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Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein–protein and protein–lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins.

Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among the few commercial polymers that are capable of forming nanodiscs, alternating diisobutylene/maleic acid (DIBMA) copolymers have gained considerable popularity as gentle and UV-transparent alternatives to aromatic polymers. However, their moderate hydrophobicities and high electric charge densities render all existing aliphatic copolymers rather inefficient under near-physiological conditions. Here, we introduce Glyco-DIBMA, a bioinspired glycopolymer that possesses increased hydrophobicity and reduced charge density but nevertheless retains excellent solubility in aqueous solutions. Glyco-DIBMA outperforms established aliphatic copolymers in that it solubilizes lipid vesicles of various compositions much more efficiently, thereby furnishing smaller, more narrowly distributed nanodiscs that preserve a bilayer architecture and exhibit rapid lipid exchange. We demonstrate the superior performance of Glyco-DIBMA in preparative and analytical applications by extracting a broad range of integral membrane proteins from cellular membranes and further by purifying a membrane-embedded voltage-gated K+ channel, which was fluorescently labeled and analyzed with the aid of microfluidic diffusional sizing (MDS) directly within native-like lipid-bilayer nano-discs.

Syntheses of 4-O-methyl-β-rhodomycins are described. Glycosylation (trimethylsilyl triflate, dichloromethane-acetone 10:1, -30 °C) of 4-O-methyl-10-O-p-nitrobenzoyl-β-rhodomycinone, obtained from β-rhodomycinone (βRMN) in a 6-step synthesis, with 1-O-tert-butyl(dimethyl)silylated derivatives of 4-O-acetyl- or 4-O-p-nitrobenzoyl-2,3,6-tri-deoxy-3-trifluoroacetylamino-β-L-arabino- and lyxo-hexopyranoses or 2,6-di-O-acetyl-2,6-dideoxy-β-L-lyxo-hexopyranose afforded 7-O-α-L-glycosyl-β-rhodomycinones. Removal of the O- and N-acyl groups with 0.1M and 1M NaOH gave the 7-O-(3-amino-2,3,6-trideoxy-α-L-arabino- and lyxo-hexopyranosyl)-4-O-methyl-β-rhodomycinones and 7-O-(2,6-dideoxy-α-L-lyxo-hexopyranosyl)-4-O-methyl-β-rhodomycinone.

Synthesis of 7-O-(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)-epsilon-(i so)rhodomycinones 16 and 17, and their 3'-morpholino derivatives are described. Glycosylation (trimethylsilyl triflate, 10:1 dichloro-methane-acetone, -35 degrees) of 1-O-tert-butyldimethylsilyl-2,3-6-trideoxy-4-O-p-nitrobenzoyl-3-trifl uoroacetamido-beta-L-lyxo-hexopyranose (4) with epsilon-rhodomycinone (epsilon-RMN, 5) or epsilon-isorhodomycinone (epsilon-isoRMN, 6) afforded 7-O-alpha-glycosyl-epsilon-RMN (9) and -epsilon-isoRMN (12) in high yield. The glycosyl donors 2,3,6-trideoxy-4-O-p-nitrobenzoyl-3-trifluoroacetamido-L-lyxo++ +-hexopyrano se (2) or its 1-O-trimethylsilylated alpha-anomer 3 were less suitable for the glycosylation of these aglycons. Sapinification of 9 and 12 provided 16 and 17, respectively, which reacted with various 2,2'-oxydiacetaldehydes under conditions of reductive alkylation to give 3'-morpholinyl-epsilon-(iso)rhodomycins.

Synthesis of 7- O -(4-amino-2,4,6-trideoxy- and 7- O -(3,4-diamino-2,3,4,6-tetradeoxy-α- l - lyxo -hexopyranosyl)-4- O -methyl-β-rhodomycinones ( 31 and 33 ) are described. The glycosyl donors 2,4,6-trideoxy-1,3-di- O-p -nitrobenzoyl-4-trifluoroacetamido-β- l - lyxo -hexopyranose ( 14β ) and 2,3,4,6-tetradeoxy-1- O-p -nitrobenzoyl-3,4-bis(trifluoroacetamido)-β- l - lyxo -hexopyranose ( 23β ) were suitable for the glycosylation of 4- O -methyl-10- O-p -nitrobenzoyl-β-rhodomycinone by the trimethylsilyl triflate method. Saponification of the resulting 7- O -α-glycosyl-4- O -methyl-β-rhodomycinones gave 31 and 33 .

Glycosylation of benzyl 2,3,6-trideoxy-3-trifluoroacetamido-α- l - lyxo -hexopyranoside ( 6 ) with 3,4-di- O -acetyl-1,5-anhydro-2,6-dideoxy- l - lyxo -hex-1-enitol ( 1 ) or 4- O -acetyl-1,5-anhydro-3- O -benzyl-2,6-dideoxy- l - lyxo -hex-1-enitol ( 2 ) in the presence of trimethylsilyl triflate/triethylamine gave α-(1→4)-linked disaccharide derivatives 7 and 8 , respectively. In the presence of trimethylsilyl triflate only, 3,4-di- O -acetyl-1- O - tert -butyldimethylsilyl-2,6-dideoxy-β- l - lyxo -hexopyranose ( 3 ) and 6 gave mainly 7 . Condensation of 1 or 2 with 9 , obtained by O -deacylation of 8 , afforded benzyl [ O -(3,4-di- O -acetyl-2,6-dideoxy-α- l - lyxo -hexopyranosyl)-(1→4)- O -(3- O -benzyl-2,6-dideoxy-α- l - lyxo -hexopyranosyl)- (1→4)-(2,3,6-trideoxy-3-trifluoro-acetamido-α- l - lyxo -hexopyranoside)] ( 11 ) and its 3″- O -benzyl analogue 12 , respectively.

Syntheses of 4-O-methyl-β-rhodomycins are described. Glycosylation (trimethylsilyl triflate, dichloromethane-acetone 10:1, -30 °C) of 4-O-methyl-10-O-p-nitrobenzoyl-β-rhodomycinone, obtained from β-rhodomycinone (βRMN) in a 6-step synthesis, with 1-O-tert-butyl(dimethyl)silylated derivatives of 4-O-acetyl- or 4-O-p-nitrobenzoyl-2,3,6-tri-deoxy-3-trifluoroacetylamino-β-L-arabino- and lyxo-hexopyranoses or 2,6-di-O-acetyl-2,6-dideoxy-β-L-lyxo-hexopyranose afforded 7-O-α-L-glycosyl-β-rhodomycinones. Removal of the O- and N-acyl groups with 0.1M and 1M NaOH gave the 7-O-(3-amino-2,3,6-trideoxy-α-L-arabino- and lyxo-hexopyranosyl)-4-O-methyl-β-rhodomycinones and 7-O-(2,6-dideoxy-α-L-lyxo-hexopyranosyl)-4-O-methyl-β-rhodomycinone.

It was found that rhodosaminylanthraclinone-type anthracyclines are readily N-mono-demethylated upon irradiation with visible light, leading to 3′-N-methyl-α-L-daunosarninylanthracyclinones in good yields. The reaction is preferentially observed with rhodosaminylanthracyclinones in which OH-4′ of the sugar (rhodosamine) moiety is not further glycosylated. 3′-N-methyl-α-L-daunosaminyl anthracyclinones provide key compounds that can readily be further derivatized.

The synthesis of 7-O-α-L-daunosaminyl-4-O-methyl-β-rhodomycinone (3) and the determination of its cytotoxic potency compared to that of the natural 7-O-α-L-dau-nosaminyl-β-rhodomycinone (4) are described. Starting with natural β-rhodomycinone (7), trimethylsilyl protecting groups were attached to the hydroxy groups at position 7 and 10, and the 4-OH group was subsequently methylated (MeI/Cs2CO3), thus providing the 4-O-methyl-7, 10-bis-O-trimethylsilyl-β-rhodomycinone (10). The two TMS groups were then deblocked to give 4-O-methyl-β-rhodomycinone (12). In a 3-stage synthesis 12 was converted into 4-O-methyl-10-O-trifiuoroacetyl-s-rhodomycinone (15) to which l, 4-bis-O-p-nitrobenzoyl-3-N-trifluoroacetyl-L-daunosamine 16 was selectively linked to afford the 7-O-α-glycoside 17. The acyl protective groups are removed by treatment with 1N NaOH to give 3.

The synthesis of the glycosyl donors 2,3-di-O-acetyl- and 2,3-di-O-chloroacetyl- 4,6-O-ethylidene-s-D-glucopyranose (10β) and (11β) and their use for the glycosidation of 4'-O-benzyloxycarbonyl- and 4'-O-chloroacetyl-4'-O-demethyl-4-epi-podophyllotoxins (12) and (13) is described. Starting from benzyl β-D-glucopyranoside (6), benzyl 2,3-di-O-acetyl- and 2,3-di-O-chloroacetyl-4,6-O-ethylidene-s-D-glucopyranoside (8) and (9) were prepared. Hydrogenolysis of the benzyl group in 8 or 9 afforded the β-hydroxy glucopyranose donors 10β and 11β. Condensation of 10β or 11β with 4'-O-Z-epi-podophyllotoxin 12 in the presence of BF3-etherate gave selectively the 4-O-(2,3-di-O-acetyl- or -2,3-di-O-chloroacetyl-4,6-O-ethylidene-β-D-glucopyranosyl)-epi-podophyllotoxins 14β and 15β, respectively. The β-glycoside 16 was prepared in the same manner starting from 11β and 4'-O-chloroacetyl-epi-podophyllotoxin 13. By deblocking (Dowex 1X8, 3:2 methanol-chloroform) of the chloroacetyl groups in 15β and the following h...

Synthesis of 3-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene- alpha-(7 alpha) and-beta-D-glucopyranose (7 beta) and their 3-O-chloroacetyl analogues (11 alpha and 11 beta) are described. Condensation (BF3-etherate, ethyl acetate, -20 degrees) of 7 alpha with 4'-O-benzyloxycarbonyl-4'-O-demethyl-4-epipodophyllotoxin (8) afforded mainly the beta-glycoside 9 beta (alpha, beta-ratio 1:9). Condensation of 11 alpha beta with 8 or the 4'-O-chloroacetyl analogue 13 gave mainly the 4-O-(2-benzyloxycarbonylamino-3-O-chloroacetyl-2-deoxy-4,6-O-ethyl idene-beta-D- glucopyranosyl)-epipodophyllotoxin 12 beta or 15 beta. Glycosidation of podophyllotoxin (14) with 11 alpha beta (during which the aglycon epimerized at C-4 under the action of BF3-etherate) afforded alpha- (16 alpha) and beta-glycoside (16 beta) in the ratio 1:5. Removal of the chloroacetyl groups from 12 beta, its alpha analogue 12 alpha, and 15 beta gave the 4-O-(2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene-alpha-(17 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-epipodophyllotoxins (17 beta and 20 beta), respectively. Hydrogenolysis of the benzyloxycarbonyl groups then gave 4-O-(2-amino-2-deoxy-4,6-O-ethylidene-alpha- (18 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-4-epipodophyllotoxin (18 beta). Reductive alkylation of 18 beta and 18 alpha afforded the 2"-deoxy-2"-dimethylamino-etoposide 3 and its alpha analogue 19 alpha.

It was determined that β-rhodomycin-II (rhodomycin A) (1), β-rhodomycin-I (rhodomycin B, betaclamycin T) (2) and γ-rhodomycin-I (iremycin) (3) may regioselectively be acylated at OH-4′ of the sugar (rhodosamine) moiety(ies), using a two-phase (chloroform/water) solvent system and sodium bicarbonate as a base. This report exemplarily describes the synthesis of the corresponding p-methoxybenzoate esters of 1, 2, and 3, i. e. derivatives 4–7, and highlights the structure elucidation of isomers 5a and 5b.

10-O-(R/S)Tetrahydropyranosyl-β-rhodomycinone (5a,b) was prepared via 7,9-O-phenylboronyl-β-rhodomycinone (3) from β-rhodomycinone (1). Glycosidation of 5a,b with 3,4-di-O-acetyl-1,5-anhydro-2,6-dideoxy-L-arabino-hex-1-enitol (3,4-di-O-acetyl-L-rhamnal) (6) and 3,4-di-O-acetyl-1,5-anhydro-2,6-dideoxy-L-lyxo-hex-1-enitol (3,4-di-O-acetyl-L-fucal) (7) using N-iodosuccinimide gave the corresponding 7-O-glycosyl-β-rhodomycinones 8a,b, 9a,b and 10a,b, 11a,b. After cleavage of the THP-ether and O-deacetylation 7-O-(2,6-dideoxy-2-iodo-α-L-manno-hexopyranosyl)-β-rhodomycinone (14) and 7-O-(2,6-dideoxy-2-iodo-α-L-talo-hexopyranosyl)-β-rhodomycinone (16) were obtained.

The syntheses of 7-O-α-L-daunosaminyl-∊-isorhodomycinone (6) and 7-O-α-L-rhodosaminyl-∊-isorhodomycinone (7) are described. The glycosyl donors 1,4-di-O-p-nitrobenzoyl-3-N-trifluoroacetyl-α,β-L-daunosamine and 1,4-di-O-acetyl-α,β-L-rhodosamine have proven to be the most suitable for the glycosylation of ∊-isorhodomycinone (∊-iso RMN) (3), using the TMS triflate method. Deblocking (0.5 N NaOH) of the protected glycoside 4 led to the desired 7-O-glycosyl-∊-isorhodomycinones 5 and 6. The daunosaminyl glycoside 6 was methylated (CH2O, NaCNBH3) to provide the rhodosaminyl derivative 7. The photolytic demethylation of this product selectively provided the 7-O-(3′-N-methyl-α-L-daunosaminyl)-∊-isorhodomycinone (8).