obtained from Dr. Brian Matlock, AstraZeneca, Wilmington, DE) were grown in DMEM, glutamax, 4.5 g L−1 D-glucose, pyruvate (GIBCO) + pen/strep (GIBCO) + 2.5% v/v FCS (HyClone, Logan, UT) + 10% v/v horse serum (GIBCO).18 The potassium phosphate buffer (pH 7.4) was of analytical grade. The water used in the experiments was obtained from a water purification system (Elgastat Maxima). (b). Assays. The cells were plated in 96-well poly-D-lysine plates (Greiner Bio-One, Kremsmünster, Austria) and grown to 90% confluence. The medium was removed and replaced with assay buffer (HEPES solution with Ca2+, Mg2+, and 0.1% BSA) with test compounds at final concentrations of 0.5−2 mM, and it was incubated for 2 h at 37 °C. Phenylthiocarbamide (PTC) was used at 6 mM, sucralose at 25 mM, and aspartame and neotame at 2 mM final concentrations. Supernatants were removed, centrifuged to remove cell debris, and frozen at −80 °C prior to testing in the Linco GLP-1 (Active) ELISA kit (Millipore, Billerica, MA) according to the Linco protocol. Plates were read on a Spectramax Gemini XS fluorescence plate reader with an excitation/emission wavelength of 355 nm/460 nm. Sensory Evaluation. (S,R)-5·TFA, (S,S)-5·TFA, (S,R)-8, and (S,S)-8 were taste-checked by two volunteers in blind tests using dilute aqueous solutions of each test compound. Different concentrations (between 0.1 and 0.001% (w/v)) of each dipeptide were evaluated at 3−5 min intervals. About 1.5 mL of each solution was applied via pipettes and allowed to flow over the voluenteer’s tongue for several seconds. Sweetness intensities were determined relative to aqueous sucrose solutions of 0.5, 2.0, 4.0, and 8.0% (w/v) as the reference.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) (a) Handmann, V. I.; Merget, M.; Tacke, R. Z. Naturforsch. 2000, 55b, 133. (b) Tacke, R.; Merget, M.; Bertermann, R.; Bernd, M.; Beckers, T.; Reissmann, T. Organometallics 2000, 19, 3486. (c) Merget, M.; Günther, K.; Bernd, M.; Günther, E.; Tacke, R. J. Organomet. Chem. 2001, 628, 183. (d) Tacke, R.; Handmann, V. I. Organometallics 2002, 21, 2619. (e) Tacke, R.; Handmann V. I. (inventors), PCT Int. Pat. Appl. WO 03/082880 A1, October 9, 2003. (f) Tacke, R.; Schmid, T.; Merget, M. Organometallics 2005, 24, 1780. (g) Falgner, S.; Schmidt, D.; Bertermann, R.; Burschka, C.; Tacke, R. Organometallics 2009, 28, 2927. (h) Falgner, S.; Burschka, C.; Wagner, S.; Böhm, A.; Daiss, J. O.; Tacke, R. Organometallics 2009, 28, 6059. (i) Falgner, S.; Buchner, G.; Tacke, R. J. Organomet. Chem. 2010, 695, 2614. (2) Weidmann, B. Chimia 1992, 46, 312. (3) (a) Mortensen, M.; Husmann, R.; Veri, E.; Bolm, C. Chem. Soc. Rev. 2009, 38, 1002. (b) Qi, Y.; Sieburth, S. McN. In Amino Acids, Peptides and Proteins in Organic Chemistry: Modified Amino Acids, Organocatalysis and Enzyme; Hughes, A. B., Ed.; Wiley-VCH: Weinheim, Germany, 2009; Vol. 2, pp 261−280. (4) (a) Kamphuis, J.; Lelj, F.; Tancredi, T.; Tonioli, C.; Temussi, P. A. Quant. Struct.-Act. Relat. 1992, 11, 486. (b) Polinelli, S.; Broxterman, Q. B.; Schoemaker, H. E.; Boesten, W. H. J.; Crisma, M.; Valle, G.; Tonioli, C.; Kamphuis, J. Bioorg. Med. Chem. Lett. 1992, 2, 453. (c) Hooper, N. M.; Hesp, R. J.; Tieku, S.; Boesten, W. H. J.; Toniolo, C.; Kamphuis, J. J. Agric. Food Chem. 1994, 42, 1397. (d) Benedetti, E.; Gavuzzo, E.; Santini, A.; Kent, D. R.; Zhu, Y.-F.; Zhu, Q.; Mahr, C.; Goodman, M. J. Pept. Sci. 1995, 1, 349. (e) Mossel, E.; Formaggio, F.; Crisma, M.; Toniolo, C.; Broxterman, Q. B.; Boesten, W. H. J.; Kamphuis, J.; Quaedflieg, P. J. L. M.; Temussi, P. Tetrahedron: Asymmetry 1997, 8, 1305. (f) Mossel, E.; Formaggio, F.; Valle, G.; Crisma, M.; Toniolo, C.; Doi, M.; Ishida, T.; Broxterman, Q. B.; Kamphuis, J. Lett. Pept. Sci. 1998, 5, 223. (g) Goodman, M.; Del Valle, J. R.; Amino, Y.; Benedetti, E. Pure Appl. Chem. 2002, 74, 1109. (5) It is worth noting that (R)-4 and (S)-4 are bioisosteric with Lphenylalanine ((S)-phenylalanine) and D-phenylalanine ((R)-phenylalanine), respectively. Due to changed atom priorities according to the Cahn−Ingold−Prelog rules, the configuration symbols (R, S) of the respective bioisosters are different. (6) Recent reviews dealing with α,α-dialkylated α-amino acids: (a) Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem. Rev. 2001, 101, 3131. (b) Cativiela, C.; Díaz-de-Villegas, M. D. Tetrahedron: Asymmetry 2007, 18, 569. (c) Tanaka, M. Chem. Pharm. Bull. 2007, 55, 349. (d) Vogt, H.; Bräse, S. Org. Biomol. Chem. 2007, 5, 406. (7) Reviews dealing with artificial sweeteners and the theory of sweet taste: (a) Yamazaki, T.; Benedetti, E.; Kent, D.; Goodman, M. Angew. Chem. 1994, 106, 1502; Angew. Chem., Int. Ed. Engl. 1994, 33, 1437. (b) Temussi, P. J. Mol. Recognit. 2006, 19, 188. (c) Meyers, B.; Brewer, M. J. Food Sci. 2008, 73, R81. (8) Reviews dealing with silicon-based drugs: (a) Bains, W.; Tacke, R. Curr. Opin. Drug Discovery Dev. 2003, 6, 526. (b) Showell, G. A.; Mills, J. S. Drug Discovery Today 2003, 8, 551. (c) Mills, J. S.; Showell, G. A. Expert Opin. Investig. Drugs 2004, 13, 1149. (d) Pooni, P. K.; Showell, G. A. Mini-Rev. Med. Chem. 2006, 6, 1169. (e) Sieburth, S. McN.; Chen, C.-A. Eur. J. Org. Chem. 2006, 311. (f) Gately, S.; West, R. Drug

ASSOCIATED CONTENT

S Supporting Information *

Figures showing the determination of the enantiomeric excess of (R)-4 and (S)-4, figures giving the 1H and 13C NMR spectra of rac-15·HCl, (R)-15·HCl, (S)-15·HCl, (R)-16·HCl, (S,R)-23, 5916


Organometallics

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(R)-4 or (S)-4 ((R)-4 → (R)-15·HCl → (R,R)-29; (S)-4 → (S)15·HCl → (R,S)-29). (29) Assignment of the respective signal was accomplished by comparison with the gas chromatographic data of the single diastereomers (R,R)-29 and (R,S)-29, which were synthesized from (R)-4 or (S)-4 ((R)-4 → (R)-15·HCl → (R,R)-29; (S)-4 → (S)15·HCl → (R,S)-29). (30) Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112. (31) Masimirembwa, C. M.; Otter, C.; Berg, M.; Jönsson, M.; Leidvik, B.; Jonsson, E.; Johansson, T.; Bäckman, A.; Edlund, A.; Andersson, T. B. Drug Metab. Dispos. 1999, 27, 1117. (32) Crespi, C. L.; Miller, V. P.; Penman, B. W. Anal. Biochem. 1997, 248, 188. (33) Ernster, L.; Siekevitz, P.; Palade, G. E. J. Cell Biol. 1962, 15, 541. (34) Baranczewski, P.; Stańczak, A.; Sundberg, K.; Svensson, R.; Wallin, Å.; Jansson, J.; Garberg, P.; Postlind, H. Pharmacol. Rep. 2006, 58, 453.

Dev. Res. 2007, 68, 156. (g) Franz, A. K. Curr. Opin. Drug Discovery Dev. 2007, 10, 654. (h) Meanwell, N. A. J. Med. Chem. 2011, 54, 2529. (9) Recent publications dealing with silicon-based drugs: (a) Johansson, T.; Weidolf, L.; Popp, F.; Tacke, R.; Jurva, U. Drug Metab. Dispos. 2010, 38, 73. (b) Tacke, R.; Nguyen, B.; Burschka, C.; Lippert, W. P.; Hamacher, A.; Urban, C.; Kassack, M. U. Organometallics 2010, 29, 1652. (c) Bauer, J. B.; Lippert, W. P.; Dörrich, S.; Tebbe, D.; Burschka, C.; Christie, V. B.; Tams, D. M.; Henderson, A. P.; Murray, B. A.; Marder, T. B.; Przyborski, S. A.; Tacke, R. ChemMedChem 2011, 6, 1509. (d) Barnes, M. J.; Burschka, C.; Büttner, M. W.; Conroy, R.; Daiss, J. O.; Gray, I. C.; Hendrick, A. G.; Tam, L. H.; Kuehn, D.; Miller, D. J.; Mills, J. S.; Mitchell, P.; Montana, J. G.; Muniandy, P. A.; Rapley, H.; Showell, G. A.; Tebbe, D.; Tacke, R.; Warneck, J. B. H.; Zhu, B. ChemMedChem 2011, 6, 2070. (e) Tacke, R.; Bertermann, R.; Burschka, C.; Dörrich, S.; Fischer, M.; Müller, B.; Meyerhans, G.; Schepmann, D.; Wünsch, B.; Arnason, I.; Bjornsson, R. ChemMedChem 2012, 7, 523. (10) Review dealing with silicon-based odorants: Tacke, R.; Metz, S. Chem. Biodiversity 2008, 5, 920. (11) Recent publications dealing with silicon-based odorants: (a) Nätscher, J. B.; Laskowski, N.; Kraft, P.; Tacke, R. ChemBioChem 2010, 11, 315. (b) Sunderkötter, A.; Lorenzen, S.; Tacke, R.; Kraft, P. Chem. Eur. J. 2010, 16, 7404. (c) Geyer, M.; Bauer, J.; Burschka, C.; Kraft, P.; Tacke, R. Eur. J. Inorg. Chem. 2011, 2769. (12) Kaiser, E., Sr.; Picart, F.; Kubiak, T.; Tam, J. P.; Merrifield, R. B. J. Org. Chem. 1993, 58, 5167. (13) (a) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543. (b) Bringmann, G.; God, R.; Schäffer, M. Phytochemistry 1996, 43, 1393. (14) The hydrogen-bonding systems were analyzed by using the program system PLATON: Spek, A. L. PLATON; University of Utrecht, Utrecht, The Netherlands, 2008. (15) Hatada, M.; Jancarik, J.; Graves, B.; Kim, S.-H. J. Am. Chem. Soc. 1985, 107, 4279. (16) Wink, D. J.; Schroeder, S. A.; Prakash, I.; Lam, K.-C.; Rheingold, A. L. Acta Crystallogr., Sect. C 1999, C55, 1365. (17) The molecular structure of (S,S)-11 in Figure 1 was generated from data given in ref 4b. (18) Rindi, G.; Grant, S. G. N.; Yiangou, Y.; Ghatei, M. A.; Bloom, S. R.; Bautch, V. L.; Solcia, E.; Polak, J. M. Am. J. Pathol. 1990, 136, 1349. (19) Wu, S. V.; Rozengurt, N.; Yang, M.; Young, S. H.; SinnettSmith, J.; Rozengurt, E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2392. (20) Saitoh, O.; Hirano, A.; Nishimura, Y. NeuroReport 2007, 18, 1991. (21) Li, X.; Staszewski, L.; Xu, H.; Durick, K.; Zoller, M.; Adler, E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4692. (22) Reimer, R. A.; Darimont, C.; Gremlich, S.; Nicolas-Métral, V.; Rüegg, U. T.; Macé, K. Endocrinology 2001, 142, 4522. (23) (a) Mazur, R. H.; Schlatter, J. M.; Goldkamp, A. H. J. Am. Chem. Soc. 1969, 91, 2684. (b) Suami, T.; Hough, L. Food Chem. 1993, 46, 235. (24) (a) Nofre, C.; Tinti, J.-M. (inventors) U.S. Pat. Appl. 5 480 668, January 2, 1996. (b) Prakash, I.; Bishay, I.; Schroeder, S. Synth. Commun. 1999, 29, 4461. (25) (a) Program WIN-DAISY 4.05; Bruker-Franzen, Bremen, Germany, 1998. (b) Weber, U.; Germanus, A.; Thiele, H. Fresenius J. Anal. Chem. 1997, 359, 46. (26) The molar ratio (R)-4·HCl/NaOH or (S)-4·HCl/NaOH takes into account the amount of the crude product (R)-4·HCl or (S)-4·HCl obtained in the former step. (27) The ammonium salt (15·HCl or 16·HCl) was dissolved in a saturated aqueous sodium carbonate solution (700 μL), the resulting free base (15 or 16) was extracted with diethyl ether (700 μL), and the resulting organic solution was then injected into the mass spectrometer. (28) Assignment of the respective resonance signals was accomplished by comparison with the NMR spectroscopic data of the single diastereomers (R,R)-29 and (R,S)-29, which were synthesized from 5917


Silicon-Containing Dipeptidic Aspartame and Neotame Analogues

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