Synthesis and Evaluation of 3′-Oleyl–Oligonucleotide Conjugates as Potential Cellular Uptake Enhancers

Abstract

The field of therapeutic oligonucleotides has experienced significant growth in recent years, both in terms of approved drugs and those undergoing clinical trials. This expansion has transformed it into a rapidly evolving area of research. However, their cellular internalization remains a major limitation for the clinical application of oligonucleotides. To address this limitation, we report different strategies for the synthesis of specialized solid supports for the direct synthesis of 3′-oleyl-oligonucleotides by means of an l-threoninol derivative. A series of in vitro cell experiments were conducted to evaluate the potential of this strategy for enhanced cellular uptake. The results suggest that lipid conjugation enhances cellular uptake and facilitates oligonucleotide intracellular trafficking. Given these findings, the modification of therapeutic oligonucleotides through the attachment of lipidic moieties using a threoninol linker emerges as a valuable strategy to enhance their cellular internalization.

In recent years, oligonucleotide therapeutics have garnered significant interest in the pharmaceutical field, resulting in the approval of nearly 15 drugs for human treatment.[2] Since oligonucleotides interfere mostly at protein expression levels, they need to reach their targets in the cell cytoplasm to exert their inhibitory actions.[3] To achieve potent inhibitory properties, the chemical modification of oligonucleotides is essential. Among the various modifications, lipid-oligonucleotide conjugates have shown promising results.[4] It has been reported that the covalent attachment of lipids such as cholesterol[5] and fatty acids,[6] and hydrophobic compounds like vitamin E[7] and folic acid[8] to oligonucleotides yields novel conjugates with enhanced biomedical properties[9] such as improved cellular uptake,[10] enhanced pharmacodynamic properties through binding to serum proteins,[11] and increased extrahepatic distribution.[12]

Although efficient lipid formulations have been discovered for systemic use, most of them result in their accumulation in the liver and consequently in toxicities. Interestingly, the modification of oligonucleotides with specific lipids, especially at the 3′ end, has been reported to result in higher retention in extrahepatic tissues.[13] For example, the conjugation of docosanoic acid to an oligonucleotide enabled successful delivery in skeletal and cardiac muscles after systemic injection,[14] whilst neuro-active steroids, endocannabinoid-like lipids, and gangliosides have been identified as conjugates to improve neuronal uptake and the distribution of oligonucleotides throughout the central nervous system.[15]

The incorporation of lipid molecules into oligonucleotides is typically performed at the 3′ or 5′ terminus of oligonucleotides to preserve their hybridization properties between strands.[16] The introduction of lipids at the 5′ end is relatively easier due to the synthesis of oligonucleotides occurring in the 3′ to 5′ direction. Consequently, the addition of the lipid can be accomplished using simple linear linkers such as 6-aminohexanol derivatives or similar compounds.[17] However, introducing lipids at the 3′ end requires linkers that connect the lipid to the solid support in a manner that allows for the subsequent assembly of the oligonucleotide, which begins at the 3′ end. These requirements are addressed through two main strategies: (1) The preparation of 3′-modified oligonucleotides with reactive moieties such as amino groups, followed by post-synthetic conjugation with fatty acids,[13] or (2) the preparation of a solid support functionalized with lipid derivatives through aminodiol linkers.[18] [19] [20] [21] This latter approach solves the challenge by providing a solid support that facilitates the assembly of the oligonucleotide while connecting the lipid to it.

Oligomers made of antiproliferative nucleosides have been shown to act as prodrugs that are activated by nuclease degradation inside the cells.[4] [22] In a recent study, the addition of palmitic acid at the 3′ end of a pentanucleotide of floxuridine showed the highest antiproliferative activity,[10] whereas cholesterol at the same position gave the worst derivative, despite its higher cellular uptake. In order to study the role of lipids in cellular uptake and endosomal escape,[23] we are interested in the production of oligonucleotides bearing different fatty acids at the 3′ end.

In the present communication, we describe the synthesis of solid supports functionalized with an l-threoninol derivative for the preparation of 3′-oleyl-oligonucleotides. Threoninol is a commercially available aminodiol that can be obtained in a pure enantiomeric form. It has been previously employed for introducing azobenzene derivatives for the photoregulation of DNA hybridization,[24] as well as in artificial nucleic acids.[25] While oligonucleotides possessing oleic acid have been previously described by post-synthetic conjugation,[17] [23] to the best of our knowledge, the synthesis of specialized solid supports for the direct synthesis of 3′-oleyl-oligonucleotides has not been reported yet.

Strategies for the Synthesis of Threoninol Oleic Amide

The incorporation of a lipid at the 3′ end of an oligonucleotide through automated synthesis requires the preparation of an appropriate lipid-modified solid support. For this purpose, we propose l-threoninol ((2R,3R)-2-amino-1,3-butanediol) as a linker molecule for synthesizing the desired lipid conjugates based on its structural properties. As previously described by Asanuma,[25] threoninol exhibits a moderate resemblance to deoxyribose, the pentose present in DNA molecules. Furthermore, threoninol fulfills the requirements of a linker molecule by possessing three reactive groups necessary for incorporating modifications at the 3′ end of oligonucleotides. Moreover, the utilization of threoninol, instead of other molecules such as serinol, enables selective trityl protection of the primary alcohol in the presence of the secondary alcohol. The amino group is essential for the introduction of the desired lipid through an amide bond, while the two reactive hydroxy groups are needed for attachment to the solid support and for the assembly of the oligonucleotide chain.

The synthesis of N-((2R,3R)-1,3-dihydroxybutan-2-yl)oleamide (N-threoninol-oleamide) (2) (Scheme [1]) was achieved by three different routes. The active reagents for the incorporation of oleic acid and the obtained yields are compiled in Table [1].

In the first strategy, the l-threoninol alcohol functions were protected with trimethylsilyl (TMS) groups (transient protection) prior to the reaction of the amine group with the oleyl chloride.[26] In this regard, threoninol was reacted with hexamethyldisilazane to generate bis(TMS)-threoninol, which was then reacted with oleyl chloride to form the amide. Finally, the TMS groups were eliminated by means of ammonia solution to obtain the desired threoninol oleamide. However, hydrolysis of the TMS ethers, during formation of the amide bond, resulted in a mixture of products such as threoninol oleamide contaminated with the corresponding di- and trioleate derivatives of threoninol. The desired compound was isolated from the mixture using a silica gel column, but a yield of only 29% of the desired compound was obtained.

Due to the moderate yield and the formation of these two side products, we designed a second route, which consisted of activation of oleic acid with 1-ethyl-3-(3-dimethylaminoproypl)carbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt), prior to the reaction with the threoninol moiety.[27] This route generated the desired product in a better yield (around 62%), although small amounts of secondary products were also observed.

Finally, the last route relied on the preparation of the 4-nitrophenyl oleate by reacting oleyl chloride with 4-nitrophenol.[28] The resulting compound was used directly in the reaction with l-threoninol in dioxane at 40 °C. This method allowed us to obtain the desired product with the highest yield (72%) among the three studied procedures, and prevented the formation of side products without the need for protection of the hydroxy groups of l-threoninol.

Synthesis of the Solid Support Functionalized with Oleic Acid

Before the incorporation of the threoninol-oleamide on the solid support, its primary hydroxy group was protected. Two different groups were incorporated at position 1 of the threoninol-oleamide: 4,4′-dimethoxytrityl (DMT) and trityl (Scheme [1]).[29] In both cases, the corresponding chloride derivative in the presence of 4-dimethylaminopyridine (DMAP) or Et₃N at 45 °C afforded the respective 1-protected compounds. Although the reaction with DMT chloride gave the desired product, we do not recommend the use of the DMT group since we observed that it is very unstable and great care had to be taken during the silica gel purification step to prevent its removal.

Incorporation of the 1-trityl-threoninol-oleamide 3 to the controlled pore glass (CPG) solid support allows its use in oligonucleotide solid-phase synthesis, and thus for the preparation of the 3′-end-lipid-modified oligonucleotides. To this end, the hemisuccinate derivative 4 of 1-trityl-threoninol-oleamide was prepared.[30] This process took place by reacting the secondary alcohol at position 3 of 1-trityl-threoninol-oleamide 3 with succinic anhydride in the presence of DMAP in anhydrous CH₂Cl₂ at room temperature overnight (Scheme [1]).[31] This compound was used to functionalize the long-chain alkylamine–controlled pore glass support (LCAA–CPG) yielding the desired solid support functionalized with oleic acid. The trityl loading on the derivatized support was determined by the acid treatment method and the degree of functionalization of the support was 60 μmol/g, which represents a yield of around 60%.

Synthesis of Oligonucleotides

Penta or decafloxuridine oligomers were developed by Gmeiner as prodrugs that generate floxuridine monophosphate inside cells after nuclease digestion,[4] [22] being a solution to the cellular resistance observed with 5-fluorouracil (5FU).[32] The action of 2′-deoxy-5-fluorouridine (FdU) is based on the inhibition of both DNA topoisomerase I and thymidylate synthase, showing higher effectiveness than 5-FU in the treatment of cancer.[33]

Aiming to improve the cellular uptake of FdU therapeutic oligonucleotides, we prepared a series of different FdU oligonucleotides modified with oleyl at the 3′ end (Scheme [2]) and the corresponding control sequences (Table [2]). Furthermore, some oligonucleotides were fluorescently labelled at the 5′ end with fluorescein (FAM) to study their cellular internalization. The modified oligonucleotides were synthetized in-house with an automated DNA synthesizer using the prepared oleyl-functionalized CPG solid support and the standard floxuridine phosphoramidite.

The corresponding lipid oligonucleotide conjugates were detached from the solid support under ammonia treatment and purified according DMT-on protocols.

The purities of the oligonucleotides were subsequently assessed by HPLC (Figure [1]), expecting higher retention times for the lipid conjugates (derived from the hydrophobicity conferred by the oleyl moiety). The identities of the purified conjugates were confirmed by MALDI-TOF (Table [2]).

Cellular Uptake of the Different Oleyl Conjugates

Next, the biological properties of 3′-oleyl conjugates were studied. Firstly, the internalization of the 3′-oleyl-oligonucleotide conjugates, along with those of the corresponding control oligonucleotides, was assessed by flow cytometry in HCC2998 (colorectal cancer) and HepG2 (hepatic cancer) cell lines. The results revealed that FdU_n-oleyl conjugates were better internalized than T_n-oleyl and unmodified oligonucleotides in both cell lines (Figure [2]). Moreover, longer oligonucleotide (n = 10) oleyl conjugates were better internalized than their unmodified analogues, while shorter oligonucleotides (n = 5) had a similar behavior in HepG2 cells, although barely any differences were observed in the HCC2998 cell line. There is a plausible explanation to this finding, as it has been reported that the internalization of shorter oligonucleotides can be facilitated by gymnosis.[34] Consequently, although the incorporation of oleyl into the oligonucleotide could improve its cellular entry by other means, it may have no relevant impact on this cell line, as gymnosis prevails. In addition, we also found that the cellular uptake of T₁₀-oleyl and T₅-oleyl was lower than that of their FdU analogues. This suggests that the cellular uptake of oligonucleotides containing FdU is more efficient than that of deoxythymidine oligonucleotides. This finding is in good agreement with previous studies, in which fluorinated oligonucleotides also showed increased internalization.[35] Therefore, the presence of fluorine atoms in oligonucleotides bearing FdU might enhance their cellular uptake.

The cellular uptake of oleyl-oligonucleotide conjugates was studied by fluorescence microscopy (Figure [3]). Higher green fluorescence signals, associated with the cellular internalization of the FAM-oligonucleotides, were observed when cells were treated with the oleyl-oligonucleotide conjugates, in both HCC2998 and HepG2 cell lines. Interestingly, an intracellular accumulation of the green fluorescence signal in the perinuclear region indicates the presence of oligonucleotides in the cell nuclei, where thymidylate synthase is found. Finally, the different cellular distribution of the oleyl-oligonucleotide conjugates, scattered in HepG2 and forming aggregates in HCC2998, suggests that their endocytosis is accomplished via different mechanisms depending on the cell line.

Cytotoxicity of the FdU-Oleyl Conjugates

After the internalization studies, we further studied the cytotoxicity of the different conjugates by the MTT assay (Figure [4]). In previous studies, we have already shown that the lipid-FdU conjugates in the presence of nucleases are degraded after 24 hours, liberating the active antimetabolite.[10] The T₅-oleyl and T₁₀-oleyl oligonucleotide conjugates were nontoxic (data not shown), indicating that oleyl is not cytotoxic, as the viability of both cell lines remained unaltered after 48 hours of exposure.

In contrast, FdU_n oligonucleotides showed clear antiproliferative activity, as expected, and some remarkable differences among them were observed. We detected a lower mortality associated with FdU₅-oleyl, when compared to FdU₁₀-oleyl. This situation can be rationally understood because both oligonucleotides yield the cytotoxic agent FdUMP upon cellular degradation. Thus, if the same concentration of FdU₅-oleyl and FdU₁₀-oleyl is added, FdU₁₀-oleyl can provide double the quantity of FdUMP compared to FdU₅-oleyl, resulting in the observed higher cytotoxicity.

Moreover, statistically significant differences between the antiproliferative activity of the FdU₁₀ and FdU₁₀-oleyl oligonucleotides were identified on the nanomolar scale in the studied cell lines (see Figure S1 in the Supporting Information). In both cases, the lipid-oligonucleotide conjugate was more cytotoxic than its unmodified analogue, which is consistent with the aforementioned higher cellular uptake of the oleyl-oligonucleotide conjugates.

Finally, a different sensitivity of the cell lines to the therapeutic oligonucleotides is recognized. If we consider HepG2 cells and the 100 nM oligonucleotide concentration, FdU₁₀ causes no mortality and FdU₁₀-oleyl shows moderate cytotoxicity. Besides, in the HCC2998 cell line, FdU₁₀ already results in some mortality, and FdU₁₀-oleyl has a much more pronounced cytotoxic activity. The different antiproliferative behavior could be related to a previously reported higher cytotoxicity in colorectal cancer cells.[22]

In conclusion, we have described, for the first time, three distinct methodologies to prepare customized solid supports for the direct synthesis of 3′-oleyl-oligonucleotides. The threoninol linker described herein is enantiomerically pure and can be prepared from commercially available reagents. However, its smaller size compared with other lipid reagents may add distinctive properties. In the case of necessity, longer spacers such as polyethylene glycol can be introduced between the lipid moiety and the oligonucleotide. The in vitro cell experiments revealed an enhanced cellular uptake of oleyl-modified oligonucleotides, consequently leading to improved therapeutic efficacy of FdU₅ and FdU₁₀ oligonucleotides. These findings provide evidence supporting the fact that modifying therapeutic oligonucleotides with lipid moieties represents a promising approach to enhance their cellular internalization. For these reasons, new strategies to facilitate the incorporation of different moieties, such as lipids, to oligonucleotides in a straightforward manner by providing lipid-functionalized solid supports will simplify the challenge of assembling oligonucleotide-3′ lipid conjugates.

Conflict of Interest

The authors declare no conflict of interest.

• References and Notes

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• 27 Oleic acid (2.19 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (3.29 mmol), 1-hydroxybenzotriazole (HOBt) (3.29 mmol), and diisopropylethylamine (DIPEA) (3.29 mmol) in anhydrous DMF (20 mL) were mixed together under argon. After 5 minutes of stirring, l-threoninol (1.35 mmol) was added and the mixture was stirred overnight at rt. The solvent was evaporated under reduced pressure. The crude was dissolved in CH₂Cl₂ and washed twice with 10% aqueous NaHCO₃ and brine, dried and evaporated. The crude product was purified using flash chromatography on silica gel with a methanol gradient (1% to 4% in CH₂Cl₂) to yield N-threoninol-oleamide 2 as a yellowish oil.
• 28 4-Nitrophenol (10 mmol) was dissolved in CH₂Cl₂ (10 mL) with Et₃N (40 mmol) and oleyl chloride (10 mmol) added under ice-bath conditions. The resulting mixture was stirred at rt for 1 h after removal of the ice bath and then washed sequentially with water, NaHCO₃, and water again. The organic layer was separated, dried and concentrated to give the desired product in 85% yield.¹H NMR (400 MHz, CDCl₃): δ = 8 2 (d, 2 H, HAr), 7.2 (d, 2 H, HAr), 5.2 (m, 2 H, CH=CH), 2.5 (t, 2 H, CH₂-CO), 2 (m, 4 H, CH₂-alkene), 1.7 (t, 2 H, CH₂-CH₂-CO), 1.3-1.2 (n, dd, m, 20H, alkyl chain), 0.8 (t, 3 H, CH₃). 0,82-0,81 (m, 3 H, CH3), C NMR (101 MHz, CDCl₃): δ = 171.29 (CO), 155.54 (Ar-O), 145.26 (Ar-NO₂), 130.11–129.66 (CH=CH), 125.19 (Ar), 122.43 (Ar), 34.34 (CH₂-CO), 31.92 (CH₂-alkene), 29.77, 29.67, 29.54, 29.34, 29.13, 29.07, 29.03, 27.24, 27.23, 27.15, 24.74, 22.69 (CH₂-CH₃, alkyl chain), 14.12 (CH₃). MS (MALDI-TOF): m/z = 503.5 (M + Et₃N).l-threoninol (1 equiv) was dissolved in dioxane (5 mL) under an Ar atmosphere, and Et₃N (4 equiv) and the above-prepared 4-nitrophenyl oleate (2 equiv) were added. The reaction was left to stir overnight at 40 °C. After work-up, the crude product was purified via silica gel column chromatography (gradient 0–3% MeOH/CH₂Cl₂) to afford product 2 as a yellowish oil [R_f = 0 15 (MeOH/CH₂Cl₂, 5:95)].
• 29a Product 2 (1 equiv) and DMAP (0.5 equiv) were dissolved in pyridine (5 mL). DIPEA (2 equiv) was added dropwise and the resulting mixture was stirred for 5 min at rt. Trityl chloride (TrCl) (1.5 equiv) was added and the mixture was heated to 45 °C and stirred overnight in the dark. Further TrCl (0.5 equiv) was added to ensure a complete reaction. Following this, MeOH (1 mL) was added and the mixture was stirred for 5 min. The pyridine was evaporated and work-up was performed. The solution medium was neutralized using saturated NaHCO₃ (30 mL), and subsequently washed with DCM (3 x 30 mL). The organic layer was separated, dried out with MgSO₄, filtered, and concentrated to dryness. The resulting crude residue was purified by flash column chromatography (CH₂Cl₂ to CH₂Cl₂/MeOH 4%), resulting in the expected trityl derivative 3 as a white solid in a yield of 78%.¹H NMR (400 MHz, CDCl₃): δ = 8.54 (s, 1 H, NH), 7.35–7.29 (m, 6 H), 7.27–7.13 (m, 9 H), 6.01 (d, J = 8.7 Hz, 1 H, CH₃CH-OH), 5.31–5.24 (m, 2 H, CH=CH), 4.03–3.98 (m, 1 H, CH₃CH-OH), 3.88–3.83 (m, 1 H, CH-NH), 3.71–3.64 (m, 2 H, CH₂OH), 3.36 (dd, J = 9.6, 4.4 Hz, 1 H), 3.20 (dd, J = 9.6, 3.5 Hz, 1 H), 2.15 (dd, J = 8.5, 6.8 Hz, 4 H, CH₂CH=CH), 1.94–1.93 (m, 2 H, CO-CH₂-CH₂), 1.83–1.75 (m, 2 H, CO-CH₂-CH₂), 1.27–1.20 (m, 20 H, alkyl chain), 1.05 (d, J = 6.3 Hz, 3 H, CH₃-threoninol), 0.86–0.72 (m, 3 H, CH₃-oleyl). ¹³C NMR (101 MHz, CDCl₃): δ = 172.53 (CO), 148.76 , 142.28 (Carom), 134.98 (Carom), 128.98–128.72 (CH=CH), 127.43 (Carom), 127.04 (Carom), 126.88 (Carom), 126.33 (Carom), 122.74 (Carom), 86.28 (Cq), 76.21, 67.67 (CH-NH), 66.95 (CH₃CH-OH), 64.47 (CH₂-O), 52.16 (CH₂-CO), 35.90 (CH₂-CH=CH), 30.88, 28.75, 28.71, 28.68, 28.50, 28.33, 28.30, 28.13, 26.20, 26.17, 24.86, 24.58 (CH₂, alkyl chain), 21.67 (CH₂-CH₃), 18.86 (CH₃-threoninol), 13.11 (CH₃-oleyl).
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Corresponding Authors

Publication History

Received: 20 July 2023

Accepted after revision: 30 October 2023

Article published online:
01 December 2023

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• References and Notes

• 1 Present address: Department of Organic Chemistry Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Santiago de Compostela University CIQUS, Spain.
• 2 Curreri A, Sankholkar D, Mitragotri S, Zhao Z. Bioeng. Transl. Med. 2023; 8: e10374
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• 3 Egli M, Manoharan M. Nucleic Acids Res. 2023; 51: 2529
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• 26 l-Threoninol (5 mmol) was dissolved in DMF (5 mL), treated with HMDS (10 mmol) and then stirred at rt for 30 min. The solution was evaporated to dryness. The crude residue was dissolved in pyridine (15 mL), oleic chloride (2 mL) was added dropwise and the mixture was stirred at rt for 1 h. The pyridine was evaporated and the crude residue was dissolved in CH₂Cl₂ and washed with brine. After concentration of the organic layer, the residue was dissolved in dioxane/MeOH (1:1 + NH₃ 25%) and evaporated. Purification by flash chromatography (CH₂Cl₂/MeOH 1% to 4%) yielded N-threoninol-oleamide 2. ¹H NMR (400 MHz, CDCl₃): δ = 6.34 (d, J = 8.5 Hz, 1 H, CH-OH), 5.39–5.25 (m, 2 H, CH=CH), 4.20 (s, 1 H, CH₃-CH-OH), 3.81 (s, 4 H, CH₂-NH, CH₂-OH), 3.04 (s, 1 H, NH), 2.27 (t, 4 H, CH₂CH=CH), 2.02 (m, 2 H, CH₂CO), 1.64 (m, 2 H, CH₂CH₂CO), 1.37–1.29 (m, 20 H, CH₂-oleic), 1.21 (d, J = 6.4 Hz, 3 H, CH₃-threoninol), 0.91(t, J = 13.9 Hz, 3 H, CH₃-oleyl). ¹³C NMR (125 MHz, CDCl₃): δ = 174.42 (CO), 130.0–129.72 (CH=CH), 68.53 (CH-NH), 64.85 (CH₃CH-OH), 54.63 (CH₂-O), 36.9 (CH₂-CO), 31.91 (CH₂-CH=CH), 29.78, 29.73, 29.34, 29.32, 29.29, 29.18, 27.24, 27.19, 25.88 (CH₂, alkyl chain), 22.69 (CH₂-CH₃), 20.50 (CH₃-threoninol), 14.13 (CH₃-oleyl).
• 27 Oleic acid (2.19 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (3.29 mmol), 1-hydroxybenzotriazole (HOBt) (3.29 mmol), and diisopropylethylamine (DIPEA) (3.29 mmol) in anhydrous DMF (20 mL) were mixed together under argon. After 5 minutes of stirring, l-threoninol (1.35 mmol) was added and the mixture was stirred overnight at rt. The solvent was evaporated under reduced pressure. The crude was dissolved in CH₂Cl₂ and washed twice with 10% aqueous NaHCO₃ and brine, dried and evaporated. The crude product was purified using flash chromatography on silica gel with a methanol gradient (1% to 4% in CH₂Cl₂) to yield N-threoninol-oleamide 2 as a yellowish oil.
• 28 4-Nitrophenol (10 mmol) was dissolved in CH₂Cl₂ (10 mL) with Et₃N (40 mmol) and oleyl chloride (10 mmol) added under ice-bath conditions. The resulting mixture was stirred at rt for 1 h after removal of the ice bath and then washed sequentially with water, NaHCO₃, and water again. The organic layer was separated, dried and concentrated to give the desired product in 85% yield.¹H NMR (400 MHz, CDCl₃): δ = 8 2 (d, 2 H, HAr), 7.2 (d, 2 H, HAr), 5.2 (m, 2 H, CH=CH), 2.5 (t, 2 H, CH₂-CO), 2 (m, 4 H, CH₂-alkene), 1.7 (t, 2 H, CH₂-CH₂-CO), 1.3-1.2 (n, dd, m, 20H, alkyl chain), 0.8 (t, 3 H, CH₃). 0,82-0,81 (m, 3 H, CH3), C NMR (101 MHz, CDCl₃): δ = 171.29 (CO), 155.54 (Ar-O), 145.26 (Ar-NO₂), 130.11–129.66 (CH=CH), 125.19 (Ar), 122.43 (Ar), 34.34 (CH₂-CO), 31.92 (CH₂-alkene), 29.77, 29.67, 29.54, 29.34, 29.13, 29.07, 29.03, 27.24, 27.23, 27.15, 24.74, 22.69 (CH₂-CH₃, alkyl chain), 14.12 (CH₃). MS (MALDI-TOF): m/z = 503.5 (M + Et₃N).l-threoninol (1 equiv) was dissolved in dioxane (5 mL) under an Ar atmosphere, and Et₃N (4 equiv) and the above-prepared 4-nitrophenyl oleate (2 equiv) were added. The reaction was left to stir overnight at 40 °C. After work-up, the crude product was purified via silica gel column chromatography (gradient 0–3% MeOH/CH₂Cl₂) to afford product 2 as a yellowish oil [R_f = 0 15 (MeOH/CH₂Cl₂, 5:95)].
• 29a Product 2 (1 equiv) and DMAP (0.5 equiv) were dissolved in pyridine (5 mL). DIPEA (2 equiv) was added dropwise and the resulting mixture was stirred for 5 min at rt. Trityl chloride (TrCl) (1.5 equiv) was added and the mixture was heated to 45 °C and stirred overnight in the dark. Further TrCl (0.5 equiv) was added to ensure a complete reaction. Following this, MeOH (1 mL) was added and the mixture was stirred for 5 min. The pyridine was evaporated and work-up was performed. The solution medium was neutralized using saturated NaHCO₃ (30 mL), and subsequently washed with DCM (3 x 30 mL). The organic layer was separated, dried out with MgSO₄, filtered, and concentrated to dryness. The resulting crude residue was purified by flash column chromatography (CH₂Cl₂ to CH₂Cl₂/MeOH 4%), resulting in the expected trityl derivative 3 as a white solid in a yield of 78%.¹H NMR (400 MHz, CDCl₃): δ = 8.54 (s, 1 H, NH), 7.35–7.29 (m, 6 H), 7.27–7.13 (m, 9 H), 6.01 (d, J = 8.7 Hz, 1 H, CH₃CH-OH), 5.31–5.24 (m, 2 H, CH=CH), 4.03–3.98 (m, 1 H, CH₃CH-OH), 3.88–3.83 (m, 1 H, CH-NH), 3.71–3.64 (m, 2 H, CH₂OH), 3.36 (dd, J = 9.6, 4.4 Hz, 1 H), 3.20 (dd, J = 9.6, 3.5 Hz, 1 H), 2.15 (dd, J = 8.5, 6.8 Hz, 4 H, CH₂CH=CH), 1.94–1.93 (m, 2 H, CO-CH₂-CH₂), 1.83–1.75 (m, 2 H, CO-CH₂-CH₂), 1.27–1.20 (m, 20 H, alkyl chain), 1.05 (d, J = 6.3 Hz, 3 H, CH₃-threoninol), 0.86–0.72 (m, 3 H, CH₃-oleyl). ¹³C NMR (101 MHz, CDCl₃): δ = 172.53 (CO), 148.76 , 142.28 (Carom), 134.98 (Carom), 128.98–128.72 (CH=CH), 127.43 (Carom), 127.04 (Carom), 126.88 (Carom), 126.33 (Carom), 122.74 (Carom), 86.28 (Cq), 76.21, 67.67 (CH-NH), 66.95 (CH₃CH-OH), 64.47 (CH₂-O), 52.16 (CH₂-CO), 35.90 (CH₂-CH=CH), 30.88, 28.75, 28.71, 28.68, 28.50, 28.33, 28.30, 28.13, 26.20, 26.17, 24.86, 24.58 (CH₂, alkyl chain), 21.67 (CH₂-CH₃), 18.86 (CH₃-threoninol), 13.11 (CH₃-oleyl).
• 30 Compound 3 (1 equiv) was dried (2 × toluene, 2 × MeCN) and dissolved in anhydrous CH₂Cl₂. Succinic anhydride (1.3 equiv) and DMAP (1.3 equiv) were added and the reaction mixture was stirred overnight at rt. The organic layer was washed with NaH₂PO₃ (0.1 M) and dried. The resulting crude residue was used in the next step without further purification.
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• 31b In brief, hemisuccinate 4 (1 equiv) and DMAP (1.3 equiv) were dissolved in MeCN. This solution was mixed with 2,2-dithiobis(5-nitropyridine) (1 equiv) dissolved in MeCN/CH₂Cl₂ (1:3). Finally, PPh₃ (1.2 equiv) dissolved in MeCN was added. The resulting mixture was vortexed briefly and then poured into a vial containing LCAA-CPG (70 μmol/g), which had been pre-washed with MeCN. After a 2-hour reaction, the support was washed with methanol and diethyl ether and then dried.
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