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Article

N-Alkylated Iminosugar Based Ligands: Synthesis and Inhibition of Human Lysosomal β-Glucocerebrosidase

by
Andreas Wolfsgruber
1,
Martin Thonhofer
1,
Patrick Weber
1,
Seyed A. Nasseri
2,
Roland Fischer
3,
Michael Schalli
1,
Arnold E. Stütz
1,
Stephen G. Withers
2 and
Tanja M. Wrodnigg
1,*
1
Glycogroup, Institute of Chemistry and Technology of Biobased Systems, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria
2
Chemistry Department, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada
3
Institute of Inorganic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria
*
Author to whom correspondence should be addressed.
Molecules 2020, 25(20), 4618; https://doi.org/10.3390/molecules25204618
Submission received: 8 September 2020 / Revised: 23 September 2020 / Accepted: 25 September 2020 / Published: 11 October 2020
(This article belongs to the Special Issue Targeting Carbohydrate–Protein Interactions)
Browse Figures
Versions Notes

Abstract

:
The scope of a series of N-alkylated iminosugar based inhibitors in the d-gluco as well as d-xylo configuration towards their interaction with human lysosomal β-glucocerebrosidase has been evaluated. A versatile synthetic toolbox has been developed for the synthesis of N-alkylated iminosugar scaffolds conjugated to a variety of terminal groups via a benzoic acid ester linker. The terminal groups such as nitrile, azide, alkyne, nonafluoro-tert-butyl and amino substituents enable follow-up chemistry as well as visualisation experiments. All compounds showed promising inhibitory properties as well as selectivities for β-glucosidases, some exhibiting activities in the low nanomolar range for β-glucocerebrosidase.

Graphical Abstract

1. Introduction

Iminoalditols, also called iminosugars, represent polyhydroxylated alkaloids and are structurally related to common carbohydrates in which the endocyclic oxygen atom is replaced by a basic trivalent nitrogen atom. This exchange endows this class of glycomimetics with basic properties that are responsible for their remarkable biological activity. Iminoalditols are widely distributed in nature and can be found in bacteria, fungi or plants [1]. Common structural features are monocyclic systems such as piperidines (e.g., compounds 1 and 2) as well as pyrrolidines (e.g., 3) and bicyclic scaffolds, for example indolizidines (e.g., 4), pyrrolizidines (e.g., 5), and nortropanes (e.g., 6) (see Figure 1).
The wide variety of iminosugars known today is also the result of synthetic efforts to replace the ring oxygen with heteroatoms such as nitrogen, sulfur or phosphorus in the early 1960s. In 1966, the first synthetically achieved iminosugar 1,5-dideoxy-1,5-imino-d-glucitol (1), better known as 1-deoxynojirimycin (1-DNJ), was reported [2,3] and with this structure, an entire interdisciplinary scientific research field was born [4,5,6].
In general, representatives of this substance class are known as ligands for carbohydrate-processing enzymes (CPEs), as they interact with the active site of the respective protein as mimics of the natural substrate [7,8,9,10]. Iminosugar-based structures are versatile probes and have been used in several different applications as inhibitors, [11,12] therapeutics [8,9,10] and probes for activity-based protein profiling [13,14]. In particular, these structures have proved interesting for the therapy of CPE- associated diseases such as cancer [15], bacterial infections [16], HIV [17], HPV [18], influenza [19], hepatitis [20], the dengue virus [21], malaria [22] and fungal infections [23]. In addition, iminosugar based glycomimetics have shown encouraging results as so-called pharmacological chaperones (PCs) in the treatment of lysosomal storage diseases (LSDs) [24,25,26]. Due to their strong interaction with the active site of their respective enzyme, they can stabilize the correctly folded structures of mutant lysosomal enzymes, thereby obviating their cellular clearance [24,27].
An interesting enzyme in this respect is human lysosomal β-glucocerebrosidase (glucosylceramidase, GCase, EC 3.2.1.45, glycosidase family 30 [28]). This enzyme catalyses the degradation of the β-glycosidic linkage of glucosylceramide or glucosylsphingosine into d-glucose units and ceramide or sphingosine, respectively, which is the last step of the lysosomal catabolism of glycosylsphingosines in the autophagy-lysosomal pathway. A malfunction of GCase causes Gaucher disease (GD), a severe lysosomal storage disorder with an incidence of approx. 1 in every 50,000 people worldwide [29]. Additionally, Parkinson’s as well as Alzheimer’s disease have been linked to GCase deficiency [30,31].
In this context, a large variety of iminosugar based glycomimetics have been investigated [4,32,33,34,35,36,37]. Paradigmatic examples are shown in Figure 2. Compounds 7 [38] have been introduced by Mellet and coworkers, d-xylo configured 1-C-alkyl iminosugars 8 [39] represent another important family which is also true for compounds 9 [40], all showing significant properties as pharmacological chaperones for mutant GCases.
We are particularly interested in iminoalditol structures, where the modification is located at the ring nitrogen in order to install customised features for different research related properties of GCase. In this context, well-known N-alkylated examples are compounds 10 and 11, which inhibit GCase with Ki values of 116 µM and 0.3 µM, respectively [41] (Figure 3).A large variety of N-substituted iminosugar based glycomimetics was contributed by Overkleeft and co-workers, their compound collection of 1-DNJ derivatives contain various hydrophobic groups at the ring nitrogen such as alkyl chains 12 [42], different biphenyl derivatives 13 [43], an adamantly group 14 [44] and carborane-modified moieties 15 [45], just to mention a few. All of these compounds show Ki-values in the low micro to nanomolar range against GCase (respective Ki values see references [42,43,44,45]). Additionally, N,O-dialkylated derivatives of 1-DNJ (e.g., compound 16) were reported recently as potent ligands of ceramide transport protein [46]. Furthermore, multivalent iminosugar-based structures [47] (e.g., compounds 17 and 18) were reported to exhibit multivalency effects on GCase inhibition with Ki values of 55 nM and 285 nM, respectively [48]. 1,5-Dideoxy-1,5-imino-d-xylitol-based N-alkylated derivatives 19 and 20 were found as selective inhibitors of GCase with Ki values of 57 µM and 4.1 µM, respectively. [36].
Now, we present a robust synthetic method for the simple and concise composition of diversity driven N-alkylated iminosugar based inhibitors, which have the potential to be used as powerful tools such as inhibitors, pharmacological chaperones or probes for investigations of glycoprocessing enzymes in general and GCase in particular.

2. Results

2.1. Synthesis

The developed synthetic concept relies on the coupling of three main building blocks, as shown in Figure 4: The iminosugar scaffold (A) acts as an active site ligand; the interface moiety (B) enables variation in length and consequently properties of the final product; the terminal building block (tag) (C) provides various functional groups that can be customised for further applications.
Since we are interested in inhibiting β-glucosidases, we employed d-gluco as well as d-xylo configured iminosugar scaffolds. The component constructed from the interface (B) and terminal building block or tag (C), including a six-carbon alkyl spacer for constant conjugation to the iminosugar (A), was intended to react either through an N-alkylation reaction or via reductive amination with the ring nitrogen of the respective iminosugar moiety. Therefore, various esters of modified benzoic acids and different ω-halogen alcohols (6-bromo-, and 6-chlorohexanol) act as suitable building blocks. The alkyl halide employed can either react directly in N-alkylation reactions, or can alternatively be converted into the corresponding aldehyde functionality through a Kornblum oxidation, as required for reductive amination reactions. As terminal tags we have chosen nitrile, azide, alkyne, nonafluoro-tert-butyl and N-dansyl functionalities which will enable follow-up chemistry as well as visualisation experiments in further investigations.
To keep the synthetic approach as flexible as possible, the conceptual synthetic strategy has been designed to start with the central hydroxybenzoic acid motif, to which the two handles are introduced. The installation of the terminal nitrile via an ether bond can be realised by a simple Williamson ether synthesis employing 6-bromohexanenitrile and potassium carbonate with methyl hydroxybenzoate (21) to obtain compound 22 (Scheme 1). Saponification of the methyl ester leads to the previously reported benzoic acid derivative 23 [49]. Subsequent esterification under standard Mitsunobu conditions with 6-chlorohexanol, PPh3 and DIAD in THF provided compound 24. In light of the poor reactivity of alkyl chlorides in N-alkylation reactions, a second parallel series of building blocks containing a more reactive aldehyde functionality was prepared via a Kornblum reaction [50,51]. This would allow coupling to the amine via a reductive amination reaction in the event that the alkyl halide route was unsuccessful. Therefore, compound 24 was treated with sodium hydrogen carbonate and DMSO at 120 °C to give aldehyde building block 25. The nitrile can be used in orthogonal follow up chemistry such as (2+3) cycloaddition [52,53] for further labelling of the enzyme-ligand complex.
Additionally, the reaction sequence can be rearranged in order to start with the installation of the ester bond in the first step followed by formation of the ether (cf. Scheme 1 and Scheme 2). To effect this the hydroxyl group of hydroxybenzoic acid was protected with a THP group under standard reaction conditions, giving compound 26 [54] which was then converted into benzoic ester 27 using 6-bromohexanol, PPh3 and DIAD in THF. Removal of the THP group under acidic conditions provided known compound 28 [55]. Subsequent etherification under standard Mitsunobu conditions with 2-(2-azidoethoxy)ethanol [56] gave intermediate 29. Finally, the bromine functionality was transformed into an aldehyde by a Kornblum oxidation to give compound 30. In order to evaluate different types of spacers, and to demonstrate the diversity of the presented synthetic tool box, a 2-(2-hydroxyethoxy)ethanol chain has been introduced in this interface (compound 30). The azide group allows for subsequent click chemistry tagging of the ligand-enzyme complex after incubation for labelling or quantification experiments [57].
The same approach has been followed for the introduction of a terminal alkyne group to enable orthogonal follow up click chemistry (Scheme 3). Therefore, 6-chlorohexanol has been employed in a Mitsunobu reaction with THP protected hydroxybenzoic acid 26 to yield ester 31. Removal of the THP group under acidic conditions using ion exchange resin liberated alcohol 32. Subsequent etherification with 3-bromoprop-1-yne and potassium carbonate in acetone led to structure 33. To demonstrate an additional procedure for the preparation of the desired aldehyde, the alkyl chloride was first hydrolysed to alcohol 34 using modified Kornblum reaction conditions. Therefore, compound 33 was treated with NaHCO3 in a mixture of DMSO and water at 100 °C to yield alcohol 34 which, in a second step, was oxidized with Dess Martin’s reagent (DMP) to desired component 35. X-ray diffraction (XRD) studies unambiguously confirmed the structure of 35 (CCDC 2021385, see SM Figure S1).
Next, we wanted to introduce a reporter group to allow subsequent mass spectrometric monitoring of reactions by introducing a heavy substituent, in particular a nonafluoro-tert-butoxy group (Scheme 4) [58]. Therefore, diethylenoxy benzoic acid methyl ester 36 was synthesised as described previously from methyl hydroxybenzoate (21) and 2-(2-chloroethoxy)ethanol [59]. Compound 36 underwent a Mitsunobu reaction employing nonafluoro-tert-butanol, PPh3 and DIAD in THF to give compound 37. Follow-up chemistry for the introduction of the aldehyde at the other terminus of the hydroxybenzoic ester moiety was performed as described before. Saponification of the methyl ester provided benzoic acid derivative 38, followed by esterification under Mitsunobu conditions using 6-bromohexanol to obtain alkyl bromide 39. As seen in the 2 step conversion of the halocarbon into the corresponding aldehyde (compare Scheme 3), the bromine (compound 39) could be successfully hydrolyzed to alcohol 40 then oxidized to aldehyde 41 using Dess Martin´s reagent. Alcohol 40 can be formed starting from either the bromide or chloride of type 39.
In order to gain access to an amino substituent at the terminal end ready for further functionalisations with for example fluorescent dyes, benzoic acid moiety 42 [60] carrying a Cbz-protected aminoethyl spacer-arm in its para position was introduced (Scheme 5). Esterification of compound 42 to compound 43 followed by oxidation, as before, yielded aldehyde 44.
Assembly of the different building blocks via halides 24, 29, 33, 39 and 43, turned out to be problematic since the halides were rather unreactive, requiring elevated temperatures and extended reaction times, hence preparatively unsatisfying yields below 10% were obtained. Therefore, a reductive amination reaction between the aldehydes of the respective components 25, 30, 35, 41 and 44 and the ring nitrogen of the iminosugars 1-DNJ (1) and 1,5-dideoxy-1,5-imino-d-xylitol (DIX, 2) was employed since this method allowed smoother and faster reactions leading to far better yields compared to the N-alkylation approach (Scheme 6). The two chosen iminosugars, 1 [61] and 2 [62] were synthesised as previously described by our group.
Two different reducing methods were employed for the reductive amination reaction between the aldehyde building block and the iminosugar scaffold, depending on the nature of the functional group on the phenyl linker. Reaction of the cyano group-containing building block 25 with iminosugars 1 and 2 was carried out under an atmosphere of H2 with Pd/C (10%) as catalyst (variant a, Scheme 6), yielding the corresponding d-gluco and d-xylo configured structures 45 (63%) and 46 (48%), respectively.
All other reductive aminations were performed with NaBH3CN in methanol (variant b, Scheme 6) to avoid reduction of other substituents present in the respective molecules. In this way compounds 47 and 48 were obtained in yields of 56% and 54%, respectively. Likewise the alkyne-containing building block 35 and NHCbz-carrying component 44 reacted with iminosugars 1 and 2 to give compounds 51 (60%), 52 (35%), 53 (55%) and 54 (27%) in the yields shown using NaBH3CN. Indeed the same method also worked well with nonafluoro-tert-butyl group reagent 41 yielding iminosugar derivatives 49 and 50 in yields of 52% and 62% respectively while the yield of compound 46 was increased up to 71% employing this methodology, compared to 48% with Pd/C and H2.
N-Cbz-Deprotection of compounds 53 and 54 was achieved using Pd/C (10%) under an atmosphere of H2, allowing reaction of the amines with dansyl chloride in the presence of triethylamine in MeOH to give the desired dansylated inhibitors of both d-gluco and d-xylo configuration, compounds 55 and 56, respectively (Scheme 7).

2.2. Biological Evaluation

Inhibition constants were determined for the interaction of each of a selected set of glycoside hydrolases (GHs) with the N-alkylated iminosugar based glycomimetics 45–56 (Table 1). With the exception of the terminal alkyne 51, all compounds were better inhibitors of the human lysosomal β-glucosidase GCase (GH30) than any of the other enzymes, including the bacterial β-glucosidase from Agrobacterium species. (Abg, GH1). In all cases, the d-gluco configured iminosugar based compounds showed better activities against both β-glucosidases GCase and Abg compared to the d-xylo analogs. None of the compounds were particularly useful inhibitors of either the human lysosomal α-galactosidase (Fabrazyme, GH27) or the β-galactosidase from Escherichia. coli (E. coli, GH2). This is not surprising since these enzymes are fairly specific galactosidases. However, all compounds turned out to inhibit the GH35 bovine liver β-galactosidase in the micromolar range. The presence of aromatic substituents caused some unexpected inhibition, most notably with α-glucosidase from Saccharomyces cerevisiae (S.cer., GH13) and β-galactosidase from E. coli likely due to adventitious interactions. This additional binding interaction is also seen with the “productive” inhibitor/enzyme combinations, with the 1-DNJ derivatives 53 (NHCbz) and 55 (NHdansyl), increasing affinity for GCase down to Ki values of 22 and 18 nM, respectively. This follows a trend we have observed with most of our compounds, that the dansyl moiety contributes significantly to a better interaction with β-glucosidases [37,63]. Interestingly, β-glucosidase from Abg shows a slight preference for a shorter handle between the interface and the terminal building block as in compounds 51–56 with Ki values in the nanomolar range (Table 1) compared to compounds 45–50 wherein the handle contains a six membered chain and has Ki values in the low micromolar range. This trend cannot be detected with GCase.

3. Materials and Methods

3.1. General Methods

NMR Spectra were recorded on an INOVA 500 spectrometer (Varian, Palo Alto, CA, USA) operating at 499.82 MHz (1H), and at 470.3 MHz (19F) or on a Bruker (Billerica, MA, USA) Ultrashield spectrometer at 300.36 (1H) and 75.53 (13C) MHz, respectively. CDCl3 was employed for aromatic compounds and CD3OD for unprotected ligands as indicated. Chemical shifts are listed in ppm employing residual, non-deuterated solvent as the internal standard. Structures of crucial intermediates were unambiguously assigned APT, COSY and HSQC experiments. Carbon and hydrogen numbering in NMR spectra was conducted as indicated in representative structures shown above (Scheme 1, Scheme 7). Optical rotations were measured on a Perkin Elmer 341 polarimeter (Perkin Elmer, Waltham, MA, USA) at the wavelength of 589 nm and a path length of 10 cm at 20 °C. MALDI-TOF Mass Spectrometry was performed on a Micromass TofSpec 2E Time-of-Flight Mass Spectrometer (Waters Corporation, Milford, MA, USA). Analytical TLC was performed on precoated aluminum plates Silica Gel 60 F254 (E. Merck 5554, E. Merck, Darmstadt, Germany), detected with UV light (254 nm). For staining, a solution of vanillin (9 g) in a mixture of H2O (950 mL)/EtOH (750 mL)/H2SO4 (120 mL) or ceric ammonium molybdate (100 g ammonium molybdate/8 g ceric sulphate in 1 L 10% H2SO4) were employed followed by heating on a hotplate. For column chromatography, silica gel 60 (230–400 mesh, E. Merck 9385) or silica gel 60 (Acros Organics, AC 24036, Thermo Fisher Scientific Inc., Waltham, MA, USA) were used. Unless otherwise specified, all starting materials, reagents and solvents are commercially available and were used without further purification. Reactions were performed at ambient temperature and ambient pressure. Otherwise, conditions are explicitly specified. Reaction monitoring was performed by TLC. NMR spectra for new compounds and XRD data for structure 35 are presented in the Supplementary Materials.

3.2. General Synthetic Procedures

3.2.1. General Procedure A: (Mitsunobu Reaction)

A 10% solution of the respective starting material (1.0 equiv.) in THF, Ph3P (1.0 equiv.), diisopropyl azodicarboxylate (DIAD, 1.0 equiv.) and the respective alcohol (1.0 equiv.) was stirred until completed conversion of the reactants was detected. Subsequently, the reaction mixture was diluted with CH2Cl2 and washed consecutively with aqueous HCl (2 N) and saturated NaHCO3. After drying over Na2SO4, the filtrate was concentrated under reduced pressure to provide the corresponding crude product.

3.2.2. General Procedure B: (Kornblum Oxidation)

Variant 1: (Conversion of a halocarbon to the corresponding aldehyde)
A 10% solution of the respective halocarbon (1.0 equiv.) in DMSO was stirred with NaHCO3 (4.0-6.0 equiv.) at 120 °C until completed conversion of the starting material was detected. After allowing the system to cool to room temperature, the reaction mixture was diluted with CH2Cl2 and subsequently washed with water. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure providing the corresponding crude product.
Variant 2: (Conversion of a halocarbon to the corresponding alcohol)
Alternatively, the respective halocarbon was dissolved in a mixture of DMSO and water (10:1 v/v) instead of pure DMSO. The remaining protocol is identical to variant 1.

3.2.3. General Procedure C: (Dess-Martin Oxidation)

To a 10% solution of the respective alcohol (1 equiv.) in CH2Cl2, Dess-Martin periodinane (1.1 equiv.) was added. After completed conversion of the starting material, the reaction mixture was carefully quenched with saturated NaHCO3. After separation and drying over Na2SO4, the organic layers were filtered off and concentrated under reduced pressure to obtain the corresponding crude aldehyde.

3.2.4. General Procedure D: (Reductive Amination employing NaBH3CN)

A 20% solution of the respective aldehyde (1.0 equiv.) and iminosugar (1.0–1.2 equiv.) in MeOH (containing a catalytic amount of AcOH) was stirred for 15 min before NaBH3CN (1.5–3.0 equiv.) was added. After completed conversion of the starting materials was detected, the reaction mixture was concentrated under reduced pressure to provide the corresponding crude title compound.
Methyl 4-((5-cyanopentyl)oxy)benzoate (22). To a stirred solution of commercially available methyl 4-hydroxybenzoate (21, 2.01 g, 13.2 mmol) in acetone (30 mL), K2CO3 (4.40 g, 31.8 mmol) and 6-bromohexanenitrile (2.61 mL, 19.7 mmol) were added. After complete conversion of the starting material, the reaction mixture was filtered off, diluted with CH2Cl2 and washed three times with H2O. The combined organic layers were dried over Na2SO4, filtered off and the solvent was removed under reduced pressure. Purification on silica gel (cyclohexane-ethyl acetate 12:1) provided compound 22 (2.86 g, 11.6 mmol, 87.5%) as a colourless syrup. 1H-NMR (300 MHz, CDCl3) δ = 7.97 (d, 2 H, Ar), 6.89 (d, 2 H, Ar), 4.02 (t, 2 H, H-1), 3.87 (s, 3 H, OCH3), 2.38 (t, 2 H, H-5), 1.88–1.59 (po, 6 H, H-2, H-3, H-4); 13C-NMR (75.5 MHz, CDCl3) δ = 166.9 (OC = O), 162.7 (ipso), 131.7 (Ar), 122.7 (ipso), 119.6 (CN), 114.1 (Ar), 67.6 (C-1), 51.9 (OCH3), 28.5 (C-2), 25.5, 25.3 (C-3, C-4), 17.2 (C-5); MS: Calcd for [C14H17NO3Na]: m/z 270.1106 [M + Na]+; Found [M + Na]+ 270.1106.
6-Chlorohexyl 4-((5-cyanopentyl)oxy)benzoate (24). A solution of 22 (509 mg, 2.06 mmol) in dioxane/H2O (3:1 v/v, 40 mL) was treated with solid NaOH (329 mg, 8.23 mmol) and stirred for 3 h. After completed conversion of the starting material, the reaction mixture was neutralised by addition of acidic ion exchange resin (Amberlite® IR-120H+ washed with H2O), filtrated and evaporated to dryness. The resulting intermediate 23 [49] was immediately used in the next step without further purification. Following general procedure A, 23 was treated with 6-chlorohexanol (275 µL, 2.06 mmol), PPh3 (540 mg, 2.06 mmol) and DIAD (404 µL, 2.06 mmol). Purification on silica gel (cyclohexane-ethyl acetate 10:1) afforded title compound 24 (659 mg, 1.87 mmol, 90.8% over two steps) as a colourless wax. 1H-NMR (300 MHz, CDCl3) δ = 7.94 (d, 2 H, Ar), 6.86 (d, 2 H, Ar), 4.24 (t, 2 H, H-1´), 3.98 (t, 2 H, H-1), 3.52 (t, 2 H, H-6´), 2.35 (t, 2 H, H-5), 1.83-1.35 (po, 14 H, H-2´, H-3´, H-4´, H-5´, H-2, H-3, H-4); 13C-APT NMR (75.5 MHz, CDCl3) δ = 166.3 (OC = O), 162.6 (ipso), 131.5 (Ar), 122.8 (ipso), 119.5 (CN), 114.0 (Ar), 67.5 (C-1), 64.5 (C-1´), 44.9 (C-6´), 32.4, 28.6, 28.3, 26.5, 25.4, 25.3, 25.1 (C-2´, C-3´, C-4´, C-5´, C-2, C-3, C-4), 17.0 (C-5); MS: Calcd for [C19H26ClNO3Na]: m/z 374.1499 [M + Na]+; Found [M + Na]+ 374.1496.
6-Oxohexyl-4-((5-cyanopentyl)oxy)benzoate (25). Following general procedure B1, 24 (659 mg, 1.87 mmol) was reacted with NaHCO3 (630 mg, 7.50 mmol). Purification on silica gel (cyclohexane-ethyl acetate 5:1) afforded compound 25 (438 mg, 1.32 mmol, 70.6%) as a colourless oil. 1H-NMR (300 MHz, CDCl3) δ = 9.77 (s, 1 H, HC = O), 7.97 (d, 2 H, Ar), 6.89 (d, 2 H, Ar), 4.28 (t, 2 H, H-1´), 4.02 (t, 2 H, H-1), 2.47 (m, 2 H, H-5´), 2.39 (t, 2 H, H-5), 1.89–1.40 (po, 12 H, H-2´, H-3´, H-4´, H-2, H-3, H-4); 13C-NMR (75.5 MHz, CDCl3) δ = 202.5 (HC = O), 166.5 (OC = O), 162.8 (ipso), 131.7 (Ar), 122.9 (ipso), 119.6 (CN), 114.2 (Ar), 67.6 (C-1), 64.5 (C-1´), 43.9 (C-5´), 28.7, 28.5, 25.8, 25.5, 25.3, 21.8 (C-2´, C-3´, C-4´, C-2, C-3, C-4), 17.3 (C-5); MS: Calcd for [C19H25NO4Na]: m/z 354.1681 [M + Na]+; Found [M + Na]+ 354.1666.
6-Bromohexyl 4-((tetrahydro-2H-pyran-2-yl)oxy)benzoate (27). Following general procedure A, compound 26 [54 (982 mg, 4.42 mmol) was treated with 6-bromohexanol (578 µL, 4.42 mmol), PPh3 (1.16 g, 4.42 mmol) and DIAD (868 µL, 4.42 mmol). Purification on silica gel (cyclohexane-ethyl acetate 20:1) provided compound 27 (1.45 g, 3.76 mmol, 85.1%) as a colourless syrup. 1H-NMR (300 MHz, CDCl3) δ = 7.97 (d, 2 H, Ar), 7.06 (d, 2 H, Ar), 5.50 (dd, 1 H, THP), 4.28 (t, 2 H, H-1´), 3.85 (m, 1 H, THP), 3.61 (m, 1 H, THP), 3.41 (t, 2 H, H-6´), 2.06-1.37 (po, 14 H, H-2´, H-3´, H-4´, H-5´, 3× CH2-THP); 13C-NMR (75.5 MHz, CDCl3) δ = 166.5 (OC = O), 161.0 (ipso), 131.5 (Ar), 123.6 (ipso), 116.0 (Ar), 96.2 (THP), 64.6 (C-1´), 62.2 (THP), 33.8 (C-6´), 32.8, 30.3, 28.8, 28.0, 25.4, 25.2 (C-2´, C-3´, C-4´, C-5´, 2× THP), 18.7 (THP); MS: Calcd for [C18H25BrO4Na]: m/z 407.0834 [M + Na]+; Found [M + Na]+ 407.0840.
6-Bromohexyl 4-hydroxybenzoate (28). A solution of 27 (1.05 g, 2.73 mmol) in 40 mL dioxane/H2O (3:1 v/v) was treated with acidic ion exchange resin (Amberlite® IR-120H+ washed with H2O) at 45 °C. After completed conversion of the starting material, the reaction mixture was filtered and the solvents were removed under reduced pressure. Purification on silica gel (cyclohexane-ethyl acetate 10:1) provided known compound 28 [55] (622 mg, 2.07 mmol, 75.8%) as a colourless wax. 1H-NMR (300 MHz, CDCl3) δ = 7.94 (d, 2 H, Ar), 7.76 (s, 1 H, OH), 6.92 (d, 2 H, Ar), 4.30 (t, 2 H, H-1´), 3.41 (t, 2 H, H-6´), 1.84-1.72 (m, 2 H, H-5´), 1.71-1.59 (m, 2 H, H-2´), 1.54-1.34 (po, 4 H, H-3´, H-4´); 13C-NMR (75.5 MHz, CDCl3) δ = 167.6 (OC = O), 161.0 (ipso), 132.0 (Ar), 122.0 (ipso), 115.5 (Ar), 65.1 (C-1´), 33.8 (C-6´), 32.7 (C-5´), 28.6 (C-2´), 27.9, 25.3 (C-3´, C-4´); MS: Calcd for [C13H17BrO3Na]: m/z 323.0259 [M + Na]+; Found [M + Na]+ 323.0258.
6-Bromohexyl 4-(2-(2-azidoethoxy)ethoxy)benzoate (29). According to general procedure A, compound 28 (290 mg, 0.962 mmol) was reacted with 2-(2-azidoethoxy)ethanol [56] (126 mg, 0.962 mmol), PPh3 (252 mg, 0.962 mmol) and DIAD (189 µL, 0.962 mmol). Silica gel chromatography (cyclohexane-ethyl acetate 20:1) of the resulting mixture gave structure 29 (325 mg, 0.784 mmol, 81.5%) as a colourless syrup. 1H-NMR (300 MHz, CDCl3) δ = 7.98 (d, 2 H, Ar), 6.94 (d, 2 H, Ar), 4.28 (t, 2 H, H-1´), 4.19 (m, 2 H, H-1), 3.89 (m, 2 H, H-2), 3.75 (m, 2 H, H-3), 3.41 (po, 4 H, H-6´, H-4), 1.87 (m, 2 H, H-5´), 1.78 (m, 2 H, H-2´), 1.55-1.40 (po, 4 H, H-3´, H-4´); 13C-NMR (75.5 MHz, CDCl3) δ = 166.5 (OC = O), 162.6 (ipso), 131.7 (Ar), 123.3 (ipso), 114.3 (Ar), 70.5 (C-3), 69.7 (C-2), 67.7 (C-1), 64.7 (C-1´), 50.8 (C-4), 33.8 (C-6´), 32.8 (C-5´), 28.8 (C-2´), 28.0, 25.4 (C-3´, C-4´); MS: Calcd for [C17H24BrN3O4Na]: m/z 436.0848 [M + Na]+; Found [M + Na]+ 436.0860.
6-Oxohexyl 4-(2-(2-azidoethoxy)ethoxy)benzoate (30). Following general procedure B1, 29 (223 mg, 0.538 mmol) was reacted with NaHCO3 (226 mg, 2.69 mmol). Purification on silica gel (cyclohexane-ethyl acetate 5:1) afforded compound 30 (161 mg, 0.461 mmol, 85.7%) as a pale yellow syrup. 1H-NMR (300 MHz, CDCl3) δ = 9.75 (s, 1 H, HC = O), 7.96 (d, 2 H, Ar), 6.92 (d, 2 H, Ar), 4.25 (t, 2 H, H-1´), 4.17 (m, 2 H, H-1), 3.87 (m, 2 H, H-2), 3.73 (m, 2 H, H-3), 3.40 (m, 2 H, H-4), 2.45 (m, 2 H, H-5´), 1.82-1.63 (po, 4 H, H-2´, H-4´), 1.46 (m, 2 H, H-3´); 13C-NMR (75.5 MHz, CDCl3) δ = 202.4 (HC = O), 166.4 (OC = O), 162.5 (ipso), 131.6 (Ar), 123.1 (ipso), 114.3 (Ar), 70.4 (C-3), 69.6 (C-2), 67.6 (C-1), 64.4 (C-1´), 50.8 (C-4), 43.8 (C-5´), 28.7 (C-2´), 25.8, 21.8 (C-3´, C-4´); MS: Calcd for [C17H23N3O5Na]: m/z 372.1535 [M + Na]+; Found [M + Na]+ 372.1557.
6-Chlorohexyl 4-((tetrahydro-2H-pyran-2-yl)oxy)benzoate (31). According to general procedure A, carboxylic acid 26 [54] (305 mg, 1.37 mmol) was reacted with 6-chlorohexanol (183 µL, 1.37 mmol), PPh3 (359 mg, 1.37 mmol) and DIAD (269 µL, 1.37 mmol). Silica gel chromatography (cyclohexane-ethyl acetate 13:1) afforded structure 31 (240 mg, 0.704 mmol, 51.4%) as a colourless syrup. 1H-NMR (300 MHz, CDCl3) δ = 7.97 (d, 2 H, Ar), 7.06 (d, 2 H, Ar), 5.49 (dd, 1 H, THP), 4.28 (t, 2 H, H-1´), 3.84 (m, 1 H, THP), 3.59 (m, 1 H, THP), 3.52 (t, 2 H, H-6´), 2.10-1.40 (po, 14 H, H-2´, H-3´, H-4´, H-5´, 3x CH2-THP); 13C-NMR (75.5 MHz, CDCl3) δ = 166.4 (OC = O), 160.9 (ipso), 131.5 (Ar), 123.6 (ipso), 115.9 (Ar), 96.1 (THP), 64.6 (C-1´), 62.1 (THP), 45.0 (C-6´), 32.6, 30.2, 28.7, 26.6, 25.5, 25.2 (C-2´, C-3´, C-4´, C-5´, 2x THP), 18.6 (THP); MS: Calcd for [C18H25ClO4Na]: m/z 363.1339 [M + Na]+; Found [M + Na]+ 363.1337.
6-Chlorohexyl 4-hydroxybenzoate (32). A stirred solution of 31 (102 mg, 0.299 mmol) in dioxane/H2O (1:1 v/v, 6 mL) was treated with acidic ion exchange resin (Amberlite® IR-120H+ washed with H2O) at 40 °C. After completed removal of the THP group was detected, the reaction mixture was filtered off and the solvents were removed in vacuo. Purification on silica gel (cyclohexane-ethyl acetate 10:1) afforded 32 (73.7 mg, 0.287 mmol, 96.0%) as a colourless wax. 1H-NMR (300 MHz, CDCl3) δ = 7.95 (d, 2 H, Ar), 6.89 (d, 2 H, Ar), 6.52 (s, 1 H, OH), 4.30 (t, 2 H, H-1´), 3.54 (t, 2 H, H-6´), 1.79 (po, 4 H, H-2´, H-5´) 1.49 (po, 4 H, H-3´, H-4´); 13C-NMR (75.5 MHz, CDCl3) δ = 167.2 (OC = O), 160.4 (ipso), 132.1 (Ar), 122.7 (ipso), 115.4 (Ar), 65.0 (C-1´), 45.1 (C-6´), 32.6 (C-5´), 28.7 (C-2´), 26.7, 25.5 (C-3´, C-4´); MS: Calcd for [C13H17ClO3Na]: m/z 279.0764 [M + Na]+; Found [M + Na]+ 279.0765.
6-Chlorohexyl 4-(propargyloxy)benzoate (33). To a stirred solution of 32 (1.33 g, 5.18 mmol) in acetone (40 mL), K2CO3 (2.86 g, 20.7 mmol) and 3-bromoprop-1-yne (669 µL, 6.21 mmol, 80% in toluene) were added. After completed conversion of the starting material, the reaction mixture was filtrated, diluted with CH2Cl2 and washed consecutively with HCl (2 N) and saturated aqueous NaHCO3. After drying over Na2SO4, the filtrate was evaporated to dryness. Purification on silica gel (cyclohexane-ethyl acetate 15:1) provided compound 33 (1.25 g, 4.24 mmol, 81.9%) as a colourless syrup. 1H-NMR (300 MHz, CDCl3) δ = 8.00 (d, 2 H, Ar), 6.99 (d, 2 H, Ar), 4.74 (d, 2 H, H-1), 4.29 (t, 2 H, H-1´), 3.54 (t, 2 H, H-6´), 2.55 (s, 1 H, H-3), 1.77 (po, 4 H, H-2´, H-5´) 1.49 (po, 4 H, H-3´, H-4´); 13C-NMR (75.5 MHz, CDCl3) δ = 166.3 (OC = O), 161.3 (ipso), 131.6 (Ar), 123.9 (ipso), 114.6 (Ar), 78.0 (C-2), 76.2 (C-3), 64.7 (C-1´), 56.0 (C-1), 45.1 (C-6´), 32.6 (C-5´), 28.8 (C-2´), 26.7, 25.6 (C-3´, C-4´); MS: Calcd for [C16H19ClO3Na]: m/z 317.0920 [M + Na]+; Found [M + Na]+ 317.0922.
6-Hydroxyhexyl 4-(propargyloxy)benzoate (34). Following general procedure B2, halocarbon 33 (451 mg, 1.53 mmol) was treated with NaHCO3 (514 mg, 6.12 mmol). Separation on silica gel (cyclohexane-ethyl acetate 10:1) provided alcohol 34 (322 mg, 1.17 mmol, 76.5%) as a colourless oil. 1H-NMR (300 MHz, CDCl3) δ = 7.98 (d, 2 H, Ar), 6.97 (d, 2 H, Ar), 4.71 (d, 2 H, H-1), 4.26 (t, 2 H, H-1´), 3.61 (t, 2 H, H-6´), 2.55 (s, 1 H, H-3), 2.00 (s, 1 H, OH), 1.74 (m, 2 H, H-2´), 1.55 (m, 2 H, H-5´), 1.42 (po, 4 H, H-3´, H-4´); 13C-NMR (75.5 MHz, CDCl3) δ = 166.4 (OC = O), 161.2 (ipso), 131.6 (Ar), 123.8 (ipso), 114.5 (Ar), 77.9 (C-2), 76.2 (C-3), 64.8 (C-1´), 62.7 (C-6´), 55.9 (C-1), 32.6 (C-5´), 28.8 (C-2´), 25.9, 25.5 (C-3´, C-4´); MS: Calcd for [C16H20O4Na]: m/z 299.1259 [M + Na]+; Found [M + Na]+ 299.1259.
6-Oxohexyl 4-(propargyloxy)benzoate (35). According to general procedure C, alcohol 34 (355 mg, 1.28 mmol) was oxidised employing Dess- Martin periodinane (598 mg, 1.41 mmol). Purification on silica gel (cyclohexane-ethyl acetate 10:1) realised aldehyde 35 (243 mg, 0.886 mmol, 69.2%) as amorphous solid. Recrystallisation from cyclohexane/ethyl acetate afforded colourless crystals (CCDC 2021385, see SM Figure S1). m.p.: 64-66 °C; 1H-NMR (300 MHz, CDCl3) δ = 9.76 (s, 1 H, HC = O), 7.99 (d, 2 H, Ar), 6.99 (d, 2 H, Ar), 4.73 (d, 2 H, H-1), 4.28 (t, 2 H, H-1´), 2.54 (s, 1 H, H-3), 2.46 (m, 2 H, H-5´), 1.80-1.65 (po, 4 H, H-2´, H-4´), 1.47 (m, 2 H, H-3´); 13C-NMR (75.5 MHz, CDCl3) δ = 202.4 (HC = O), 166.3 (OC = O), 161.3 (ipso), 131.6 (Ar), 123.8 (ipso), 114.6 (Ar), 77.9 (C-2), 76.2 (C-3), 64.5 (C-1´), 55.9 (C-1), 43.8 (C-5´), 28.7 (C-2´), 25.8 (C-3´), 21.8 (C-4´); MS: Calcd for [C16H18O4Na]: m/z 297.1103 [M + Na]+; Found [M + Na]+ 297.1104.
Methyl 4-(2-(2-((nonafluoro-tert-butyl)oxy)ethoxy)ethoxy)benzoate (37). Following general procedure A, compound 36 [59] (1.30 g, 5.41 mmol) was treated with nonafluoro-tert-butyl alcohol (754 µL, 5.41 mmol), PPh3 (1.42 g, 5.41 mmol) and DIAD (1.06 mL, 5.41 mmol). Purification on silica gel (cyclohexane-ethyl acetate 20:1) provided compound 37 (1.92 g, 4.19 mmol, 77.4%) as a colourless syrup. 1H-NMR (300 MHz, CDCl3) δ = 7.93 (d, 2 H, Ar), 6.86 (d, 2 H, Ar), 4.12 (po, 2 H, H-1), 4.08 (po, 2 H, H-4), 3.82 (po, 5 H, H-2, OCH3), 3.74 (m, 2 H, H-3); 13C-NMR (75.5 MHz, CDCl3) δ = 166.8 (OC = O), 162.6 (ipso), 131.6 (Ar), 122.9 (ipso), 120.4 (q, 3 C, JC,F 292.5 Hz, CF3), 114.1 (Ar), 79.9 (q, 1 C, JC,F 29.7 Hz, C(CF3)3), 69.9, 69.7, 69.4, 67.6 (C-1, C-2, C-3, C-4), 51.6 (OCH3).
6-Hydroxyhexyl 4-(2-(2-((nonafluoro-tert-butyl)oxy)ethoxy)ethoxy)benzoate (40). A solution of 37 (700 mg, 1.53 mmol) in 40 mL dioxane/H2O (3:1 v/v) was treated with solid NaOH (306 mg, 7.64 mmol) and stirred until completed conversion of the starting material was detected. After neutralisation with acidic ion exchange resin (Amberlite® IR-120H+ washed with H2O), the reaction mixture was filtrated and evaporated to dryness providing intermediate 38. This compound was directly converted to halocarbon 39 following general procedure A employing 6-bromohexanol (200 µL, 1.53 mmol), PPh3 (401 mg, 1.53 mmol) and DIAD (300 µL, 1.53 mmol). Due to problems during the separation of the resulting reaction mixture, crude product 39 was immediately hydrolysed according to general procedure B2 employing NaHCO3 (514 mg, 6.12 mmol). Purification on silica gel (cyclohexane-ethyl acetate 10:1) provided alcohol 40 (412 mg, 0.757 mmol, 49.5% over 3 steps) as a colourless oil. 1H-NMR (300 MHz, CDCl3) δ = 7.98 (d, 2 H, Ar), 6.92 (d, 2 H, Ar), 4.28 (t, 2 H, H-1´), 4.16 (po, 4 H, H-1, H-4), 3.89 (m, 2 H, H-2), 3.80 (m, 2 H, H-3), 3.64 (t, 2 H, H-6´), 1.94 (bs, 1 H, OH), 1.76 (m, 2 H, H-2´), 1.59 (m, 2 H, H-5´), 1.45 (po, 4 H, H-3´, H-4´); 13C-NMR (75.5 MHz, CDCl3) δ = 166.6 (OC = O), 162.6 (ipso), 131.7 (Ar), 123.3 (ipso), 120.3 (q, 3 C, JC,F 293.0 Hz, CF3), 114.3 (Ar), 79.8 (q, 1 C, JC,F 28.9 Hz, C(CF3)3), 70.1 (C-3), 69.8 (C-2), 69.5, 67.7 (C-1, C-4), 64.8 (C-1´), 62.9 (C-6´), 32.8 (C-5´), 28.9 (C-2´), 26.0, 25.6 (C-3´, C-4´).
6-Oxohexyl 4-(2-(2-((nonafluoro-tert-butyl)oxy)ethoxy)ethoxy)benzoate (41). Following general procedure C, alcohol 40 (91.0 mg, 0.167 mmol) was treated with Dess-Martin periodinane (78.0 mg, 0.184 mmol). Purification on silica gel (cyclohexane-ethyl acetate 15:1) afforded aldehyde 41 (76.2 mg, 0.140 mmol, 83.8%) as a colourless oil. 1H-NMR (300 MHz, CDCl3) δ = 9.75 (s, 1 H, HC = O), 7.96 (d, 2 H, Ar), 6.91 (d, 2 H, Ar), 4.27 (t, 2 H, H-1´), 4.15 (po, 4 H, H-1, H-4), 3.88 (m, 2 H, H-2), 3.79 (m, 2 H, H-3), 2.45 (m, 2 H, H-5´), 1.83-1.63 (po, 4 H, H-2´, H-4´) 1.46 (m, 2 H, H-3´); 13C-NMR (75.5 MHz, CDCl3) δ = 202.4 (HC = O), 166.4 (OC = O), 162.6 (ipso), 131.6 (Ar), 123.2 (ipso), 120.4 (q, 3 C, JC,F 292.6 Hz, CF3), 114.3 (Ar), 79.9 (q, 1 C, JC,F 29.7 Hz, C(CF3)3), 70.1 (C-3), 69.8 (C-2), 69.5, 67.7 (C-1, C-4), 64.5 (C-1´), 43.8 (C-5´), 28.7 (C-2´), 25.8 (C-3´), 21.8 (C-4´).
6-Bromohexyl 4-(2-(((benzyloxy)carbonyl)amino)ethyl)benzoate (43). According to general procedure A, literature known compound 42 [60] (585 mg, 1.96 mmol) was treated with 6-bromohexanol (256 µl, 1.96 mmol), PPh3 (514 mg, 1.96 mmol) and DIAD (385 µl, 1.96 mmol). Silica gel chromatography (cyclohexane-ethyl acetate 13:1) provided structure 43 (615 mg, 1.33 mmol, 67.9%) as a slightly yellow syrup. 1H-NMR (300 MHz, CDCl3) δ = 7.89 (d, 2 H, Ar), 7.26 (s, 5 H, NH-COOCH2Ph), 7.17 (d, 2 H, Ar), 5.02 (s, 2 H, NH-COOCH2Ph), 4.72 (bs, 1 H, NH), 4.24 (t, 2 H, H-1´), 3.37 (po, 4 H, H-2, H-6´), 2.80 (m, 2 H, H-1), 1.80 (m, 2 H, H-5´), 1.69 (m, 2 H, H-2´), 1.38 (po, 4 H, H-3´, H-4´); 13C-NMR (75.5 MHz, CDCl3) δ = 166.6 (OC = O), 156.4 (NH-COOCH2Ph), 144.2 (ipso), 136. 6 (ipso), 130.0-128.2 (Ar), 66.9 (NH-COOCH2Ph), 64.9 (C-1´), 42.0 (C-2), 36.3 (C-1), 33.8 (C-6´), 32.8 (C-5´), 28.7 (C-2´), 28.0, 25.4 (C-3´, C-4´); MS: Calcd for [C23H28BrNO4Na]: m/z 484.1099 [M + Na]+; Found [M + Na]+ 484.1098.
6-Oxohexyl 4-(2-(((benzyloxy)carbonyl)amino)ethyl)benzoate (44). Following general procedure B1, halocarbon 43 (798 mg, 1.73 mmol) was treated with NaHCO3 (874 mg, 10.4 mmol). Purification on silicagel (cyclohexane-ethyl acetate 8:1) gave aldehyde 44 (335 mg, 0.843 mmol, 48.7%) as a colourless oil. 1H-NMR (300 MHz, CDCl3) δ = 9.70 (s, 1 H, HC = O), 7.89 (d, 2 H, Ar), 7.27 (s, 5 H, NH-COOCH2Ph), 7.18 (d, 2 H, Ar), 5.02 (s, 2 H, NH-COOCH2Ph), 4.71 (bs, 1 H, NH), 4.24 (t, 2 H, H-1´), 3.40 (m, 2 H, H-2), 2.81 (m, 2 H, H-1), 2.40 (t, 2 H, H-5´), 1.75-1.57 (po, 4 H, H-2´, H-4´), 1.41 (m, 2 H, H-3´); 13C-NMR (75.5 MHz, CDCl3) δ = 202.4 (HC = O), 166.6 (OC = O), 156.4 (NH-COOCH2Ph), 144.3 (ipso), 136.6 (ipso), 130.0-128.2 (Ar), 66.9 (NH-COOCH2Ph), 64.7 (C-1´), 43.9 (C-5´), 42.0 (C-2), 36.3 (C-1), 28.7 (C-2´), 25.8 (C-3´), 21.8 (C-4´); MS: Calcd for [C23H27NO5Na]: m/z 420.1787 [M + Na]+; Found [M + Na]+ 420.1783.
N-(6-((4-((5-Cyanopentyl)oxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-glucitol (45). A solution of aldehyde 25 (60.2 mg 0.182 mmol), AcOH (25 µL) and 1-deoxynojirimycin (1 [61], 38.6 mg, 0.237 mmol) in 3 mL MeOH was stirred with Pd/C (10%) under an atmosphere of H2 at ambient pressure until completed conversion of the starting material. After removal of the catalyst, the filtrate was concentrated under reduced pressure. Purification on silica gel (ethyl acetate-MeOH 8:1) provided compound 45 (55.1 mg, 0.115 mmol, 63.2%) as amorphous white solid. [ a ] D 20 : −5.4 (c = 1.22, MeOH); 1H-NMR (300 MHz, CD3OD) δ = 7.91 (d, 2 H, Ar), 6.94 (d, 2 H, Ar), 4.25 (t, 2 H, H-6´), 4.02 (t, 2 H, H-1´´), 3.89 (m, 2 H, H-6a, H-6b), 3.55 (ddd, 1 H, J1a,2 4.9 Hz, J1b,2 9.8 Hz, J2,3 8.7 Hz, H-2), 3.44 (dd, 1 H, J4,5 9.3 Hz, H-4), 3.18 (po, 2 H, H-1a, H-3), 3.01 (m, 1 H, H-1´), 2.82 (m, 1 H, H-1´), 2.46 (po, 4 H, H-1b, H-5, H-5´´), 1.87-1.29 (po, 14 H, H-2´, H-3´, H-4´, H-5´, H-2´´, H-3´´, H-4´´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 164.5 (ipso), 132.5 (Ar), 123.6 (ipso), 121.1 (CN), 115.3 (Ar), 79.5 (C-3), 70.7 (C-4), 69.5 (C-2), 68.9 (C-1´´), 67.4 (C-5), 65.7 (C-6´), 57.7 (C-6), 56.4 (C-1), 53.8 (C-1´), 29.7, 29.4, 27.8, 26.9, 26.3, 26.2, 24.7 (C-2´, C-3´, C-4´, C-5´, C-2´´, C-3´´, C-4´´), 17.3 (C-5´´); MS: Calcd for [C25H38N2O7Na]: m/z 501.2577 [M + Na]+; Found [M + Na]+ 501.2578.
N-(6-((4-((5-Cyanopentyl)oxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-xylitol (46). Method A: Following general procedure D, aldehyde 25 (30.5 mg, 92.0 µmol) was treated with 1,5-imino-1,5-dideoxy-d-xylitol (2 [62], 13.4 mg, 0.101 mmol) and NaBH3CN (17.3 mg, 0.276 mmol). Purification on silica gel (ethyl acetate-MeOH 8:1) provided compound 46 (29.3 mg, 65.3 µmol, 71.0%) as a white solid. 1H-NMR (300 MHz, CD3OD) δ = 7.96 (d, 2 H, Ar), 6.99 (d, 2 H, Ar), 4.29 (t, 2 H, H-6´), 4.08 (m, 2 H, H-1´´), 3.50 (ddd, 2 H, J1a/5a,2/4 4.5 Hz, J1b/5b,2/4 10.0 Hz J2/4,3 8.9 Hz, H-2/4), 3.11 (dd, 1 H, H-3), 2.99 (dd, 2 H, J1a/1b,5a/5b 10.6 Hz H-1a/5a), 2.46 (po, 4 H, H-1´, H-5´´), 1.94 (dd, 2 H, H-1b/5b), 1.88-1.36 (po, 14 H, H-2´, H-3´, H-4´, H-5´, H-2´´, H-3´´, H-4´´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 164.5 (ipso), 132.5 (Ar), 123.7 (ipso), 121.1 (CN), 115.3 (Ar), 80.3 (C-3), 71.4 (C-2/4), 68.9 (C-1´´), 65.8 (C-6´), 59.4 (C-1/5), 58.9 (C-1´), 29.8, 29.4, 28.1, 27.6, 27.0, 26.4, 26.3, (C-2´, C-3´, C-4´, C-5´, C-2´´, C-3´´, C-4´´), 17.3 (C-5´´); MS: Calcd for [C24H36N2O6H]: m/z 449.2652 [M + H]+; Found [M + H]+ 449.2649.
Method B: A solution of aldehyde 25 (51.0 mg 0.154 mmol), AcOH (20 µL) and iminosugar 2 (24.6 mg, 0.185 mmol) in 2.5 mL MeOH was stirred with Pd/C (10%) under an atmosphere of H2 at ambient pressure. After completed conversion of the starting material, the reaction mixture was filtrated and concentrated under reduced pressure. Purification on silica gel (ethyl acetate-MeOH 8:1) gave compound 46 (33.0 mg, 73.6 µmol, 47.8%) as a white solid.
N-(6-((4-(2-(2-Azidoethoxy)ethoxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-glucitol (47). Following general procedure D, aldehyde 30 (98.0 mg, 0.280 mmol) was treated with iminosugar 1 (54.8 mg, 0.336 mmol) and NaBH3CN (35.2 mg, 0.560 mmol). Purification on silica gel (ethyl acetate-MeOH 8:1) provided compound 47 (78.1 mg, 0.157 mmol, 56.1%) as a white solid. [ a ] D 20 : -3.4 (c = 1.47, MeOH); 1H-NMR (300 MHz, CD3OD) δ = 7.98 (d, 2 H, Ar), 7.04 (d, 2 H, Ar), 4.30 (t, 2H, H-6´), 4.23 (m, 2 H, H-1´´), 3.89 (po, 4 H, H-2´´,H-6a, H-6b), 3.76 (m, 2 H, H-3´´), 3.52 (ddd, 1 H, J1a,2 4.7 Hz, J1b,2 10.2 Hz, H-2), 3.40 (po, 3 H, H-4, H-4´´), 3.19 (dd, 1 H, J2,3 = J3,4 9.1 Hz, H-3), 3.08 (dd, 1 H, H-1a), 2.91 (m, 1 H, H-1´), 2.70 (m, 1 H, H-1´), 2.32 (po, 2 H, H-1b, H-5), 1.80 (m, 2 H, H-5´), 1.68-1.34 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 164.3 (ipso), 132.5 (Ar), 123.9 (ipso), 115.4 (Ar), 80.2 (C-3), 71.6 (C-4), 71.4 (C-3´´), 70.6 (C-2´´), 70.3 (C-2), 68.9 (C-1´´), 67.4 (C-5), 65.8 (C-6´), 58.9 (C-6), 57.3 (C-1), 53.7 (C-1´), 51.8 (C-4´´), 29.8 (C-5´), 28.1, 27.0, 25.1 (C-2´, C-3´, C-4´); MS: Calcd for [C23H36N4O8Na]: m/z 519.2431 [M + Na]+; Found [M + Na]+ 519.2431.
N-(6-((4-(2-(2-Azidoethoxy)ethoxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-xylitol (48). According to general procedure D, aldehyde 30 (25.0 mg, 71.6 µmol) was treated with iminosugar 2 (11.4 mg, 85.9 µmol) and NaBH3CN (13.5 mg, 0.215 mmol). Purification on silica gel (ethyl acetate-MeOH 8:1) afforded xylitol derivative 48 (18.1 mg, 38.8 µmol, 54.2%) as a white solid. 1H-NMR (300 MHz, CD3OD) δ = 8.01 (d, 2 H, Ar), 7.05 (d, 2 H, Ar), 4.31 (t, 2 H, H-6´), 4.24 (m, 2 H, H-1´´), 3.90 (m, 2 H, H-2´´), 3.77 (m, 2 H, H-3´´), 3.53 (ddd, 2 H, J2/4,1a/5a 9.6 Hz, J2/4,1b/5b 3.3 Hz, J2/4,3 8.5 Hz, H-2/4), 3.42 (m, 2 H, H-4´´), 3.15 (dd, 1 H, H-3), 3.03 (dd, 2 H, J1a/5a,1b/5b 10.9 Hz, H-1a/5a), 2.51 (m, 2 H, H-1´), 2.04 (dd, 2 H, H-1b/5b), 1.81 (m, 2 H, H-5´), 1.66-1.37 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 164.3 (ipso), 132.5 (Ar), 124.0 (ipso), 115.4 (Ar), 79.1 (C-3), 71.4 (C-3´´), 71.2 (C-2/4), 70.6 (C-2´´), 68.9 (C-1´´), 65.8 (C-6´), 59.1 (C-1/5), 58.8 (C-1´), 51.8 (C-4´´), 29.8 (C-5´), 28.1, 27.4, 27.0 (C-2´, C-3´, C-4´); MS: Calcd for [C22H34N4O7H]: m/z 467.2506 [M + H]+; Found [M + H]+ 467.2507.
N-(6-((4-(2-(2-(Nonafluoro-tert-butyloxy)ethoxy)ethoxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-glucitol (49). Following general procedure D, aldehyde 41 (52.0 mg, 95.9 µmol) was treated with iminosugar 1 (18.8 mg, 0.115 mmol) and NaBH3CN (18.1 mg, 0.288 mmol). Purification on silica gel (ethyl acetate-MeOH 8:1) gave structure 49 (34.3 mg, 49.7 µmol, 51.8%) as a white solid. [ a ] D 20 : -4.4 (c = 1.17, MeOH); 1H-NMR (300 MHz, CD3OD) δ = 7.96 (d, 2 H, Ar), 7.03 (d, 2 H, Ar), 4.31 (m, 2 H, H-6´), 4.22 (po, 4 H, H-1´´, H-4´´), 3.85 (po, 6 H, H-2´´, H-3´´, H-6a, H-6b), 3.51 (ddd, 1 H, J1a, 2 4.8 Hz, J1b,2 10.1 Hz, H-2), 3.38 (dd, 1 H, J3,4 = J4,5 9.2Hz, H-4), 3.17 (dd, 1 H, J2,3 9 Hz, H-3), 3.05 (dd, 1 H, H-1), 2.88 (m, 1 H, H-1´), 2.67 (m, 1 H, H-1´), 2.25 (po, 2 H, H-1b, H-5), 1.80 (m, 2 H, H-5´), 1.66-1.34 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 164.3 (ipso), 132.5 (Ar), 124.0 (ipso), 123.7, 121.8 (q, 3 C, JC,F 291.9 Hz, CF3), 115.4 (Ar), 80.4 (C-3), 71.8 (C-4), 71.2, 70.9, 70.7, 68.9 (C-1´´, C-2´´, C-3´´, C-4´´), 70.5 (C-2), 67.4 (C-5), 65.8 (C-6´), 59.2 (C-6), 57.5 (C-1), 53.7 (C-1´), 29.8 (C-5´), 28.2, 27.0, 25.1 (C-2´, C-3´, C-4´); MS: Calcd for [C27H36F9NO9H]: m/z 690.2325 [M + H]+; Found [M + H]+ 690.2316.
N-(6-((4-(2-(2-(Nonafluoro-tert-butyloxy)ethoxy)ethoxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-xylitol (50). Following general procedure D, aldehyde 41 (43.0 mg, 79.3 µmol) was treated with iminosugar 2 (11.6 mg, 95.2 µmol) and NaBH3CN (15.0 mg, 0.238 mmol). Purification on silica gel (ethyl acetate-MeOH 8:1) provided compound 50 (32.4 mg, 49.2 µmol, 62.0%) as a white solid.1H-NMR (300 MHz, CD3OD) δ = 7.97 (d, 2 H, Ar), 7.02 (d, 2 H, Ar), 4.31 (t, 2H, H-6´), 4.22 (po, 4 H, H-1´´, H-4´´), 3.87 (t, 2 H, H-2´´), 3.82 (t, 2 H, H-3´´), 3.56 (ddd, 2 H, J1a/5a,2 3.5 Hz, J1b/5b,2 9.1 Hz, J2/4,3 8.4 Hz, H-2/4), 3.21 (dd, 1 H, H-3), 3.07 (dd, 2 H, J1a/5a,1b/5b 10.9 Hz, H-1a/5a), 2.60 (m, 2 H, H-1´), 2.18 (dd, 2 H, H-1b/5b), 1.80 (m, 2 H, H-5´), 1.68-1.35 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 164.3 (ipso), 132.5 (Ar), 124.0 (ipso), 121.8 (q, 3 C, JC,F 290.9 Hz, CF3), 115.4 (Ar), 79.1 (C-3), 71.1, 70.9, 70.9, 70.7, 68.9 (C-2/4, C-1´´, C-2´´, C-3´´, C-4´´), 65.8 (C-6´), 58.7, 58.6 (C-1/5, C-1´), 29.8 (C-5´), 28.0, 27.1, 26.9 (C-2´, C-3´, C-4´); MS: Calcd for [C26H34F9NO8Na]: m/z 682.2039 [M + Na]+; Found [M + Na]+ 682.2039.
N-(6-((4-(Propargyloxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-glucitol (51). Following general procedure D, aldehyde 35 (62.6 mg 0.228 mmol) was treated with iminosugar 1 (37.2 mg, 0.228 mmol) and NaBH3CN (21.5 mg, 0.342 mmol). Silica gel chromatography (ethyl acetate-MeOH 7:1) gave structure 51 (57.4 mg, 0.136 mmol, 59.6%) as a white solid. [ a ] D 20 : -6.0 (c = 1.08, MeOH); 1H-NMR (300 MHz, CD3OD) δ = 7.98 (d, 2 H, Ar), 7.07 (d, 2 H, Ar), 4.82 (m, 2 H, H-1´´), 4.30 (m, 2 H, H-6´), 3.88 (m, 2 H, H-6a, H-6b), 3.52 (ddd, 1 H, J1a,2 4.9 Hz, J1b,2 9.7 Hz, H-2), 3.39 (dd, 1 H, J3,4 = J4,5 9.3 Hz, H-4), 3.18 (dd, 1 H, J2,3 9.0 Hz, H-3), 3.11 (dd, 1 H, J1a,1b 11.3 Hz, J1a,2 4.9 Hz, H-1a), 3.02-2.90 (m, 2 H, H-1´, H-3´´), 2.72 (m, 1 H, H-1´), 2.35 (po, 2 H, H-1b, H-5), 1.78 (m, 2 H, H-5´), 1.69-1.32 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 167.9 (OC = O), 163.0 (ipso), 132.4 (Ar), 124.5 (ipso), 115.8 (Ar), 80.0 (C-3), 79.1 (C-2´´), 77.3 (C-3´´), 71.3 (C-4), 70.1 (C-2), 67.4 (C-5), 65.8 (C-6´), 58.5 (C-6), 57.0 (C-1), 56.8 (C-1´´), 53.7 (C-1´), 29.8 (C-5´), 28.0, 27.0, 25.0 (C-2´, C-3´, C-4´); MS: Calcd for [C22H31NO7H]: m/z 422.2179 [M + H]+; Found [M + H]+ 422.2179.
N-(6-((4-(Propargyloxy)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-xylitol (52). Following general procedure D, aldehyde 35 (104 mg, 0.379 mmol) was treated with iminosugar 2 (50.5 mg, 0.379 mmol) and NaBH3CN (35.8 mg, 0.569 mmol). Purification on silica gel (ethyl acetate-MeOH 10:1) afforded compound 52 (52.4 mg, 0.134 mmol, 35.4%) as a white solid.1H-NMR (300 MHz, CD3OD) δ = 7.99 (d, 2 H, Ar), 7.08 (d, 2 H, Ar), 4.83 (m, 2 H, H-1´´), 4.30 (t, 2 H, H-6´), 3.54 (m, 2 H, H-2/4), 3.16 (dd, 1 H, J2/4,3 8.1 Hz, H-3), 3.02 (po, 3 H, H-1a/5a, H-3´´), 2.48 (m, 2 H, H-1´), 2.02 (m, 2 H, H-1b/5b), 1.79 (m, 2 H, H-5´), 1.66-1.33 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 167.8 (OC = O), 163.0 (ipso), 132.4 (Ar), 124.5 (ipso), 115.8 (Ar), 79.8 (C-3), 79.2 (C-2´´), 77.3 (C-3´´), 71.1 (C-2/4), 65.8 (C-6´), 59.1 (C-1/5), 58.8 (C-1´), 56.8 (C-1´´), 29.7 (C-5´), 28.1, 27.4, 27.0 (C-2´, C-3´, C-4´); MS: Calcd for [C21H29NO6Na]: m/z 414.1893 [M + Na]+; Found [M + Na]+ 414.1894.
N-(6-((4-(2-(((Benzyloxy)carbonyl)amino)ethyl)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-glucitol (53). Following general procedure D, aldehyde 44 (92.2 mg, 0.232 mmol) was treated with iminosugar 1 (37.9 mg, 0.232 mmol) and NaBH3CN (21.9 mg, 0.348 mmol). Purification on silica gel (ethyl acetate-MeOH 10:1) provided compound 53 (69.8 mg, 0.128 mmol, 55.2%) as a white solid. [ a ] D 20 : -5.0 (c = 1.04, MeOH); 1H-NMR (300 MHz, CD3OD) δ = 7.87 (d, 2 H, Ar), 7.27 (m, 7 H, Ar), 4.97 (s, 2 H, NH-COOCH2Ph), 4.27 (t, 2 H, H-6´), 3.93 (dd, 1 H, J5,6a 2.5 Hz, J6a,6b 12.3 Hz, H-6a,), 3.82 (dd, 1 H, J5,6b 2.5 Hz, H-6b), 3.56 (ddd, 1 H, J1a,2 3.9 Hz, J2,3 9.9 Hz, H-2), 3.47 (dd, 1 H, J3,4 = J4,5 9.5 Hz, H-4), 3.27 (po, 4 H, H-1a, H-3, H-1´´), 3.07 (m, 1 H, H-1´a), 2.90 (m, 1 H, H-1´b), 2.81 (m, 2 H, H-2´´), 2.62 (po, 2 H, H-1b, H-5), 1.73 (m, 2 H, H-5´), 1.64 (m, 2 H, H-2´), 1.55–1.29 (po, 4 H, H-3´,H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.1 (OC = O), 158.8 (NH-COOCH2Ph), 146.4 (ipso), 138.4 (ipso), 130.6-128.7 (Ar), 79.1 (C-3), 70.1 (C-4), 69.0 (C-2), 67.4 (C-5), 67.3 (NH-COOCH2Ph), 65.9 (C-6´), 57.0 (C-6), 55.9 (C-1), 53.8 (C-1´), 42.9 (C-1´´), 37.0 (C-2´´), 29.7 (C-5´), 27.7, 26.8, (C-3´, C-4´), 24.5 (C-5´); MS: Calcd for [C29H40N2O8Na]: m/z 567.2682 [M + Na]+; Found [M + Na]+ 567.2681.
N-(6-((4-(2-(((Benzyloxy)carbonyl)amino)ethyl)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-D-xylitol (54). Following general procedure D, aldehyde 44 (88.3 mg, 0.222 mmol) was treated with iminosugar 2 (29.6 mg, 0.222 mmol) and NaBH3CN (20.9 mg, 0.333 mmol). Silica gel chromatography (ethyl acetate-MeOH 7:1) provided compound 54 (30.6 mg, 59.5 µmol, 26.8%) as a white solid.1H-NMR (300 MHz, CD3OD) δ = 7.94 (d, 2 H, Ar), 7.33 (m, 7 H, Ar), 5.06 (s, 2 H, NH-COOCH2Ph), 4.32 (m, 2 H, H-6´), 3.51 (m, 2 H, J1a/5a,2/4 4.4 Hz, J1b/5b,2/4 9.8 Hz, J2/4,3 8.9 Hz, H-2/4), 3.38 (m, 2 H, H-1´´), 3.10 (dd, 1 H, H-3), 2.99 (m, 2 H, J1a/5a,1b/5b 10.7 Hz, H-1a/5a), 2.87 (m, 2 H, H-2´´), 2.43 (m, 2 H, H-1´), 1.94 (dd, 2 H, H-1b/5b), 1.80 (m, 2 H, H-5´), 1.63-1.35 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.1 (OC = O), 158.8 (NH-COOCH2Ph), 146.4 (ipso), 138.5 (ipso), 130.6-128.7 (Ar), 80.4 (C-3), 71.4 (C-2/4), 67.3 (NH-COOCH2Ph), 66.0 (C-6´), 59.4 (C-1/5), 58.8 (C-1´), 42.9 (C-1´´), 37.0 (C-2´´), 29.7 (C-5´), 28.1, 27.6, 27.0 (C-2´, C-3´, C-4´); MS: Calcd for [C28H38N2O7Na]: m/z 537.2577 [M + Na]+; Found [M + Na]+ 537.2578.
N-(6-((4-(2-(Dansylamino)ethyl)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-glucitol (55). A 10% solution of N-Cbz protected compound 53 (84.9 mg, 0.156 mmol) in MeOH containing AcOH (20 µL) was stirred with Pd/C (10%) under an atmosphere of H2 at ambient pressure until completed liberation of the corresponding amine was detected. After removal of the catalyst, the filtrate was immediately treated with Et3N (100 µL) and dansyl chloride (46.4 mg, 0.172 mmol). Additional stirring for 90 min, evaporation of the solvents followed by purification on silica gel (ethyl acetate-MeOH 3:1) provided title compound 55 (50.2 mg, 78.0 µmol, 50.0% over 2 steps) as a slightly yellow wax. [ a ] D 20 : -5.6 (c = 1.08, MeOH); 1H-NMR (300 MHz, CD3OD) δ = 8.53 (d, 1 H, dansyl), 8.19 (m, 2 H, dansyl), 7.67 (d, 2 H, Ar), 7.52 (m, 2 H, dansyl), 7.20 (d, 1 H, dansyl), 7.00 (d, 2 H, Ar), 4.29 (t, 2H, H-6´), 3.88 (m, 2 H, H-6a, H-6b), 3.51 (ddd, 1 H, J1b,2 10.0 Hz, J2,3 9.3 Hz, H-2), 3.35 (m, 1 H, H-4), 3.16 (po, 3 H, H-3, H-1´´), 3.01 (dd, 1 H, J1a,1b 11.0 Hz, J1b,2 4.7 Hz, H-1a), 2.84 (po, 7 H, H-1´, dansyl), 2.65 (po, 3 H, H-1´, H-2´´), 2.16 (po, 2 H, H-1b, H-5), 1.76 (m, 2 H, H-5´), 1.63-1.31 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 153.1-116.3 (dansyl, Ar), 80.5 (C-3), 72.0 (C-4), 70.7 (C-2), 67.4 (C-5), 66.0 (C-6´), 59.4 (C-6), 57.6 (C-1), 53.7 (C-1´), 45.8 (dansyl), 44.9 (C-1´´), 36.7 (C-2´´), 29.8 (C-5´), 28.2, 27.1, 25.2 (C-2´, C-3´, C-4´); MS: Calcd for [C33H45N3O8SNa]: m/z 666.2825 [M + Na]+; Found [M + Na]+ 666.2825.
N-(6-((4-(2-(Dansylamino)ethyl)benzoyl)oxy)hexyl)-1,5-dideoxy-1,5-imino-d-xylitol (56). A 10% solution of N-Cbz protected compound 54 (54.0 mg, 0.105 mmol) in MeOH containing AcOH (20 µL) was stirred with Pd/C (10%) under an atmosphere of H2 at ambient pressure until completed liberation of the corresponding amine was detected. After removal of the catalyst, the filtrate was immediately treated with Et3N (100 µL) and dansyl chloride (31.3 mg, 0.116 mmol). Additional stirring for 90 min, evaporation of the solvents followed by purification on silica gel (ethyl acetate-MeOH 3:1) provided title compound 56 (28.7 mg, 46.8 µmol, 44.6% over 2 steps) as a slightly yellow wax. 1H-NMR (500 MHz, CD3OD) δ = 8.54 (d, 1 H, dansyl), 8.19 (m, 2 H, dansyl), 7.67 (d, 2 H, Ar), 7.52 (m, 2 H, dansyl), 7.23 (d, 1 H, dansyl), 7.00 (d, 2 H, Ar), 4.35 (t, 2 H, H-6´), 3.55 (ddd, 2 H, J1a/5a,2/4 4.8 Hz, J1b/5b,2/4 9.7 Hz, H-2/4), 3.24 (m, 2 H, H-1´´), 3.15 (dd, 1 H, J2/4,3 8.8 Hz, H-3), 3.03 (dd, 2 H, J1a/5a,1b/5b 10.8 Hz, H-1b/5b), 2.94 (s, 6 H, dansyl), 2.73 (m, 2 H, H-2´´), 2.47 (m, 2 H, H-1´), 1.97 (m, 2 H, H-1b, H-5b), 1.84 (m, 2 H, H-5´), 1.63-1.36 (po, 6 H, H-2´, H-3´, H-4´); 13C-NMR (75.5 MHz, CD3OD) δ = 168.0 (OC = O), 153.1-116.3 (dansyl, Ar), 80.5 (C-3), 71.4 (C-2/4), 66.0 (C-6´), 59.5 (C-1/5), 58.9 (C-1´), 45.8 (dansyl), 44.9 (C-1´´), 36.7 (C-2´´), 29.8 (C-5´), 28.2, 27.7, 27.0 (C-2´, C-3´, C-4´); MS: Calcd for [C32H43N3O7SNa]: m/z 636.2720 [M + Na]+; Found [M + Na]+ 636.2720.

3.3. Kinetic Studies

Kinetic studies were performed at room temperature in an appropriate buffer for each enzyme (specific conditions can be found below). All the reactions were performed in half-area 96-well-plates (Corning, Corning, NY, USA) and monitored with a Synergy H1 plate reader (BioTek Instruments, Winooski, VT, USA). In each experiment, the appropriate concentration of the enzyme was incubated with different concentrations of the inhibitors for 2–5 min before initiating the reaction by the addition of substrate. The initial rate was then measured by monitoring the increase in absorbance as a result of the release of 4-nitrophenol at 405 nm for up to five minutes. Ki determinations were performed using two different substrate concentrations. For each substrate concentration, a range of three to six inhibitor concentrations was used. Dixon plots (1/v vs. [I]) were constructed to validate the use of competitive inhibition model and to assess the fit of the data. The data were then fit to a competitive inhibition model using non-linear regression analysis with Grafit 7.0.3 (Erithacus Software, East Grinstead, UK).

Specific Assay Conditions for Each Enzyme:

Agrobacterium sp. β-glucosidase (Abg): [64,65] 50 mM sodium phosphate buffer (pH 7). Substrate: pNP β-Gal, Km = 4.1 mM.
E. coli lac z β-galactosidase: 50 mM sodium phosphate, 1.0 mM MgCl2 (pH 7). Substrate: pNP β-Gal, Km = 60 µM.
Bovine liver β-galactosidase: 50 mM sodium phosphate buffer (pH 7). Substrate: pNP β-Gal, Km = 0.65 mM.
Fabrazyme (Acid α-galactosidase): 20 mM sodium citrate, 50 mM sodium phosphate, 1.0 mM tetrasodium EDTA, 0.25% v/v Triton X-100® and 0.25% w/v taurocholic acid buffer (pH 5.5). Substrate: 2,4-DNP α-Gal, Km = 0.65 mM.
S. cerevisiae α-Glucosidase: 50 mM sodium phosphate buffer (pH 7.0). Substrate: pNP α-Glc, Km = 0.75 mM.
GCase (β-glucocerebrosidase): 20 mM sodium citrate, 50 mM sodium phosphate, 1.0 mM tetrasodium EDTA, 0.25% v/v Triton X-100® and 0.25% w/v taurocholic acid buffer (pH 7). Substrate: 2,4-DNP β-Glc, Km = 2.7 mM.

4. Conclusions

We have developed a robust and flexible conceptual synthetic protocol towards N-alkylated iminosugar based inhibitors for glycoside hydrolases and have probed this concept on D-gluco (45, 47, 49, 51, 53, 55) and d-xylo (46, 48, 50, 52, 54, 56) configured iminosugar scaffolds. The sequence of the composition of the different building blocks allows for flexibility in choosing the spacer length and terminal tag on the non iminosugar hemisphere of the compound. Furthermore, we introduced different terminal tags such as nitrile, azide, alkyne, nonafluoro-tert-butyl and amino substituents, which allows for simple follow-up chemistry customised for different applications such as orthogonal labelling with fluorescent dyes as reporter groups or ligation reactions. The biological evaluation with a set of different glycoside hydrolases showed that all synthesised compounds proved to bind tightly to GCase with Ki values in the low micro and nanomolar range. Most of them exhibit also good selectivities, thereby clearly underlining the potential of this compound class to be used as tools and therapeutics in the context of human lysosomal β-glucocerebrosidase. Potential applications are their use as enzyme inhibitors, pharmacological chaperons and active site directed ligands for enzyme labelling.

Supplementary Materials

The following are available online, NMR spectra for new compounds. Figure S1: Structure of compound 35 confirmed by XRD analysis (CCDC 2021385).

Author Contributions

A.W. and M.S. have performed the syntheses, M.T. and P.W. contribute and assisted with the development and the syntheses; R.F. provided crystal structure for compound 35; S.A.N. performed biochemical experiments; A.E.S. conceived the synthetic experiments; S.G.W. supervised and evaluated biochemical studies; T.M.W. supervised the study. All authors have read and agreed to the published version of the manuscript.

Funding

Financial support by the Austrian Science Funds FWF (P30372-B21). Support from NAWI Graz is gratefully acknowledged (A.W., M.S., M.T., T.M.W). S.G.W thanks GlycoNet, the Canadian Network of Centres of Excellence in glycoscience, for financial support. P.W. is recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Institute of Chemistry and Technology of Biobased Systems, Graz University of Technology.

Acknowledgments

We thank T. Chalupka and F. Bobner for contributing to the synthesis of spacer building blocks during their Bachelor Thesis.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
Molecules 25 04618 g001 550
Figure 1. Paradigmatic structural examples of selected iminosugars 1–6.
Figure 1. Paradigmatic structural examples of selected iminosugars 1–6.
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Molecules 25 04618 g002 550
Figure 2. Selected structures of iminosugar based glycomimetics.
Figure 2. Selected structures of iminosugar based glycomimetics.
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Figure 3. Selected structures of N-alkylated iminosugars.
Figure 3. Selected structures of N-alkylated iminosugars.
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Figure 4. Building block concept for iminosugar-based ligands (R = CH2OH or H).
Figure 4. Building block concept for iminosugar-based ligands (R = CH2OH or H).
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Molecules 25 04618 sch001 550
Scheme 1. Reagents and Conditions: (a) 6-Bromohexanenitrile, K2CO3, acetone, 86%; (b) NaOH, dioxane/H2O; (c) 6-chlorohexanol, PPh3, DIAD, THF, 91% over 2 steps; (d) NaHCO3, DMSO, 120 °C, 71%.
Scheme 1. Reagents and Conditions: (a) 6-Bromohexanenitrile, K2CO3, acetone, 86%; (b) NaOH, dioxane/H2O; (c) 6-chlorohexanol, PPh3, DIAD, THF, 91% over 2 steps; (d) NaHCO3, DMSO, 120 °C, 71%.
Molecules 25 04618 sch001
Molecules 25 04618 sch002 550
Scheme 2. Reagents and Conditions: (a) 6-Bromohexanol, PPh3, DIAD, THF, 85%; (b) Amberlite® IR-120H+, dioxane/H2O, 45 °C, 76%; (c) N3(CH2CH2O)2H, PPh3, DIAD, THF, 82%; (d) NaHCO3, DMSO, 120 °C, 86%.
Scheme 2. Reagents and Conditions: (a) 6-Bromohexanol, PPh3, DIAD, THF, 85%; (b) Amberlite® IR-120H+, dioxane/H2O, 45 °C, 76%; (c) N3(CH2CH2O)2H, PPh3, DIAD, THF, 82%; (d) NaHCO3, DMSO, 120 °C, 86%.
Molecules 25 04618 sch002
Molecules 25 04618 sch003 550
Scheme 3. Reagents and Conditions: (a) 6-Chlorohexanol, PPh3, DIAD, THF, 51%; (b) Amberlite® IR-120H+, dioxane/H2O, 40 °C, 96%; (c) 3-bromoprop-1-yne, K2CO3, acetone, 82%; (d) NaHCO3, DMSO/H2O, 100 °C, 77%; (e) DMP, CH2Cl2, 69%.
Scheme 3. Reagents and Conditions: (a) 6-Chlorohexanol, PPh3, DIAD, THF, 51%; (b) Amberlite® IR-120H+, dioxane/H2O, 40 °C, 96%; (c) 3-bromoprop-1-yne, K2CO3, acetone, 82%; (d) NaHCO3, DMSO/H2O, 100 °C, 77%; (e) DMP, CH2Cl2, 69%.
Molecules 25 04618 sch003
Molecules 25 04618 sch004 550
Scheme 4. Reagents and Conditions: (a) (CF3)3COH, PPh3, DIAD, THF, 77%; (b) NaOH, dioxane/H2O; (c) 6-bromohexanol, PPh3, DIAD, THF; (d) NaHCO3, DMSO/H2O, 100 °C, 49% over 3 steps; (e) DMP, CH2Cl2, 84%.
Scheme 4. Reagents and Conditions: (a) (CF3)3COH, PPh3, DIAD, THF, 77%; (b) NaOH, dioxane/H2O; (c) 6-bromohexanol, PPh3, DIAD, THF; (d) NaHCO3, DMSO/H2O, 100 °C, 49% over 3 steps; (e) DMP, CH2Cl2, 84%.
Molecules 25 04618 sch004
Molecules 25 04618 sch005 550
Scheme 5. Reagents and Conditions: (a) 6-Bromohexanol, PPh3, DIAD, THF, 68%; (b) NaHCO3, DMSO, 120 °C, 49%.
Scheme 5. Reagents and Conditions: (a) 6-Bromohexanol, PPh3, DIAD, THF, 68%; (b) NaHCO3, DMSO, 120 °C, 49%.
Molecules 25 04618 sch005
Molecules 25 04618 sch006 550
Scheme 6. Reagents and Conditions: Overview of various reductive amination reactions and respective conditions: (a) Pd/C (10%), H2-atm., AcOH cat., MeOH; (b) NaBH3CN, AcOH cat., MeOH.
Scheme 6. Reagents and Conditions: Overview of various reductive amination reactions and respective conditions: (a) Pd/C (10%), H2-atm., AcOH cat., MeOH; (b) NaBH3CN, AcOH cat., MeOH.
Molecules 25 04618 sch006
Molecules 25 04618 sch007 550
Scheme 7. Reagents and Conditions: (a) Pd (10%), H2-atm., MeOH, AcOH then dansyl chloride, Et3N, MeOH (50% for 55; 45% for 56, over two steps).
Scheme 7. Reagents and Conditions: (a) Pd (10%), H2-atm., MeOH, AcOH then dansyl chloride, Et3N, MeOH (50% for 55; 45% for 56, over two steps).
Molecules 25 04618 sch007
Table
Table 1. Ki-values [µM] of compounds 45–56 with Abg = β-glucosidase/β-galactosidase from Agrobacterium sp.; E. coli = lac Z β-galactosidase from E. coli; Bovine liv. = β-galactosidase from bovine liver; Fabrazyme = commercial recombinant human lysosomal α-galactosidase; S. cer. = α-glucosidase from Saccharomyces cerevisiae; GCase = recombinant human lysosomal β-glucocerebrosidase; N.I. = no or weak inhibition, with Ki > 1 mM;
Table 1. Ki-values [µM] of compounds 45–56 with Abg = β-glucosidase/β-galactosidase from Agrobacterium sp.; E. coli = lac Z β-galactosidase from E. coli; Bovine liv. = β-galactosidase from bovine liver; Fabrazyme = commercial recombinant human lysosomal α-galactosidase; S. cer. = α-glucosidase from Saccharomyces cerevisiae; GCase = recombinant human lysosomal β-glucocerebrosidase; N.I. = no or weak inhibition, with Ki > 1 mM;
Compound Enzyme (GH Family)
Molecules 25 04618 i001
β-Glc/Galβ-Galβ-Galα-Galα-Glcβ-Glc
R =Config.Nr.Abg (GH1)E. coli (GH2)Bovine liv. (GH35)Fabrazyme (GH27)S. cer. (GH13)GCase (GH30)
Molecules 25 04618 i002Glc451.31N.I.2.42N.I.23.70.28
Xyl4612.10N.I.7.76N.I.N.I.0.62
Molecules 25 04618 i003Glc471.70N.I.1.74N.I.2210.09
Xyl4832.6N.I.11.76N.I.N.I.1.40
Molecules 25 04618 i004Glc493.60N.I.6.94N.I.N.I.0.35
Xyl5023.702558.36N.I.N.I.0.40
Molecules 25 04618 i005Glc510.0022N.I.3.25N.I.4.90.37
Xyl5217N.I.7.41N.I.2670.74
Molecules 25 04618 i006Glc530.064242.13N.I.8.10.022
Xyl540.58N.I.4.65N.I.N.I.0.31
Molecules 25 04618 i007Glc550.06891.82N.I.8.70.018
Xyl560.7619810.36N.I.N.I.2.60

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Wolfsgruber, A.; Thonhofer, M.; Weber, P.; Nasseri, S.A.; Fischer, R.; Schalli, M.; Stütz, A.E.; Withers, S.G.; Wrodnigg, T.M. N-Alkylated Iminosugar Based Ligands: Synthesis and Inhibition of Human Lysosomal β-Glucocerebrosidase. Molecules 2020, 25, 4618. https://doi.org/10.3390/molecules25204618

AMA Style

Wolfsgruber A, Thonhofer M, Weber P, Nasseri SA, Fischer R, Schalli M, Stütz AE, Withers SG, Wrodnigg TM. N-Alkylated Iminosugar Based Ligands: Synthesis and Inhibition of Human Lysosomal β-Glucocerebrosidase. Molecules. 2020; 25(20):4618. https://doi.org/10.3390/molecules25204618

Chicago/Turabian Style

Wolfsgruber, Andreas, Martin Thonhofer, Patrick Weber, Seyed A. Nasseri, Roland Fischer, Michael Schalli, Arnold E. Stütz, Stephen G. Withers, and Tanja M. Wrodnigg. 2020. "N-Alkylated Iminosugar Based Ligands: Synthesis and Inhibition of Human Lysosomal β-Glucocerebrosidase" Molecules 25, no. 20: 4618. https://doi.org/10.3390/molecules25204618

APA Style

Wolfsgruber, A., Thonhofer, M., Weber, P., Nasseri, S. A., Fischer, R., Schalli, M., Stütz, A. E., Withers, S. G., & Wrodnigg, T. M. (2020). N-Alkylated Iminosugar Based Ligands: Synthesis and Inhibition of Human Lysosomal β-Glucocerebrosidase. Molecules, 25(20), 4618. https://doi.org/10.3390/molecules25204618

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