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. 2023 Apr 6;88(9):5597–5608. doi: 10.1021/acs.joc.3c00094

Total Synthesis of GE81112A: An Orthoester-Based Approach

Scherin Fayad †,‡, Ardalan Jafari ‡, Sören M M Schuler §,∥, Michael Kurz †, Oliver Plettenburg ‡, Peter E Hammann ∥,⊥, Armin Bauer †, Gerrit Jürjens ‡,§,*, Christoph Pöverlein †,*
PMCID: PMC10167690  PMID: 37023463

Abstract

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The GE81112 series, consisting of three naturally occurring tetrapeptides and synthetic derivatives, is evaluated as a potential lead structure for the development of a new antibacterial drug. Although the first total synthesis of GE81112A reported by our group provided sufficient amounts of material for an initial in depth biological profiling of the compound, improvements of the routes toward the key building blocks were needed for further upscaling and structure–activity relationship studies. The major challenges identified were poor stereoselectivity in the synthesis of the C-terminal β-hydroxy histidine intermediate and a concise access to all four isomers of the 3-hydroxy pipecolic acid. Herein, we report a second-generation synthesis of GE81112A, which is also applicable to access further representatives of this series. Based on Lajoie’s ortho-ester-protected serine aldehydes as key building blocks, the described route provides both a satisfactory improvement in stereoselectivity of the β-hydroxy histidine intermediate synthesis and a stereoselective approach toward both orthogonally protected cis and trans-3-hydroxy pipecolic acid.

Introduction

The worldwide problem of increasing multidrug resistance is compounded by the decline in new classes of antimicrobials entering into clinical practice. This fact led to renewed interest in novel scaffolds as starting points for new antimicrobial agents. GE81112A (1),1,2 a nonribosomally synthesized tetrapeptide, and its congeners GE81112B (2) and GE81112B1 (3) (Figure 1) represent such a novel chemical structure. Since the anti-Gram-negative activity is based on ribosome targeting,2,3 the GE81112 series gained high interest as a potential lead.

Figure 1.

Figure 1

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Structure of the GE81112 series.

The first total synthesis of GE81112A (1) accomplished by our group led to the revision of the originally published structure4 and provided first batches of 1 on a 100 mg scale for in depth biological profiling.5 Structure–activity relationships were expanded by the Renata group which developed a concise total synthesis of GE81112B1 (3) and 10 non-natural analogues thereof.6,7 After careful analysis of our (first-generation) route toward 1 and Renata’s synthesis of 3, we identified the syntheses of both β-hydroxy amino acid moieties as major areas to be improved.

While a nonstereoselective approach toward all four stereoisomers of the 3-hydroxy pipecolic acid moiety was considered acceptable in the first total synthesis of GE81112A with regard to ambiguity concerning the absolute configuration and planned SAR studies (Scheme 1, entry G), an adapted route featuring Teoc-protected (2S,3R)-hydroxy pipecolic acid 5 in a stereocontrolled manner was required for scale-up and synthesis of derivatives. In the same line, poor stereoselectivity experienced in the synthesis of the C-terminal β-hydroxy histidine building block 22 using Garner’s aldehyde (dr 2:1) was identified as an additional issue (Scheme 2). In the total synthesis of GE81112B1 (3) by the Renata group,6,7 (2S,3R)-hydroxy pipecolic acid derivative 6 was prepared from (2S)-pipecolic acid (14) by chemoenzymatic oxidation8 in two steps (Scheme 1, entry H).

Scheme 1. Selected Examples of Reported Syntheses of cis-3-Hydroxy Pipecolic Acid Derivatives.

Scheme 1

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Scheme 2. Retrosynthetic Analysis of trans- and cis-3-Hydroxy Pipecolic Acids 15 (A) and 4 (B) and β-Hydroxy Histidine Building Block 22 (C).

Scheme 2

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Our strategy for a concise stereoselective synthesis of GE81112A (1) is based on a detailed analysis of its revised structure revealing an intriguing feature: both the β-hydroxy pipecolic acid and the β-hydroxy histidine display the same threo configuration. Therefore, we reasoned that it should be feasible to deliver intermediates for both moieties from one common building block defining the required threo configuration in a highly selective manner. We first focused on the β-hydroxy pipecolic acid targeting the Teoc-protected cis-(2S,3R)-configured product 5 as key intermediate. Due to the total synthesis of GE81112B1 (3) by the Renata group, Boc-protected compound 6 became a target as well. Besides high yields, technical feasibility, and low number of synthetic steps, the high degree of stereoselectivity and enantiomeric excess (ee) represent important criteria for such an alternative route. While numerous synthetic protocols for 3-hydroxy pipecolic acid derivatives had been reported, our search for cis-selective methods resulted in surprisingly few convincing examples (Scheme 1): In a synthesis from Chavan et al. starting from readily available chiral pool material l-ascorbic acid (Scheme 1, entry A9), the advantage of cheap starting materials is completely outweighed by in total 18 synthetic steps and one racemization event. Syntheses from d-sugars such as d-glucose (not shown)10 or d-glycals (Scheme 1, entry B11) are attractive for the enantiomeric cis-(2R,3S)-configured product ent-4, but unsuitable for our target molecules. The reported syntheses from Cochi et al. utilizing enantioselective ring expansion of proline-derived intermediate 9 (Scheme 1, entry C12), from Chavan et al. with a key aziridine intermediate 10 (Scheme 1, entry D13), and from Marjanovic et al. using dioxone intermediate 11 accessible via organocatalysis (Scheme 1, entry E14) gave access to 4 in up to 10 synthetic steps. The shortest route employs hydrogenation of 3-hydroxypicolic acid (12) affording cis-selectively rac-4 (Scheme 1, entry F15), but separation of the enantiomers remains an issue.16

In comparison, synthetic strategies based on nucleophilic addition to serine derivatives such as Garner’s aldehyde,17,18 other serinals19 or a serine Weinreb amide20 seemed to be more attractive since serine is a cheap chiral starting material readily available in both d- and l-configurations. Since we4 and others18,21 experienced only low to moderate diastereoselectivities in addition reactions of various nucleophiles to Garner’s aldehyde, we were looking for an alternative approach. In this context, Lajoie’s orthoester-protected serine aldehydes 21a-c(22) gained our attention (Scheme 2). Transformation of the carboxylic acid into its corresponding trioxabicyclo[2.2.2] ortho (OBO) ester installs a very bulky group enabling the attack of the nucleophile to the aldehyde moiety in the preferred orientation and at the same time keeping the oxidation state constant. The formed product possesses the syn (or threo) selectivity, which is fully in line with the nonchelation-controlled Felkin–Anh model. Consequently, Lajoie’s aldehyde has been successfully applied to the synthesis of several β-hydroxy,23,24 β-methoxy,25 and other unusual amino acids.26

In 2014, Karjalainen and Koskinen reported a scalable route based on Garner’s aldehyde 18 yielding trans-(2R,3R)-3-hydroxy pipecolic acid 15 in 11 steps with an overall yield of 31% and without the need for chromatographical purification of intermediates (A, Scheme 2).27 Our initial idea was to adapt the concept of a stereoselective addition of a metal alkyne species to Lajoie’s aldehyde 21c combined with a stringent strategy of functionalization and ring closure to minimize the number of necessary steps. Since reductive amination is an established method of piperidine ring formation,28 we envisaged an addition reaction of an acetal-protected propiolaldehyde 20 to orthoester 21c as the key step to deliver intermediate 19, the precursor for the ring closure to 4.29 After ring formation under reductive hydrogenation conditions, the free carboxylic acid 4 would be obtained directly after a hydrolysis-saponification sequence (B, Scheme 2) in contrast to the Garner’s aldehyde based route A which requires an additional oxidation step.

Furthermore, we planned to investigate serinals 21a and 21b as a potential substitute for Garner’s aldehyde 18 employed in the first total synthesis of GE81112A for the preparation of β-hydroxy histidine intermediate 22. For this purpose, we chose the Fmoc- or Boc-protecting groups since hydrogenolytic Cbz removal would be incompatible with the chlorine present in the histidine moiety (C, Scheme 2).

Results and Discussion

Starting from alkyne 20,30 metalation with ethyl magnesium bromide followed by reaction with aldehyde 21c at low temperature delivered products 19 and 25 with yields up to 39% and a diastereomeric ratio (dr) of merely 1.8:1 for the desired syn (threo) isomer 19 (Scheme 3).31 The low yield could be linked to an incomplete deprotonation of the terminal alkyne, which required extensive periods of time. In order to curb the long deprotonation periods, we substituted the Grignard species by a lithium organyl resulting in an increase of the yield to 81% and of the dr of >20:1 for the syn-product 19. The reaction was upscaled to 2g resulting in a yield of 71% and a constant ee compared to utilizing aldehyde 21c (ee = 97%). After recrystallization target, compound 19 was obtained in 60% yield and with an ee > 99%.

Scheme 3. Stereoselective Alkynyl Addition and Synthesis of cis-3-Hydroxypipecolic Acid Derivatives 5 and 6.

Scheme 3

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Conditions: (a) (i) 20 (4 equiv), EtMgBr (4 equiv), THF, rt, 13 h; (ii) 21c, CH2Cl2/Et2O (1:1), −78 °C, 6.5 h, (19, 25% and 25, 14%, dr 1.8:1); (b) (i) 20 (4 equiv), n-BuLi (4 equiv), THF, −78 °C, 10 min; rt, 10 min; (ii) 21c, THF, −78 °C, 30 min, 81% (dr > 20:1); (c) H2, 85 bar), Pd(OH)2, THF, 70 °C; (d) AcCl, MeOH, 70 °C, 79% over two steps; (e) Teoc-OSu, NaHCO3, 1,4-dioxane/H2O (2:1), 0 °C to rt, 24 h, 42%; (f) 1 M LiOH(aq.), THF, rt, 5 h, (5, 79%); (g) H2 (1 bar), 20 mol % Pd(OH)2, 10 mol % Pd/C, 20% AcOH(aq.), 1,4-dioxane, rt, 24 h; (h) 1 M HCl(aq.), 1,4-dioxane, rt, 2 h; (i) Teoc-OSu, NaHCO3, 1,4-dioxane/H2O (2:1), 0 °C to rt, 24 h, (30, 47% over three steps); (j) Boc2O, NaHCO3, 1,4-dioxane/H2O (2:1), 0 °C to rt, (31, 44% over three steps); (k) 1 M LiOH(aq.), THF, rt, 6 h, (5, 95%), (6, 89%).

The conversion of intermediate 19 into literature-known cis-3-hydroxypipecolic acid methyl ester 27 was achieved by hydrogenation under high pressure (85 bar) in a Thales Nano H-Cube at 70 °C using Pearlman’s catalyst followed by OBO ester opening and transesterification under acidic conditions (Scheme 3). Reaction control (LC/MS) indicated that hydrogenation of the 1,5-dihydro-3H-2,4-benzodioxepin protecting group is the rate-limiting step which required a hydrogen pressure of 85 bar to obtain full conversion. Additionally, compound 27 was converted to target molecule 5 by Teoc-protection and saponification. Notably, the hydrogenation of 19 was possible under milder conditions (room temperature, atmospheric pressure) when diluted acetic acid was added. We hypothesized that protonation of the basic amine may prevent partial catalyst poisoning and facilitate hydrogenetic cleavage of the 1,5-dihydro-3H-2,4-benzodioxepin protecting group. The crude mixture was treated with aqueous hydrochloric acid to drive the orthoester hydrolysis to completion. The synthesis of the desired derivatives 5 and 6 was completed by carbamate protection of intermediate 29 and saponification (Scheme 3). Analogously, isomer 25 was successfully converted to the trans-3-hydroxypipecolic acid methyl ester 32.32

In comparison with the successfully established entry to the pipecolic acids 5 and 6, an effective synthesis of β-hydroxy histidine intermediate 22 depended on the yield and stereoselectivity of the addition step of 2-chloro imidazole derivative 24 (prepared by lithiation of commercially available imidazole 33 and trapping of the lithium species with C2Cl6) to serinal 21a (Scheme 4). Applying conditions similar to those of the previously described syntheses of alkyne addition products 19/25 gave access to desired stereoisomer 23a and minor diastereomer 34a as an inseparable mixture with a preparative yield of 67% and a synthetically useful selectivity (dr > 5:1). Since the separation of the diastereomers could not be achieved and test reactions demonstrated that the Fmoc-protecting group was not compatible to the basic transesterification conditions required for the synthesis of target compound 22 (Scheme 5), we switched to Boc as the protecting group. We were able to increase the yields while reducing the required equivalents of iodo-imidazole 24 which is irreversibly consumed by the iodine-magnesium exchange and cannot be recycled (in contrast to alkyne building block 20 that is metalated by deprotonation cf. Scheme 3). However, the selectivity dropped significantly (dr 1.8:1). Switching to the corresponding lithiated imidazole as a nucleophile retained the selectivity at a dr of 5:1. In contrast to the Fmoc-series, the isolation of pure diastereomer 23b was possible by careful chromatography.33 To obtain 23b in a preparative useful yield of 62% (after two chromatography runs), careful control of time, temperature, and equivalents of n-BuLi employed was required since the reaction turned out to be prone to decomposition when reaction conditions were modified. The remaining mixture of 23a/34a was converted to 23b and 34b by a one-pot Fmoc-deprotection Boc-protection sequence, and the desired isomer 23b was isolated as a pure compound after chromatography.

Scheme 4. Addition of Metalated Imidazole 24 to Aldehydes 21a and 21b.

Scheme 4

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Conditions: (a) 33, LDA (1.05 equiv), C2Cl6 (1.1 equiv), THF, −78 °C, 1 h, 86%; (b) (i) 24 (4.0 equiv), EtMgBr (4.0 equiv), CH2Cl2, 0 °C, 1 h; (ii) 21a, CH2Cl2, −78 °C, 2 h, (67%, mixture 23a and 34a, ratio > 5:1); (c) (i) 24 (2.2 equiv), i-BuMgBr (2.2 equiv), CH2Cl2, 0 °C, 1 h; (ii) 21b, CH2Cl2, −78 °C, 2 h, (83%, mixture 23b and 34b, ratio 1.8:1); (d) (i) 24 (3.0 equiv), n-BuLi (3.0 equiv), THF, −78 °C, 30 min; (ii) 21b, THF, −78 °C, 5 h, (62% of pure 23b) (e) (i) NHMe2, THF, rt; (ii) evaporation; (iii) Boc2O, THF, NaHCO3(aq.), rt (65% of pure 23b).

Scheme 5. Synthesis of β-Hydroxy Histidine Building Block 22 and Formal Total Synthesis of GE81112A (1).

Scheme 5

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Conditions: (a) TIPSOTf, 2,6-lutidine, CH2Cl2, −78 °C, 94%; (b) 80% AcOH(aq.), TFE, 30 °C, 24 h; (c) MeOH, K2HPO4, 40 °C, 48 h, 83% over 2 steps; (d) 4 N HCl in 1,4-dioxane, THF, rt, 24 h, crude product 22 used without purification (ref (4)).

Intermediate 23b was converted to TIPS-protected compound 35 followed by OBO ester opening and simultaneous trityl deprotection. The transesterification with K2HPO4 in methanol resulted in methyl ester 37 in good overall yield.24 Final Boc deprotection completed the synthesis of target compound 22 (Scheme 5). The synthesis of key intermediate 22 was thereby shortened, and the yields were increased from 15% over six steps to 48% over five steps. The sensitivity of OBO-protected intermediates 23b and 35 toward ortho-ester hydrolysis was identified as the only disadvantage hampering chromatography, analytics, and storage of these intermediates.

Additionally, a second-generation synthesis based on the highly stereoselective addition (dr 10:1) of trityl-protected imidazole 24 to Garner’s aldehyde (18) was developed by us which gave a significant improvement in overall yield (cf. SI). The desired stereoisomer was obtained in high purity by crystallization on a 20 g scale. Unfortunately, even though the trityl group gave a higher selectivity, a change to the SO2NMe2 protection group was nonetheless required since it was more acid-tolerant. Thus, it was able to withstand the following acetonide cleavage and alcohol oxidation to the carboxylic acid 22 after initial TIPS protection. With these two improved synthetic routes toward intermediate 22 in hand, we not only achieved the formal total syntheses of GE81112A (1), but also secured a robust and scalable material supply of 22, which is crucial for further SAR investigations. Compared to the remarkably short and fully diastereoselective synthetic access to β-hydroxy histidine intermediate 42 by Renata et al.7 (Scheme 6), our improved routes reached a similar level of convenience while relying solely on chemical means.

Scheme 6. Comparison of Different Synthetic Routes toward β-Hydroxy Histidine Building Blocks 22 and 42.

Scheme 6

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The most critical step within the endgame of our first-generation synthesis of GE81112A (1) was the saponification of methyl ester 49 (Scheme 7). Intermediates 49 and 50 were quite sensitive to basic conditions (resulting in partial decomposition). Only under well monitored conditions, a moderate yield of 42% for the saponification was achieved, and the reproducibility of that reaction—especially on scale—turned out to be difficult to control.4 These findings motivated us to modify the protection group strategy and to utilize TMSE-protected building block 44 (for details regarding the synthetic procedure cf. SI) for the endgame (Scheme 7). After coupling 44 with dipeptide 43 (derived in 14 steps from d-xylose and l-His(Trt)-OMe) to 45, the reduced amine 46 could be coupled with building block 5, derived now from the OBO route. With the TMSE ester in place on the C-terminus, the separate saponification step could be omitted, and all fluoride labile protecting groups (TMSE, TIPS, TBS, and Teoc) of protected tetrapeptide 47 were removed in one step by TAS-F under mild and neutral conditions. Thus, compound 48 could be isolated in an acceptable yield.34 After final trityl deprotection, GE81112A (1) was obtained in 67% yield. Overall, this modification shortened the endgame by one synthetic step, facilitated chromatographic purification due to less side product formation, and improved the yield significantly from 9 to 29% over five steps.

Scheme 7. Synthesis of GE81112A (1) with a Modified Protecting Group Strategy.

Scheme 7

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Conditions: (a) EDC·HCl, Oxyma, NaHCO3, DMF/CH2Cl2 (1:1), −40 °C to rt, 48 h, 80%; (b) 1 M Me3P in THF, THF/H2O (5:1), rt, 12 h; (c) Acid 5, EDC·HCl, Oxyma, NaHCO3, DMF/CH2Cl2 (1:1), 0 °C to rt, 12 h, 82% over 2 steps; (d) 1 M TAS-F in DMF, H2O, DMF, rt, 48 h, 67%; (e) 10% formic acid in TFE, rt, 12 h, 67%; (f) LiOH, THF/H2O, rt, 40% (ref (4)).

Conclusions

We have demonstrated that Lajoie’s OBO-protected serinals 21a-c are readily available and synthetically useful building blocks for the synthesis of β-hydroxy amino acids in threo configuration. Compared to the widely used Garner’s aldehyde 18, the high threo selectivity caused by the steric bulk of the OBO-orthoester (in line with the nonchelation-controlled Felkin–Anh model) and no need for a final oxidation of the addition product to the carboxylic acid are prime advantages of the corresponding routes over the established ones. In the synthesis of cis-3-hydroxy pipecolic acids 5 and 6, a concise and selective access was developed, which was in our hands more convenient than other literature-known strategies. Furthermore, Boc-protected serinal 21b enabled us to improve our first-generation synthesis of the β-hydroxy histidine building block 22 significantly. As main drawbacks, the tendency of aldehydes 21a-c to epimerize and the sensitivity of the OBO-orthoester protecting group toward hydrolysis were identified, which require increased care during experimentation and precautionary measures and may be a reason why the use of 21a-c remained a niche in the synthesis of β-hydroxy amino acids compared to Garner’s aldehyde (18). Nevertheless, our synthetic studies enabled us to improve the sophisticated total synthesis of GE81112A (1) allowing us to provide the building blocks for synthetic analogues of the GE-series. Since key questions concerning the biological activity and the pharmacological and safety profile of GE81112 remain to be adressed,5 modification of the structural backbone and therefore synthetical access is of utmost importance to answer these. The reported results helped us to tackle a number of these issues by generating a number of new derivatives which will be reported in due course.

Experimental Section

General Information

All chemicals and solvents/anhydrous solvents were commercially supplied and used without further purification. For heating of reaction mixtures, aluminum flask carriers in different sizes from IKA were used. Reactions were monitored using thin layer chromatography (TLC) or using one of the following LCMS systems: 1100 HPLC (Agilent) with DAD equipped with an Esquire 3000plus MS detector (Bruker) or 1200 HPLC (Agilent) with DAD equipped with MSD (Agilent) ESI quadrupole MS. TLC was performed on precoated silica gel glass plates (Merck TLC Silica gel 60 F254), and compounds were detected under UV light (254 nm) and/or by staining with an aqueous solution of KMnO4 with K2CO3 and NaOH, or an aqueous solution of phosphomolybdic acid, cerium(IV) sulfate, and H2SO4, or a solution of ninhydrin in n-BuOH/AcOH (100:3) followed by heating with a heat gun. Products were purified by flash column chromatography using silica gel 60 M (Macherey-Nagel) or silica gel from Merck (particle size: 40–63 μm, 60 Å average diameter) or by using automated flash column chromatography systems (puriFlash XS 520Plus from Interchim or Reverlis PREP device from Büchi) equipped with ISOLUTE Flash SI II columns of different sizes from Biotage or PF-15SIHC flash columns of different sizes from Interchim (eluants are given in parentheses). For all OBO-orthoester-containing compounds, conditioning with 2% NEt3 in n-heptane prevented OBO-orthoester hydrolysis by the slightly acidic silica. Reverse-phase flash column chromatography was performed on a FlashPure EcoFlex C18 (Bruker) equipped with FlashPure EcoFlex C18 end capped columns (eluants are given in parentheses). Preparative HPLC was performed on a WATERS AutoPurification HPLC/MS system equipped with a WATERS Sunfire Prep C18 OBD 5 μm 50 × 100 mm column with water/TFA 0.1% as mobile phase A and acetonitrile as mobile phase B (eluents are given in parentheses). The product-containing fractions were collected and freeze-dried to yield the final product. NMR spectra were recorded on a Bruker AVANCE II spectrometer (400 MHz), a Bruker AVANCE III spectrometer (600 MHz), a Bruker AMX400 spectrometer (400 MHz), or a Bruker DPX400 spectrometer (400 MHz) with CDCl3, C6D6, DMDO-d6, or D2O as the solvent with chemical shifts (δ) quoted in parts per million (ppm) and referenced to the solvent signal (δ1H/13C: CDCl3 7.26/77.2, C6D6 7.16/128.1, DMDO-d6 2.50/39.5). For all OBO-orthoester-containing compounds, CDCl3 was filtered through a small plug of basic Al2O3 prior to sample preparation in order to prevent OBO-orthoester hydrolysis by traces of acid. Assignment was confirmed based on COSY, HSQC, HMBC, and NOESY correlations. High-resolution mass spectrometry was performed on a 1290 UPLC (Agilent) with DAD and ELSD equipped with maXis II (Bruker) ESI TOF MS or Alliance 2695 (Waters) with DAD equipped with a Micromass LCT device (Waters) or an Acquity UPLC (Waters) with DAD equipped with a Micromass Q TOF Premier mass spectrometer (Waters). Specific rotation was measured by a polarimeter (MCP 100 polarimeter) from Anton Paar or a polarimeter (P 3000 series) from Krüss. Some hydrogenations were performed with an H-Cube Mini Plus from Thales Nano with a flow rate of 0.3 mL/min.

Benzyl ((1S,2R)-4-(1,5-Dihydrobenzo[e][1,3]dioxepin-3-yl)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)but-3-yn-1-yl)carbamate (19) and Benzyl ((1S,2S)-4-(1,5-Dihydrobenzo[e][1,3]dioxepin-3-yl)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)but-3-yn-1-yl)carbamate (25)

Condition 1 (Deprotonation with EtMgBr; Scheme 3, Condition a)

Alkyne 20 (5.77 g, 33.1 mmol, 4.00 equiv) was dissolved in THF (40 mL). EtMgBr (0.9 M in tert-butylmethylether, 36.8 mL, 33.1 mmol, 4.00 equiv) was added dropwise at room temperature and stirred for 1 h. The reaction mixture was then cooled to −78 °C. Aldehyde 21c (2.69 g, 8.21 mmol, 1.00 equiv) was dissolved in Et2O/CH2Cl2 (30 mL, 1:1) in a separate flask, cooled to −78 °C, and then added dropwise to the alkyne-solution. The reaction mixture was stirred at −78 °C for 6.5 h and was then diluted with saturated aqueous NH4Cl (40 mL) and CH2Cl2 (100 mL). The layers were separated, and the aqueous layer was re-extracted with CH2Cl2 (3 × 25 mL). The combined organic layers were washed with saturated aqueous NH4Cl (20 mL), H2O (20 mL), and saturated aqueous NaCl (20 mL) and were dried over MgSO4 and filtered, and the solvent was removed under reduced pressure. The crude product was purified via column chromatography (silica, conditioning with 2% NEt3 in n-heptane, 0–100% ethyl acetate in n-heptane) to obtain the separated diastereomers 19 (1.03 g, 2.07 mmol, 25%) and 25 (562 mg, 1.13 mmol, 14%) as colorless solids.

Condition 2 (Deprotonation with n-BuLi, Scheme 3, Condition b)

Alkyne 20 (0.79 g, 4.5 mmol, 4.0 equiv) was dissolved in THF (15 mL) and then cooled to −78 °C. Then n-BuLi (2.5 M in n-hexane, 1.8 mL, 4.5 mmol, 4.0 equiv) was added dropwise, and the reaction mixture was stirred for 10 min. The reaction mixture was then stirred without a cooling bath for 10 min and afterward cooled to −78 °C again. Aldehyde 21c (0.36 g, 1.1 mmol, 1.0 equiv) was dissolved in THF (10 mL) in a separate flask, cooled to −78 °C, and then added dropwise to the alkyne-solution. The reaction mixture was stirred at −78 °C for 0.5 h and was then diluted with saturated aqueous NH4Cl (25 mL) and ethyl acetate (80 mL). The layers were separated, and the organic layer was washed with saturated aqueous NaHCO3 (20 mL) and saturated aqueous NaCl (30 mL) and was dried over MgSO4 and filtered, and the solvent was removed under reduced pressure. The crude product was purified via column chromatography (silica, conditioning with 2% NEt3 in n-heptane, 0–100% ethyl acetate in n-heptane) to obtain the diastereomeric mixture of 19 and 25 (0.45 g, 0.91 mmol, 81%, dr > 20:1; dr determined by 1H-NMR) as a colorless solid.

To gain access to the pure diastereomer 19, the reaction was repeated in a bigger scale and under the same reaction conditions: Starting from alkyne 20 (4.75 g, 27.3 mmol, 4.00 equiv) and aldehyde 21c (2.19 g, 6.82 mmol, 1.00 equiv), the diastereomeric mixture of 19 and 25 (2.40 g, 4.85 mmol, 71%, dr > 20:1; dr determined by 1H-NMR) was recrystallized from n-heptane/ethyl acetate (10:1) to obtain pure diastereomer 19 (1.81 g, 3.66 mmol, 54%, ee > 99%) as a colorless solid. Major diastereomer 19: 1H-NMR (CDCl3, 600 MHz): 7.335 (bs, 2H, Cbz-aryl-H, 7.332 (bs, 2H, Cbz-aryl-H), 7.29 (bs, 1H, Cbz-aryl-H), 7.20–7.17 (m, 2H, Aryl-H), 7.09–7.07 (m, 2H, Aryl-H), 5.58 (s, 1H, CH-OCH2), 5.47 (d, J = 10.2 Hz, 1H, NH), 5.12 (d, J = 14.4 Hz, 1H, CH-OCH2), 5.10 (d, J = 14.4 Hz, 1H, CH-OCH2), 5.09 (bs, 2H, Cbz-CH2), 4.98 (bs, 1H, CH-OH), 4.73 (d, J = 14.4 Hz, 1H, CH-OCH2), 4.71 (d, J = 14.4 Hz, 1H, CH-OCH2), 4.17 (dd, J = 10.2 Hz, 1.5 Hz, 1H, CαH), 3.93 (s, 6H, OBO-CH2), 3.16 (d, J = 3.1 Hz, 1H, OH), 0.82 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 150 MHz): 156.6 (s, C=O), 138.42 (s, Ar-Cq), 138.40 (s, Ar-Cq), 136.6 (s, Cbz-Ar-Cq), 128.6 (d, Cbz-Ar-CH), 128.16 (d, Cbz-Ar-CH), 128.13 (d, Cbz-Ar-CH), 127.16 (d, Ar-CH), 126.89 (d, Ar-CH), 126.9 (d, Ar-CH), 126.9 (d, Ar-CH), 108.4 (s, OBO-Cq), 93.3 (d, CH-OCH2), 83.1 (s, HO-CH-C≡C), 80.5 (s, HO-CH-C≡C), 72.9 (t, OBO-CH2), 68.8 (t, CH2-OCH), 68.7 (t, CH2-OCH), 67.1 (t, Cbz-CH2), 62.0 (d, CH-OH), 57.6 (d, CαH), 30.8 (s, OBO-Cq), 14.4 (q, CH3); HRMS (ESI) m/z: [M + H]+ calcd for C27H30NO8 496.1966; found: 496.1971; Rf (n-heptane/ethyl acetate 3:1): 0.48; specific rotation [α]D20 = −7.5° (c = 0.03; CH2Cl2); HPLC (Chiralcel OJ-H, EtOH/MeOH = 50/50, flow rate = 1.0 mL/min, l = 210 nm) tR = 5.9 min (19). Minor diastereomer 25: 1H-NMR (CDCl3, 600 MHz): 7.33 (bs, 2H, Cbz-aryl-H), 7.33 (bs, 2H, Cbz-aryl-H), 7.31 (bs, 1H, Cbz-aryl-H), 7.20–7.17 (m, 2H, Aryl-H), 7.10–7.06 (m, 2H, Aryl-H), 5.60 (s, 1H, CH-OCH2), 5.30 (d, J = 9.2 Hz, 1H, NH), 5.14 (d, J = 14.4 Hz, 1H, CH-OCH2), 5.11 (d, J = 14.4 Hz, 1H, CH-OCH2), 5.09–5.06 (m, 2H, Cbz-CH2), 4.78 (dd, J = 6.7 Hz, 4.8 Hz, 1H, CH-OH), 4.75 (d, J = 14.4 Hz, 1H, CH-OCH2), 4.73 (d, J = 14.4 Hz, 1H, CH-OCH2), 4.13 (dd, J = 9.2 Hz, 4.8 Hz, 1H, CαH), 3.91 (s, 6H, OBO-CH2), 3.64 (d, J = 6.8 Hz, 1H, OH), 0.81 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 150 MHz): 157.1 (s, C=O), 138.5 (s, Ar-Cq), 138.4 (s, Ar-Cq), 136.3 (s, Cbz-Ar-Cq), 128.6 (d, Cbz-Ar-CH), 128.3 (d, Cbz-Ar-CH), 128.3 (d, Cbz-Ar-CH), 127.2 (d, Ar-CH), 127.2 (d, Ar-CH), 126.9 (d, Ar-CH), 107.7 (s, OBO-Cq), 93.5 (d, CH-OCH2), 83.5 (s, HO-CH-C≡C), 81.3 (s, HO-CH-C≡C), 72.9 (t, OBO-CH2), 68.7 (t, CH2-OCH), 68.7 (t, CH2-OCH), 67.4 (t, Cbz-CH2), 63.4 (d, CH-OH), 59.3 (d, CαH), 30.8 (s, OBO-Cq), 14.4 (q, CH3); HRMS (ESI) m/z: [M + H]+ calcd for C27H30NO8 496.1966; found: 496.1971; Rf (n-heptane/ethyl acetate 3:1): 0.6; HPLC (Chiralcel OJ-H, EtOH/MeOH = 50/50, flow rate = 1.0 mL/min, l = 210 nm) tR = 11.1 min (25).35

(2S,3R)-2-(4-Methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)piperidin-3-ol (26)

Alkyne 19 (70 mg, 0.14 mmol, 1.0 equiv) was dissolved in THF (28 mL). The reaction mixture was hydrogenated by the H-Cube at 70 °C and 85 bar in the presence of Pd(OH)2 (20 mol %). After completion of the reaction, the solvent was removed under reduced pressure. Amine 26 (32 mg, 0.14 mmol, quant.) was obtained as a yellowish oil. The acid-labile and highly polar crude product was used without further purification and analytics. HRMS (ESI) m/z: [M + H]+ calcd for C11H20NO4: 230.1387; found: 230.1396.

(2S,3R)-3-Hydroxy-2-(methoxycarbonyl)piperidine Hydrochloride (27)

Dry MeOH (2.3 mL) was cooled to 0 °C. Acetyl chloride (1 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 20 min. Afterward, OBO ester 26 (32 mg, 0.14 mmol, 1.0 equiv) was dissolved in MeOH (1 mL) and added dropwise. The reaction mixture was heated to 70 °C and was stirred for 48 h. The solvent was removed under reduced pressure, and the crude product was applied to cation exchange chromatography (Dowex 50WX8 200–400, MeOH) in order to obtain methylester hydrochloride 27 (39 mg; 0.20 mmol; 79% over 2 steps) as a colorless oil. 1H-NMR (D2O, 400 MHz): 4.57 (m, 1H, CH-OH), 4.18 (m, 1H, CαH), 3.85 (s, 3H, CH3), 3.47 (m, 1H, CH2-NH), 3.05 (m, 1H, CH2-NH), 1.98 (m, 2H, CH2-OH), 1.81 (m, 2H, CH2-CH2-NH); 13C{1H}-NMR (D2O, 100 MHz): 168.7 (s, C=O), 63.6 (d, CH-OH), 60.5 (d, Cα), 53.6 (q, CH3), 43.7 (t, CH2-NH), 28.0 (t, CH2-CH-OH), 15.6 (t, CH2-CH2-NH); HRMS (ESI) m/z: [M + H]+ calcd for C7H14NO3 160.0968; found: 160.0967; Rf (15% MeOH in CH2Cl2): 0.3; specific rotation [α]D25 = −15.88° (c = 0.6; H2O).

2-Methyl 1-(2-(Trimethylsilyl)ethyl) (2R,3S)-3-Hydroxypiperidine-1,2-dicarboxylate (28)

Amine 27 (30 mg, 0.19 mmol, 1.0 equiv) was dissolved in 1,4-dioxane/H2O (2:1, 6 mL). The reaction mixture was cooled to 0 °C. Solid NaHCO3 (475 mg, 5.60 mmol, 30.0 equiv) and water (3 mL) were added to adjust a pH of 9. Afterward, Teoc-OSu (49 mg, 0.19 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (1 mL) and added dropwise. The reaction mixture was stirred at room temperature overnight. The reaction mixture was extracted with ethyl acetate (3 × 15 mL). The combined organic layers were dried over Na2SO4 and filtered. The solvent was removed under reduced pressure, and the crude product was purified via column chromatography (silica, 1% MeOH in CH2Cl2) to obtain carbamate 28 (24 mg, 0.080 mmol, 42%) as a yellowish oil. 1H-NMR (CDCl3, 400 MHz): 5.17–5.06 (m, 1H, CαH), 4.26–4.15 (m, 2H, TMS-CH2-CH2), 4.10–3.86 (m, 1H, CH2-NH), 3.77 (s, 3H, CH3), 3.78–3.69 (m, 1H, CH-OH), 2.81–2.58 (m, 1H, CH2-NH), 2.07–1.93 (m, 1H, CH2-CH-OH), 1.76–1.64 (m, 1H, CH2-CH2-NH), 1.56–1.43 (m, 2H, CH2-CH2-NH, CH2-CH-OH), 1.05–0.92 (m, 2H, CH2-TMS), 0.03 (s, 9H, TMS); 13C{1H}-NMR (CDCl3, 100 MHz): 172.4 (s, CH3O-C=O), 156.4 (s, N-C=O), 68.9 (d, CH-OH), 64.5 (t, TMS-CH2-CH2), 57.7 (d, Cα), 52.5 (q, CH3), 41.1 (t, CH2-N), 30.4 (t, CH2-CH-OH), 23.9 (t, CH2-CH2-N), 17.8 (t, TMS-CH2), −1.4 (q, TMS); HRMS (ESI) m/z: [M + Na]+ calcd for C13H25NO5SiNa 326.1394; found: 326.1399; Rf (5% MeOH in CH2Cl2): 0.56; specific rotation [α]D25 = −23.11° (c = 1.2; CHCl3).

3-Hydroxy-2-(hydroxymethyl)-2-methylpropyl (2S,3R)-3-Hydroxypiperidine-2-carboxylate Hydrochloride (29)

Alkyne 19 (155 mg, 0.313 mmol, 1.00 equiv) was dissolved in 1,4-dioxane (10 mL). Pd(OH)2 (20 mol %, 220 mg, 0.313 mmol, 1.00 equiv) and Pd/C (10 mol %, 333 mg, 0.313 mmol, 1.00 equiv) were added. Aqueous AcOH (20%, 0,5 mL) was added, and the reaction mixture was hydrogenated at room temperature under a hydrogen atmosphere (1 bar). After completion of the reaction, the reaction mixture was filtered by Celite, and the solvent was removed under reduced pressure. The crude product was dissolved in 1,4-dioxane (2 mL), and aqueous HCl (1 M, 0.300 mL, 0.391 mmol, 1.25 equiv) was added. The reaction mixture was stirred for 2 h at room temperature and then neutralized by solid NaHCO3. The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over Na2SO4 and filtered. The solvent was removed under reduced pressure to obtain amine 29 (32 mg, 0.14 mmol, quant.) as colorless oil. The highly polar crude product was utilized in the next step without further purification. HRMS (ESI) m/z: [M + H]+ for C11H22NO5 248.1492; found: 248.1501.

2-(3-Hydroxy-2-(hydroxymethyl)-2-methylpropyl) 1-(2-(Trimethylsilyl)ethyl) (2S,3R)-3-Hydroxypiperidine-1,2-dicarboxylate (30)

Amine 29 (19 mg, 0.077 mmol, 1.0 equiv) was carbamate protected analogously to 28. The resulting crude product was purified via column chromatography (silica, 100% ethyl acetate) to obtain Teoc-protected 3-hydroxypipecolic ester 30 (14 mg, 0.033 mmol, 47%) as yellowish oil. 1H-NMR (CDCl3, 400 MHz): 5.03–4.96 (m, 1H, CαH), 4.31–4.24 (m, 1H, CO2-CH2), 4.24–4.14 (m, 2H, TMS-CH2-CH2), 4.14–4.08 (m, 1H, CO2-CH2), 3.94–3.84 (m, 1H, CH2-N), 3.85–3.74 (m, 1H, CH-OH), 3.59–3.54 (m, 4H, CH2-OH), 3.00 (t, J = 11.2 Hz, 1H, CH2-N), 2.00–1.89 (m, 1H, CH2-CH2-N), 1.78–1.69 (m, 1H, CH2-CH2-N), 1.59–1.47 (m, 2H, CH2-CH-OH), 0.99 (t, J = 8.3 Hz, 2H, TMS-CH2), 0.83 (s, 3H, CH3), 0.03 (s, 9H, TMS); 13C{1H}-NMR (CDCl3, 100 MHz): 171.8 (s, CαH-C=O), 156.9 (s, N-C=O), 68.6 (d, CH-OH), 68.2 (t, CαH-CO2-CH2), 67.4 (t, CH2-OH), 67.1 (t, CH2-OH), 64.6 (t, TMS-CH2-CH2), 58.3 (d, Cα), 41.2 (s, Cq), 40.6 (t, CH2-N), 29.7 (t, CH2-CH-OH), 23.4 (t, CH2-CH2-N), 17.8 (t, TMS-CH2), 17.2 (q, CH3), −1.4 (q, TMS); HRMS (ESI) m/z: [M + Na]+ calcd for C17H33NO7SiNa 414.1919; found: 414.1925; Rf (ethyl acetate): 0.3; specific rotation [α]D20 = −33.4° (c = 1.0; CHCl3).

1-(tert-Butyl) 2-(3-Hydroxy-2-(hydroxymethyl)-2-methylpropyl) (2S,3R)-3-Hydroxypiperidine-1,2-dicarboxylate (31)

Amine 29 (77 mg, 0.31 mmol, 1.0 equiv) was dissolved in 1,4-dioxane/H2O (3 mL, 2:1). The reaction mixture was cooled to 0 °C. Solid NaHCO3 (789 mg, 9.40 mmol, 30.0 equiv) and water (3 mL) were added to adjust a pH of 9. Afterward, Boc2O (68 mg, 0.31 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (1 mL) and was added dropwise at 0 °C. The reaction mixture was stirred at room temperature overnight. The reaction mixture was extracted with ethyl acetate (3 × 10 mL), the combined organic layers were dried over Na2SO4 and filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified via column chromatography (silica, 100% ethyl acetate) to obtain Boc-protected 3-hydroxypipecolic ester 31 (48 mg, 0.14 mmol, 44%) as a yellowish oil. Main conformer: 1H-NMR (CDCl3, 400 MHz): 4.96–4.93 (m, 1H, CαH), 4.24 (s, 1H, CO2-CH2), 4.17–4.04 (m, 1H, CO2-CH2), 4.02–3.84 (m, 1H, CH2-N), 3.81–3.76 (m, 1H, CH-OH), 3.59–3.48 (m, 4H, CH2-OH), 2.99–2.86 (m, 1H, CH2-N), 1.98–1.87 (m, 1H, CH2-CH-OH), 1.77–1.65 (m, 1H, CH2-CH2-N), 1.55–1.47 (m, 2H, CH2-CH2-N, CH2-CH-OH), 1.44 (s, 9H, Boc), 0.83 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 100 MHz): 172.0 (s, CαH-C=O), 154.9 (s, Boc-C=O), 81.0 (s, Boc-Cq), 72.9 (d, CH-OH), 68.1 (t, CH2-OH), 66.9 (t, CO2-CH2), 57.8 (d, Cα), 41.4 (s, CH3-Cq), 40.6 (t, CH2-N), 29.7 (t, CH2-CH-OH), 28.4 (q, Boc), 23.5 (t, CH2-CH2-N), 17.2 (q, CH3). Minor conformer: 1H-NMR (CDCl3, 400 MHz): 4.93–4.86 (m, 1H, CαH), 4.27 (s, 1H, CO2-CH2), 4.17–4.04 (m, 1H, CO2-CH2), 4.03–3.91 (m, 1H, CH2-N), 3.82–3.75 (m, 1H, CH-OH), 3.59–3.48 (m, 4H, CH2-OH), 2.84–2.70 (m, 1H, CH2-N), 1.98–1.87 (m, 1H, CH2-CH-OH), 1.77–1.65 (m, 1H, CH2-CH2-N), 1.55–1.47 (m, 2H, CH2-CH2-N, CH2-CH-OH), 1.44 (s, 9H, Boc), 0.83 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 100 MHz): 172.0 (s, CαH-C=O), 154.9 (s, Boc-C=O), 81.0 (s, Boc-Cq), 72.9 (d, CH-OH), 68.6 (t, CH2-OH), 67.1 (t, CO2-CH2), 57.8 (d, Cα), 41.4 (s, CH3-Cq), 40.6 (t, CH2-N), 29.7 (t, CH2-CH-OH), 28.4 (q, Boc), 23.5 (t, CH2-CH2-N), 17.2 (q, CH3); HRMS (ESI) m/z: [M + Na]+ calcd for C16H29NO7Na, 370.1836; found: 370.1839; Rf (ethyl acetate): 0.25; specific rotation [α]D20 = −35° (c = 1.0; CHCl3).

(2S,3R)-3-Hydroxy-1-((2-(trimethylsilyl)ethoxy)carbonyl)piperidine-2-carboxylic Acid (5)

From intermediate 28. Ester 28 (24 mg, 0.079 mmol, 1.0 equiv) was dissolved in THF (0.8 mL). Aqueous LiOH (1 M, 0.079 mL, 0.079 mmol, 1.0 equiv) was added, and the reaction mixture was stirred for 5 h at room temperature. The reaction mixture was neutralized by addition of AcOH (4.5 μL, 0.079 mmol, 1.0 equiv), and the solvent was removed under reduced pressure. The crude product was purified via MPLC (RP-C18, 4 g, 20 min, 0.05% TFA in CH3CN/H2O, 50–60% CH3CN) to obtain Teoc-protected 3-hydroxypipecolic acid 5 (18 mg, 0.062 mmol, 79%) as colorless oil.

From intermediate 30: Ester 30 (780 mg, 1.90 mmol, 1.00 equiv) was dissolved in THF (20 mL). Aqueous LiOH (1 M, 2.79 mL, 2.79 mmol, 1.40 equiv) was added, and the reaction mixture was stirred for 6 h at room temperature. The reaction mixture was then neutralized by addition of AcOH (0.160 mL, 2.79 mmol, 1.40 equiv), and the solvent was removed under reduced pressure. The crude product was purified via MPLC (RP-C18, 12 g, 20 min, 0.05% TFA in CH3CN/H2O, 50–60% CH3CN) to obtain Teoc-protected 3-hydroxypipecolic acid 5 (550 mg, 1.90 mmol, 95%) as colorless oil. 1H-NMR (C6D6, 400 MHz): 5.40–5.21 (m, 1H, CαH), 4.22 (t, J = 7.7 Hz, 2H, TMS-CH2-CH2), 3.87–3.86 (m, 2H, CH-OH, CH2-N), 2.97 (t, J = 12.1 Hz, 1H, CH2-N), 1.80 (d, J = 11.8 Hz, 1H, CH2-CH-OH), 1.47 (q, J = 11.8 Hz, 1H, CH2-CH-OH), 1.27–1.21 (m, 2H, CH2-CH2-N), 0.92 (t, J = 7.7 Hz, 2H, TMS-CH2), −0.04 (s, 9H, TMS); 13C{1H}-NMR (C6D6, 100 MHz): 172.7 (s, CO2H), 157.5 (s, Teoc-C=O), 68.5 (d, CH-OH), 64.9 (t, CO2-CH2), 58.3 (d, Cα), 41.0 (t, CH2-N), 29.7 (t, CH2-CH-OH), 23.6 (t, CH2-CH2-N), 17.8 (t, TMS-CH2), −1.5 (q, TMS); HRMS (ESI) m/z: [M + Na]+ calcd for C12H23NO5SiNa, 312.1238; found: 312.1248; specific rotation [α]D20 = −22.3° (c = 1.0; MeOH).

(2S,3R)-1-(tert-Butoxycarbonyl)-3-hydroxypiperidine-2-carboxylic Acid (6)

Ester 31 (27 mg, 0.080 mmol, 1.0 equiv) was saponified analogously to ester 30. The crude product was purified by MPLC (RP-C18, 4 g, 15 min, 0.05% TFA in CH3CN/H2O, 35–45% CH3CN) to obtain Boc-protected 3-hydroxypipecolic acid 6 (17 mg, 0.070 mmol, 89%) as a colorless oil. Main conformer: 1H-NMR (CDCl3, 400 MHz): 5.38–5.22 (m, 1H, CαH), 3.76–3.86 (m, 2H, CH2-N), 2.98–2.77 (m, 1H, CH-OH), 1.77–1.63 (m, 1H, CH2-CH-OH), 1.51–1.45 (m, 1H, CH2-CH2-N), 1.38 (s, 9H, Boc), 1.26–1.06 (m, 2H, CH2-CH-OH, CH2-CH2-N); 13C{1H}-NMR (CDCl3, 100 MHz): 172.5 (s, CO2H), 156.6 (s, Boc-C=O), 81.2 (s, Boc-Cq), 68.5 (d, CH-OH), 57.9 (d, Cα), 41.3 (t, CH2-N), 29.7 (t, CH2-CH-OH), 28.3 (q, Boc), 23.6 (t, CH2-CH2-N). Minor conformer: 1H-NMR (CDCl3, 400 MHz): 5.13–4.98 (m, 1H, CαH), 4.19–4.01 (m, 2H, CH2-N), 2.98–2.77 (m, 1H, CH-OH), 1.77–1.63 (m, 1H, CH2-CH-OH), 1.51–1.45 (m, 1H, H-CH2-CH2-N), 1.38 (s, 9H, Boc), 1.26–1.06 (m, 2H, CH2-CH-OH, CH2-CH2-N); 13C{1H}-NMR (CDCl3, 100 MHz): 172.5 (s, CO2H), 156.6 (s, Boc-C=O), 81.2 (s, Boc-Cq), 68.5 (d, CH-OH), 57.9 (d, Cα), 41.3 (t, CH2-N), 29.7 (t, CH2-CH-OH), 28.3 (q, Boc), 23.6 (t, CH2-CH2-N); HRMS (ESI) m/z: [M + Na]+ calcd for C11H19NO5Na 268.1155; found: 268.1169; specific rotation [α]D20 = −30.4° (c = 1.0; CHCl3).

2-Chloro-4-iodo-1-trityl-1H-imidazole (24)

Imidazole 33 (19.1 g, 43.1 mmol, 1.00 equiv) was dissolved in THF (450 mL) and cooled to −78 °C. Then LDA (1 M in THF, 45.0 mL, 45.0 mmol, 1.05 equiv) was added dropwise. The reaction mixture was stirred at −78 °C for 1 h. C2Cl6 (11.3 g, 47.6 mmol, 1.10 equiv) was added portion wise to the reaction solution at −78 °C. After 10 min, saturated aqueous NH4Cl (150 mL) was added. The solution was warmed to room temperature, and the THF was removed under reduced pressure. Water (150 mL) was added, and the mixture was extracted with ethyl acetate (3 × 400 mL). The combined organic layers were dried over Na2SO4 and filtered, and the solvent was removed under reduced pressure. The residue was recrystallized from ethanol (250 mL) to obtain pure imidazole 24 (17.5 g, 37.1 mmol, 86%) as a colorless solid. 1H-NMR (CDCl3, 400 MHz): 7.39–7.37 (m, 9H, Trt), 7.17–7.12 (m, 6H, Trt), 6.94 (s, 1H, Im-H); 13C{1H}-NMR (CDCl3, 100 MHz): 141.0 (Trt), 134.6 (Im-C2), 130.0 (Trt), 129.9 (Trt), 129.1 (Im-C5), 128.2 (Trt), 78.0 (Trt), 77.0 (Im-C4); HRMS (ESI) m/z: [M + Na]+ calcd for C22H16ClIN2Na 492.9944; found: 492.9949; Rf (petroleum ether:ethyl acetate 9:1): 0.50; melting point: 224 °C.

(9H-Fluoren-9-yl)methyl ((1S,2S)-2-(2-Chloro-1-trityl-1H-imidazol-4-yl)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)carbamate (23a) and (9H-Fluoren-9-yl)methyl ((1S,2R)-2-(2-Chloro-1-trityl-1H-imidazol-4-yl)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)carbamate (34a)

Iodo-imidazole 24 (3.68 g, 7.82 mmol, 4.00 equiv) was dissolved in CH2Cl2 (40 mL) and cooled to 0 °C. Then EtMgBr (1 M in THF, 7.82 mL, 7.82 mmol, 4.00 equiv) was added dropwise. After 1 h, the solution was cooled to −78 °C. Aldehyde 21a (800 mg, 1.95 mmol, 1.00 equiv) was dissolved in CH2Cl2 (10 mL) in a separate flask, cooled to −78 °C, and added to the Grignard reaction solution at −78 °C. After 2 h, saturated aqueous NH4Cl (40 mL) was added. The solution was warmed to room temperature and diluted with CH2Cl2 (25 mL). The aqueous layer was extracted with CH2Cl2 (3 × 25 mL). The combined organic layers were washed with saturated aqueous NH4Cl (20 mL), dried over MgSO4, and filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified via column chromatography (silica, conditioning with 2% NEt3 in n-heptane, n-heptane to n-heptane/ethyl acetate 1:3) to obtain a diastereomeric mixture of 23a and 34a (980 mg, 1.3 mmol, 67%, dr > 5:1 by 1H-NMR and >10:1 by chiral HPLC) as a colorless solid.

HPLC (Chiralpak IE, n-heptane/EtOH/MeOH = 72/14/14, flow rate = 1.0 mL/min, l = 265 nm) tR = 12.1 (23a), 17.7 (34a); Main diastereomer 23a, main conformer: 1H-NMR (CDCl3, 600 MHz): 7.77–7.28 (m, 8H, aryl-H), 7.35–7.06 (m, 15H, Trt), 6.84 (s, 1H, Im-H), 5.55 (d, J = 10.3 Hz, 1H, NH), 5.30 (bs, 1H, CH-OH), 4.22/4.17 (d, 2H, Fmoc-CH2), 4.12 (bs, 1H, Fmoc-CH), 4.24 (bs, 1H, CαH), 3.97 (s, 6H, OBO-CH2), 3.28 (d, J = 2.2 Hz, 1H, OH), 0.83 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 150 MHz): 156.5 (s, C=O), 144.5 (s, Cq-Fmoc), 144.1 (s, Cq-Fmoc), 141.6 (s, Cq-Trt), 141.4 (s, Cq-Fmoc), 141.3 (s, Cq-Fmoc), 138.6 (s, Im-Cq), 133.4 (s, Im-CCl), 130.1 (d, CH-Trt), 127.9 (d, CH-Trt), 127.7 (d, CH-Trt), 127.22 (d, CH-Fmoc), 127.16 (d, CH-Fmoc), 125.6 (d, CH-Fmoc), 125.5 (d, CH-Fmoc), 119.9 (d, Im-CH), 108.8 (s, OBO-Cq), 76.3 (s, Trt-Cq), 73.0 (t, OBO-CH2), 67.30 (t, Fmoc-CH2), 67.25 (d, CHOH), 57.2 (d, Cα), 47.3 (d, Fmoc-CH), 30.9 (s, OBO-Cq-CH3), 14.5 (q, CH3); Main diastereomer 23a, minor conformer: 1H-NMR (CDCl3, 600 MHz): 7.77–7.28 (m, 8H, aryl-H), 7.35–7.06 (m, 15H, Trt), 6.88 (s, 1H, Im-H), 5.18 (d, J = 10.5 Hz, 1H, NH), 5.34 (bs, 1H, CH-OH), 4.22/4.17 (d, 2H, Fmoc-CH2), 4.11 (bs, 1H, Fmoc-CH), 4.39 (bs, 1H, CαH), 3.97 (s, 6H, OBO-CH2), 3.22 (bs, 1H, OH), 0.83 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 150 MHz): 156.3 (s, C=O), 144.5 (s, Cq-Fmoc), 144.1 (s, Cq-Fmoc), 141.6 (s, Cq-Trt), 141.4 (s, Cq-Fmoc), 141.3 (s, Cq-Fmoc), 138.8 (s, Im-Cq), 133.1 (s, Im-CCl), 130.1 (d, CH-Trt), 127.9 (d, CH-Trt), 127.7 (d, CH-Trt), 127.22 (d, CH-Fmoc), 127.16 (d, CH-Fmoc), 125.6 (d, CH-Fmoc), 125.5 (d, CH-Fmoc), 120.0 (d, Im-CH), 108.8 (s, OBO-Cq), 76.3 (s, Trt-Cq), 73.0 (t, OBO-CH2), 67.73 (d, CHOH), 67.30 (t, Fmoc-CH2), 57.7 (d, Cα), 47.3 (d, Fmoc-CH), 30.9 (s, OBO-Cq-CH3), 14.5 (q, CH3); Minor diastereomer 34a, main conformer: 1H-NMR (CDCl3, 600 MHz): 7.77–7.28 (m, 8H, aryl-H), 7.35–7.06 (m, 15H, Trt), 6.85 (s, 1H, Im-H), 5.46 (d, J = 10.3 Hz, 1H, NH), 4.87 (dd, J = 7.6, 3.7 Hz, 1H, CH-OH), 4.22/4.17 (d, 2H, Fmoc-CH2), 4.12 (bs, 1H, CαH), 4.11 (bs, 1H, Fmoc-CH), 3.97 (s, 6H, OBO-CH2), 3.76 (d, J = 3.7 Hz, 1H, OH), 0.83 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 150 MHz): 156.5 (s, C=O), 144.5 (s, Cq-Fmoc), 144.1 (s, Cq-Fmoc), 141.6 (s, Cq-Trt), 141.4 (s, Cq-Fmoc), 141.3 (s, Cq-Fmoc), 139.2 (s, Im-Cq), 132.5 (s, Im-CCl), 130.1 (d, CH-Trt), 127.9 (d, CH-Trt), 127.7 (d, CH-Trt), 127.22 (d, CH-Fmoc), 127.16 (d, CH-Fmoc), 125.6 (d, CH-Fmoc), 125.5 (d, CH-Fmoc), 120.7 (d, Im-CH), 108.8 (s, OBO-Cq), 108.6 s, OBO-Cq), 76.3 (s, Trt-Cq), 73.0 (t, OBO-CH2), 68.2 (d, CHOH), 67.30 (t, Fmoc-CH2), 59.7 (d, Cα), 47.3 (d, Fmoc-CH), 30.9 (s, OBO-Cq-CH3), 14.5 (q, CH3); Minor diastereomer 34a, minor conformer: 1H-NMR (CDCl3, 600 MHz): 7.77–7.28 (m, 8H, aryl-H), 7.35–7.06 (m, 15H, Trt), 6.77 (s, 1H, Im-H), 4.95 (d, J = 10.3 Hz, 1H, NH), 4.81 (bs, 1H, CH-OH), 4.22/4.17 (d, 2H, Fmoc-CH2), 4.12 (bs, 1H, CαH), 4.11 (bs, 1H, Fmoc-CH), 3.97 (s, 6H, OBO-CH2), 3.83 (d, J = 3.7 Hz, 1H, OH), 0.83 (s, 3H, CH3); 13C{1H}-NMR (CDCl3, 150 MHz): 156.5 (s, C=O), 144.5 (s, Cq-Fmoc), 144.1 (s, Cq-Fmoc), 141.6 (s, Cq-Trt), 141.4 (s, Cq-Fmoc), 141.3 (s, Cq-Fmoc), 139.2 (s, Im-Cq), 132.5 (s, Im-CCl), 130.1 (d, CH-Trt), 127.9 (d, CH-Trt), 127.7 (d, CH-Trt), 127.22 (d, CH-Fmoc), 127.16 (d, CH-Fmoc), 125.6 (d, CH-Fmoc), 125.5 (d, CH-Fmoc), 120.7 (d, Im-CH), 108.6 (s, OBO-Cq), 108.8 (s, OBO-Cq), 76.3 (s, Trt-Cq), 73.0 (t, OBO-CH2), 68.2 (d, CHOH), 67.30 (t, Fmoc-CH2), 59.7 (d, Cα), 47.3 (d, Fmoc-CH), 30.9 (s, OBO-Cq-CH3), 14.5 (q, CH3); 776.2498; found: 776.2499 (M + Na)+; Rf (n-heptane/ethyl acetate 3:1): 0.47.

tert-Butyl ((1S,2S)-2-(2-Chloro-1-trityl-1H-imidazol-4-yl)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)carbamate (23b) and tert-Butyl ((1S,2R)-2-(2-Chloro-1-trityl-1H-imidazol-4-yl)-2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)ethyl)carbamate (34b)

Conditions 1 (Scheme 4, Conditions c)

Iodo-imidazole 24 (324 mg, 0.743 mmol, 2.20 equiv) was dissolved in CH2Cl2 (5 mL) and cooled to 0 °C. Then iBuMgBr (2 M in n-hexane, 0.370 mL, 0.743 mmol, 2.20 equiv) was added dropwise. After 1 h, the solution was cooled to −78 °C. Aldehyde 21b (97.0 mg, 0.340 mmol, 1.00 equiv) was dissolved in CH2Cl2 (5 mL) in a separate flask, cooled to −78 °C, and added to the Grignard reaction solution at −78 °C. After 2 h, saturated aqueous NH4Cl (15 mL) was added. The solution was warmed to room temperature and diluted with CH2Cl2 (10 mL). The aqueous layer was extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were washed with saturated aqueous NH4Cl (2 × 10 mL) and saturated aqueous NaCl (10 mL), dried over Na2SO4, and filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified via column chromatography (silica, conditioning with 2% NEt3 in petroleum ether, petroleum ether to petroleum ether/ethyl acetate 1:2) to obtain a diastereomeric mixture of 23b and 34b (177 mg, 0.280 mmol, 83%, dr 1.8:1 by 1H-NMR) as a colorless solid. HPLC (Chiralpak IE, n-heptane/EtOH/MeOH = 72/14/14, flow rate = 1.0 mL/min, l = 202 nm) tR = 9.3 (23b), 11.5 (34b).

Conditions 2 (Scheme 4, Conditions d)

Iodo-imidazole 24 (836 mg, 1.78 mmol, 3.00 equiv) was dissolved in THF (9 mL) and cooled to −78 °C. Then n-BuLi (1.54 M in n-hexane, 1.15 mL, 1.18 mmol, 3.00 equiv) was added dropwise, and the reaction mixture was stirred for 30 min. Aldehyde 21b (170 mg, 0.592 mmol, 1.00 equiv) was dissolved in THF (6 mL) in a separate flask, cooled to −78 °C, and added to the lithium reaction solution at −78 °C. After 5 h, saturated aqueous NH4Cl (10 mL) was added. The solution was warmed to room temperature and diluted with CH2Cl2 (10 mL). The aqueous layer was extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were washed with saturated aqueous NH4Cl (10 mL) and saturated aqueous NaCl (10 mL), dried over Na2SO4, and filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified via column chromatography (silica, conditioning with 2% NEt3 in petroleum ether, petroleum ether/ethyl acetate 4:1 to 100% ethyl acetate) to obtain 23b and 34b as a colorless solid and a mixture of diastereomers with a dr of 5:1(determined by 1H-NMR). The diastereomers were separated by a second purification via flash chromatography (25 g PF-15SIHC flash column, conditioning with 2% NEt3 in petroleum ether, 0–100% ethyl acetate in n-heptane in 60 min) to obtain the desired main diastereomer 23b (231 mg, 0.364 mmol, 62%) as a colorless solid (all fractions containing traces of 23b and the undesired minor diastereomer 34b were discarded).

Conditions 3 (Scheme 4, Conditions e)

To a solution of the diastereomeric mixture of 23a and 34a (282 mg, 0.374 mmol, dr > 5:1, 1.00 equiv) in THF (10 mL) was added aqueous NHMe2 (40%, 1.00 mL, 7.93 mmol, 21.2 equiv), and the reaction mixture was stirred at room temperature for 30 min until LC/MS indicated complete Fmoc-deprotection. Toluene (30 mL) was added, and all volatiles were removed in vacuo. The crude product was further dried in high vacuum (0.1 mbar) overnight to remove all traces of NHMe2. The crude mixture was dissolved in THF (10 mL) and saturated aqueous NaHCO3 (1 mL), followed by Boc2O (245 mg, 1.12 mmol, 3.00 equiv) were added and the reaction mixture was stirred at room temperature overnight. Ethyl acetate (30 mL) and water (5 mL) were added, and the layers were separated. The organic layer was washed with saturated aqueous NaCl (5 mL) and dried over Na2SO4. After filtration the solvent was removed under reduced pressure and the resulting crude product was purified via flash chromatography (25 g PF-15SIHC flash column, conditioning with 2% NEt3 in petroleum ether, 0%–100% ethyl acetate in n-heptane in 60 min) to obtain the desired main diastereomer 23b (153 mg, 0.242 mmol, 65%) as a colorless solid (all fractions containing traces of 23b and the undesired minor diastereomer 34b were discarded). Main diastereomer 23b: 1H-NMR (CDCl3, 400 MHz): 7.31–7.27 (m, 9H, Trt), 7.17–7.09 (m, 6H, Trt), 6.82 (s, 1H, Im-H), 5.24 (d, J = 10.4 Hz, 1H, CH-OTIPS), 5.21 (bs, 1H, NH), 4.03 (d, J = 10.1 Hz, 1H, CαH), 3.93 (s, 6H, OBO-CH2), 3.23 (d, J = 1.9 Hz, 1H. OH), 1.35 (s, 9H, Boc), 0.80 (s, 3H, OBO-CH3); 13C{1H}-NMR (CDCl3, 100 MHz): 155.8 (s, Boc-C=O), 141.7 (s, Trt), 138.8 (s, Im), 133.2 (s, OBO-Cq), 130.1 (d, Trt), 128.0 (d, Trt), 127.9 (d, Trt), 119.8 (d, Im-CH), 108.9 (s, Im), 79.1 (s, Trt-Cq), 76.3 (s, Boc-Cq), 72.8 (t, OBO-CH2), 67.2 (d, C-OH), 56.6 (d, Cα), 30.8 (s, OBO-Cq), 28.5 (q, Boc-CH3), 14.5 (q, OBO-CH3); HRMS (ESI) m/z: [M + H]+ calcd for C35H39N3O6Cl 632.2527; found: 632.2525; Rf (3% MeOH in CH2Cl2): 0.25; specific rotation [α]D20 = −7° (c = 1.0; CHCl3). Minor diastereomer 34b (the chemical shifts for 34b were determined from the mixture derived from conditions 1): 1H-NMR (CDCl3, 400 MHz): 7.31–7.27 (m, 9H, Trt), 7.17–7.09 (m, 6H, Trt), 6.80 (s, 1H, Im-H), 5.21 (bs, 1H, NH), 5.13 (d, J = 10.04 Hz, 1H, CH-OTIPS), 4.03 (d, J = 10.1 Hz, 1H, CαH), 3.85 (s, 6H, OBO-CH2), 3.23 (s, 1H. OH), 1.35 (s, 9H, Boc), 0.78 (s, 3H, OBO-CH3); 13C{1H}-NMR (CDCl3, 100 MHz): 155.8 (s, Boc-C=O), 141.7 (s, Trt), 138.8 (s, Im), 133.2 (s, OBO-Cq), 130.1 (d, Trt), 128.0 (d, Trt), 127.9 (d, Trt), 119.8 (d, Im-CH), 108.5 (s, Im), 79.3 (s, Trt-Cq), 76.2 (s, Boc-Cq), 72.6 (t, OBO-CH2), 68.2 (d, C-OH), 58.4 (d, Cα), 30.7 (s, OBO-Cq), 28.5 (q, Boc-CH3), 14.5 (q, OBO-CH3).

tert-Butyl ((1S,2S)-2-(2-Chloro-1-trityl-1H-imidazol-4-yl)-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)-2-((triisopropylsilyl)oxy)ethyl)carbamate (35)

Alcohol 23b (100 mg, 0.158 mmol, 1.00 equiv) was dissolved in CH2Cl2 (1.6 mL) and cooled to −78 °C. 2,6-Lutidine (129 μL, 1.27 mmol, 8.00 equiv) and TIPS-OTf (170 μL, 0.633 mmol, 4.00 equiv) were added dropwise, and the reaction mixture was stirred for 5 h at −78 °C. Saturated aqueous NH4Cl (3 mL) was added. The aqueous layer was extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were dried over Na2SO4 and filtered, and the solvent was removed under reduced pressure. The resulting crude product was purified via column chromatography (silica, 1% MeOH in CH2Cl2) to obtain 35 (117 mg, 0.148 mmol, 94%) as a colorless solid. 1H-NMR (CDCl3, 400 MHz): 7.32–7.26 (m, 9H, Trt), 7.15–7.08 (m, 6H, Trt), 6.70 (s, 1H, Im-H), 5.36 (m, 1H, CH-OTIPS), 5.12 (d, J = 10.3 Hz, 1H, NH), 3.93 (d, J = 10.3 Hz, 1H, CαH), 3.84 (s, 6H, OBO-CH2), 1.34 (s, 9H, Boc-CH3), 0.97 (s, 21H, TIPS), 0.76 (s, 3H, OBO-CH3); 13C{1H}-NMR (CDCl3, 100 MHz): 155.8 (s, Boc-C=O), 142.3 (s, Im-CCl), 141.8 (s, Trt), 132.5 (s, OBO-Cq), 130.1 (d, Trt), 128.0 (d, Trt), 127.8 (d, Trt), 120.2 (d, Im-CH), 108.2 (s, Im), 78.6 (s, Trt-Cq), 76.0 (s, Boc-Cq), 72.6 (t, OBO-CH2), 68.4 (d, CH-OTIPS), 58.6 (d, CαH), 30.6 (s, OBO-Cq), 28.6 (q, Boc-CH3), 18.13 (q, TIPS-CH3), 18.10 (q, TIPS-CH3), 14.6 (q, OBO-CH3), 12.9 (d, TIPS-CH); HRMS (ESI) m/z: [M + H]+ calcd for C44H59N3O6SiCl 788.3862; found: 788.3862; Rf (petroleum ether/ethyl acetate 1:1): 0.63; specific rotation [α]D20 = −24.7° (c = 1.0; CHCl3).

3-Hydroxy-2-(hydroxymethyl)-2-methylpropyl (2S,3S)-2-((tert-Butoxycarbonyl)amino)-3-(2-chloro-1H-imidazol-4-yl)-3-((triisopropylsilyl)oxy)propanoate (36)

OBO ester 35 (44 mg, 0.056 mmol, 1.0 equiv) was dissolved in 2,2,2-trifluoroethanol (0.6 mL), and aqueous AcOH (80%, 0.6 mL) was added subsequently. The reaction mixture was warmed to 30 °C and stirred overnight. Then the mixture was cooled to room temperature, and aqueous saturated NaHCO3 (2 mL) was added. After dilution with ethyl acetate (8 mL), the layers were separated. The organic layer was dried over Na2SO4 and filtered, and the solvent was removed under reduced pressure. Diol 36 (32 mg, 0.056 mmol, quant.) was obtained as a yellowish oil. The highly polar crude product was utilized in the next step without further purification.

Methyl (2S,3S)-2-((tert-Butoxycarbonyl)amino)-3-(2-chloro-1H-imidazol-4-yl)-3-((triisopropylsilyl)oxy)propanoate (37)

Diol 36 (32 mg, 0.056 mmol, 1.0 equiv) was dissolved in MeOH (1.9 mL), and K2HPO4 (291 mg, 1.67 mmol, 30.0 equiv) was added subsequently. The reaction mixture was warmed to 40 °C and stirred for 48 h. Then the reaction mixture was cooled to room temperature, diluted with CH2Cl2 (10 mL), and filtered over Celite. The solvent was removed under reduced pressure, and the crude product was purified via column chromatography (silica, 1–3% MeOH in CH2Cl2) to obtain methylester 37 (22 mg, 0.046 mmol, 83%) as a colorless solid. 1H-NMR (CDCl3, 400 MHz): 6.88 (s, 1H, Im-H), 5.67–5.36 (m, 1H, NH), 5.37–5.09 (m, 1H, CH-OTIPS), 4.56–4.38 (m, 1H, CαH), 3.75 (s, 3H, CH3), 1.43 (s, 9H, Boc), 0.99 (s, 21H, TIPS); 13C{1H}-NMR (CDCl3, 100 MHz): 171.5 (s, CH3O-C=O), 156.0 (s, Boc-C=O), 142.6 (s, Im-Cq), 130.0 (s, Im-CCl), 115.1 (d, Im-CH), 80.6 (s, Boc-Cq), 70.5 (d, C-OTIPS), 59.2 (d, Cα), 52.5 (q, CH3), 28.4 (q, Boc-CH3), 18.0 (q, TIPS-CH3), 17.9 (q, TIPS-CH3), 12.5 (d, TIPS-CH).; HRMS (ESI) m/z: [M + Na]+ calcd for C21H38N3O5SiClNa 498.2161; found: 498.2169; Rf (5% MeOH in CH2Cl2): 0.45; specific rotation [α]D25 = −18.4° (c = 1.0; CHCl3); HPLC (Chiralpak IC, n-heptane/EtOH/MeOH = 94/3/3, flow rate = 1.0 mL/min, l = 212 nm) tR = 6.1 min.

Methyl (2S,3S)-2-Amino-3-(2-chloro-1H-imidazol-4-yl)-3-((triisopropylsilyl)oxy)propanoate (22)

The deprotection of methylester 37 was performed based on our previously reported procedure.4 Dihydrochloride 22 was obtained as a colorless solid and was used without further purification.

2-(Trimethylsilyl)ethyl (5S,7S,10S,13S)-7-Azido-5-((carbamoyloxy)methyl)-13-((S)-(2-chloro-1H-imidazol-4-yl)((triisopropylsilyl)oxy)methyl)-2,2,3,3-tetramethyl-8,11-dioxo-10-((1-trityl-1H-imidazol-4-yl)methyl)-4-oxa-9,12-diaza-3-silatetradecan-14-oate (45)

Carboxylic acid 43 (99 mg, 0.14 mmol, 1.0 equiv) was dissolved in CH2Cl2/DMF (1.4 mL, 1:1), and the resulting reaction mixture was cooled to −40 °C. Oxyma (44 mg, 0.31 mmol, 2.2 equiv), EDC · HCl (59 mg, 0.31 mmol, 2.2 equiv), and NaHCO3 (118 mg, 1.41 mmol, 10.0 equiv) were added, and the suspension was stirred for 5 min before hydrochloride 44(4) (90 mg, 0.17 mmol, 1.2 equiv) was added in one portion. The suspension was slowly warmed to room temperature and stirred for 48 h. After addition of water (30 mL) and ethyl acetate (40 mL), the layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 40 mL). The combined organic layers were washed with saturated aqueous NH4Cl (70 mL), H2O (70 mL), saturated aqueous NaHCO3 (70 mL), and saturated aqueous NaCl (70 mL), and then dried over Na2SO4. After filtration, the solvent was removed under reduced pressure, and the resulting crude product was purified via column chromatography (silica, petroleum ether:ethyl acetate 5:1) to obtain 45 (130 mg, 0.112 mmol, 80% over two steps) as a light orange foam. 1H-NMR (CDCl3, 400 MHz): 12.58 (bs, 1H, Im-NH), 7.87 (m, 1H, Im), 7.82–7.72 (m, 1H, NH), 7.57–7.40 (m, 1H, NH) 7.38–7.30 (m, 9H, Trt), 7.15–7.06 (m, 6H, Trt), 6.82 (m, 1H, Cl-Im), 6.66 (s, 1H, Im), 5.32 (m, 1H, TIPSO-CH), 4.93 (dd, 1H, J = 6.9, 4.3 Hz, CH-CH2-Im), 4.78 (br s, 2H CONH2), 4.64–4.39 (m, 1H, CH-CO2TMSE), 4.14–3.91 (m, 5H, CH2-CHOTBS, CH2-CH2-TMS), 3.16–2.98 (m, 2H, CH2-Im), 2.09–1.97 (m, 1H, N3-CH-CH2), 1.86–1.71 (m, 1H, N3-CH-CH2), 1.05–0.86 (m, 32H, TIPS, TBS, CH2-TMS), 0.14–0.06 (m, 6H, TBS), 0.05–0.02 (m, 9H, TMS); 13C{1H}-NMR (CDCl3, 100 MHz): 170.6 (N3-CH-CO-NH), 170.1 (CH-CO-NH), 169.3 (CO2TMSE), 156.4 (s, CONH2), 141.9 (Trt), 138.8 (Im), 136.7 (Im), 129.7 (Trt), 128.3 (Trt), 128.2 (Trt), 120.3 (Cl-Im), 68.1 (NH-CO2-CH2), 66.9 (CH-OTIPS), 64.2 (CH-OTBS), 60.4 (N3-CH), 59.1 (NH-CH-CO2TMSE), 55.1 (NH-CH-CO-NH), 45.8 (CO2-CH2), 37.0 (CH2-CH-OTBS), 25.83 (TBS), 25.79 (CH2-TMS), 18.1 (s, TBS), 17.8 (TIPS), 17.7 (TIPS), 12.0 (TIPS), −1.5 (TMS), −4.3 (q, TBS), −4.9 (q, TBS); HRMS (ESI) m/z: [M + H]+ calcd for C57H84ClN10O8Si3 1155.5470; found: 1155.5475; Rf (8% MeOH in CH2Cl2): 0.55.

2-(Trimethylsilyl)ethyl (5S,7S,10S,13S)-7-Amino-5-((carbamoyloxy)methyl)-13-((S)-(2-chloro-1H-imidazol-4-yl)((triisopropylsilyl)oxy)methyl)-2,2,3,3-tetramethyl-8,11-dioxo-10-((1-trityl-1H-imidazol-4-yl)methyl)-4-oxa-9,12-diaza-3-silatetradecan-14-oate (46)

Azide 45 (130 mg, 0.112 mmol, 1.00 equiv) was dissolved in degassed THF/H2O (1.5 mL, 5:1). Trimethylphosphine (1 M in THF, 134 μL, 0.134 mmol, 1.20 equiv) was added, and the resulting reaction mixture was stirred for 12 h at room temperature. All volatiles were removed under reduced pressure and the resulting crude product 46 was used in the following peptide coupling without further purification.

2-(Trimethylsilyl)ethyl (2R,3S)-2-(((7S,10S,13S,15S)-15-((Carbamoyloxy)methyl)-7-((S)-(2-chloro-1H-imidazol-4-yl)((triisopropylsilyl)oxy)methyl)-2,2,17,17,18,18-hexamethyl-6,9,12-trioxo-10-((1-trityl-1H-imidazol-4-yl)methyl)-5,16-dioxa-8,11-diaza-2,17-disilanonadecan-13-yl)carbamoyl)-3-hydroxypiperidine-1-carboxylate (47)

Acid 5 (39 mg, 0.13 mmol, 1.2 equiv) was dissolved in CH2Cl2/DMF (1.2 mL, 1:1), and the resulting mixture was cooled to 0 °C. After addition of Oxyma (35 mg, 0.25 mmol, 2.2 equiv), EDC·HCl (47 mg, 0.25 mmol, 2.2 equiv), and NaHCO3 (49 mg, 0.56 mmol; 5.0 equiv), the suspension was stirred for 5 min before amine 46 (crude product from previous reaction, 0.112 mmol, 1.00 equiv) dissolved in CH2Cl2/DMF (1.0 mL, 1:1) was added dropwise. The suspension was slowly warmed to room temperature and was stirred for 12 h. After hydrolysis by addition of water (20 mL) and ethyl acetate (30 mL), the layers were separated, and the aqueous layer was extracted with ethyl acetate (3 × 40 mL). The combined organic layers were washed with saturated aqueous NH4Cl (40 mL), water (40 mL), saturated aqueous NaHCO3 (40 mL), and saturated aqueous NaCl (40 mL) and finally dried over Na2SO4. After filtration, the solvent was removed under reduced pressure, and the resulting crude product was purified via column chromatography (silica, 2–6% MeOH in CH2Cl2) to obtain tetrapeptide 47 (129 mg, 0.0920 mmol, 82% over 2 steps) as a colorless solid. 1H-NMR and 13C{1H}-NMR: broad signals even by utilizing a variety of solvents, no meaningful spectra; HRMS (ESI):m/z: [M + H]+ calcd for C69H107ClN9O12Si4 1400.6799; found: 1400.6809; Rf (6% MeOH in CH2Cl2): 0.45.

(2S,3S)-2-((S)-2-((2S,4S)-5-(Carbamoyloxy)-4-hydroxy-2-((2S,3R)-3-hydroxypiperidine-2-carboxamido)pentanamido)-3-(1-trityl-1H-imidazol-4-yl)propanamido)-3-(2-chloro-1H-imidazol-4-yl)-3-hydroxypropanoic Acid (48)

Tetrapeptide 47 (129 mg, 0.0920 mmol, 1.00 equiv) was dissolved in DMF (0.6 mL) and water (33.1 μL, 1.84 mmol, 20.0 equiv), and then TAS-F (1 M in DMF, 1.84 mL, 1.84 mmol, 20.0 equiv) was added. The reaction mixture was stirred for 48 h. The reaction mixture was directly applied to MPLC (RP-C18, 4 g, 20 min, 0.1% FA in CH3CN/H2O, 0–80% CH3CN) in order to obtain tetrapeptide 48 (55 mg, 0.062 mmol, 67%) as a colorless solid. 1H-NMR and 13C{1H}-NMR: broad signals even by utilizing a variety of solvents, no meaningful spectra; HRMS (ESI) m/z: [M + H]+ calcd for C43H49ClN9O10 886.3285; found: 886.3285; Rf (20% MeOH in CH2Cl2): 0.21.

(2S,3S)-2-((S)-2-((2S,4S)-5-(Carbamoyloxy)-4-hydroxy-2-((2S,3R)-3-hydroxypiperidine-2-carboxamido)pentanamido)-3-(1H-imidazol-4-yl)propanamido)-3-(2-chloro-1H-imidazol-4-yl)-3-hydroxypropanoic Acid (GE81112A, 1)

Tetrapeptide 48 (55 mg, 0.062 mmol, 1.0 equiv) was dissolved in TFE/formic acid (1 mL, 9:1), and the resulting solution was stirred for 12 h. The reaction was stopped by dilution with water (5 mL) and direct freeze drying. The crude product was purified via flash chromatography (RP-18, 15 min, 0–80% MeCN (0.1% formic acid)) to obtain GE81112A (1, 27 mg, 0.042 mmol, 67%) as a colorless solid. To generate the TFA salt, 1 was lyophilized with a solution of 0.05% TFA in water. 1H-NMR (DMSO-d6, 500 MHz) and 13C{1H}-NMR (DMSO-d6, 100 MHz) are in full accordance with the previously reported data4 (spectra see the Supporting Information); HRMS (ESI) m/z: [M + H]+ calcd for C24H35ClN9O10 644.2190; found: 644.2186; specific rotation [α]D25 = +21.4° (c = 1.1; MeOH; Lit.: +23.7°4).

Acknowledgments

This study was financially supported by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK) via generous grant for the LOEWE Research Center Insect Biotechnology and Bioresources. Sanofi contributed in the framework of the Sanofi-Fraunhofer Natural Product Center of Excellence. We thank Chiara Presenti, Silke Herok-Schöpper, Yolanda Kleiner, Thorsten Schäfer, Heiko Heese, Joachim Kluge, Karin Rahn-Hotze, Manuela Schnierer, and Christoph Hartwig for technical assistance. We thank Prof. Dr. Andreas Vilcinskas for general support and Dr. Cédric Couturier, Dr. Frédéric Jeannot, and Dr. Eric Bacqué for valuable discussions.

Data Availability Statement

The data underlying this study are available in the published article and its online Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.3c00094.

  • Detailed experimental procedures including spectroscopic data and determination of enantiomeric excess (PDF)

The authors declare the following competing financial interest(s): P. Hammann, C. Pverlein, M. Kurz and A. Bauer are or have been employed by Sanofi S.A. or one of its affiliates and are shareholders of Sanofi S.A. O. Plettenburg has been employed by Sanofi S.A. S. Schuler and P. Hammann is or has been employed by Evotec International GmbH.

Supplementary Material

jo3c00094_si_001.pdf (4.3MB, pdf)

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  30. For preparation of alkyne 20 and aldehyde 21a-c see Supporting Information.
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  32. Details about a more selective formation of 25 and its conversion to methyl ester 32 are reported in the Supporting Information.
  33. For details see Experimental Section. Alternatively, the separation of the diastereomers was possible on the stage of compound 37 by preparative HPLC (for details see Supporting Information).
  34. The crude mixture of the TAS-F mediated deprotection was directly subjected to HPLC-purification (RP-18), for details see Experimental Section.
  35. Compound 25 was only obtained starting from a partially racemic aldehyde 21c. Therefore, no specific rotation was measured.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jo3c00094_si_001.pdf (4.3MB, pdf)

Data Availability Statement

The data underlying this study are available in the published article and its online Supporting Information.

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