A Versatile Method of Tethering Biomolecules to Pyrrole Precursors for Functionalized Magnetic Polypyrrole Core-Shell Nanoparticles

Abstract

A mild and versatile method based on copper-catalyzed [3+2] cycloaddition (Meldal-Sharpless reaction) was developed to tether biomolecules, such as monosaccharides, biotin, cholesterol, or uridine to the N-atom of pyrrole. The required azido and alkyne functions can be placed in either reactant, i.e. in the pyrrole or the biomolecule. The products are interesting precursors for functionalized superparamagnetic polypyrrole core-shell nanoparticles.

Superparamagnetic nanoparticles, in particular iron(II,III) oxide (Fe₃O₄) nanoparticles, have gained wide interest in biological and medical applications, such as in cancer treatment by hyperthermia, as contrast reagents in diagnosis, and in the separation of biomaterials. [¹-8] In all of these applications the nanoparticles are exposed to body fluids and it is desirable that they are inert under these conditions and preferably locate at the target tissues. The stability against the biological environment can be improved by covering Fe₃O₄ nanoparticles with polymer shells leading to so-called core-shell nanoparticles. [²] [4] [7] [9] Tethering biological molecules to the polymer shell can make the core-shell nanoparticles more tolerable to the biological system and can help to target locations of interest if the biomolecules contain recognition functions. [4] [6] [7] Several polymers, e.g. polyvinyl alcohol, polyvinyl acetate, polyethylene­glycol (PEG), dextran, or silica have been utilized for this purpose. Polypyrroles (PPy) have rarely been used in this field although they are known to be biocompatible [¹0] [¹¹] and can form proper shells with Fe₃O₄ nanoparticles and, thus, protect them very well. [¹²-¹4] There are only a few reports where PPy-Fe₃O₄ core-shell nanoparticles were obtained in which the polypyrrole shell is decorated with biomolecules. [¹5] In these cases, amino acids or folic acid were tethered to pyrrole rings by EDC coupling.

We became interested in how far the popular and widely used copper-catalyzed [3+2] cycloaddition of azides and alkynes to give 1,2,3-triazoles (Meldal-Sharpless ‘click’ reaction) can be applied for tethering biomolecules to pyrroles, which can later be used for the preparation of functionalized shells of magnetic core-shell Fe₃O₄ nanoparticles. As compared with other tethering tools, the Meldal-Sharpless cycloaddition method tolerates many functional groups allowing the omission of protective groups and, thus, it has also been widely applied for biological molecules. So far, the copper-catalyzed [3+2] cycloaddition of azides with alkynes has been employed in PPy chemistry only once when 1-decylpyrroles with a terminal azido or propargyl ether moiety were electro-polymerized. The resulting PPy electrode surfaces were further functionalized by click reactions with histidine tags and biomolecules. [¹6]

We demonstrate here that the Meldal-Sharpless reaction gives straightforward access to pyrrole monomers equipped with carbohydrate, nucleoside, biotin, and steroid moieties and we provide proof that the products can, in principle, be used for the preparation of functionalized magnetic core-shell Fe₃O₄ nanoparticles.

To maintain the capability of pyrrole monomers to undergo proper oxidative polymerization to polypyrroles, positions 2 and 5 of the pyrrole ring must be free, i.e. positions 3 and 4 and the ring nitrogen atom are available as tethering sites for biomolecules. Since the introduction of functional groups into positions 3 and 4 can suffer from the formation of regioisomers (competing formation of 2- or 5-substituted pyrroles) the nitrogen atom of the pyrrole ring is often the favored site for the attachment of substituents and, thus, it was also chosen in the synthesis presented here. Either functionality, azido or alkyne moiety, necessary for Meldal-Sharpless cycloaddition could be introduced at the pyrrole nitrogen atom. Thus 1-(6-bromohexyl)-1H-pyrrole (1) was transformed into the corresponding hitherto unknown azide 2 by nucleophilic substitution with sodium azide in acetonitrile. On the other hand 3-(1H-pyrrol-1-yl)propanoic acid (5), [¹7] which is easily available via addition of pyrrole to acrylonitrile and subsequent basic hydrolysis, was transformed into the N-propargylamide 6. Surprisingly, the very versatile alkyne 6 is previously unknown; it can be expected to play an important role in the chemistry of functionalized pyrroles in the future. The biomolecules containing reactants 3 and 7 are known and all of them were easy to attain.

The [3+2]-cycloaddition reactions (Schemes  [¹] and  [²] , Table  [¹] ) were performed by applying established procedures with copper sulfate as catalyst in the presence of sodium ascorbate (Method A). High yields of products 4 and 8 were achieved in the majority of cases after long reaction times (several days). Sometimes, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine [¹8] [¹9] (TBTA) had to be used as a ligand for copper (Method B) in order to accelerate the reaction and to obtain reasonable yields in acceptable reaction times (within hours). However, this procedure has the disadvantage of causing difficulties during the separation of the polar [3+2]-cycloaddition product from the ligand (see product 4b).

The 1,2,3-triazole-biomolecule pyrrole conjugates 4 and 8 appeared as colorless, stable products that are solids in most cases. Their structures were confirmed by spectroscopic methods. In addition to the signals of the pyrrole, the biomolecule, and the linker moieties, diagnostic signals for the 1,2,3-triazole ring were found in the NMR spectra [δ = 7.4-7.9 (^¹H NMR), δ = 121-124 and 144-146 (^¹³C NMR)]. For later applications, e.g. as a recognition function, it is essential that the biomolecule moiety in the polypyrrole copolymer is unprotected. Thus, the acetyl groups found in the sugar units of 4d, 8b, and 8c, remaining from the preparation of the starting 1-azido saccharides, had to be removed. This could be achieved by transesterification with methanol in the presence of sodium methoxide providing the unprotected glycosides 9 in high yields (Scheme  [³] ).

Table 1 [3+2]-Cycloaddition Products 4 and 8

Product		Methoda	Time	Yieldb (%)
4a		A B	2 d 3 h	71 88
4b		A B	5 d 3 h	52 35
4c		A B	2 d 2 h	45 77
4d		A B	2 d 3 h	65 81
8a		B	1 h	87
8b		A B	4 d 3 h	99 92
8c		A B	9 d 3 h	68 88
a Method A: CuSO4˙5 H2O (cat.), Na ascorbate, H2O-t-BuOH or H2O-THF, r.t., 2-9 d; Method B: CuSO4˙5 H2O (cat.), Na ascorbate, TBTA, H2O-t-BuOH or H2O-THF, r.t., 1-3 h. b Yield of isolated product.

From our results it can be concluded that the copper-catalyzed azide-alkyne cycloaddition is a versatile method for tethering biomolecules to pyrrole that is likely to be successful also with other, more complex biological structures. As proof of the principle, the cholesterol-functionalized pyrrole 4a was applied to the preparation of functionalized magnetic core-shell nanoparticles. Pyrrole 4a was copolymerized with unsubstituted pyrrole (4a/pyrrole, 1:2) by oxidation with ammonium persulfate in the presence of a water-based magnetic Fe₃O₄-nanofluid stabilized by a double layer of lauric acid (Scheme  [4] ). The envisaged functionalized core-shell nanoparticles 10 were obtained as a black powder after magnetic separation and drying. The FT-IR spectrum clearly shows the incorporation of the cholesterol moiety (bands at 2931 and 2853 cm^-¹ ascribed to CH₂ and CH₃ asymmetric stretching) [²0] [²¹] in addition to the typical polypyrrole bands at 1598-1681 cm^-¹ (collective mode of intra- and inter-ring vibration) and the strong Fe₃O₄ band at 592 cm^-¹ (Figure  [¹] ).

This example demonstrates that pyrroles, wherein the tethering position of biomolecules is the ring N-atom and the tether unit contains a 1,2,3-triazole are suitable for the preparation of functionalized magnetic core-shell nanoparticles. We presently apply the method to other functionalized pyrrole monomers and approach investigations of the resulting materials in biological systems, such in therapy and diagnosis or in the separation of biological materials.

3-(1H-Pyrrol-1-yl)propanoic acid (5), [¹7] N,N,N-tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), [¹8] [¹9] and the biomolecules 3 [cholesterol propargyl ether (3a), [²²] [²³] N-(prop-2-ynyl)biotinamide (3b), [²4] 2,3,4,6-tetra-O-acetyl-1-(prop-2-ynyl)-β-d-glucose (3c), [²5] [²6] 2,3,4,6-tetra-O-acetyl-1-(prop-2-ynyl)-β-d-galactose (3d) [²7] ] and 7 [2′-azido-2′-deoxyuridine (7a), [²8] 2,3,4,6-tetra-O-acetyl-1-azido-1-deoxy-β-d-glucose (7b), [²9] 1-azido-2,3,4,6-tetra-O-acetyl-1-deoxy-β-d-galactose (7c) [²9] ] were prepared by the described procedures and all analytical data of the products matched with those reported in the literature. Other starting materials were purchased from Aldrich­ and Acros. TLC analysis was performed on Merck silica gel 60 F254 plates and visualized by UV illumination and by charring with phosphomolybdic acid, KMnO₄, or ninhydrin. Silica gel 60 (0.035-0.070 mm, Acros) was used for preparative column chromatography. Melting points were determined on a Boetius hot-stage apparatus and are uncorrected. ^¹H and ^¹³C NMR spectra were recorded at 300 and 75.5 MHz, respectively, on a Bruker AC-300 with TMS as internal standard. Elemental analyses were ascertained with a Euro EA analyzer. HRMS (ESI) were measured with a Thermo Finnigan LTQ-FT-ICR-MS with MeOH as solvent. Optical rotations were determined with a Perkin-Elmer-241 polarimeter. FT-IR spectra were obtained with a Jasco FTIR 610 spectrophotometer.

1-(6-Bromohexyl)-1 H -pyrrole (1)

A 50% aq NaOH soln (25 mL) was added to a vigorously stirred soln of pyrrole (671 mg, 10.0 mmol) and Bu₄NHSO₄ (340 mg, 1.0 mmol) in CH₂Cl₂ (30 mL) at 0 ˚C. After a few min, 1,6-dibromohexane (6.10 mmol, 25.0 mmol) was added dropwise and the resultant soln was stirred at 0 ˚C for 30 min. After this time the reaction was allowed to come up to r.t. and stirred for 21 h. To this mixture was added 1 M HCl (30 mL) and CH₂Cl₂ (20 mL) and the organic layer was separated and extracted with CH₂Cl₂ (2 × 15 mL). The combined organic layers were washed with sat. aq NaHCO₃ (2 × 30 mL), H₂O (30 mL), and brine (30 mL), dried (MgSO₄), and evaporated under reduced pressure. The crude product was purified by flash column chromatography (silica gel) to give 1 (1.38 g, 6.0 mmol, 60%) as a red oil; R _f  = 0.35 (cyclohexane-EtOAc, 95:5).

^¹H NMR (CDCl₃): δ = 1.30-1.40 (m, 2 H, CH₂), 1.45-1.55 (m, 2 H, CH₂), 1.78-1.95 (m, 4 H, 2 CH₂), 3.43 (t, J = 6.8 Hz, 2 H, CH₂Br), 3.91 (t, J = 7.1 Hz, 2 H, N_py-CH₂), 6.18 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.68 (t, J = 2.1 Hz, 2 H, 2 CH_py).

^¹³C NMR (CDCl₃): δ = 25.9 (CH₂), 27.8 (CH₂), 31.4 (CH₂), 32.6 (CH₂), 33.8 (CH₂Br), 49.5 (N_py-CH₂), 108.0 (2 CH_py), 120.5 (2 CH_py).

1-(6-Azidohexyl)-1 H -pyrrole (2)

NaN₃ (452 mg, 6.95 mmol) was added to a soln of 1 (1.07 g, 4.63 mmol) in MeCN (12 mL). The mixture was heated to reflux for 21 h. The soln was allowed to cool to r.t. and H₂O (100 mL) was added. The aqueous layer was extracted with CH₂Cl₂ (3 × 100 mL) and the combined organic layers were dried (Na₂SO₄) and evaporated under reduced pressure. Purification by flash column chromatography (silica gel) gave 2 (879 mg, 4.57 mmol, 99%) as a clear yellow oil; R _f  = 0.40 (cyclohexane-EtOAc, 9:1).

^¹H NMR (CDCl₃): δ = 1.30-1.48 (m, 4 H, 2 CH₂), 1.55-1.68 (m, 2 H, CH₂), 1.77-1.87 (m, 2 H, CH₂), 3.28 (t, J = 6.8 Hz, 2 H, CH₂N₃), 3.90 (t, J = 7.1 Hz, 2 H, N_py-CH₂), 6.17 (t, J = 1.9 Hz, 2 H, 2 CH_py), 6.68 (t, J = 2.1 Hz, 2 H, 2 CH_py).

^¹³C NMR (CDCl₃): δ = 26.4 (2 CH₂), 28.8 (CH₂), 31.5 (CH₂), 49.5 (N_py-CH₂), 51.4 (CH₂N₃), 107.9 (2 CH_py), 120.5 (2 CH_py).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₀H₁₇N₄: 193.1448; found: 193.1457.

N -(Prop-2-ynyl)-3-(1 H -pyrrol-1-yl)propanamide (6)

To a stirred soln of 3-(1H-pyrrol-1-yl)propanic acid (5, 1.39 g, 10.0 mmol) and propargylamine (606 mg, 11.0 mmol) in anhyd CH₂Cl₂ (100 mL) was added 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) (2.02 g, 13.0 mmol), HOBt (2.03 g, 15.0 mmol), and DIPEA (1.42 g, 11.0 mmol) under argon at r.t. The mixture was stirred 19 h and then diluted with the same amount of CH₂Cl₂. The organic layer was separated and washed with 3 M HCl (2 × 50 mL) and H₂O (2 × 65 mL), dried (MgSO₄), and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel) to give 6 (1.58 g, 8.97 mmol, 90%) as an ivory-colored solid; mp 64-65 ˚C; R _f  = 0.47 (CH₂Cl₂ -MeOH, 95:5).

^¹H NMR (CDCl₃): δ = 2.21 (t, J = 2.6 Hz, 1 H, CH₂C≡CH), 2.59 (t, J = 6.7 Hz, 2 H, CH₂CO), 3.97 (dd, J ₁ = 2.6 Hz, J ₂ = 5.3 Hz, 2 H, CH ₂C≡CH), 4.21 (t, J = 6.7 Hz, 2 H, N_py-CH₂), 6.02 (br s, 1 H, CONH), 6.13 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.64 (t, J = 2.1 Hz, 2 H, 2 CH_py).

^¹³C NMR (CDCl₃): δ = 29.3 (CH₂C≡CH), 38.6 (CH₂CO), 45.4 (N_py-CH₂), 71.7 (CH₂C≡CH), 79.3 (CH₂ C≡CH), 108.5 (2 CH_py), 120.6 (2 CH_py), 170.1 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₀H₁₃N₂O: 177.1022; found: 177.1015.

Anal. Calcd for C₁₀H₁₂N₂O: C, 68.16; H, 6.86; N, 15.90. Found: C, 68.22; H, 6.96; N, 15.68.

1,3-Dipolar Cycloaddition; General Procedure

Method A: A mixture of azide 2 or 7 (1.0 equiv) and alkyne 3 or 6 (1.0 equiv) was dissolved in distilled H₂O-t-BuOH or H₂O-THF (1:1) (depending on solubility). To the stirred and degassed soln was added successively CuSO₄˙5 H₂O (0.2 equiv) and sodium ascorbate (0.4 equiv) added at r.t. The resulting soln was stirred until TLC indicated completion of the reaction. The soln was diluted with CH₂Cl₂ and the aqueous layer was extracted with CH₂Cl₂ (3 ×) The combined organic layers were washed with H₂O, dried (Na₂SO₄), and evaporated under reduced pressure. Purification by flash column chromatography (silica gel) gave the desired compounds 4a-d and 8a-c.

Method B: Analogous to Method A, but using additional TBTA (0.2 equiv).

4-[(Cholest-5-en-3β-yloxy)methyl]-1-[6-(1 H -pyrrol-1-yl)hexyl]-1 H -1,2,3-triazole (4a)

According to the general procedure, method A, using azide 2 (288 mg, 1.50 mmol), alkyne 3a (637 mg, 1.50 mmol), CuSO₄˙5 H₂O (75 mg, 0.30 mmol), and sodium ascorbate (119 mg, 0.60 mmol) in THF-H₂O (1:1, 60 mL) for 2 d; separation [CH₂Cl₂ (50 mL), CH₂Cl₂ (3 × 20 mL), H₂O (2 × 35 mL)] and column chromatography gave 4a (656 mg, 1.06 mmol, 71%).

According to the general procedure, method B, using azide 2 (288 mg, 1.50 mmol), alkyne 3a (637 mg, 1.50 mmol), CuSO₄˙5 H₂O (75 mg, 0.30 mmol), sodium ascorbate (119 mg, 0.60 mmol), and TBTA (159 mg, 0.30 mmol) in THF-H₂O (1:1, 60 mL) for 3 h; separation [CH₂Cl₂ (50 mL), CH₂Cl₂ (3 × 20 mL), H₂O (2 × 35 mL)] and column chromatography gave 4a (813 mg, 1.32 mmol, 88%).

Waxy white solid; mp 88-89 ˚C; R _f  = 0.47 (cyclohexane-EtOAc, 1:1).

[α]_D ^²² -17.1 (c 1.01, CHCl₃).

^¹H NMR (CDCl₃): δ = 0.67 (s, 3 H, CCH₃), 0.86 (dd, J ₁ = 1.3 Hz, J ₂ = 6.6 Hz, 6 H, CHCH ₃), 0.88-2.31 (m, 41 H, OCHCH ₂C, 2 CCH₃, 14 CH₂, 6 CH), 2.38 (ddd, J ₁ = 2.1 Hz, J ₂ = 4.7 Hz, J ₃ = 13.2 Hz, 1 H, OCHCH ₂C), 3.23-3.42 (m, 1 H, OCHCH₂C), 3.85 (t, J = 7.0 Hz, 2 H, N_py-CH₂), 4.30 (t, J = 7.1 Hz, 2 H, CH₂N_triazole), 4.68 (s, 2 H, OCH₂C_triazole), 5.35 (d, J = 5.2 Hz, 1 H, CCHCH₂C), 6.13 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.62 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.49 (s, 1 H, CH_triazole).

^¹³C NMR (CDCl₃): δ = 12.0 (CH₃), 18.8 (CH₃), 19.5 (CH₃), 21.2 (CH₂), 22.7 (CH₃), 22.9 (CH₃), 23.9 (CH₂), 24.4 (CH₂), 26.2 (2 CH₂), 28.1 [CH(CH₃)₂], 28.3 (CH₂), 28.4 (CH₂), 30.2 (CH₂), 31.4 (CH₂CH₂N_triazole), 32.0 (CH), 32.1 (CH₂), 35.9 (CH), 36.3 (CHCH₂), 37.0 (CCH₃), 37.3 (CH₂), 39.2 (CHCH₂), 39.6 (CH₂), 39.9 (CH₂), 42.4 (CCH₃), 49.5 (N_py-CH₂), 50.3 (CH, CH₂N_triazole), 56.3 (CH), 56.9 (CH), 61.8 (OCH₂C_triazole), 79.1 (CHOCH₂C_triazole), 108.0 (2 CH_py), 120.5 (2 CH_py), 122.0 (CCHCH₂), 122.2 (CH_triazole), 140.8 (CCHCH₂), 146.1 (C_triazole).

HRMS (ESI): m/z [M + H]⁺ calcd for C₄₀H₆₅N₄O: 617.5153; found: 617.5156.

Anal. Calcd for C₄₀H₆₄N₄O: C, 77.87; H, 10.46; N, 9.08. Found: C, 77.69; H, 10.46; N, 9.03.

N -({1-[6-(1 H -Pyrrol-1-yl)hexyl]-1 H -1,2,3-triazol-4-yl}methyl)biotinamide (4b)

According to the general procedure, method A, using azide 2 (304 mg, 1.58 mmol), alkyne 3b (445 mg, 1.58 mmol), CuSO₄˙5 H₂O (79 mg, 0.32 mmol), and sodium ascorbate (125 mg, 0.63 mmol) in t-BuOH-H₂O (1:1, 60 mL) for 5 d; separation [CH₂Cl₂ (50 mL), CH₂Cl₂ (3 × 20 mL), H₂O (2 × 35 mL)] and column chromatography gave 4b (390 mg, 0.82 mmol, 52%).

According to the general procedure, method B, using azide 2 (96 mg, 0.50 mmol), alkyne 3b (141 mg, 0.50 mmol), CuSO₄˙5 H₂O (25 mg, 0.10 mmol), sodium ascorbate (40 mg, 0.20 mmol), and TBTA (53 mg, 0.10 mmol) in t-BuOH-H₂O (1:1, 26 mL) for 3 h; separation [CH₂Cl₂ (20 mL), CH₂Cl₂ (3 × 10 mL), H₂O (2 × 10 mL)] and column chromatography gave 4b (84 mg, 0.18 mmol, 35%); complete removal of TBTA failed.

Cream-colored solid; mp 163-164 ˚C; R _f  = 0.22 (CHCl₃ -MeOH, 9:1).

^¹H NMR (CDCl₃): δ = 1.26-1.33 (m, 4 H, 2 CH₂), 1.35-1.48 (m, 2 H, CH₂), 1.56-1.78 (m, 6 H, 2 CH₂, CH ₂CH₂N_triazole), 1.79-1.90 (m, 2 H, CH_biotin-CH ₂), 2.18 (t, J = 6.4 Hz, 2 H, CH ₂CONH), 2.74 (d, J = 12.7 Hz, 1 H, CH_biotin-CH ₂S), 2.90 (dd, J ₁ = 3.5 Hz, J ₂ = 12.5 Hz, 1 H, CH_biotin-CH ₂S), 3.11 (d, J = 3.1 Hz, 1 H, CH _biotin-CH₂), 3.84 (t, J = 7.0 Hz, 2 H, N_py-CH₂), 4.26 (t, J = 7.0 Hz, 2 H, CH₂N_triazole), 4.29-4.42 (m, 2 H, NH_biotin-CHCHS, NHCH ₂C_triazole), 4.45-4.55 (m, 2 H, NH_biotin-CHCH₂S, NHCH ₂C_triazole), 6.11 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.61 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.75 (br s, 1 H, CONH), 7.28 (s, 1 H, CONH_biotin), 7.57 (s, 1 H, CH_triazole), 7.95 (s, 1 H, CONH_biotin).

^¹³C NMR (CDCl₃): δ = 25.5 (CH₂), 26.2 (2 CH₂), 28.1 (CH₂), 28.3 (CH₂), 30.1 (CH_biotin-CH₂), 31.3 (CH₂CH₂N_triazole), 34.4 (NHCH₂C_triazole), 35.9 (CH₂CONH), 40.8 (CH_biotin-CH₂S), 49.4 (N_py-CH₂), 50.3 (CH₂N_triazole), 56.0 (CH_biotin-CH₂CH₂), 60.4 (NH_biotin­-CHCH₂S), 61.8 (NH_biotin-CHCHS), 108.0 (2 CH_py), 120.5 (2 CH_py). 122.6 (CH_triazole), 145.3 (C_triazole), 164.9 (C=O), 173.6 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₃₆N₇O₂S: 474.2646; found: 474.2646.

1-[6-(1 H -Pyrrol-1-yl)hexyl]-4-[(2,3,4,6-tetra- O -acetyl-β- d -glucopyranosyloxy)methyl]-1 H -1,2,3-triazole (4c)

According to the general procedure, method A, using azide 2 (79 mg, 0.41 mmol), alkyne 3c (158 mg, 0.41 mmol), CuSO₄˙5 H₂O (20 mg, 0.08 mmol), and sodium ascorbate (32 mg, 0.16 mmol) in t-BuOH-H₂O (1:1, 25 mL) for 2 d; separation [CH₂Cl₂ (20 mL), CH₂Cl₂ (3 × 10 mL), H₂O (2 × 10 mL)] and column chromatography gave 4c (107 mg, 0.18 mmol, 45%).

According to the general procedure, method B, using azide 2 (79 mg, 0.41 mmol), alkyne 3c (158 mg, 0.41 mmol), CuSO₄˙5 H₂O (20 mg, 0.08 mmol), sodium ascorbate (32 mg, 0.16 mmol), and TBTA (44 mg, 0.08 mmol) in t-BuOH-H₂O (1:1, 26 mL) for 2 h; separation [CH₂Cl₂ (20 mL), CH₂Cl₂ (3 × 10 mL), H₂O (2 × 10 mL)] and column chromatography gave 4c (183 mg, 0.32 mmol, 77%).

Viscous yellow oil; R _f  = 0.14 (cyclohexane-EtOAc, 1:4).

[α]_D ^²² -14.7 (c 1.00 CHCl₃).

^¹H NMR (CDCl₃): δ = 1.28-1.34 (m, 4 H, 2 CH₂), 1.70-1.79 (m, 2 H, CH₂), 1.83-1.91 (m, 2 H, CH₂), 1.96 (s, 3 H, CH₃), 1.98 (s, 3 H, CH₃), 2.01 (s, 3 H, CH₃), 2.07 (s, 3 H, CH₃), 3.72 (ddd, J ₁ = 2.4 Hz, J ₂ = 4.7 Hz, J ₃ = 9.9 Hz, 1 H, H5), 3.85 (t, J = 7.0 Hz, 2 H, N_py-CH₂), 4.13 (dd, J ₁ = 2.2 Hz, J ₂ = 12.5 Hz, 1 H, C₅H-CH ₂), 4.22-4.35 (m, 3 H, C₅H-CH ₂, CH₂N_triazole), 4.67 (d, J = 7.9 Hz, 1 H, H1), 4.80 (d, J = 12.5 Hz, 1 H, CH₂C_triazole), 4.92 (d, J = 12.6 Hz, 1 H, CH₂C_triazole), 5.00 (dd, J ₁ = 8.0 Hz, J ₂ = 9.4 Hz, 1 H, H2), 5.08 (t, J = 9.6 Hz, 1 H, H4), 5.19 (t, J = 9.4 Hz, 1 H, H3), 6.11 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.61 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.47 (s, 1 H, CH_triazole).

^¹³C NMR (CDCl₃): δ = 20.7 (2 CH₃), 20.8 (CH₃), 20.9 (CH₃), 26.1 (CH₂), 26.2 (CH₂), 30.2 (CH₂), 31.3 (CH₂), 49.4 (N_py-CH₂), 50.3 (CH₂N_triazole), 61.9 (CH₂C_triazole), 63.1 (CH₂O), 68.4 (C4), 71.3 (C2), 72.0 (C5), 72.8 (C3), 100.0 (C1), 108.0 (2 CH_py), 120.5 (2 CH_py), 122.7 (CH_triazole), 144.2 (C_triazole), 169.4 (C=O), 169.5 (C=O), 170.3 (C=O), 170.7 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₇H₃₉N₄O₁₀: 579.2661; found: 579.2676.

1-[6-(1 H -Pyrrol-1-yl)hexyl]-4-[(2,3,4,6-tetra- O -acetyl-β- d -galactopyranosyloxy)methyl]-1 H -1,2,3-triazole (4d)

According to the general procedure, method A, using azide 2 (192 mg, 1.00 mmol), alkyne 3d (386 mg, 1.00 mmol), CuSO₄˙5 H₂O (50 mg, 0.20 mmol), and sodium ascorbate (79 mg, 0.40 mmol) in t-BuOH-H₂O (1:1, 50 mL) for 2 d; separation [CH₂Cl₂ (45 mL), CH₂Cl₂ (3 × 18 mL), H₂O (2 × 30 mL)] and column chromatography gave 4d (375 mg, 0.65 mmol, 65%).

According to the general procedure, method B, using azide 2 (481 mg, 2.50 mmol), alkyne 3d (966 mg, 2.50 mmol), CuSO₄˙5 H₂O (125 mg, 0.50 mmol), sodium ascorbate (198 mg, 1.00 mmol), and TBTA (265 mg, 0.50 mmol) in t-BuOH-H₂O (1:1, 120 mL) for 3 h; separation [CH₂Cl₂ (100 mL), CH₂Cl₂ (3 × 40 mL), H₂O (2 × 70 mL)] and column chromatography gave 4d (1.17 g, 2.02 mmol, 81%).

Viscous yellow oil; R _f  = 0.22 (cyclohexane-EtOAc, 3:7).

[α]_D ^²² -22.2 (c 1.01, CHCl₃).

^¹H NMR (CDCl₃): δ = 1.26-1.34 (m, 4 H, 2 CH₂), 1.67-1.78 (m, 2 H, CH₂), 1.80-1.91 (m, 2 H, CH₂), 1.95 (s, 3 H, CH₃), 1.96 (s, 3 H, CH₃), 2.03 (s, 3 H, CH₃), 2.12 (s, 3 H, CH₃), 3.85 (t, J = 7.0 Hz, 2 H, N_py-CH₂), 3.93 (t, J = 6.5 Hz, 1 H, H5), 4.11-4.19 (m, 2 H, C₅H-CH ₂), 4.29 (t, J = 7.1 Hz, 2 H, CH₂N_triazole), 4.63 (d, J = 7.9 Hz, 1 H, H1), 4.78 (d, J = 12.5 Hz, 1 H, CH₂C_triazole), 4.95 (d, J = 12.5 Hz, 1 H, CH₂C_triazole), 5.00 (dd, J ₁ = 3.4 Hz, J ₂ = 10.5 Hz, 1 H, H3), 5.20 (dd, J ₁ = 7.9 Hz, J ₂ = 10.5 Hz, 1 H, H2), 5.38 (d, J = 2.9 Hz, 1 H, H4), 6.09 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.60 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.46 (s, 1 H, CH_triazole).

^¹³C NMR (CDCl₃): δ = 20.6 (CH₃), 20.7 (2 CH₃), 20.8 (CH₃), 26.1 (CH₂), 26.2 (CH₂), 30.2 (CH₂), 31.3 (CH₂), 49.4 (N_py-CH₂), 50.2 (CH₂N_triazole), 61.3 (CH₂C_triazole), 63.6 (CHCH₂), 67.1 (C4), 68.8 (C2), 70.8 (C3, C5), 100.5 (C1), 108.0 (2 CH_py), 120.5 (2 CH_py), 122.6 (CH_triazole), 144.2 (C_triazole), 169.5 (C=O), 170.1 (C=O), 170.3 (C=O), 170.4 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₇H₃₉N₄O₁₀: 579.2661; found: 579.2666.

N -[1-(2′-Deoxyuridin-2′-yl)]-1 H -1,2,3-triazol-4-methyl)-3-(1 H -pyrrol-1-yl)propanamide (8a)

According to the general procedure, method B, using azide 7a (242 mg, 0.90 mmol), alkyne 6 (159 mg, 0.90 mmol), CuSO₄˙5 H₂O (45 mg, 0.18 mmol), sodium ascorbate (71 mg, 0.38 mmol), and TBTA (96 mg, 0.18 mmol) in THF-H₂O (1:1, 3 mL) for 1 h; column chromatography gave 8a (355 mg, 0.78 mmol, 87%) as a white foam; mp 88-92 ˚C; R _f  = 0.27 (EtOAc-MeOH, 5:1).

^¹H NMR (CD₃OD): δ = 2.61 (t, J = 6.6 Hz, 2 H, CH₂CO), 3.65-3.82 (m, 2 H, 5′-CH₂), 4.14 (t, J = 6.6 Hz, 2 H, N_py-CH₂), 4.17-4.25 (m, 1 H, H4′), 4.35 (s, 2 H, CH₂C_triazole), 4.47-4.57 (m, 1 H, H3′), 4.55 (s, 1 H, CONH), 5.34 (t, J = 6.0 Hz, 1 H, H2′), 5.70 (d, J = 8.0 Hz, 1 H, CHCHN_uridine), 5.96 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.52 (d, J = 9.0 Hz, 1 H, H1′), 6.59 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.73 (s, 1 H, CH_triazole­), 8.07 (d, J = 8.0 Hz, 1 H, CHN_uridine).

^¹³C NMR (CD₃OD): δ = 35.7 (CH₂C_triazole), 39.2 (CH₂), 46.5 (N_py-CH₂), 62.2 (5′-CH₂), 67.3 (C2′), 71.4 (C3′), 87.9 (C4′), 88.0 (C1′), 103.4 (CHCHN_uridine), 109.1 (2 CH_py), 121.6 (2 CH_py), 125.6 (CH_triazole­), 141.9 (CHN_uridine), 145.9 (C_triazole), 152.1 (C=O), 165.9 (C=O), 173.4 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₂₄N₇O₆: 446.1783; found: 446.1773.

3-(1 H -Pyrrol-1-yl)- N -{[1-(2,3,4,6-tetra- O -acetyl-β- d -gluco­pyranosyl)-1 H -1,2,3-triazol-4-yl]methyl}propanamide (8b)

According to the general procedure, method A, using azide 7b (343 mg, 0.92 mmol), alkyne 6 (162 mg, 0.92 mmol), CuSO₄˙5 H₂O (46 mg, 0.18 mmol), and sodium ascorbate (73 mg, 0.37 mmol) in t-BuOH-H₂O (1:1, 44 mL) for 4 d; separation [CH₂Cl₂ (32 mL), CH₂Cl₂ (3 × 20 mL), H₂O (2 × 15 mL)] and column chromatography gave 8b (502 mg, 0.91 mmol, 99%).

According to the general procedure, method B, using azide 7b (343 mg, 0.92 mmol), alkyne 6 (162 mg, 0.92 mmol), CuSO₄˙5 H₂O (46 mg, 0.18 mmol), sodium ascorbate (73 mg, 0.37 mmol), and TBTA (98 mg, 0.18 mmol) in t-BuOH-H₂O (1:1, 50 mL) for 3 h; separation [CH₂Cl₂ (35 mL), CH₂Cl₂ (3 × 20 mL), H₂O (2 × 15 mL)] and column chromatography. To remove the ligand completely a second column chromatography (EtOAc-MeOH, 98:2) was necessary to give 8b (465 mg, 0.85 mmol, 92%).

White solid; mp 172-173 ˚C; R _f  = 0.39 (CH₂Cl₂ -MeOH, 9:1).

[α]_D ^²² -28.1 (c 1.02, CHCl₃).

^¹H NMR (CDCl₃): δ = 1.84 (s, 3 H, CH₃), 2.02 (s, 3 H, CH₃), 2.06 (s, 3 H, CH₃), 2.07 (s, 3 H, CH₃), 2.61 (t, J = 6.8 Hz, 2 H, CH₂CO), 4.00 (ddd, J ₁ = 2.1 Hz, J ₂ = 4.8 Hz, J ₃ = 10.1 Hz, 1 H, H5), 4.14 (dd, J ₁ = 2.1 Hz, J ₂ = 12.6 Hz, 1 H, C₅H-CH ₂), 4.22 (t, J = 6.7 Hz, 2 H, N_py-CH₂), 4.31 (dd, J ₁ = 4.8 Hz, J ₂ = 12.7 Hz, 1 H, C₅H-CH ₂), 4.45 (d, J = 5.8 Hz, 2 H, CH₂C_triazole), 5.22-5.30 (m, 1 H, H3), 5.38-5.46 (m, 2 H, H2, H4), 5.83 (dd, J ₁ = 2.7 Hz, J ₂ = 6.6 Hz, 1 H, H1), 6.10 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.31 (t, J = 5.7 Hz, 1 H, CONH), 6.62 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.64 (s, 1 H, CH_triazole).

^¹³C NMR (CDCl₃): δ = 20.3 (CH₃), 20.6 (CH₃), 20.7 (CH₃), 20.8 (CH₃), 35.0 (CH₂C_triazole), 38.7 (CH₂CO), 45.6 (N_py-CH₂), 61.6 (C₅H-CH₂), 67.7 (C3), 70.5 (C4), 72.7 (C2), 75.2 (C5), 85.8 (C1), 108.5 (2 CH_py), 120.7 (2 CH_py), 121.0 (CH_triazole), 145.4 (C_triazole), 168.9 (C=O), 169.4 (C=O), 170.0 (C=O), 170.3 (C=O), 170.6 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₂N₅O₁₀: 550.2144; found: 550.2150.

Anal. Calcd for C₂₄H₃₁N₅O₁₀: C, 52.46; H, 5.69; N, 12.74. Found: C, 52.62; H, 5.80; N, 12.56.

3-(1 H -Pyrrol-1-yl)- N -{[1-(2,3,4,6-tetra- O -acetyl-β- d -galacto­pyranosyl)-1 H -1,2,3-triazol-4-yl]methyl}propanamide (8c)

According to the general procedure, method A, using azide 7c (188 mg, 0.50 mmol), alkyne 6 (88 mg, 0.50 mmol), CuSO₄˙5 H₂O (25 mg, 0.10 mmol), and sodium ascorbate (40 mg, 0.20 mmol) in t-BuOH-H₂O (1:1, 26 mL) for 9 d; separation [CH₂Cl₂ (20 mL), CH₂Cl₂ (3 × 10 mL), H₂O (2 × 10 mL)] and column chromatography gave 8c (188 mg, 0.34 mmol, 68%) as a foam.

According to the general procedure, method B, using azide 7c (392 mg, 1.05 mmol), alkyne 6 (185 mg, 1.05 mmol), CuSO₄˙5 H₂O (52 mg, 0.21 mmol), sodium ascorbate (83 mg, 0.42 mmol), and TBTA (111 mg, 0.21 mmol) in t-BuOH-H₂O (1:1, 60 mL) for 3 h; separation [CH₂Cl₂ (35 mL), CH₂Cl₂ (3 × 20 mL), H₂O (2 × 15 mL)] and column chromatography. To remove the ligand completely a second column chromatography (EtOAc-MeOH, 98:2) was necessary to give 8c (507 mg, 0.92 mmol, 88%) as a foam.

Foam; mp 69-71 ˚C; R _f  = 0.20 (CH₂Cl₂ -MeOH, 95:5).

[α]_D ^²² -7.1 (c 1.01, CHCl₃).

^¹H NMR (CDCl₃): δ = 1.87 (s, 3 H, CH₃), 2.01 (s, 3 H, CH₃), 2.03 (s, 3 H, CH₃), 2.24 (s, 3 H, CH₃), 2.61 (t, J = 6.6 Hz, 2 H, CH₂CO), 4.06-4.28 (m, 5 H, H5, CH5-CH ₂, N_py-CH₂), 4.36-4.57 (m, 2 H, CH₂C_triazole), 5.25 (dd, J ₁ = 3.4 Hz, J ₂ = 10.2 Hz, 1 H, H3), 5.47-5.58 (m, 2 H, H2, H4), 5.80 (d, J = 9.3 Hz, 1 H, H1), 6.13 (m, 3 H, 2 CH_py, CONH), 6.63 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.71 (s, 1 H, CH_triazole­).

^¹³C NMR (CDCl₃): δ = 20.4 (CH₃), 20.6 (CH₃), 20.7 (CH₃), 20.8 (CH₃), 35.1 (CH₂C_triazole), 39.0 (CH₂CO), 45.7 (N_py-CH₂), 61.4 (C₅H-CH₂), 67.0 (C4), 68.1 (C2), 70.9 (C3), 74.2 (C5), 86.5 (C1), 108.6 (2 CH_py), 120.7 (2 CH_py), 121.0 (CH_triazole), 145.3 (C_triazole), 169.1 (C=O), 170.0 (C=O), 170.2 (C=O), 170.3 (C=O), 170.5 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₂N₅O₁₀: 550.2144; found: 550.2128.

Deprotection of Tetraacetates 4d and 8b,c; General Procedure

Dry NaOMe-MeOH soln (0.1 equiv) (freshly prepared 1.0 M soln) was added to a stirred (0.1 M) soln of the glycoside tetraacetate 4 or 8 (1.0 equiv) in MeOH at r.t. The mixture was stirred for 30 min (TLC showed complete conversion). Neutralization by addition of DOWEX 50 × 8 ion-exchange resin (pH 6), followed by filtration and evaporation of the filtrate to dryness, afforded the pure unprotected products 9a-c.

4-[(β- d -Galactopyranosyloxy)methyl]-1-[6-(1 H -pyrrol-1-yl)hexyl]-1 H -1,2,3-triazole (9a)

Following the general procedure using 4d (579 mg, 1.00 mmol) gave 9a (355 mg, 0.86 mmol, 86%) as a brown oil; R _f  = 0.24 (MeCN-H₂O, 9:1).

[α]_D ^²0 -12.4 (c 1.01, CH₂Cl₂).

^¹H NMR (CD₃CN): δ = 1.14-1.40 (m, 4 H, 2 CH₂), 1.63-1.75 (m, 2 H, CH₂), 1.76-1.88 (m, 2 H, CH₂), 3.36 (br s, 4 H, 4 OH), 3.43-3.53 (m, 3 H), 3.62-3.76 (m, 2 H), 3.81-3.85 (m, 3 H), 4.25-4.35 (m, 3 H, CH₂N_triazole, H1), 4.72 (d, J = 12.5 Hz, 1 H, CH₂C_triazole), 4.89 (d, J = 12.5 Hz, 1 H, CH₂C_triazole), 6.00 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.63 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.79 (s, 1 H, CH_triazole).

^¹³C NMR (CD₃CN): δ = 26.4 (CH₂), 26.6 (CH₂), 30.6 (CH₂), 31.9 (CH₂), 49.7 (N_py-CH₂), 50.8 (CH₂N_triazole), 62.2 (C₅H-CH₂), 62.6 (CH₂C_triazole), 69.7 (C4), 72.0 (C2), 74.3 (C3), 75.8 (C5), 103.4 (C1), 108.3 (2 CH_py), 121.2 (2 CH_py), 124.5 (CH_triazole), 144.8 (C_triazole).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₉H₃₁N₄O₆: 411.2238; found: 411.2237.

N -{[1-(β- d -Glucopyranosyl)-1 H -1,2,3-triazol-4-yl]methyl}-3-(1 H -pyrrol-1-yl)propanamide (9b)

Following the general procedure using 8b (1.18 g, 2.14 mmol) gave 9b (801 mg, 2.10 mmol, 98%) as a white solid; mp 94-97 ˚C (dec.); R _f  = 0.27 (MeCN-H₂O, 9:1).

[α]_D ^²² +1.5 (c 1.01, MeOH).

^¹H NMR (DMSO-d ₆): δ = 2.58 (t, J = 6.9 Hz, 2 H, CH₂CO), 3.20-3.26 (m, 1 H), 3.35-3.49 (m, 3 H), 3.65-3.76 (m, 2 H, C₅H-CH ₂, H2), 4.12 (t, J = 6.9 Hz, 2 H, N_py-CH₂), 4.31 (d, J = 5.4 Hz, 2 H, CH₂C_triazole), 5.48 (d, J = 9.3 Hz, 1 H, H1), 5.97 (t, J = 2.1 Hz, 2 H, 2 CH_py), 6.71 (t, J = 2.1 Hz, 2 H, 2 CH_py), 7.85 (s, 1 H, CH_triazole), 8.45 (t, J = 5.7 Hz, 1 H, CONH).

^¹³C NMR (DMSO-d ₆): δ = 34.2 (CH₂C_triazole), 37.3 (CH₂), 44.9 (N_py-CH₂), 60.8 (C₅H-CH₂), 69.6 (C4), 72.1 (C2), 77.0 (C3), 80.0 (C5), 87.5 (C1), 107.6 (2 CH_py), 120.6 (2 CH_py), 121.7 (CH_triazole), 144.9 (C_triazole), 169.8 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₆H₂₄N₅O₆: 382.1721; found: 382.1727.

N -{[1-(β- d -Galactopyranosyl)-1 H -1,2,3-triazol-4-yl]methyl}-3-(1 H -pyrrol-1-yl)-propanamide (9c)

Following the general procedure using 8c (448 mg, 0.82 mmol) gave 9c (252 mg, 0.66 mmol, 81%) as a light brown hygroscopic solid; mp 126-130 ˚C (dec); R _f  = 0.15 (MeCN-H₂O, 9:1).

[α]_D ^²4 +15.2 (c 1.00, MeOH).

^¹H NMR (DMSO-d ₆-D₂O, 14:1): δ = 2.56 (t, J = 6.9 Hz, 2 H, CH₂CO), 3.44-3.60 (m, 3 H), 3.68-3.72 (m, 1 H), 3.75-3.77 (m, 1 H), 3.97-4.03 (m, 1 H), 4.11 (t, J = 6.9 Hz, 2 H, N_py-CH₂), 4.23-4.39 (m, 2 H, CH₂C_triazole), 5.44 (d, J = 9.2 Hz, 1 H, H1), 5.97 (t, J = 2.0 Hz, 2 H, 2 CH_py), 6.70 (t, J = 2.0 Hz, 2 H, 2 CH_py), 7.88 (s, 1 H, CH_triazole), 8.40-8.48 (m, 1 H, CONH).

^¹³C NMR (DMSO-d ₆-D₂O, 14:1): δ = 34.3 (CH₂C_triazole), 37.4 (CH₂), 44.9 (N_py-CH₂), 60.5 (C₅H-CH₂), 68.5 (C4), 69.4 (C2), 73.8 (C3), 78.4 (C5), 88.1 (C1), 107.7 (2 CH_py), 120.5 (2 CH_py), 121.5 (CH_triazole), 144.9 (C_triazole), 169.9 (C=O).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₆H₂₄N₅O₆: 382.1721; found: 382.1724.

Acknowledgment

We thank Prof. Dr. Ladislau Vekas, Romanian Academy of Science, Timisoara Branch, for providing magnetic Fe₃O₄ nanofluid. We further acknowledge Saltigo GmbH, Bayer Services GmbH & Co. OHG, BASF AG, and Sasol GmbH for the donation of chemicals.

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