Protocol for the Incorporation of Î³-Amino Acids into Peptides

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A protocol for the incorporation of #-amino acids into peptides: application to (-)-shikimic acid based 2-amino-methylcyclohexanecarboxylic acids Marcos A. González, Amalia M Estévez, María Campos, Juan C Estévez, and Ramon J. Estevez J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02671 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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The Journal of Organic Chemistry

A protocol for the incorporation of γ-amino acids into peptides: application to (-)-shikimic acid based 2-aminomethylcyclohexanecarboxylic acids Marcos A. González, Amalia M. Estévez, María Campos, Juan C. Estévez* and Ramón J. Estévez Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) and Departamento de Química Orgánica. Campus Vida. Universidade de Santiago de Compostela, 15782 Santiago de Compostela, SPAIN TOC

The first example of a new protocol for the incorporation of γ-amino acids into peptides is reported. It

involved

a

shikimic

acid

based

stereoselective

synthesis

polyhydroxylated-2-

nitromethylcyclohexanecarboxylic acids, which were directly incorporated into peptides.

Currently, there is considerable interest on the stereoselective synthesis of non-natural amino acids, mainly β- and γ-amino acids.1 This constitutes the first step for the access to diverse conformationally strained peptidomimetics that can improve the limitations of α-peptides and their derived proteins, mainly their conformational flexibility and their metabolic instability.2 In fact, although less considered than β-amino acids,1a,g the search for efficient and versatile strategies for the synthesis of γ-amino acids became an active research field,1c,f on account of their biological activity as neurotransmitters3 and their application in the design and synthesis of peptides and nanotubes. Specific interest of alicyclic γ-amino acids lie in the possibilities offered for chemical diversity and for modulation of the conformational constrains in γ- and αγ-peptides,4 In fact, four structural motifs are present in this kind of γ-amino acids, depending on the number of carbon atoms in their backbone chain that are embedded in their rings.5,6 1 Environment ACS Paragon Plus

The Journal of Organic Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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This article deals with 2-aminomethylcyclohexanecarboxylic acids, a class of alicyclic γ-amino acids that have received some attention in recent times.6f-g But the range of their possible applications in γ-peptide chemistry is limited by their current small amount and the few methods reported for their selective, efficient preparation.7 In fact, a limited number of studies on their peptides have been reported.7c,8 Accordingly, there is some urgency in the search of methods for the selective preparation of these targets, that additionally allow to substantially increase their present amount, by the introduction of substituents on the ring and/or at the γ-carbon atom. Specifically, polyhydroxylated 2-aminomethylcyclohexanecarboxylic acids should give access to lipophilic or hydrophilic γ-peptides, by protection or deprotection of the hydroxy substituents. Herein we report the first synthesis of polyhydroxylated 2-aminomethylcyclohexane carboxylic acids from (-)-shikimic acid, a suitable starting material, on account of its convenient structural properties: a cyclohexane ring bearing an αβ-unsaturated carboxylic acid moiety and three hydroxy substituents with well defined spatial orientations.9 Our synthetic plan involved a Michael addition of nitromethane to an alkyl shikimate, followed by reduction of the nitro group of the resulting 2-nitromethylcarboxylic acids to amino.7b-c,10 A proper protocol for the incorporation of 2aminomethylcyclohexanecarboxylic acids into peptides was also developed. Michael addition of nitromethane to (-)-shikimic acid derivative 1a11 using DBU as a base, provided a mixture of γ-nitroester 2a (35%) and bicyclic lactone 4 (45%) (Scheme 1), whose respective absolute stereochemistries were unambiguously established by X-ray experiments (Figure 1).12 Alternatively, when this reaction was carried out by refluxing a mixture of shikimate 1a, nitromethane and TBAF in THF, the only reaction product was 2a. Formation of lactone 4 might be explained in terms of a spontaneous lactonization of γ-nitroester 3a under the reaction conditions. In a third experiment, aimed at avoiding this undesired lactonization, orthogonal protection of the free OH group of shikimic acid derivative 1a13 by treatment with TBDMSCl, was followed by reaction of the resulting derivative 1b with nitromethane, under the same conditions as for 1a. This provided a mixture of epimeric γ-nitroester 2b (35%) and 3b (45%) (Scheme 1). Structures of 2b and 3b were established as follows: treatment of trans-γ-nitroester 2a with TBDMSCl provided 2-nitromethylcyclohexanecarboxylic acid ester 2b.

On the other hand,

reaction of cis-γ-nitroester 3b with TBAF gave lactone 4 via γ-nitroester 3a, as expected.

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Scheme 1. Conditions: i) CH3NO2, DBU, rt, 48 h (45% 4, 35% 2a). ii) CH3NO2, TBAF 1 M in THF, THF, reflux, 16 h, (56%). iii) TBDMSCl, imidazole, DMF, rt, 12 h (86%). iv) CH3NO2, DBU, rt, 48 h (45% 3b, 35% 2b). v) TBDMSCl, imidazole, DMF, rt, 12 h (90%). vi) TBAF 1M in THF, rt, 12 h, (quantitative).

2a

4

Figure 1: ORTED diagrams ofγ-nitroester 2a and nitrolactone 4. It was assumed that the Michael addition of the nitronate anion of nitromethane to the αβunsaturated carboxylic acid ester moiety of 1a results in a stereospecific formation of adduct 5a and that the protonation of its enolate moiety can occur both according to trajectory a (less favoured) or trajectory b (more favoured), the result being the formation of an equilibrium mixture of 2a (thermodynamic isomer, minor component) and 3a (kinetic isomer, major component) (Scheme 2). Formation of the mixture 2b+3b from 1b might be explained in similar terms. This hypothesis is in accordance with the fact that when 1a was reacted with nitromethane, using TBAF as the base and THF as the solvent, the only reaction product was 2a. It is probable that in this case a similar mixture 2a+3a resulted, but the higher temperature favours the irreversible formation of the thermodynamically more stabilized isomer 2a.



Scheme 2 According to our plan, the transformation of γ-nitroester 2b into the corresponding orthogonally protected γ-amino acid 6b was next attempted (Scheme 3). As expected,10 catalytic hydrogenation of 2b directly provided γ-lactam 7a, as a result of the spontaneous cyclization of the γ-amino acid 6a initially formed. Reaction of 7a with Boc2O provided its N-protected derivative 7b, which was next subjected to basic hydrolysis conditions in order to transform it into the corresponding γ-amino acid 6b. But an inseparable mixture of 6b and its epimer 8 was obtained, as a result of the epimerization of the stereogenic center contiguous to the carbonyl group, probably before the opening of the lactam ring.

Scheme 3. Conditions: i) H2 (P=1 atm), Raney-Ni, MeOH, rt, 12 h, (98%). ii) Boc2O, DMPA, Cl2CH2, rt, 2 h, (95%). iii) LiOH(aq) 1M, THF, rt, 6 h, (91%). This side process was easily avoided by basic hydrolysis of 2b to its γ-nitro acid 2c (Scheme 4). Subsequent catalytic hydrogenation of 2c, in the presence of Boc2O, gave a mixture of the orthogonally protected γ-amino acid 6b (40% yield) and its γ-lactam 7b (35% yield). These compounds probably resulted from partial transformation of 6c into its N-unprotected lactam 7a and subsequent reaction of both compounds with Boc2O.


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Scheme 4. Conditions: i) LiOH(aq) 1M, THF, rt, 6 h (92%). ii) H2 (P=1 atm), Raney-Ni, MeOH, rt, 12 h; then: (Boc)2O, DMPA, Cl2CH2, rt, 1 h (40 % 6c, 35% 7b, two steps). iii) ClH3NCH2CO2Me, DIEA, coupling agent (see Table 1), DMF, rt, 24 h. In order to prevent the partial lactamization leading to 7b, incorporation of γ-amino acid 6b into peptides was directly assayed from its precursor 2c (Scheme 4). Accordingly, the reaction of 2c with glycine methyl ester hydrochloride, using EDCI as a coupling agent, gave the expected dipeptide-like compound 9, which in part underwent a retro-Michael reaction leading to the shikimic acid derivative 10 (Table 1, entry 1). Other coupling agents (TBTU, HATU, PyBOP) provided poorer results (Table 1, entries 2-4). Table 1 Starting material Coupling reagent 9 (% yield) 10 (% yield) EDCI 50 30 2c TBTU traces 20 2c HATU traces 16 2c PyBOP traces 15 2c Pentafluorophenol 90 0 2c

As shown in Table 1 (entries 1-4), the better results were achieved with the softer coupling agent (EDCI).14 Taking this into account, we hypothesized that activating the carboxylic acid moiety of compound 2c as a pentafluorophenol ester (a coupling agent softer than EDCI), the coupling reaction should improve. In confirming this hypothesis, reaction of acid 2c with DIC and pentafluorophenol provided 2d, with its carboxylic acid moiety activated as a pentafluorophenol ester (Scheme 5). Subsequent reaction of 2d with glycine methyl ester hydrochloride, using DIEA



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as the base, provided the dipeptide-like compound 9 (90%). This protocol allowed us to avoid a retro-Michael process, similar to those leading to compound 10. According to our plan, catalytic hydrogenation of 9, using Raney-Ni as the catalyst, gave the expected dipeptide 11, which directly provided tripeptide 12 (70% yield, two steps), after reaction with DIEA and the pentafluorophenol ester of N-benzyloxycarbonylglycine.15

Scheme 5. Conditions: i) Pentafluorophenol, DIC, Cl2CH2, rt, 12 h. ii) glycine methylester hydrochloride, DIEA, DMF, rt, 2 h (90% 9, 88% 13, two steps). iii) H2 (P=1 atm), Raney-Ni, MeOH, rt, 12 h. iv) perfluorophenyl 2-(((benzyloxy)carbonyl)amino)acetate, DIEA, DMF, rt, 3 h (70% 12, 68% 15, two steps). v) LiOH(aq), THF, rt, 6 h (95%). On the other hand, when the γ-nitro acid ester 3b was subjected to the protocol leading to tripeptide 12, tripeptide 15 was obtained via compounds 3c, 3d, 13 and 14. In summary, (-)-shikimic acid proved to be a suitable raw material for a simple, efficient and selective synthesis of 2-aminomethylcyclohexanecarboxylic acids, by a protocol involving a Michael addition of nitromethane to its αβ-unsaturated carboxylic acid moiety, followed by reduction of the nitro group to an amine. Following this protocol we have developed a simple and efficient synthesis of the two first reported polyhydroxylated 2-aminomethylcyclohexane carboxylic acids

[(3aR,4R,5R,7R,7aR)-7-((tert-Butyldimethylsilyl)oxy)-2,2-dimethyl-4-

(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylic acid

(2c) and (3aR,4R,5S,7R,7aR)-7-

((tert-Butyldimethylsilyl)oxy)-2,2-dimethyl-4-(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5carboxylic acid (3c). This (-)-shikimic acid based synthesis of 2-aminomethylcyclohexanecarboxylic acids expands the 6 Environment ACS Paragon Plus

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inventory of available building blocks for peptidomimetic foldamer construction.16 In addition, a protocol for the incorporation of these amino acids into peptides has been developed. Interest of this new protocol for the incorporation of γ-amino acids into peptides lies on that it allows to prevent their undesired high propensity to lactamization and epimerization. Work is now in progress aimed at the preparation of more complex peptides based on these γ-amino acids, in order to have access to lipo or hydrosoluble peptides, by protecting or deprotecting the OH substituents on the cyclohexane ring. They are promising candidates for biological applications, for a generation of new materials and for their potential applications as catalysts.

Experimental section General considerations Melting points were determined using a Kofler Thermogerate apparatus and are uncorrected. Specific rotations were recorded on a JASCO DIP-370 optical polarimeter. Infrared spectra were recorded on a MIDAC FTIR spectrophotometer. Nuclear magnetic resonance spectra were recorded on a Bruker WM-250 or a Varian Mercury 300 apparatus. Mass spectra were obtained on a Kratos MS 50 TC mass spectrometer. X-ray experiments were obtained with a Bruker Appex II apparatous. Elemental analyses were obtained from the Elemental Analysis Service at the University of Santiago de Compostela. Thin layer chromatography (tlc) was performed using Merck GF-254 type 60 silica gel and ethyl EtOAc/hexane mixtures as eluents; the tlc spots were visualized with a Hanessian stain (dipping into a solution of 12.5 g of (NH4)4Mo7O24.4H2O, 5 g of Ce(SO4)2.4H2O and 50 mL of H2SO4 in 450 mL of H2O, and warming). Column chromatography was carried out using Merck type 9385 silica gel. Solvents were purified as in reference 17.

(3aR,4R,5R,7R,7aS)-Methyl 7-hydroxy-2,2-dimethyl-4(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylate (2a) and (3aS,4R,7S,8R,8aR)-2,2dimethyl-8-(nitromethyl)tetrahydro-4,7-methano[1,3]dioxolo[4,5-c]oxepin-6(7H)-one (4) DBU (1.60 mL, 10.7 mmol) was added to a solution of 1a (2.0 g, 8.8 mmol) in nitromethane (160 mL, 2.42 mol) and the resulting solution was stirred at room temperature for 48 h. The liquids were then evaporated in vacuo and the residue was purified by flash column chromatography (EtOAc/hexane 1:1), to give 0.89 g, (35% yield) of γ-nitro ester 2a and 1.02 g (45% yield) of bicyclic nitrolactone 4, as pale yellow solids that were crystallized from a mixture of ethyl ether and hexane.



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Compound 2a: Rf (eluent: EtOAc/hexane 1:1): 0.40. [α]D20 +160° (c 1.0, CHCl3). mp 138-140 ºC (ethyl ether/hexane). IR (CHCl3) ν 3528, 1734, 1547, 1386 cm-1. 1H NMR (250 MHz, CDCl3) δ 1.36 (s, 3H, -CH3); 1.49 (s, 3H, -CH3); 1.91-2.09 (m, 2H, H-6, H-6´); 2.17 (d, 1H, J=3.3 Hz, -OH); 2.41-2.51 (m, 1H, H-1); 2.72-2.81 (m, 1H, H-2); 3.70 (s, 3H, -OCH3); 4.11-4.18 (m, 2H, -CHHNO2 and H3); 4.30-4.33 (m, 1H, -CHHNO2); 4.59-4.61 (m, 2H, H-4, and H-5) ppm.

13

C-NMR (62.5

MHz, CDCl3) δ 26.0 (CH3); 28.0 (CH3); 31.8 (CH2); 37.3 (CH); 40.5 (CH); 52.1 (CH3); 66.2 (CH); 73.8 (CH); 75.4 (CH2); 77.0 (CH); 109.6 (C); 174.1 (C) ppm. MS (CI) m/z 290 (100, [M+H]+). Anal calcd for C12H19NO7: C, 49.82; H, 6.62; N, 4.84. Found C, 49.61; H, 6.79; N, 5.02. Compound 4: Rf (eluent: EtOAc/hexane 1:1): 0.60. [α]D20 +37.5° (c 1.0, CHCl3). mp 109-111 ºC (ethyl ether/hexane). IR (CHCl3) ν 1777, 1552, 1383 cm-1. 1H NMR (250 MHz, CDCl3) δ 1.35 (s, 3H, -CH3); 1.54 (s, 3H, -CH3); 2.36-2.42 (m, 2H, H-1, and H-4); 2.67-2.74 (m, 2H, H-1´,and H-5); 3.93-3.97 (m, 1H, H-6); 4.46-4.54 (m, 3H, -CH2NO2 and H-7); 4.98-5.01 (m, 1H, H-2) ppm.

13

C-

NMR (62.5 MHz, CDCl3) δ 25.6 (CH3); 27.6 (CH3); 30.4 (CH2); 38.0 (CH); 42.5 (CH); 72.6 (CH); 74.2 (CH); 75.3 (CH2); 77.2 (CH); 112.3 (C); 175.1 (C) ppm. MS (CI) m/z 258 (100, [M+H]+); 200 (93, [MH-C3H6O]+); 153 (60, [MH-C8H9]+). Anal calcd for C11H15NO6: C, 51.36; H, 5.88; N, 5.44. Found C, 51.31; H, 6.01; N, 5.21.

(3aR,4R,5R,7R,7aS)-Methyl 7-hydroxy-2,2-dimethyl-4(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylate (2a) TBAF (0.76 mL, 1M in THF) was added to a solution of 1a (116 mg, 0.51 mmol) and nitromethane (55 µL, 1.02 mmol)) in dry THF (2 mL). The resulting mixture was refluxed for 17 h, under argon, and then concentrated under reduced pressure. The residue was taken up in EtOAc (30 mL), washed with aqueous 1M HCl (3x10 mL) and brine (3x10 mL), dried with anhydrous sodium sulphate and concentrated to dryness under reduced pressure. Flash column chromatography of residue (EtOAc/hexane 2:3) gave 82 mg (56% yield) of γ-nitro ester 2a, as a pale yellow solid.

(3aR,7R,7aR)-Methyl 7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-3a,6,7,7atetrahydrobenzo[d][1,3]dioxole-5-carboxylate (1b) tert-Butyldimethylsilyl chloride (6.72 g, 44.8 mmol) and imidazole (5.89 g, 88 mmol) were added to a solution of 1a (4.5 g, 19.7 mmol) in dry DMF (100 mL) and the resulting mixture was stirred at room temperature, under argon, for 12 h. Then, brine (20 mL) was added and the stirring was continued for 30 min. The reaction mixture was poured into water (200 mL), the suspension was extracted with EtOAc (3x100 mL), and the joined organic layers were washed with water (200 mL),


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dried with anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Purification of residue by flash column chromatography (EtOAc/hexane 1:10) gave 5.8 g (86% yield) of (-)-shikimic acid derivative 1b, as an oil: Rf (eluent: EtOAc/hexane 1:1): 0.90. [α]D20 -84.3 ° (c , 2.1 in CHCl3). IR (CHCl3) ν 1736 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.06 (s, 3H, -SiCH3); 0.08 (s, 3H, -SiCH3); 0.86 (s, 9H, -C(CH3)3); 1.38 (s, 6H, 2 x -CH3); 2.26-2.36 (m, 1H, H6); 2.492.61 (m, 1H, H6’); 3.76 (s, 3H, -OCH3); 4.02-4.09 (m, 2H); 4.68-4.71 (m, 1H); 6.82-6.85 (m, 1H) ppm. 13C NMR (62.5 MHz, CDCl3) δ -4.7 (CH3); -4.6 (CH3); 18.2 (C); 25.9 (3 x CH3); 26.3 (CH3); 28.1 (CH3); 29.6 (CH2); 52.1 (CH3); 68.8 (CH); 72.5 (CH); 77.0 (CH); 109.6 (C); 129.3 (C); 134.7 (CH); 167.2 (C) ppm. MS (ESI+) m/z 365 (100, [M+Na]+). Anal calcd for C17H30O5Si: C, 59.61; H, 8.83. Found C, 59.87; H, 8.98.

(3aR,4R,5R,7R,7aR)-Methyl 7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-4(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylate (2b) and (3aR,4R,5S,7R,7aR)methyl 7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-4(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylate (3b) Compound 1b was subjected to the conditions for the transformation of 1a into the mixture 2a+4. Work-up provided a solid residue which was subjected to flash column chromatography (EtOAc/hexane 1:12). This provided trans-γ-nitro ester 2b (1.89 g, 35%) and cis-γ-nitro ester 3b (2.30 g, 45%), as clear oils. Compound 2b: Rf (eluent: EtOAc/hexane 1:10): 0.40. [α]D20 +38.4 ° (c, 1 in CHCl3). IR (CHCl3) ν 1736, 1554, 1382 cm-1. 1H NMR (250MHz, CDCl3) δ 0.09 (s, 6H, 2 x -SiCH3), 0.89 (s. 9H, C(CH3)3), 1.36 (s, 3H, -CH3); 1.50 (s, 3H, -CH3); 1.83-2.04 (m, 2H, H6 and H6’), 2.45 (tt, 1H, J=10.6 Hz, J=4.8 Hz), 2.77 (td, 1H, J=11.5 Hz, J=3.8 Hz), 3.69 (s, 3H, -OCH3), 3.99-4.02 (m, 1H), 4.12 (dd, 1H, J=4.9 Hz, J=9.5 Hz), 4.26 (q, 1H, J=2,6 Hz), 4.59-4.60 (m, 2H, -CH2-NO2) ppm. 13C NMR (62.5 MHz, CDCl3) δ -5.0 (CH3); -4.9 (CH3); 18.0 (C), 25.7 (3 x CH3); 26.4 (CH3); 28.3 (CH3); 32.7 (CH2); 37.1 (CH); 40.8 (CH); 52.1 (CH3); 66.9 (CH); 74.2 (CH); 75.4 (CH2); 77.0 (CH); 109.7 (C); 174.1 (C) ppm. MS (ESI+) m/z 426 (100, [M+Na]+), 346 (6). Anal calcd for C18H33NO7Si: C, 53.57; H, 8.24; N, 3.47. Found C, 53.31; H, 8.39; N, 3.44. Compound of 3b: Rf (eluent: EtOAc/hexane 1:10): 0.50. [α]D20 +30° (c 1.0, CHCl3). IR (CHCl3) ν 1741, 1556, 1389 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.06 (s, 3H, -SiCH3), 0.07 (s, 3H, -SiCH3), 0.87 (s. 9H, -C(CH3)3), 1.34 (s, 3H, -CH3); 1.49 (s, 3H, -CH3); 2.00-2.20 (m, 2H, H6 and H6’), 2.522.61 (m, 1H, H4), 2.79-2.83 (m, 1H, H5), 3.66 (s, 3H, -OCH3), 4.03 (dd, 1H, J=5.0 Hz, J=3.0 Hz), 4.19 (q, 1H, J=3.6 Hz), 4.43 (dd, 1H, J=9.5 Hz, J= 5.1 Hz), 4,69-4.81 (m, 2H, -CH2-NO2) ppm. 13C



NMR (62.5 MHz, CDCl3) δ -5.0 (CH3); -4.8 (CH3); 18.4 (C), 25.9 (3 x CH3); 26.3 (CH3); 28.3 (CH3); 31.9 (CH2); 37.5 (CH); 39.9 (CH); 51.8 (CH3); 68.0 (CH); 72.9 (CH); 75.7 (CH2); 77.8 (CH); 109.1 (C); 173.1 (C) ppm. MS (ESI+) m/z 426 (100, [M+Na]+). Anal calcd for C18H33NO7Si: C, 53.57; H, 8.24; N, 3.47. Found C, 53.40; H, 8.15; N, 3.57.

(3aR,4R,5R,7R,7aR)-Methyl

7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-4-

(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylate (2b) Reaction of compound 2a with tert-butyldimethylsilyl chloride (0.12 g, 0.80 mmol) and imidazole (0.11 g, 1.62 mmol), under the same conditions as for the transformation of 1a into 1b, provided trans-γ-nitro ester 2b (125 mg, 90% yield) as an oil.

(3aS,4R,7S,8R,8aR)-2,2-dimethyl-8-(nitromethyl)tetrahydro-4,7-methano[1,3]dioxolo[4,5c]oxepin-6(7H)-one (4) A solution of 3b (100 mg, 0.25 mmol) in a 1 M solution of TBAF in THF (2 mL) was stirred at room temperature for 12 hours. The reaction mixture was then concentrated under reduced pressure, the residue dissolved in dichloromethane (10 mL) and washed with water (10 mL). The organic layer was then dried with anhydrous sodium sulfate, filtered and the liquids removed under reduced pressure, to give bicyclic nitro lactone 4 quantitatively (63 mg), as a pale yellow solid.

(3aR,4R,5aR,8aR,8bR)-4-((tert-butyldimethylsilyl)oxy)-2,2-dimethyloctahydro-6H[1,3]dioxolo[4,5-e]isoindol-6-one (7a). Raney-Ni (15 mL, 10% in weight) was added to a deoxygenated solution of 2b (0.36 g, 0.72 mmol) in methanol (30 mL). The resulting suspension was stirred at room temperature under hydrogen (P=1 atm) for 12 h, and then filtered through a Celite pad, which was washed with methanol and the filtrate was concentrated under reduced pressure. Flash column chromatography (CHCl3/MeOH 8:1) to gave 0.3 g (98%) of 7a, as a white solid that was crystallized from a mixture of EtOAc and hexane: Rf (eluent: EtOAc/hexane 1:1): 0.10. [α]D20 +67.8° (c 0.25, CHCl3). mp 120-122 °C (EtOAc/hexane). IR (CHCl3) ν 3230, 1709, 1083 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.05 (s, 3H, SiCH3); 0.06 (s, 3H, -SiCH3); 0.84 (s, 9H, -C(CH3)3); 1.32 (s, 3H, -CH3); 1.43 (s, 3H, -CH3); 1.541.66 (m, 1H, H-5); 1.94-2.14 (m, 2H, H-5’, H-8a); 2.25-2.36 (m, 1H, H-5a); 3.02-3.10 (m, 1H, H8); 3.49-3.56 (m, 1H, H-8’); 3.94-3.97 (m, 1H, H-3a); 4.14 (dd, 1H, J=4.9 Hz, J=9.6 Hz, H-8b); 4.30-4.34 (m, 1H, H-4); 7.16 (s, 1H, -NH) ppm.

13

C NMR (62.5 MHz, CDCl3) δ -5.1 (CH3); -4.9

(CH3); 18.0 (C); 25.8 (3 x CH3); 26.4 (CH3); 28.6 (CH3); 30.1 (CH2); 37.8 (CH); 45.3 (CH2); 45.8


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(CH); 68.8 (CH); 76.4 (CH); 78.6 (CH); 108.9 (C); 179.1 (C) ppm. HRMS (ESI+) m/z, calcd. for C17H32NO4Si [M+H]+: 342.2101. Found 342.2100.

(3aR,4R,5aR,8aR,8bR)-4-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-6-oxohexahydro-3aH[1,3]dioxolo[4,5-e]isoindole-7(8bH)-carboxylate (7b). Di-tert-butyl dicarbonate (0.26 g, 1.16 mmol) and DMAP (0.08 g. 0.65 mmol) were added to a solution of 7a (0.2 g, 0.58 mmol) in dry dichoromethane (4.5 mL) and the resulting mixture was stirred at room temperature, under argon, for 2 h. The solvent was then evaporated in vacuo and the resulting residue submitted to flash column chromatography (EtOAc/ hexane 1:3) to give 102 mg (95%) of 7b, as a solid that was crystallized from a mixture of EtOAc and hexane: Rf (eluent: EtOAc/hexane 1:2): 0.70. [α]D20 +87.6° (c 0.3, CHCl3). mp 115-117 °C (EtOAc/hexane). IR (CHCl3) ν 1747, 1713 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.05 (s, 3H, -SiCH3); 0.06 (s, 3H, SiCH3); 0.84 (s, 9H, -C(CH3)3); 1.33 (s, 3H, -CH3); 1.45 (s, 3H, -CH3); 1.48 (s, 9H, -C(CH3)3); 1.63 (ddd, 1H, J=13.4 Hz, J=11.8 Hz, J=2.9 Hz, H-5); 1.84-2.07 (m, 2H); 2.45 (ddd, 1H, J=14.6 Hz, J=11.8 Hz, J=3.1 Hz); 3.25-3.36 (m, 1H); 3.94-4.14 (m, 3H); 4.31-4.34 (m, 1H) ppm.

13

C NMR

(62.5 MHz, CDCl3) δ -5.0 (CH3); -4.9 (CH3); 18.0 (C); 25.8 (3 x CH3); 26.4 (CH3); 28.1 (3 x CH3); 28.6 (CH3); 29.9 (CH2); 40.1 (CH); 41.4 (CH); 49.3 (CH2); 68.4 (CH); 76.2 (CH); 78.4 (CH); 82.9 (C); 109.2 (C); 150.2 (C); 174.2 (C) ppm. HRMS (ESI+) m/z [M+Na]+ calcd. for C22H39NNaO6Si 464.2444. Found 464.2439.

(3aR,4R,5R,7R,7aR)-4-(((tert-butoxycarbonyl)amino)methyl)-7-((tert-butyldimethylsilyl)oxy)2,2-dimethylhexahydrobenzo[d][1,3]dioxole-5-carboxylic acid (6b) and (3aR,4R,5S,7R,7aR)-4(((tert-butoxycarbonyl)amino)methyl)-7-((tert-butyldimethylsilyl)oxy)-2,2dimethylhexahydrobenzo[d][1,3]dioxole-5-carboxylic acid (8) 1M LiOH aqueous solution (1.2 mL, 1.2 mmol) was added to a solution of 7b (0.15 g, 0.35 mmoles) in THF (2 mL) and the resulting solution was stirred at room temperature for 2 h. The THF was then evaporated and the resulting suspension acidified with 10% aqueous solution of acetic acid (0.75 mL). The mixture was extracted with ethyl acetate (3x10 mL), the organic layers dried with anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Flash column chromatography of the obtained residue (CH2Cl2/MeOH 97:3) provided (0.14 g, 91%) of a 1:1 mixture of 6b and 8, as a white solid mixture, which showed the following spectral data: IR (CHCl3) ν 3331, 1711, 1064 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.04 (s, 6H, 2 x -SiCH3); 0.06 (s, 6H, 2 x -SiCH3); 0.89 (s, 18H, 2 -C(CH3)3); 1.33 (s, 6H, 2 x -CH3); 1.41 (s, 12H, 4 x -CH3); 1.44 (s,



Page 12 of 19

6H, 2 x -CH3); 1.46 (s, 6H, 2 x -CH3); 1.65-1.96 (m, 3H); 2.05-2.20 (m, 1H); 2.40-2.57 (m, 1H); 2.69-2.84 (m, 2H); 3.04-3.26 (m, 2H); 3.28-3.51 (m, 1H); 3.67-3.96 (m, 4H); 4.05-4.14 (m, 1H); 4.23-4.35 (m, 1H); 4.98-5.12 (m, 1H); 5.22-5.25 (m, 1H); 6.52 (bs, 2H, -NH); 10.2 (bs, 2H, COOH) ppm. 13C NMR (62.5 MHz, CDCl3) δ -5.0 (2 x CH3); -4.8 (2 x CH3); 18.0 (2 x C); 25.7 (8 x CH3); 26.2 (2 x CH3); 28.3 (6 x CH3); 30.2 (CH2); 31.0 (CH2); 37.5 (CH); 38.8 (CH); 39.2 (CH); 39.5 (CH); 40.8 (CH2); 40.9 (CH2); 70.6 (CH); 71.5 (CH); 75.8 (C); 75.9 (C); 78.9 (CH); 79.1 (CH); 79.3 (CH); 80.9 (CH); 108.3 (2 x C); 156.2 (C); 157.5 (C); 178.2 (C); 178.6 (C) ppm. HRMS (ESI+): calculated for C22H42NO7Si [M+H]+: 460.2731. found: 460.2732.

(3aR,4R,5R,7R,7aR)-7-((tert-Butyldimethylsilyl)oxy)-2,2-dimethyl-4(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylic acid (2c) 1M LiOH aqueous solution (5 mL, 5 mmol) was added to a solution of 2b (0.5 g, 1.24 mmol) in THF (20 mL) and the resulting solution was stirred at room temperature for 6 h. The reaction was then acidified with saturated aqueous solution of ammonium chloride (5 mL), the THF removed under reduced pressure, and the residue was suspended in water (50 mL) and extracted with dichloromethane (2x20 mL). The pooled organic layers were dried with anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Flash column chromatography of the residue (EtOAc/hexane 1:1) provided trans-γγ-nitro acid 2c (0.44 g, 92% yield), as a clear oil: Rf (eluent: EtOAc/hexane 1:10): 0.10. [α]D20 +87° (c 0.3, in CHCl3). IR (CHCl3) ν 3300, 1711, 1556, 1374 cm1 1

. H NMR (250 MHz, CDCl3) δ 0.08 (s, 6H, 2 x -SiCH3), 0.88 (s, 9H, -C(CH3)3), 1.35 (s, 3H, -

CH3); 1.48 (s, 3H, -CH3); 1.92-2.01 (m, 2H, H6 and H6’), 2.42 (ddt, 1H, J=11,6 Hz, J=9.3 Hz, J=4.7 Hz, H4), 2.74-2.91 (m, 1H, H5), 4.00 (dd, 1H, J=5.0 Hz, J=2.7 Hz), 4.15 (dd, 1H, J=9.2 Hz, J=4.9 Hz), 4.25-4.28 (m, 1H), 4,62-4.64 (m, 2H, -CH2-NO2), 10.66 (bs, 1H, -COOH) ppm.

13

C NMR

(62.5 MHz, CDCl3) δ -4.9 (CH3); -4.8 (CH3); 18.0 (C), 25.8 (3 x CH3); 26.4 (CH3); 28.3 (CH3); 32.7 (CH2); 36.9 (CH); 40.4 (CH); 66.9 (CH); 74.2 (CH); 75.4 (CH2); 77.0 (CH); 109.9 (C); 179.9 (C) ppm. MS (ESI+) m/z 412 (100, [M+Na]+). Anal calcd for C17H31NO7Si: C, 52.42; H, 8.02; N, 3.60. Found C, 52.56; H, 7.91; N, 3.50.

(3aR,4R,5R,7R,7aR)-4-(((tert-butoxycarbonyl)amino)methyl)-7-((tert-butyldimethylsilyl)oxy)2,2-dimethylhexahydrobenzo[d][1,3]dioxole-5-carboxylic

acid

(6b)

(3aR,4R,5aR,8aR,8bR)-4-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-6-oxohexahydro-3aH[1,3]dioxolo[4,5-e]isoindole-7(8bH)-carboxylate (7b).


and

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Raney-Ni (0.8 mL, 10% in weight) was added to a deoxygenated solution of 2c (50 mg, 0.13 mmol) in methanol (8 mL). The resulting suspension was stirred at room temperature under hydrogen (P=1 atm) for 12 h, and then filtered through a Celite pad, which was washed with methanol, and the filtrate was concentrated under reduced pressure. The residue was directly taken in dichloromethane (5 mL), di-tert-butyl dicarbonate (0.15 g, 0.66 mmol) and DMAP (0.06 g. 0.48 mmol) were added and the resulting mixture was stirred at room temperature, under argon, for 2 h. The solvent was then evaporated in vacuo and the resulting residue submitted to flash column chromatography in a gradient (EtOAc/ hexane 1:4) to (EtOAc/ hexane 1:1) to give 23 mg (40% yield) of γ-amino acid 6b, as an amorphous solid, and 20 mg (35% yield) of bicyclic lactam 7b, as a solid that was crystallized from a mixture of EtOAc and hexane. Compound 6b: Rf (eluent: EtOAc/hexane 1:4): 0.50. [α]D20 -16.9° (c 1.4, in CHCl3). mp 104105 °C (EtOAc/hexane) IR (CHCl3) ν 3370, 1720, 1715 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.06 (s, 3H, -SiCH3); 0.08 (s, 3H, -SiCH3); 0.87 (s, 9H, -C(CH3)3); 1.34 (s, 3H, CH3); 1.43 (s, 9H, C(CH3)3); 1.48 (s, 3H, -CH3); 1.76-2.04 (m, 2H, H6 and H6’); 2.10-2.60 (m, 1H, H1); 2.79-2.86 (m, 1H, H2); 3.15-3.48 (m, 2H, -CH2N); 3.85-3.95 (m, 2H); 4.13-4.38 (m, 1H); 5.07 and 6.45 (2 x bs, 1H, NH); 8.25 (bs, 1H, -COOH) ppm. 13C NMR (62.5 MHz, CDCl3) δ -4.8 (CH3); -4.6 (CH3); 18.2 (C); 25.9 (4 x CH3); 26.4 (CH3); 28.5 (3 x CH3); 30.4 and 31.2 (CH2); 37.7 and 39.0 (CH); 39.5 and 39.8 (CH); 41.3 (CH2); 70.7 and 71.6 (CH); 76.0 (CH); 77,4 (CH); 79.1 and 79.6 (C); 108.6 (C); 156.3 and 157.6 (C); 178.3 (C) ppm. MS (ESI+) m/z 482 (18, [M+Na]+); 346 (100). Anal calcd for C22H41NO7Si: C, 57.49; H, 8.99; N, 3.05. Found C, 57.27; H, 9.11; N, 3.19.

Methyl

2-((3aR,4R,5R,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-4-

(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxamido)acetate

(9)

and

methyl

2-

((3aR,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-3a,6,7,7atetrahydrobenzo[d][1,3]dioxole-5-carboxamido)acetate (10) DIEA (0.07 mL, 0.40 mmol) was added to a solution of EDCI-HCl (39 mg, 0.20 mmol), carboxymethylglycine hydrochloride (20 mg, 0.16 mmol) and 2c (60 mg, 0.15 mmol) in dry DMF (3 mL) and the resulting solution was stirred at room temperature for 24 h. The reaction was then added to brine (30 mL) and extracted with dichloromethane (2x15 mL). The organic layers were washed with water (30 mL), dried with anhydrous sodium sulphate and filtered. Removal of the solvent under reduced pressure and subsequent flash column chromatography of the solid residue (EtOAc/hexane 1:2 to EtOAc /hexane 1:1) gave compound 9 (35 mg, 50% yield), as a clear oil, and compound 10 (18 mg, 30% yield), as a yellow oil.



Compound 9: Rf (eluent: EtOAc/hexane 1:1): 0.70. [α]D20 +49° (c 0.2, in CHCl3). IR (CHCl3) ν 3325, 1752, 1661, 1557, 1373 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.06 (s, 6H, 2 x -SiCH3), 0.86 (s. 9H, -C(CH3)3), 1.33 (s, 3H, -CH3); 1.45 (s, 3H, -CH3); 1.69-1.78 (m, 1H, H6), 2.04 (ddd, 1H, J=14.1 Hz, J=11.7 Hz, J=2.5 Hz, H6’), 2.26 (ddt, 1H, J=11.6 Hz, J=9.5 Hz, J=3.9 Hz, H4), 2.75 (td, 1H, J=11.7 Hz, J=3.3 Hz, H5), 3.72 (s, 3H, -OCH3), 3.86 (dd, 1H, J=18.7 Hz, J=5.2 Hz), 3.97-4.27 (m, 4H), 4.67 (dd, 1H, J=12.6 Hz, J=3.7 Hz, -CHH-NO2), 4.74 (dd, 1H, J=12.5 Hz, J=4.1 Hz, -CHHNO2), 6.37 (t, 1H, J=5.7 Hz, -NH) ppm.

13

C NMR (62.5 MHz, CDCl3) δ -4.9 (CH3); -4.8 (CH3);

18.0 (C), 25.8 (3 x CH3); 26.3 (CH3); 28.3 (CH3); 33.0 (CH2); 38.2 (CH); 41.2 (CH2); 41.6 (CH); 52.5 (CH3); 67.1 (CH); 73.5 (CH); 74.8 (CH2); 77.0 (CH); 109.7 (C); 170.3 (C), 173.7 (C) ppm. MS (ESI+) m/z 483 (100, [M+Na]+), 403 (7). Anal calcd for C20H36N2O8Si: C, 52.15; H, 7.88; N, 6.08. Found C, 52.27; H, 7.99; N, 6.33. Compound 10: Rf (eluent: EtOAc/hexane 1:1): 0.40. [α]D20 +9° (c 0.3, in CHCl3). IR (CHCl3) ν 3377, 1735, 1678 cm-1. 1H NMR (300 MHz, CDCl3) δ 0.07 (s, 3H, -SiCH3), 0.10 (s, 3H, -SiCH3), 0.87 (s. 9H, -C(CH3)3), 1.39 (s, 3H, -CH3); 1.40 (s, 3H, -CH3); 2,30 (dd, 1H, J=16.7 Hz, J=5.4 Hz, H6), 2,55-2.62 (m, 1H, H6’), 3.78 (s, 3H, -CH3), 4.02-4.12 (m, 4H), 4.69-4.72 (m, 1H), 6.28 (bs, 1H, NH), 6.44-6.45 (m, 1H, CH) ppm. 13C NMR (75 MHz, CDCl3) δ -4.8 (CH3); -4.6 (CH3); 18.0 (C), 25.8 (3 x CH3); 26.1 (CH3); 28.1 (CH3); 30.1 (CH2); 41.4 (CH2); 52.5 (CH3); 68.8 (CH); 72.4 (CH); 77.2 (CH); 109.4 (C); 128.7 (C); 133.0 (CH); 167.6 (C), 170.4 (C) ppm. MS (ESI+) m/z 422 (69, [M+Na]+), 400 (100, [M+1]+), 342 (56). Anal calcd for C19H33NO6Si: C, 57.11; H, 8.32; N, 3.51. Found C, 56.97; H, 8.49; N, 3.25.

Methyl

2-((3aR,4R,5R,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-4-

(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxamido)acetate (9) To a solution of 2c (0.3 g, 0.77 mmol) in dry dichloromethane (40 mL), cooled at 0 ºC, diisopropylcarbodiimide (0.15 g, 1,16 mmol) and pentafluorophenol (0.43 g, 2.31 mmol) were added and the resulting mixture was stirred for 3 h at 0 ºC and then at room temperature for 12 h. The reaction mixture was poured into a sodium bicarbonate aqueous saturated solution (50 mL), and the suspension was extracted with dichloromethane (2x20 mL). The joined organic layers were dried with anhydrous sodium sulphate, filtered and concentrated under reduced pressure. To a solution of the resulting residue in dry DMF (20 mL), DIEA (123 mg, 0.94 mmol) and then glycine methyl ester hydrochloride (111 mg, 0.86 mmol) were added and the resulting mixture was stirred at room temperature for 2 h. The mixture was then added to 100 mL of a 1:1 mixture of brine and water and extracted with EtOAc (3x25 mL). The joined organic layers were washed with water (50


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mL), dried with anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Flash column chromatography of the residue (EtOAc/hexane 1:2) provided (0.32 g, 90% yield) dipeptidelike compound 9, as a clear gum.

Methyl 2-((3aR,4R,5R,7R,7aR)-4-((2-(((benzyloxy)carbonyl)amino)acetamido)methyl)-7-((tertbutyldimethylsilyl)oxy)-2,2-dimethylhexahydrobenzo[d][1,3]dioxole-5-carboxamido)acetate (12) Raney-Ni (1,6 mL) was added to a deoxygenated solution of 9 (100 mg, 0.22 mmol) in methanol (16 mL) and the resulting mixture was stirred under a hydrogen atmosphere (P=1 atm) for 12 h. This mixture was then filtered through a Celite pad, which was washed with methanol. The filtrate was concentrated under reduced pressure. The resulting oil was taken up in dry DMF (6 mL), DIEA (30 mg, 0.22 mmol) and then perfluorophenyl 2-(((benzyloxy)carbonyl)amino)acetate (82 mg, 0.22 mmol) were added and the resulting mixture was stirred at room temperature for 3 h, and then diluted with EtOAc (80 mL) and washed with saturated aqueous solution of ammonium chloride (2x40 mL). The organic layer was washed with water (40 mL), dried with anhydrous sodium sulphate, concentrated to dryness under reduced pressure. The residue was purified by to flash column chromatography (EtOAc/hexane 3:1) to give tripeptide 12 (104 mg, 70% yield), as a clear gum: Rf (eluent: EtOAc/hexane 2:1): 0.90. [α]D20 +27° (c 0.1, in CHCl3). IR (CHCl3) ν 3322, 1733, 1662 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.07 (s, 3H, -SiCH3), 0.08 (s, 3H, -SiCH3), 0.87 (s. 9H, C(CH3)3), 1.31 (s, 3H, -CH3); 1.46 (s, 3H, -CH3); 1.61 (dt, 1H, J=13.5 H, J=4.7 Hz), 1.91-2.03 (m, 2H), 2.33-2.42 (m, 1H) 3.36-3.49 (m, 2H), 3.72 (s, 3H, -OCH3), 3.78-4.26 (m, 7H), 5.05-5.15 (m, 2H, -CH2Ph), 5.58 (t, 1H, J=5.5 Hz, -NH), 7.02 (bs, 1H, -NH), 7.13 (bs, 1H, -NH), 7.30-7.36 (m, 5H, -Ph) ppm.

13

C NMR (62.5 MHz, CDCl3) δ -4.8 (CH3); -4.7 (CH3); 18.1 (C), 25.9 (3 x CH3);

26.3 (CH3); 28.3 (CH3); 32.4 (CH2); 39.3 (CH); 40.8 (CH2); 41.1 (CH), 41.2 (CH2); 44.7 (CH2); 52.5 (CH3); 67.3 (CH2); 68.0 (CH); 76.2 (CH); 77.9 (CH); 109.3 (C); 128.2 (2 x CH); 128.4 (CH); 128.7 (2 x CH); 136.2 (C), 156.7 (C), 169.9 (C), 170.9 (C); 175.0 (C) ppm. MS (ESI+) m/z 644 (100, [M+Na]+), 622 (38, [M+H]+), 564 (10). Anal calcd for C30H47N3O9Si: C, 57.95; H, 7.62; N, 6.76. Found C, 57.87; H, 7.93; N, 6.49.

(3aR,4R,5S,7R,7aR)-7-((tert-Butyldimethylsilyl)oxy)-2,2-dimethyl-4(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxylic acid (3c) When compound 3b (0.5 g, 1.24 mmol) was subjected to the procedure for the preparation of 2c, cis-γγ-nitro acid 3c was obtained (0.46 g, 95% yield), as a clear oil: Rf (eluent: EtOAc/hexane 1:10):



0.10. [α]D20 +12° (c 0.2, in CHCl3). IR (CHCl3) ν 3300, 1708, 1555, 1382 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.08 (s, 6H, 2 x -SiCH3), 0.88 (s. 9H, -C(CH3)3), 1.35 (s, 3H, -CH3); 1.48 (s, 3H, -CH3); 1,95-2.00 (m, 2H, H6 and H6’), 2.42 (ddt, 1H, J=11.6 Hz, J=9.3 Hz, J=4.7 Hz, H4), 2.77-2.88 (m, 1H, H5), 4.00 (dd, 1H, J=5.0 Hz, J=2.7 Hz), 4.15 (dd, 1H, J=9.2 Hz, J=4.9 Hz), 4.26 (q, 1H, J=3.0 Hz), 4.62-4.64 (m, 2H, -CH2-NO2), 10.66 (bs, 1H, -COOH) ppm. 13C NMR (62.5 MHz, CDCl3) δ 5.2 (CH3); -5.1 (CH3); 18.2 (C), 25.7 (3 x CH3); 26.4 (CH3); 28.4 (CH3); 32.2 (CH2); 37.6 (CH); 39.9 (CH); 67.7 (CH); 72.8 (CH); 75.7 (CH2); 77.5 (CH); 109.4 (C); 179.3 (C) ppm. MS (ESI+) m/z 412 (100, [M+Na]+). Anal calcd for C17H31NO7Si: C, 52.42; H, 8.02; N, 3.60. Found C, 52.75; H, 8.19; N, 3.48.

Methyl

2-((3aR,4R,5S,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-2,2-dimethyl-4-

(nitromethyl)hexahydrobenzo[d][1,3]dioxole-5-carboxamido)acetate (13) Following the procedure for the preparation of compound 9, the title compound 13 was obtained (0.31 g, 88% yield) from compound 3c (0.3 g, 0.77 mmol), as a clear gum: Rf (eluent: EtOAc/hexane 1:3): 0.40. [α]D20 +14° (c 0.4, in CHCl3). IR (CHCl3) ν 3331, 1750, 1655, 1555, 1382 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.07 (s, 3H, -SiCH3), 0.09 (s, 3H, -SiCH3), 0.87 (s. 9H, C(CH3)3), 1.31 (s, 3H, -CH3); 1.46 (s, 3H, -CH3); 1.77-1.89 (m, 1H, H6), 2.01 (ddd, 1H, J=14.2 Hz, J=7.2 Hz, J=4.8 Hz), 2.62 (tt, 1H, J=8.7 Hz, J=5.4 Hz, H4), 2.92 (td, 1H, J=7.1 Hz, J=5.4 Hz, H5), 3.75 (s, 3H, -OCH3), 3.96-4.10 (m, 4H), 4.35 (dd, 1H, J=9.0 Hz, J=6.0 Hz), 4.64 (dd, 1H, J=13.7 Hz, J=5.4 Hz, -CHH-NO2), 4.76 (dd, 1H, J=13.7 Hz, J=8.7 Hz, -CHH-NO2), 6.56 (t, 1H, J=5.1 Hz, -NH) ppm. 13C NMR (62.5 MHz, CDCl3) δ -4.8 (CH3); -4.6 (CH3); 18.2 (C), 25.6 (CH3); 25.8 (3 x CH3); 27.9 (CH3); 31.4 (CH2); 38.7 (CH); 39.4 (CH); 41.4 (CH2); 52.6 (CH3); 69.7 (CH); 73.4 (CH); 75.1 (CH2); 79.3 (CH); 108.9 (C); 170.2 (C), 172.8 (C) ppm. MS (ESI+) m/z 483 (31, [M+Na]+), 369 (100). Anal calcd for C20H36N2O8Si: C, 52.15; H, 7.88; N, 6.08. Found C, 51.97; H, 7.49; N, 6.12.

Methyl 2-((3aR,4R,5S,7R,7aR)-4-((2-(((benzyloxy)carbonyl)amino)acetamido)methyl)-7-((tertbutyldimethylsilyl)oxy)-2,2-dimethylhexahydrobenzo[d][1,3]dioxole-5-carboxamido)acetate (15) Tripeptide 15 was obtained (93 mg, 68% yield) from 13, as a clear gum, following the protocol for the preparation of its analogue 12: Rf (eluent: EtOAc/hexane 2:1): 0.90. [α]D20 -6° (c 0.1, in CHCl3). IR (CHCl3) ν 3331, 1731, 1658 cm-1. 1H NMR (250 MHz, CDCl3) δ 0.07 (s, 3H, -SiCH3), 0.09 (s, 3H, -SiCH3), 0.87 (s. 9H, -C(CH3)3), 1.30 (s, 3H, -CH3); 1.46 (s, 3H, -CH3); 1.64-1.92 (m, 3H),


Page 16 of 19

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2.30-2-42 (m, 1H), 2.69 (q, 1H, J=7.6 Hz), 2.99-3.10 (m, 1H), 3.61-3.88 (m, 7H), 4.02 (t, 1H, J=6.5 Hz), 4.20-4.34 (m, 2H), 5.03-5.13 (m, 2H, -CH2Ph), 5.60 (t, 1H, J=6.0 Hz, -NH), 7.31-7.40 (m, 6H, -Ph, and -NH), 7.49 (bs, 1H, -NH) ppm.

13

C NMR (62.5 MHz, CDCl3) δ -4.8 (CH3); -4.3 (CH3);

18.2 (C), 25.6 (3xCH3); 25.9 (CH3); 27.9 (CH3); 31.3 (CH2); 37.5 (CH); 39.8 (CH); 40.3 (CH2); 41.1 (CH2); 44.9 (CH2); 52.7 (CH3); 67.4 (CH2); 71.2 (CH); 76.3 (CH); 80.5 (CH); 108.5 (C); 128.2 (2 x CH); 128.5 (CH); 128.7 (2 x CH); 136.0 (C), 157.2 (C), 169.5 (C), 172.0 (C); 175.0 (C) ppm. MS (ESI+) m/z 644 (3, [M+Na]+), 622 (38, [M+H]+), 604 (21), 582 (100). Anal calcd for C30H47N3O9Si: C, 57.95; H, 7.62; N, 6.76. Found C, 57.67; H, 7.46; N, 6.92.

ASSOCIATED CONTENT * Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxxxxxx. 1

H and 13C NMR spectra for all new products, single crystal structures and tables of single-

crystal data (PDF) Single-crystal X-ray data for 2a (CCDC 1526382) and 4 (CCDC 1526383) (CIF)

AUTHOR INFORMATION Corresponding Authors * Fax: +34-881815704. E-mail: [email protected].

ORCID Estévez, Juan C.: 0000-0002-2777-9582

Acknowledgements. This work has received financial support from the Spanish Ministry of Science and Innovation (CTQ2009 ‐ 08490), the Xunta de Galicia (Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09; Project CN2011/037 and Project GRC2014/040), the European Union (European Regional Development Fund-ERDF) and Galchimia S.A.

References and Notes. [1]. a) Asymmetric Synthesis of Non-Proteinogenic Amino Acids. Ashot S. Saghyan, Peter Langer. WILEY-VCH. 2016. b) Ordonez, M.; Cativiela, C.; Romero-Estudillo, I. Tetrahedron: Asymm.



2016, 27, 999-1055. c) Ashfaq, M.; Tabassum, R.; Ahmad, M. M.; Hassan, N. A.; Oku, H.; Rivera, G. Med. Chem. 2015, 5, 295-309. d) Weiner, B.; Szymański, W.; Janssen, D.B.; Minnaard, A.J.; Feringa, B.L. Chem. Soc. Rev. 2010, 39, 1656-91. e) Goodman, C. M.; Choi, S.; Shandler, S.; DeGrado, W. F. Nat. Chem. Biol. 2007, 3, 252-262. f) Ordonez, M.; Cativiela, C. Tetrahedron: Asymm. 2007, 18, 3–99. g) Enantioselective Synthesis of β-Amino Acids, 2nd Edition. Eusebio Juaristi (Editor), Vadim A. Soloshonok. John Wiley & sons Ed. Second Edition. 2005. [2]. a) Seebach D. I.; Beck, A. K.; Bierbaum, D. J. Chem. Biodivers. 2004, 1, 1111-239. b) Goodman, C. M.; Choi, S.; Shandler, S.; DeGrado, W. F. Nat. Chem. Biol. 2007, 3, 252-262. [3] a) Wydysh, E. A.; Vadlamudi, A.; Medghalchi, S. M.; Townsend, C. A. Bioorg. Med. Chem. 2010, 18, 6470–6479. b) Trost, B. M.; Zhang, T. Angew. Chem. Int. Ed. 2008, 47, 3759–3761. c) Satoh, N.; Akiba, T.; Yokoshima, S.; Fukuyama, T. Angew. Chem. Int. Ed. 2007, 46, 5734–5736. d) Gamma-Aminobutyric Acid (GABA). Dom Guillaume; Zoubida Charrouf. Alternative Medicine Reviews 2007, 12, 275-279. e) Johnston, G. A. R. Curr. Top. Med. Chem. 2002, 2, 903–913. f) Qiu, J.; Pingsterhaus, J. M.; Silverman, R. B. J. Med. Chem. 1999, 42, 4725–4728. [4] a) Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Chem. Rev. 2011, 111, 657–687. b) Fülöp, F.; Martinek, T. A.; Tóth, G. K. Chem. Soc. Rev. 2006, 35, 323-34. c) Kuhl, A; Hahn, M. G.; Dumić, M.; Mittendorf, J. Amino Acids 2005, 29, 89-100. [5] For references on alicyclic β-peptides, see: North, M. J. Pept .Sci. 2006, 301-13. [6] For references on alicyclic γ-peptides, see: a) Kang, Y. K.; Lee, J. Y. New J. Chem. 2015, 39, 3241-3249. b) Rodriguez-Vazquez, N.; Salzinger, S.; Silva, L. F.; Amorin, M.; Granja, J. R. Eur. J. Org. Chem. 2013, 3477-3493. c) Balaram, P. Biopolymers 2010, 94, 733-41. d) Vasudev, P.G.; Chatterjee, S.; Shamala, N., Balaram, P. Acc. Chem. Res. 2009, 42, 628-39. e) Baxendale, I. R.; Ernst, M.; Krahnert, W.-R.; Ley, S. V. Synlett. 2002, 10, 1641-1644. f) Kennewell, P. D.; Matharu, S. S.; Taylor, J. B.; Westwood, R.; Sammes, P. G. J. Chem. Soc., Perkin Trans 1 1982, 2553-62. g) Kennewell, P. D.; Matharu, S. S.; Taylor, J. B.; Westwood, R.; Sammes, P.G. J. Chem. Soc., Perkin Trans 1 1982, 2563-70. [7] a) Ordóñez, M.; Cativiela, Carlos; Romero-Estudillo, Iván. Tetrahedron: Asymm. 2016, 27, 999–1055. b) Szakonyi, Z.; Csőr, Ä.; Haukka, M.; Fülöp, F. Tetrahedron 2015, 71, 4846-4852. c) Giuliano, M.W.; Maynard, S.J.; Almeida, A.M.; Guo, L.; Guzei, I.A.; Spencer, L.C.; Gellman, S.H. J. Amer. Chem. Soc. 2014, 136, 15046-15053. d) Malihi, F.; Clive, D.L.J.; Changa, C-C.; Minaruzzaman. J. Org. Chem. 2013, 78, 996-1013. e) Guo, L.; Zhang, W.; Guzei, I.A.; Spencer, L.C.; Gellman, S.H. Org. Lett. 2012, 14, 2582-2585.


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[8] a) Kang, Y.K.; Byun, B.J. Biopolymers 2012, 97, 1018-1025 b) Gellman, S. H.; Guo, L.; Giuliano, M. PCT Int. Appl. (2011), WO 2011047190 A1 20110421. [9] For recent reviews on (-)-shikimic acid, see: a) Borah, J. C. Curr. Sci. 2015, 109, 1672-9. b) Díaz Quiroz, D. C.; Carmona, S. B.; Bolívar, F.; Escalante, A. Res. Rep. Med. Chem. 2014, 4, 35– 46. c) Ghosh, S.; Chisti, Y.; Uttam C. Banerjee, U. C. Biotechnol. Adv. 2012, 301, 425–1431. And references therein. See also: d) Gonzalez-Castro, M. A.; Poole, D. L.; Estevez, J. C.; Fleet, G. W. J.; Estevez, R. J. Tetrahedron: Asymm. 2015, 26, 320-323. e) Cuellar, M. A.; Quinones, N.; Vera, V.; Salas, C. O.; Estevez, J. C.; Estevez, R. J. Synlett 2015, 26, 552-556. [10] a) Aguilera, J.; Favier, I.; Sans, M.; Mor, A.; Alvarez-Larena, A.; Illa, O.; Gomez, M.; Ortuno, R. M. Eur. J. Org. Chem. 2015, 810-819. b) Szakonyi, Z.; Balazs, A.; Martinek, T. A.; Fülöp, Ferenc Tetrahedron: Asymm. 2010, 21, 2498-2504. c) Izquierdo, S.; Aguilera, J.; Buschmann, H. H.; Garcia, M.; Torrens, A.; Ortuno, R. M. Tetrahedron: Asymm. 2008, 19, 651-653. [11] Compound 1a was obtained in a two step sequence, strating from commercially available (-)shikimic acid (100 g, 130 $) (92% global yield). See: Chahoua, L.; Baltas, M.; Gorrichon, L.; Tisnes, P.; Zedde, C. J. Org. Chem. 1992, 57, 5798-801. [12] Crystallographic data for the structures of compounds 2a and 4 have been deposited with the Cambridge Crystallographic Data Center as supplementary publications no. CCDC-1526382 and no. 1526383, respectively. Copies of the data can be obtained free of charge on application to CCDC, 12

Union

Road,

Cambridge

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[fax

(+44)1223-336-033;

e-mail

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Protocol for the Incorporation of Î³-Amino Acids into Peptides

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