Oligonucleotides Containing 1-Aminomethyl or 1-Mercaptomethyl-2-deoxy-d-ribofuranoses: Synthesis, Purification, Characterization, and Conjugation with Fluorophores and Lipids.

Oligonucleotide conjugates are widely used as therapeutic drugs, gene analysis, and diagnostic tools. A critical step in the biologically relevant oligonucleotide conjugates is the design and synthesis of functional molecules that connect oligonucleotide with ligands. Here, we report the synthesis and application for oligonucleotide functionalization of novel tethers based on aminomethyl and mercaptomethyl sugar derivatives. Starting from a common cyano sugar precursor, three novel phosphoramidites have been prepared in the two α- and β-anomeric forms. The mercaptomethyl sugar was protected with the S-acetyl group, while two different protecting groups have been developed for the aminomethyl sugar. These two protecting groups are orthogonal, as they can be removed independently using photolysis or ammonolysis. This combination allowed the introduction of two different ligands in a single oligonucleotide.

Introduction

Therapeutic oligonucleotides have tremendous potential for treating a variety of diseases if they can reach the target cells successfully upon administration. More recently, this task has been accomplished by covalent conjugation of peptides, lipids, and GalNAc to oligonucleotides.[1,2] Often, preparation of these conjugates requires the presence of a reactive group such as an amino or thiol group within an oligonucleotide.[3−5] The therapeutic applications of oligonucleotides have triggered a high demand for oligonucleotide conjugates with enhanced active or passive targeting properties and with the possibility to achieve tissue-specific delivery.[6−8] Toward this end, researchers are developing nucleosidic and non-nucleosidic phosphoramidite derivatives that enable efficient preparation of oligonucleotide conjugates.[3,9] Some of the conventional strategies are postsynthetic protocols where a reactive group is added to the oligonucleotide. This approach has been employed for the preparation and screening of several conjugates using a common reactive species.[6] Some of the most common reactive groups used for the preparation of oligonucleotide conjugates are amino and thiol groups, although a large number of reactions using click chemistry have been also developed.[10]

Amino groups react readily with carboxylic acid derivatives via amide formation as well as with isothiocyanates to form thioureas.[11] Although nucleobases have amino functions, these groups are aromatic amines and have low reactivity. For this reason, it is possible to use primary alkylamino groups for the selective introduction of ligands to oligonucleotides. Aminoalkylalcohols, such as 6-aminohexanol[6,12] or 5′-amino-2′,5′-dideoxynucleoside[13] derivatives, are utilized for the introduction of amino groups at the 5′-end. However, the introduction of amino groups at the 3′-end or at internal positions of oligonucleotides requires the use of aminoalkyldiols such as 2-amino-1,3-propanediol[14] or 2-aminobutyl-1,3-propanediol derivatives.[15]

On the other hand, thiol groups have a selective reactivity with maleimide and haloacetamide derivatives to form thioethers.[11] The introduction of thiol groups in oligonucleotides is usually done by preparing 3-mercaptopropanol and 6-mercaptohexanol derivatives protected either by trityl[16] or disulfide groups.[17,18]

We have recently described the synthesis of novel 1′-homo-N-2′-deoxy-α-nucleosides[19] and 1β-[(thymin-1-yl)acetylaminomethyl]-1,2-dideoxy-d-erythro-pentofuranose as model compounds for nucleosides containing an extended link between the ribose and the nucleobase.[20] These nucleoside derivatives are prepared from the cyano sugar derivatives (1α or 1β) which can be used as common and valuable intermediates for the synthesis of amino (2α or 2β) and thiol (14α or 14β) linkers for the introduction of reactive groups into oligonucleotides. Aminomethyl and mercaptomethyl sugar derivatives are ideal linker molecules, because they are cyclic aminodiol or mercaptodiol compatible with oligonucleotide synthesis. These sugar derivatives can be obtained in a defined stereochemistry as single α- or β-anomers, and they can be conveniently introduced at any position within the oligonucleotide. Additionally, utilization of the 2-deoxyribose framework offers an unique advantage of maintaining normal distance between two nucleosidic units when incorporated in the middle of an oligonucleotide. Here, we describe the synthesis of several solid supports and phosphoramidite derivatives of aminomethyl and mercaptomethyl sugar derivatives and the use of these solid supports and phosphoramidites for the preparation of amino- and mercapto-oligonucleotides. Another objective of the present work is the study of orthogonal protecting groups in order to synthesize oligonucleotide conjugates carrying two or more distinct ligands. Specifically, we studied the base-labile trifluoroacetyl and the photolabile 1-(2-nitrophenyl)ethoxycarbonyl (NPEC) groups for the aminomethyl sugar derivative and the base labile acetyl group for the mercaptomethyl sugar derivative. Several oligonucleotides carrying lipid and fluorescent compounds are prepared to demonstrate the utility of the novel phosphoramidites described in this work.

Results

Synthesis of 1-Functionalized 1,2-Dideoxy-d-erythro-pentofuranose Phosphoramidites 5α/5β, 8α/8β, and 16α/16β

The synthesis of phosphoramidites was carried out starting from α- or β-cyano sugar derivative 1 (Scheme ), which is easily accessible to perform on a large scale.[21]

Scheme 1

Synthesis of 1-Trifluoroacetylaminomethyl-1,2-dideoxy-d-erythro-pentofuranosyl-3-phosphoramidites

Reagents and conditions: (a) LiAlH4, THF, reflux, 4 h; (b) Ethyl trifluoroacetate, Et3N, DMF, 80 °C, 24 h, 70% (3α) and 80% (3β) two steps; (c) DMTCl, Et3N, 1,4-dioxane, 30 °C, 2 h, 65% (4α) and 70% (4β); (d) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, Pr2NEt, CH2Cl2, rt, 1 h, 84% (5α) and 70% (5β).

Treatment of the latter with LiAlH4 in THF at reflux enabled simultaneous reduction of the cyano group and cleavage of the toluoyl groups, furnishing amino diol 2α/2β. Subsequent protection of the amino group with ethyl trifluoroacetate in Et3N and DMF at 80 °C gave 3α or 3β in 70% and 80% yield, respectively, from the starting substrate 1α/1β. Next, protection of the primary alcohol with 4,4′-dimethoxytrityl chloride in the presence of Et3N and 1,4-dioxane at 30 °C afforded the respective DMT-protected compounds 4α (65% yield) or 4β (70% yield). Phosphitylation of 4 with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite gave the desired phosphoramidite derivatives 5α or 5β in 84% and 70% yield, respectively.

Preparation of phosphoramidites of 1α- and 1β-aminomethyl-1,2-dideoxy-d-erythro-pentofuranoses bearing a photolabile protecting group at the amino function is outlined in Scheme . The amino diol 2 was reacted with 1-(2-nitrophenyl)ethyl-N-succinimidyl carbonate[22] to afford carbamates 6α (55% yield) or 6β (50% yield). As above, protection of the primary alcohol with DMT group yielded 7α/7β, and subsequent phosphitylation gave derivatives 8α or 8β in 78% and 72% yield, respectively.

Scheme 2

Synthesis of 1-Aminomethyl-1,2-dideoxy-d-erythro-pentofuranosyl-3-phosphoramidites Bearing a Photolabile Protecting Group at the Amino Function

Reagents and conditions: (a) 1-(2-Nitrophenyl)ethyl N-succinimidyl carbonate, Et3N, MeOH, 30 °C, 1 h, 55% (6α) and 50% (6β); (b) DMTCl, Et3N, 1,4-dioxane, 35 °C, 2 h, 80% (7α) and 80% (7β); (c) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, Pr2NEt, CH2Cl2, rt, 1 h, 78% (8α) and 72% (8β).

The synthetic protocol for the 1-S-mercaptomethyl-1,2-dideoxy-d-erythro-pentofuranosyl-3-O-phosphoramidites 16α or 16β is summarized in Scheme . The nitriles 1α/1β were treated with potassium hydroxide in MeOH/H2O. Under these conditions, hydrolysis of nitrile and in situ esterification in addition to the removal of the toluoyl protecting groups generated esters 9α or 9β in 85% and 75% yield, respectively. Then, alcohol groups were protected as tert-butyldimethylsilyl ether to give derivatives 10α/10β. The reduction of esters 10 with lithium aluminum hydride in THF at −45 °C afforded alcohols 11α (90% yield) or 11β (70% yield), which were transformed into the tosylates 12α/12β by treatment with p-toluensulfonyl chloride and catalytic DMAP in pyridine. The displacement of the tosylate group with potassium thioacetate in DMF afforded the thioesters 13α or 13β in 70% and 75% yields, respectively. Next, deprotection of the silyl groups with (−)-CSA in MeOH gave alcohols 14α/14β. Each isomer was transformed in the phosphoramidites 16α or 16β after DMT protection of the primary hydroxyl giving place to 15α/15β and phosphitylation of the secondary hydroxyl group.

Scheme 3

Synthesis of 1-S-Acetylmercaptomethyl-1,2-dideoxy-d-erythro-pentofuranose Phosphoramidites

Reagents and conditions: (a) KOH, MeOH, H2O, 25 °C, 3 h, 85% (9α) and 75% (9β); (b) TBSCl, Imidazole, CH2Cl2, 50 °C, 5 h, 85% (10α) and 80% (10β); (c) LiAlH4, THF, −45 °C, 0.5 h, 90% (11α) and 1 h, 70% (11β); (d) TsCl, DMAP, Py, 0 °C → rt, 9 h, 90% (12α) and 80% (12β); (e) Potassium thioacetate, DMF, 65 °C, 6 h, 70% (13α) and 75% (13β); (f) (−)-CSA, MeOH, 0 °C → rt, 2 h, 80% (14α) and 80% (14β); (g) DMTCl, Et3N, 1,4-dioxane, 30 °C, 2 h, 80% (15α) and 85% (15β); (h) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, Pr2NEt, CH2Cl2, rt, 1 h, 72% (16α) and 68% (16β).

Synthesis of Solid Supports Functionalized with 1,2-Dideoxy-d-erythro-pentofuranose Monomers 4α/4β, 7α/7β, or 15α/15β

In order to connect 1,2-dideoxy-d-erythro-pentofuranose monomers 4α/4β, 7α/7β, and 15α/15β to the oligonucleotides on their 3′-end, we prepared the appropriate solid supports carrying these different derivatives. For this reason, the secondary alcohol at position 3 of the pentafuranose ring of each one of these derivatives was reacted with succinic anhydride yielding the corresponding succinate derivatives 17α/17β, 18α/18β, and 19α/19β (Scheme ). These compounds were used to functionalize the amino-controlled pore glass support (LCAA-CPG) to yield the CPG solid supports 20α/20β, 21α/21β, and 22α/22β.

Scheme 4

Preparation of CPG Solid Supports Functionalized with 1-Aminomethyl- or 1-Mercaptomethyl-1,2-dideoxy-d-erythro-pentofuranoses

Reagents and conditions: (a) Succinic anhydride, DMAP, rt, overnight; (b) 2,2′-Dithio-bis(5-nitropyridine), Ph3P, LCAA-CPG, rt, 2 h (20-25 μmol/g).

Synthesis, Purification, and Characterization of Oligonucleotides Incorporating 4α/4β, 7α/7β, or 15α/15β 1,2-Dideoxy-d-erythro-pentofuranose Monomers

The phosphoramidites 5α/5β, 8α/8β, and 16α/16β and solid supports 20α/20β, 21α/21β, and 22α/22β were used to prepare oligonucleotides containing these modified nucleotides either at the 3′-end or at the 5′-end of the sequence. All of the sequences shown in Table were made on the automated DNA synthesizer using standard protocols.[23] The short model sequence RS carrying the four natural bases was prepared to study their stability during all of the synthesis process and to obtain the optimal cleavage conditions. Next, we used all the derivatives to prepare the gapmer oligonucleotides, which contained the complementary sequence of the Renilla luciferase gene modified at their ends with 2′-O-methyl-RNA. Often, gapmer oligonucleotides are used for antisense gene expression inhibition experiments.

Table 1

Sequence of Oligonucleotides and Its Characterization by MALDI-TOFa

code	sequences (5′ → 3′)	MW (calcd)	MW (found)
RS4α	CATTGTCCA-4α	2880.5	2880.3
RS7α	CATTGTCCA-7α	2880.5/3072.5b	2880.5/3073.5b
RS7β	CATTGTCCA-7β	2880.5/3072.5b	2880.5/3073.5b
RS15α	CATTGTCCA-15α	2897.5/5792.1c	2897.2
Gapmer4α	cguuTCCTTTGTTCugga-4α	5865	5853.8
Gapmer4β	cguuTCCTTTGTTCugga-4β	5865	5854.5
Gapmer7α	cguuTCCTTTGTTCugga-7α	5866/6061b	5864.6
Gapmer7β	cguuTCCTTTGTTCugga-7β	5866/6061b	5855/6057
Gapmer15α	cguuTCCTTTGTTCugga-15α	5884	5880.8
Gapmer15β	cguuTCCTTTGTTCugga-15β	5883	5880.5
4αGapmer	4α-cguuTCCTTTGTTCugga	5865	5864.5
4βGapmer	4β-cguuTCCTTTGTTCugga	5865	5866.8
7αGapmer	7α-cguuTCCTTTGTTCugga	5866	5865/6042
7βGapmer	7β-cguuTCCTTTGTTCugga	5866	5849/6057
15αGapmer	15α-cguuTCCTTTGTTCugga	5883	5878
15βGapmer	15β-cguuTCCTTTGTTCugga	5883	5880.8
7αGapmer4α	7β-cguuTCCTTTGTTCugga-4α	6075/6268	6074.0/6267.6

Sequences of the synthesized oligonucleotide with the 2-deoxy-d-ribofuranose derivatives. T, G, C are 2′-deoxynucleotides. a, c, g, u are 2′-OMe-nucleotides.

Expected MW with a photolabile protecting group.

Expected MW of the dimer form with a disulfide bridge.

Next, the two RS oligonucleotides containing the aminomethyl 1,2-dideoxy-d-erythro-pentofuranoses (4α and 7β) were treated with an ammonia solution overnight at 55 °C. The resulting crudes were analyzed by HPLC and characterized by MALDI-TOF. As expected, RS4α gave a unique peak with the correct mass which corresponds to the desired product deprotected. In the case of oligonucleotide RS7β, a side peak was present in the HPLC profile. Both products were collected and analyzed by mass spectrometry. The product with higher retention time corresponds to the desired product protected with the photolabile protecting group, and the minor product is the RS7β deprotected. This result indicated that the photolabile group is very sensitive to the light, and extra precautions, like working in the dark, need to be considered during deprotection in order to prevent its cleavage. The HPLC profiles are depicted in Figure , and the MW are shown in Table .

Figure 1

HPLC profiles of model oligonucleotides modified in the 3′-end with 4α, 7β, and 15α 1,2-dideoxy-d-erythro-pentofuranose derivatives. ACE 3 μm HILA-3-1546-A column was used.

In the case of the oligonucleotide RS15α, some modifications in the deprotection process were introduced to prevent side products. First, it was treated with a DBU solution followed by a wash with a 5% solution of Et3N. This treatment was necessary to remove the cyanoethyl protecting groups, as they can react with the free thiol function of the 15α sugar giving the cyanoethylmercapto derivative as a byproduct. Next, it was treated with an ammonium solution containing 0.1 M DTT overnight at 55 °C to avoid dimerization. The HPLC analysis presented a unique peak, with the mass corresponding to the correct product. The optimal deprotection conditions found for each derivative were used for the deprotection of all the other gapmer sequences. The mass for the resulting products are shown in Table . All the gapmer sequences were obtained in a good yield which ranged 42–96%. The HPLC chromatograms of the 5′- and 3′-aminomethyl-modified gapmers are shown in Figure . The two isomeric forms (α- and β-) can be perfectly distinguishes by their different retention times in the HPLC profiles. These results confirmed the enantiomeric purity of these two novel α- and β-amino-linkers.

Figure 2

HPLC profiles of the Gapmer oligonucleotides modified with the amino group: (A) 5′-modified Gapmer and (B) 3′-modified Gapmer. In blue and red are drawn the α and β isomer forms, respectively. ACE 3 μm HILA-3-1546-A column was used.

Removal of the Photolabile Protecting Group in Modified Oligonucleotides with 7α and 7β Monomers

We studied the efficiency in the removal of the photolabile protecting group NPEC of the 7α and 7β oligonucleotide derivatives attached to the solid support and when they were already cleaved from the resin in order to compare both systems. In both cases, the modified gapmers were exposed to irradiation at 340 nm for different periods of time. As shown in Table , the NPEC protecting group needed a longer time to be removed when the 7α and 7β derivatives were attached to the solid support versus in solution. However, after 2 h of reaction the NPEC group was completely removed from the solid support, and no difference was observed between 7α and 7β derivatives. These results confirmed that the presence of the solid support does not interfere in the formation of the free amino oligonucleotide derivative product attached to it, allowing further coupling reactions in the solid phase.

Table 2

Data from the Kinetic Studies for the Removal of the NPEC of 7α and 7β Gapmers

photolysis	on the CPG supporta		in solution phaseb
reaction time (min)	30	60	15	30	45	60
Gapmer7α (%)	78	91	60	89	100	100
Gapmer7β (%)	71	92	80	93	99	100

Deprotection reaction on the CPG support was realized with 2 mg of resin.

Deprotection reaction in solution was realized with 2 mg of oligonucleotide.

Preparation of Oligonucleotide Conjugates

The incorporation of fluorescent and delivery elements to oligonucleotides is important for the development of new diagnostic and therapeutic tools. The introduction of functional groups with orthogonal deprotection procedures is essential in order to incorporate multiple elements in the same oligonucleotide. In this case, the presence of NPEC in 7α- and 7β-1,2-dideoxy-d-erythro-pentofuranose derivatives allowed conjugation reaction directly on the solid support.

Prior to the incorporation of delivery elements to these modified oligonucleotides in the solid support, the gapmer4α and the RS4α oligonucleotides containing the amino derivative in the 3′-end was conjugated with fluorescein (FITC) and two different types of fatty acids (palmitic and oleic acids) in solution, respectively. The incorporation of the FITC and the two fatty acids was done by the reaction of the free amines of the modified nucleotide in the oligonucleotides with fluorescein isothiocyanate and the pentafluorophenyl ester of each one of the fatty acids. Before the conjugation reaction took place, the pentafluorophenyl esters of the oleate (25a) and palmitate (25b) were prepared as described in the literature[6,24−26] with a 93% and 96% yield, respectively (Scheme ).

Scheme 5

Synthesis of Pentafluorophenyl Esters of Fatty Acids

Next, the gapmer 4α oligonucleotide was treated with fluorescein isothiocyanate (FITC) and the RS4α was treated with the pentafluorophenyl oleate or palmitate in different buffer conditions to evaluate the influence of an organic cosolvent in the final yield of the oligonucleotide conjugate. In the case of the RS4α–fatty acids conjugation, DMF was added to the mixture of the carbonate buffer/acetonitrile solution to increase the relative amount of organic phase in the reaction. HPLC analysis revealed the presence of a product with a higher retention time then the free amino oligonucleotides in all the cases, and its mass corresponded with the desired conjugates (Table ). However, conjugate gapmer4α–FITC was only obtained in a 50% yield with respect to the 76% and 64% yield of the RS4α–fatty acid conjugates. These results indicate that the solubility of the fatty acid in the reaction conditions was crucial to improve the final yield of the conjugates.

Table 3

Oligonucleotide Conjugates and Their Characterization by MALDI-TOF

oligonucleotide-conjugates	yield (%)	MW (calc)	MW (found)
Gapmer4α-FITC	50	6256.1	6258
RS4α-Oleic	76	3146	3145.6
RS4α-Palmitic	64	3121	3123.6
RS7α-Palmitica	61	3121	3124.0
RS7α-Palmiticb	19	3121	3124.0
Palmitic-7αGapmera	72	6108	6107.5
Palmitic-7αGapmerb	66	6108	6107.5
Palmitic-7αGapmerc	46	6108	6107.5
FITC-7αGapmer4α	6	6463	6465.2
FITC-7αGapmer4α-Oleic	20	6728	6742.7

Both photolysis and conjugation in solution.

Photolysis over the solid support and conjugation in solution.

Both photolysis and conjugation on the solid support.

Next, we evaluated the conjugation of the RS7α and 7αgapmer over the solid support. These two 7α-modified oligonucleotides were incubated with pentafluorophenyl palmitate in solution or on the solid support, in order to compare the reaction efficiency between both strategies. All the products were HPLC purified and characterized by mass spectrometry. The yields obtained in the conjugation of the fatty acid with amino-oligonucleotides are shown in Table . The result showed that RS7α-palmitic is only obtained with the desired yield (61%) when the reaction was done in solution. One of the reasons for the low yields could be due to the steric hindrance of the amino at the 3′-position with the solid support which reduces the conjugation efficiency. These results were confirmed as the palmitic-7αgapmer conjugate was obtained in the solid support when the 7α-modified nucleotide was in its 5′-end. However, the conjugation reaction is less efficient (46%) than when the reaction is carried out in solution with a 66% yield. Despite this fact, solid phase is still a useful method for conjugation reactions due to its shorter reaction times and efficient removal of the excess of reagents, and because it allows orthogonal conjugation reactions with multiple elements.

Finally, to investigate the possibility of preparing an oligonucleotide with two distinct ligands, we carried out the conjugation in an orthogonal manner of a lipid and a fluorescent compound at each end of the 7αgapmer4α (Scheme ). For this purpose, the 7αgapmer4α modified at each end with the same nucleosidic derivative but with different protecting groups was used. First, irradiation of the gapmer bound to the solid support gave place selectively to the free amino group at 5′-end. Then, the resulting oligonucleotide was incubated with fluorescein (FITC) on the solid support, followed by the deprotection of 3′-trifluoroacetylamino group, which also liberated the oligo from the support. The FITC-7αgapmer4α 3′-amino was conjugated with the pentafluorophenyl oleate in solution. The final product was HPLC purified and characterized by mass spectrometry. The yield obtained is shown in Table . These results are a step forward to obtain multiple functionalized oligonucleotides for diagnostic and therapeutic applications.

Scheme 6

Synthesis of an Oligonucleotide Carrying Both a Fluorophore (FITC) and a Lipid (Oleic)

Discussion

During the past decade, we have witnessed large interest in oligonucleotide conjugates for gene analysis and therapeutic application. An important step in the production of these conjugates is the design, preparation, and functionalization of linking molecules for the connection of the ligand to the oligonucleotide. Here, we describe the synthesis of a novel series of connecting stereospecific linkers based on cyano sugar ribose precursors that can be obtained in the pure form in the two possible (α- and β-) isomeric forms. To this end, we described the synthesis of the appropriate reagents for oligonucleotide synthesis following solid-phase phosphoramidite chemistry. First, the synthesis of the aminomethyl sugar derivative from the ditoluoyl cyano-1,2-dideoxy-d-erythro-pentofuranose (1α/1β) is described. Conversion of the cyano group to the aminomethyl group is achieved in a single step that removed the toluoyl protecting group at the same time. The resulting aminomethyl sugar was protected with the base-labile trifluoroacetyl and the photolabile moieties. Second, the transformation of the cyano to the mercaptomethyl group required multiple synthesis steps. The synthesis protocol began with the conversion of the cyano group to the methyl carboxylate followed by reduction to hydroxymethyl group. Tosylation of the hydroxyl group followed by nucleophilic displacement with potassium thioacetate yielded the desired S-acetyl derivative. Then, both sugar derivatives were protected at the primary alcohol with the DMT group and were processed with the conventional methods to obtain the desired phosphoramidites and the corresponding functionalized CPG solid supports. The novel reagents are compatible with solid-phase synthesis protocols providing the desired amino or thiolated functionalized oligonucleotides (Table ).

This demonstrated the usefulness of the novel amino linkers for the preparation of lipid– and fluorescent–oligonucleotide conjugates. The development of two different and orthogonal protecting groups for the aminomethyl-oligonucleotides allows the introduction of two different ligands in a single oligonucleotide.

The novel linkers developed in this work (Figure ) are enantiomerically pure, semirigid, hydrophilic, and totally compatible with nucleic acid structural properties. Several amino and thiol linkers have been described in the literature.[5] The simplest linkers are derived from aminoalcohols or mercaptoalcohols such as 6-aminohexanol[12] or 6-mercaptohexanol.[16] 5′-Amino[13] and 5′-mercapto[27] dideoxynucleosides have also been used for the introduction of reactive groups at the 5′-position of oligonucleotides. In amino linkers, the presence of an ether function at the β-position increases the nucleophilicity of the reactive amino group and allows more efficient conjugation reactions.[28−30] However, all these linkers can only be introduced at the 5′-end of the oligonucleotides, whereas the novel linkers described in this work can be incorporated at any position in an oligonucleotide.

Figure 3

Amino and mercapto linkers for the functionalization of oligonucleotides.

The incorporation of amino groups at the 3′-end utilized aminoalkyldiols. Most of them are acyclic and nonrigid, but some of them are not enantiomerically pure such as 2-amino-1,3-propanol[14] and 2-butylamino-1,3-propanol[15] and may produce diastereoisomeric mixtures. In addition, it has been described that the 2-amino-1,3-propanol linker may produce intramolecular side reactions.[31] Threoninol derivatives have also been described for the preparation of thiolated oligonucleotides.[32] Amino-[33] and mercapto-[18]functionalized nucleosides at the nucleobases or at the 2′-position of a ribonucleotide[34] have also been reported for the incorporation of amino and thiol reactive groups. The novel linkers described herein are enantiomerically pure and are free of side reactions. They do not require the use of expensive nucleosides but can be considered similar to nucleosides functionalized at the nucleobases or at the 2′-position of a ribonucleotide. Their smaller size similar to a nucleoside would be appropriate for the introduction of local probes such as fluorescent–quencher pairs.[35] Furthermore, the cyano sugar ribose precursor could be transformed to other interesting reactive groups such as azide or alkyne groups for conjugation using cycloaddition reactions.[10] The choice of using the 2-deoxyribose framework for the attachment of the reactive group allows easy incorporation into an oligonucleotide using standard solid-phase amidite chemistry.

Conclusions

A key step in the synthesis of oligonucleotide conjugates is the preparation of the appropriate tethers that connect ligands with oligonucleotides. In this work, we provide an efficient solution to this problem that uses a common sugar precursor, cyano-2-deoxyribofuranose, for the generation of reactive aminomethyl and mercaptomethyl sugars. These intermediates have been converted to the appropriate solid supports and phosphoramidites in excellent yields for the preparation of oligonucleotides carrying amino or thiol groups at any predefined position. Oligonucleotides carrying the new tethers have been functionalized with lipids and fluoresceine demonstrating the usefulness of these enantiomerically pure, hydrophilic, and DNA-compatible linkers. Two orthogonal amino-protecting groups have been studied that can be removed under different conditions allowing the introduction of two ligands in a single oligonucleotide. The novel amidites described herein should ease the assembly of functional conjugates of oligonucleotides and pave the way for enhanced tissue targeting, cell internalization, and resistance to nucleases.

Materials and Methods

General

Reagents

Oleoyl chloride, oleic, and palmitic acids were purchased from Sigma. The standard 2′-deoxy and 2′-O-methyl-ribonucleoside phosphoramidites, reagents solutions, supports, and LCAA-CPG were purchased from Applied Biosystems (PEBiosystems Hispania S.A., Spain) and Link Technologies Ltd. (Lanarkshire, Scotland, UK). The rest of the chemicals were purchased from Aldrich, Sigma, or Fluka (Sigma-Aldrich S.A., Spain), and used without further purification. Anhydrous solvents and deuterated solvents (CDCl3 and MeOH-d4) were obtained from reputable sources and used as received. Thin-layer chromatography (TLC) was carried out on aluminum-backed Silica-Gel 60 F254 plates. The spots were visualized with UV light. Column chromatography was performed using Silica Gel (60 Å, 230 × 400 mesh). Matrix for MALDI-TOF experiments was composed of 2′,4′,6′-trihydroxyacetophenone monohydrate (THAP, Aldrich) and ammonium citrate dibasic (Fluka). Solvents for HPLC analysis were prepared using triethylammonium acetate (TEAA) and acetonitrile (Merck) as a mobile phase. The desalted columns with Sephadex G-25 (NAP-10 or NAP-5) were from GE Healthcare (Little Chalfont, UK). The rest of the chemicals were analytical reagent grade from commercial sources as specified. Ultrapure water (Millipore) was used in all experiments.

Instrumentation

NMR spectra (1H, 13C, 19F, and 31P) were measured on Bruker DPX-300 (1H 300.13 MHz, 13C 75.5 MHz, and 31P 121.5 MHz) or Varian Mercury-400 (1H 400.13 MHz, 13C 100.6 MHz, 19F 376.5 MHz, and 31P 162.0 MHz). Chemical shifts for 1H, 13C, 19F, and 31P NMR are given in parts per million (ppm) from the residual solvent signal as the reference or tetramethylsilane (TMS) and coupling constants (J) values are given in Hertz (Hz). Modified oligonucleotides were synthesized on an Applied Biosystems 3400 DNA Synthesizer (Applied Biosystems). Semipreparative and analytical reverse-phase (RP) HPLC was performed on a Waters chromatography system with a 2695 Separations Module equipped with a Waters 2998 Photodiode Array Detector using different types of semipreparative columns: column A: Nucleosil 120 C18 (250 × 8 mm), column B: Xbridge OST C18 2.5 μm (10 × 50 mm) and analytical columns: column C: XbridgeTM OST C18 2.5 μm (4.6 × 50 mm) and column D: Column ACE 3 μm HILA-3-1546-A (4.6 × 150 mm). High resolution mass spectra (HRMS) were recorded on a mass spectrometer under electron spray ionization (ESI), and mass spectra of oligos were recorded on a MALDI Voyager DETM RP time-of-flight (TOF) spectrometer (Applied Biosystems). Molecular absorption spectra between 220 and 550 nm were recorded with a Jasco V650 spectrophotometer. The temperature was controlled with an 89090A Agilent Peltier device. Hellman quartz cuvettes were used.

Synthesis of 1-Functionalized 1,2-Dideoxy-d-erythro-pentofuranose Phosphoramidites

Preparation of 1-Trifluoroacetylaminomethyl-1,2-dideoxy-d-erythro-pentofuranose Phosphoramidites 5α and 5β

Synthesis of 2α/2β

LiAlH4 (8 equiv) was added to a solution of 1α or 1β in anhydrous THF (0.15M). The reaction was stirred at reflux during 4 h. After cooling, excess of the reagent was decomposed by addition of THF and MeOH, and the mixture was filtered through Celite. The solvents were evaporated, and the crude product was subjected to column chromatography (gradient eluent MeOH–10% NH3/MeOH) to afford 2α or 2β (both contains traces of silica gel).

1α-Aminomethyl-1,2-dideoxy-d-erythro-pentofuranose (2α)

Yellowish oil. Rf: 0.20 (1% NH3/MeOH); IR (NaCl): ν 3415, 2955, 1598 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.76 (ddd, 1H, H2, J = 13.5, 4.8, 3.9 Hz), 2.41 (m, 1H, H2) 3.13 (m, 2H, H6), 3.52 (dd, 1H, H5, J = 11.7, 5.6 Hz), 3.59 (dd, 1H, H5, J = 11.7, 4.3 Hz), 3.97 (dt, 1H, H4, J = 5.5, 4.1 Hz), 4.26 (dt, 1H, H3, J = 6.6, 3.6 Hz), 4.40 (m, 1H, H1) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 38.6 (C2), 44.8 (C6), 63.4 (C5), 73.3 (C3), 76.1 (C1), 88.1 (C4) ppm; HRMS (ESI+, m/z): calcd for C6H14NO3 [M + H]+: 148.0968, found: 148.0977.

1β-Aminomethyl-1,2-dideoxy-d-erythro-pentofuranose (2β)

Yellowish oil. Rf: 0.25 (1% NH3/MeOH); IR (NaCl): ν 3420, 2953, 1644 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.88 (m, 2H, H2), 2.74 (dd, 1H, H6, J = 13.2, 6.9 Hz), 2.92 (dd, 1H, H6, J = 13.2, 3.5 Hz), 3.53 (dd, 1H, H5, J = 11.7, 5.0 Hz), 3.61 (dd, 1H, H5, J = 11.7, 4.1 Hz), 3.81 (q, 1H, H4, J = 4.4 Hz), 4.10 (m, 2H, H1 + H3) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 38.9 (C2), 45.9 (C6), 63.8 (C5), 73.9 (C3), 79.1 (C1), 88.8 (C4) ppm; HRMS (ESI+, m/z): calcd for C6H14NO3 [M + H]+: 148.0968, found: 148.0974.

Synthesis of 3α/3β

To a solution of 2α or 2β in anhydrous DMF (0.1 M) was added anhydrous Et3N (5.5 equiv) and ethyl trifluoroacetate (3.3 equiv). The mixture was stirred at 80 °C during 24 h, and then evaporated to leave a residue, which was purified by column chromatography (gradient eluent 5–20% 2-propanol/CH2Cl2) affording 3α (70% yield) or 3β (80% yield). Isolated yields are for two steps.

1,2-Dideoxy-1α-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranose (3α)

Clear oil. Rf: 0.58 (20% MeOH/CH2Cl2); IR (NaCl): ν 3404, 3302, 2940, 1713, 1216, 1191, 1160 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.69 (ddd, 1H, H2, J = 13.2, 5.8, 4.6 Hz), 2.34 (ddd, 1H, H2, J = 13.1, 7.7, 6.6 Hz), 3.45 (d, 2H, H6, J = 5.4 Hz), 3.52 (dd, 1H, H5, J = 11.7, 4.9 Hz), 3.57 (dd, 1H, H5, J = 11.7, 4.2 Hz), 3.87 (dt, 1H, H4, J = 5.3, 4.0 Hz), 4.25 (overlapped signal, 2H, H1 + H3) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 38.7 (C2), 45.3 (C6), 63.3 (C5), 73.2 (C3), 77.5 (C1), 87.6 (C4), 117.6 (q, CF3, J = 286.7 Hz), 159.2 (q, C=O, J = 36.8 Hz) ppm; HRMS (ESI+, m/z): calcd for C8H13F3NO4 [M + H]+: 244.0791, found: 244.0786, calcd for C8H12F3NNaO4 [M + Na]+: 266.0611, found: 266.0603, calcd for C8H12F3KNO4 [M+K]+: 282.0350, found: 282.0342.

1,2-Dideoxy-1β-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranose (3β)

Clear oil. Rf: 0.57 (20% MeOH/CH2Cl2); IR (NaCl): ν 3395, 3315, 2945, 1712, 1192, 1162 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.87 (m, 2H, H2), 3.44 (t, 2H, H6, J = 5.0 Hz), 3.52 (dd, 1H, H5, J = 11.7, 4.9 Hz), 3.60 (dd, 1H, H5, J = 11.7, 4.2 Hz), 3.80 (dt, 1H, H4, J = 4.5, 3.1 Hz), 4.22 (dt, 1H, H3, J = 5.9, 2.9 Hz), 4.29 (m, 1H, H1) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 38.9 (C2), 44.6 (C6), 63.6 (C5), 73.7 (C3), 77.8 (C1), 88.7 (C4), 117.6 (q, CF3, J = 286.6 Hz), 159.3 (q, C=O, J = 37.0 Hz) ppm; HRMS (ESI+, m/z): calcd for C8H13F3NO4 [M + H]+: 244.0791, found: 244.0780, calcd for C8H12F3NNaO4 [M + Na]+: 266.0611, found: 266.0599, calcd for C8H12F3KNO4 [M+K]+: 282.0350, found: 282.0337.

Synthesis of 4α/4β

Anhydrous Et3N (10 equiv) and 4,4′-dimethoxytrityl chloride (1.5 equiv) were successively added to a solution of 3α or 3β in anhydrous 1,4-dioxane (0.1 M). The mixture was stirred at 30 °C during 2 h. Then, saturated aqueous NaHCO3 was added and the solution was extracted with CH2Cl2. The organic layer was dried, filtered, and evaporated to dryness. The crude residue was purified by column chromatography (40% EtOAc/Hexane). The column was previously packed with silica gel using a 10% Et3N solution in EtOAc:Hexane (4:6, v-v). Isolated yields of 4α or 4β were 65% and 70%, respectively.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1α-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranose (4α)

Intense yellow oil. Rf: 0.22 (40% EtOAc/Hexane); IR (NaCl): ν 3414, 3282, 2934, 1715, 1509, 1252, 1202, 1177 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.68 (ddd, 1H, H2, J = 13.2, 5.8, 4.2 Hz), 2.35 (ddd, 1H, H2, J = 13.1, 7.7, 6.4 Hz), 3.07 (dd, 1H, H5, J = 9.9, 5.1 Hz), 3.14 (dd, 1H, H5, J = 10.0, 4.3 Hz), 3.47 (m, 2H, H6),3.77 (s, 6H, Me-DMT), 4.03 (q, 1H, H4, J = 4.1 Hz), 4.26 (dt, 1H, H3, J = 6.5, 3.8 Hz), 4.34 (m, 1H, H1), 6.85 (d, 4H, Hg, J = 8.9 Hz), 7.24 (m, 3H, Hc + Hd), 7.31 (d, 4H, Hf, J = 8.9 Hz), 7.43 (d, 2H, Hb, J = 7.1 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 38.7 (C2), 45.4 (C6), 55.7 (2 O-CH3), 65.5 (C5), 74.0 (C3), 77.8 (C1), 86.9 (C4), 87.5 (C10), 114.1 (4Cg), 117.6 (q, CF3, J = 286.7 Hz), 127.7 (Cd), 128.7 (2Cc), 129.3 (2Cb), 131.3 (4Cf), 137.2 (Ce), 137.3 (Ce), 146.4 (Ca), 159.2 (q, C=O, J = 37.0 Hz), 160.1 (2Ch) ppm; HRMS (ESI+, m/z): calcd for C29H30F3NNaO6 [M + Na]+: 568.1917, found: 568.1893, calcd for C29H30F3KNO6 [M+K]+: 584.1657, found: 584.1632.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1β-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranose (4β)

Yellowish oil. Rf: 0.13 (40% EtOAc/Hexane); IR (NaCl): ν 3424, 3331, 2934, 1721, 1510, 1251, 1216, 1177 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.75 (ddd, 1H, H2, J = 13.1, 10.0, 5.8 Hz), 1.90 (ddd, 1H, H2, J = 13.0, 5.5, 1.9 Hz), 3.10 (m, 2H, H5), 3.39 (dd, 1H, H6, J = 13.7, 6.3 Hz), 3.49 (dd, 1H, H6, J = 13.7, 4.6 Hz), 3.76 (s, 6H, Me-DMT), 3.94 (dt, 1H, H4, J = 5.0, 2.3 Hz), 4.23 (m, 1H, H3), 4.29 (m, 1H, H1), 6.84 (d, 4H, Hg, J = 8.9 Hz), 7.21 (m, 3H, Hc + Hd), 7.31 (d, 4H, Hf, J = 8.9 Hz), 7.44 (m, 2H, Hb) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ39.2 (C2), 44.6 (C6), 55.7 (2 O-CH3), 65.6 (C5), 74.4 (C3), 77.9 (C1), 87.4 (C10), 87.7 (C4), 114.1 (4Cg), 117.5 (q, CF3, J = 286.8 Hz), 127.8 (Cd), 128.7 (2Cc), 129.3 (2Cb), 131.3 (4Cf), 137.2 (Ce), 137.3 (Ce), 146.4 (Ca), 159.2 (q, C=O, J = 36.8 Hz), 160.1 (2Ch) ppm; HRMS (ESI+, m/z): calcd for C29H30F3NNaO6 [M + Na]+: 568.1917, found: 568.1884, calcd for C29H30F3KNO6 [M+K]+: 584.1657, found: 584.1623.

Synthesis of 5α/5β

Compound 4α or 4β was coevaporated twice with anhydrous MeCN under reduced pressure and left in a freeze-dryer overnight. Next, the product was dissolved in anhydrous CH2Cl2 (0.1 M) and anhydrous Pr2NEt (3 equiv) was added. The resulting solution was cooled in an ice bath and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.5 equiv) was added dropwise with a syringe. After 15 min, the reaction was allowed to reach rt and stirred for an additional 1 h. Then, the reaction was quenched with brine and extracted with CH2Cl2. The organic layer was dried, filtered, and evaporated to dryness. The crude residue was purified by column chromatography (40% EtOAc/Hexane) to afford 5α (84% yield) or 5β (70% yield). The column was previously packed with silica gel using a 10% Et3N solution in EtOAc:Hexane (4:6, v–v).

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1α-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (5α-A)

Clear oil. Rf: 0.63 (40% EtOAc/Hexane); IR (NaCl): ν 3318, 2966, 2254, 1723, 1509, 1251, 1213, 1179, 1033 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.15 (d, 6H, Hv, J = 6.9 Hz), 1.18 (d, 6H, Hv, J = 6.9 Hz), 1.79 (dt, 1H, H2, J = 13.1, 5.0 Hz), 2.40 (dt, 1H, H2, J = 13.6, 7.0 Hz), 2.52 (t, 2H, H, J = 6.0 Hz), 3.10 (dd, 1H, H5, J = 10.1, 4.6 Hz), 3.25 (dd, 1H, H5, J = 10.1, 4.0 Hz), 3.43 (dd, 1H, H6, J = 13.7, 4.1 Hz), 3.63 (overlapped signal, 5H, H6 + Hw + H), 3.78 (s, 6H, Me-DMT), 4.15 (q, 1H, H4, J = 4.0 Hz), 4.34 (m, 1H, H1), 4.48 (m, 1H, H3), 6.85 (d, 4H, Hg, J = 8.9 Hz), 7.24 (m, 3H, Hc + Hd), 7.30 (d, 4H, Hf, J = 8.9 Hz), 7.45 (d, 2H, Hb, J = 7.0 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 7.3 Hz), 25.0 (d, 4Cv, J = 7.4 Hz), 38.2 (d, C2, J = 4.0 Hz), 44.4 (d, 2Cw, J = 12.3 Hz), 45.2 (C6), 55.7 (2 O-CH3), 59.7 (d, C, J = 18.6 Hz), 65.1 (C5), 76.1 (d, C3, J = 16.2 Hz), 78.4 (C1), 86.0 (d, C4, J = 4.4 Hz), 87.5 (C10), 114.1 (4Cg), 117.5 (q, CF3, J = 286.8 Hz), 119.3 (CN), 127.8 (Cd), 128.8 (2Cc), 129.3 (2Cb), 131.3 (4Cf), 137.2 (Ce), 137.3 (Ce), 146.4 (Ca), 159.2 (q, C=O, J = 37.6 Hz), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ148.1 ppm; HRMS (ESI+, m/z): calcd for C38H47F3KNaO7P [M+K]+: 784.2735, found: 784.2702.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1α-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (5α-B)

Clear oil. Rf: 0.53 (40% EtOAc/Hexane); IR (NaCl): ν 3424, 3324, 2967, 2254, 1725, 1509, 1251, 1215, 1178, 1034 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.07 (d, 6H, Hv, J = 6.8 Hz), 1.17 (d, 6H, Hv, J = 6.8 Hz), 1.89 (dt, 1H, H2, J = 13.2, 4.9 Hz), 2.40 (dt, 1H, H2, J = 13.7, 7.0 Hz), 2.68 (t, 2H, H, J = 5.8 Hz), 3.08 (dd, 1H, H5, J = 10.0, 4.8 Hz), 3.19 (dd, 1H, H5, J = 10.0, 4.5 Hz), 3.54 (overlapped signal, 4H, H6 + Hw), 3.77 (s, 6H, Me-DMT), 3.78 (m, 2H, H), 4.12 (q, 1H, H4, J = 4.0 Hz), 4.33 (m, 1H, H1), 4.46 (m, 1H, H3), 6.85 (d, 4H, Hg, J = 8.9 Hz), 7.23 (m, 3H, Hc + Hd), 7.30 (d, 4H, Hf, J = 8.9 Hz), 7.43 (d, 2H, Hb, J = 7.0 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 6.8 Hz), 23.6 (d, 2Cv, J = 7.1 Hz), 23.6 (d, 2Cv, J = 7.1 Hz), 38.3 (d, C2, J = 2.8 Hz), 44.4 (d, 2Cw, J = 12.4 Hz), 45.3 (C6), 55.7 (2 O-CH3), 59.7 (d, C, J = 19.0 Hz), 65.2 (C5), 76.6 (d, C3, J = 16.8 Hz), 78.4 (C1), 85.9 (d, C4, J = 5.9 Hz), 87.5 (C10), 114.1 (4Cg), 117.5 (q, CF3, J = 286.8 Hz), 119.5 (CN), 127.8 (Cd), 128.8 (2Cc), 129.3 (2Cb), 131.3 (4Cf), 137.1 (Ce), 137.2 (Ce), 146.4 (Ca), 159.2 (q, C=O, J = 37.6 Hz), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ148.1 ppm; HRMS (ESI+, m/z): calcd for C38H48F3N3O7P [M + H]+: 746.3176, found: 746.3151, calcd for C38H47F3N3NaO7P [M + Na]+: 768.2996, found: 768.2965, calcd for C38H47F3KN3O7P [M+K]+: 784.2735, found: 784.2709.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1β-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (5β-A)

Clear oil. Rf: 0.34 (40% EtOAc/Hexane); IR (NaCl): ν 3425, 3324, 2968, 2254, 1726, 1509, 1251, 1215, 1178, 1034 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.16 (d, 6H, Hv, J = 6.8 Hz), 1.19 (d, 6H, Hv, J = 6.8 Hz), 1.83 (m, 1H, H2), 2.01 (m, 1H, H2), 2.54 (t, 2H, H, J = 6.0 Hz), 3.15 (m, 2H, H5), 3.47 (m, 2H, H6), 3.65 (overlapped signal, 4H, H + Hw), 3.78 (s, 6H, Me-DMT), 4.06 (m, 1H, H4), 4.27 (m, 1H, H1), 4.43 (m, 1H, H3), 6.86 (d, 4H, Hg, J = 8.9 Hz), 7.22 (m, 3H, Hc + Hd), 7.32 (d, 4H, Hf, J = 7.5 Hz), 7.45 (d, 2H, Hb, J = 7.3 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 6.8 Hz), 25.0 (d, 2Cv, J = 7.5 Hz), 25.0 (d, 2Cv, J = 7.5 Hz), 38.5 (d, C2, J = 4.6 Hz), 44.4 (d, 2Cw, J = 12.0 Hz), 44.5 (C6), 55.7 (2 O-CH3), 59.7 (d, C, J = 18.6 Hz), 65.1 (C5), 76.5 (d, C3, J = 16.4 Hz), 78.2 (C1), 87.1 (d, C4, J = 4.0 Hz), 87.5 (C10), 114.1 (4Cg), 117.5 (q, CF3, J = 286.8 Hz), 119.3 (CN), 127.8 (Cd), 128.7 (2Cc), 129.3 (2Cb), 131.3 (4Cf), 137.2 (Ce), 137.3 (Ce), 146.4 (Ca), 159.2 (q, C=O, J = 36.8 Hz), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ148.1 ppm; HRMS (ESI+, m/z): calcd for C38H47F3N3NaO7P [M + Na]+: 768.2996, found: 768.2968.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1β-[N-(trifluoroacetyl)aminomethyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (5β-B)

Clear oil. Rf: 0.28 (30% EtOAc/Hexane); IR (NaCl): ν 3424, 3322, 2967, 2254, 1726, 1510, 1251, 1215, 1179, 1034 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.09 (d, 6H, Hv, J = 6.8 Hz), 1.18 (d, 6H, Hv, J = 6.8 Hz), 1.80 (m, 1H, H2), 2.12 (dd, 1H, H2, J = 12.9, 4.7 Hz), 2.68 (t, 2H, H, J = 6.0 Hz), 3.12 (d, 2H, H5, J = 4.9 Hz), 3.46 (m, 2H, H6), 3.59 (m, 2H, Hw), 3.77 (m, 8H, Me-DMT + H), 4.02 (m, 1H, H4), 4.28 (dq, 1H, H1, J = 10.0, 5.5 Hz), 4.42 (m, 1H, H3), 6.85 (d, 4H, Hg, J = 8.9 Hz), 7.23 (m, 3H, Hc + Hd), 7.31 (d, 4H, Hf, J = 8.9 Hz), 7.43 (d, 2H, Hb, J = 7.2 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 7.1 Hz), 25.0 (d, 2Cv, J = 7.0 Hz), 25.0 (d, 2Cv, J = 7.0 Hz), 38.5 (d, C2, J = 3.6 Hz), 44.4 (d, 2Cw, J = 12.7 Hz), 44.5 (C6), 55.7 (2 O-CH3), 59.8 (d, C, J = 18.9 Hz), 65.2 (C5), 76.8 (d, C3, J = 17.0 Hz), 78.1 (C1), 86.9 (d, C4, J = 5.6 Hz), 87.5 (C10), 114.1 (4Cg), 117.5 (q, CF3, J = 286.8 Hz), 119.5 (CN), 127.8 (Cd), 128.8 (2Cc), 129.3 (2Cb), 131.3 (4Cf), 137.1 (Ce), 137.2 (Ce), 146.4 (Ca), 159.2 (q, C=O, J = 37.6 Hz), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ147.7 ppm; HRMS (ESI+, m/z): calcd for C38H48F3N3O7P [M + H]+: 746.3176, found: 746.3156, calcd for C38H47F3N3NaO7P [M + Na]+: 768.2996, found: 768.2972.

Preparation of 1-NPEC-aminomethyl-1,2-dideoxy-d-erythro-pentofuranose Phosphoramidites 8α and 8β

Synthesis of 6α/6β

To a solution of 2α or 2β in anhydrous MeOH (0.1M) was added anhydrous Et3N (1.5 equiv) and 1-(2-nitrophenyl)ethyl-N-succinimidyl carbonate[22] (1 equiv). The mixture was stirred at 30 °C during 1 h, and then evaporated to leave a residue, which was poured into saturated aqueous NaCl and extracted with EtOAc. The organic layer was dried, filtered, and evaporated to dryness. The crude residue was purified by column chromatography (5% MeOH/CH2Cl2) to afford 6α (55% yield from 1) or 6β (50% yield from 1).

1,2-Dideoxy-1α-[(1-(2-nitrophenyl)ethoxy)carbonylaminomethyl]-d-erythro-pentofuranose (6α)

Yellow oil. Rf: 0.29 (10% MeOH/CH2Cl2); IR (NaCl): ν 3360, 2939, 1694, 1538, 1259 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.60 (d, 3H, H11, J = 6.8 Hz), 1.64 (m, 1H, H2), 2.24 (m, 1H, H2), 3.19 (m, 2H, H6), 3.50 (dd, 1H, H5, J = 11.7, 5.6 Hz), 3.59 (dd, 1H, H5, J = 11.7, 3.9 Hz), 3.79 (m, 1H, H4), 4.09 (m, 1H, H1), 4.19 (m, 1H, H3), 6.14 (q, 1H, H10, J = 6.8 Hz), 7.48 (m, 1H, Harom), 7.72 (m, 2H, Harom), 7.94 (d, 1H, Harom, J = 7.7 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 22.5 (C11), 38.6 (C2), 46.1 (C6), 46.1 (C6), 63.3 (C5), 69.6 (C10), 73.2 (C3), 78.2 (C1), 78.3 (C1), 87.0 (C4), 125.2 (CHarom), 128.3 (CHarom), 129.5 (CHarom), 134.8 (CHarom), 134.9 (CHarom), 139.9 (C12), 149.1 (C13), 157.9 (C=O), 158.0 (C=O) ppm; HRMS (ESI+, m/z): calcd for C15H21N2O7 [M + H]+: 341.1343, found: 341.1332, calcd for C15H20N2NaO7 [M + Na]+: 363.1163, found: 363.1153, calcd for C15H20KN2O7 [M+K]+: 379.0902, found: 379.0891.

1,2-Dideoxy-1β-[(1-(2-nitrophenyl)ethoxy)carbonylaminomethyl]-d-erythro-pentofuranose (6β)

Light brown oil. Rf: 0.26 (10% MeOH/CH2Cl2); IR (NaCl): ν 3355, 2937, 1703, 1525, 1261 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.61 (d, 3H, H11, J = 6.6 Hz), 1.63–1.84 (several m, 2H, H2), 3.19 (m, 2H, H6), 3.53 (m, 2H, H5), 3.75 (m, 1H, H4), 4.16 (m, 2H, H1 + H3), 6.13 (q, 1H, H10, J = 6.5 Hz), 7.50 (m, 1H, Harom), 7.72 (m, 2H, Harom), 7.95 (d, 1H, Harom, J = 7.6 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 22.3 (C11), 22.4 (C11), 38.4 (C2), 38.6 (C2), 45.2 (C6), 45.5 (C6), 63.6 (C5), 63.7 (C5), 69.6 (C10), 73.6 (C3), 78.7 (C1), 78.8 (C1), 88.5 (C4),88.6 (C4), 125.3 (CHarom), 128.2 (CHarom), 129.6 (CHarom), 134.8 (CHarom), 139.8 (C12), 149.2 (C13), 158.2 (C=O) ppm; HRMS (ESI+, m/z): calcd for C15H21N2O7 [M + H]+: 341.1343, found: 341.1339, calcd for C15H20N2NaO7 [M + Na]+: 363.1163, found: 363.1157, calcd for C15H20KN2O7 [M+K]+: 379.0902, found: 379.0896.

Synthesis of 7α/7β

A procedure similar to that described for the synthesis of 4α/4β, starting from 6α/6β and with a reaction temperature of 35 °C, gave 7α (80% yield) or 7β (80% yield).

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1α-[(1-(2-nitrophenyl)ethoxy)carbonylamino-methyl]-d-erythro-pentofuranose (7α)

Yellowish oil. Rf: 0.19 (50% EtOAc/Hexane); IR (NaCl): ν 3422, 2932, 1719, 1525, 1508, 1252 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.58 (d, 3H, H11, J = 6.5 Hz), 1.59 (m, 1H, H2), 2.23 (m, 1H, H2), 3.10 (m, 2H, H5), 3.23 (m, 2H, H6), 3.74 and 3.75 (2s, 6H, Me-DMT), 6.15 (q, 1H, H4, J = 6.4 Hz), 4.14 (m, 1H, H1), 4.22 (m, 1H, H3), 6.15 (q, 1H, H10, J = 6.4 Hz), 6.82 (m, 4H, Harom), 7.14–7.36 (several m, 8H, Harom), 7.44 (m, 2H, Harom), 7.67 (m, 2H, Harom), 7.91 (m, 1H, Harom) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 22.5 (C11), 38.7 (C2), 38.8 (C2), 46.3 (C6), 55.7 (2 O-CH3), 65.4 (C5), 65.5 (C5), 69.6 (C10), 74.1 (C3), 74.2 (C3), 78.7 (C1), 78.9 (C1), 86.4 (C4), 87.4 (C18), 114.0 (4Cg), 125.2 (CHarom), 127.7 (Cd), 128.1 (CHarom), 128.2 (CHarom), 128.7 (2Cc), 129.4 (2Cb), 129.4 (CHarom), 131.3 (4Cf), 134.9 (CHarom), 137.3 (2Ce), 139.9 (C12), 146.5 (Ca), 149.0 (C13), 158.0 (C=O), 160.0 (2Ch) ppm; HRMS (ESI+, m/z): calcd for C36H38N2NaO9 [M + Na]+: 665.2470, found: 665.2462, calcd for C36H38KN2O9 [M+K]+: 681.2209, found: 681.2201.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1β-[(1-(2-nitrophenyl)ethoxy)carbonylamino-methyl]-d-erythro-pentofuranose (7β)

Yellowish oil. Rf: 0.16 (50% EtOAc/Hexane); IR (NaCl): ν 3424, 2931, 1722, 1525, 1510, 1252 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.52 and 1.53 (2d, 3H, H11, J = 6.4 Hz), 1.65–1.85 (several m, 2H, H2), 3.12 (m, 2H, H5), 3.23 (m, 2H, H6), 3.77 and 3.78 (2s, 6H, Me-DMT), 3.91 (m, 1H, H4), 4.19 (m, 2H, H1 + H3), 6.13 (m, 1H, H10), 6.86 (m, 4H, Harom), 7.17–7.65 (several m, 12H, Harom), 7.93 (m, 1H, Harom) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 22.4 (C11), 22.5 (C11), 38.6 (C2), 38.9 (C2), 45.3 (C6), 45.5 (C6), 55.7 (2 O-CH3), 65.6 (C5), 65.71 (C5), 69.6 (C10), 74.4 (C3), 74.5 (C3), 78.6 (C1), 78.7 (C1), 87.4 (C18), 87.6 (C4), 114.1 (4Cg), 125.2 (CHarom), 127.8 (Cd), 128.2 (CHarom), 128.8 (2Cc), 129.4 (2Cb), 129.5 (CHarom), 131.3 (4Cf), 134.8 (CHarom), 137.3 (Ce), 137.4 (Ce), 139.8 (C12), 146.5 (Ca), 149.0 (C13), 157.9 (C=O), 160.1 (2Ch) ppm; HRMS (ESI+, m/z): calcd for C36H38N2NaO9 [M + Na]+: 665.2470, found: 665.2454, calcd for C36H38KN2O9 [M+K]+: 681.2209, found: 681.2192.

Synthesis of 8α/8β

A procedure analogous to that described for the synthesis of 5α/5β, starting from 7α/7β, gave 8α (78% yield) or 8β (72% yield).

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1α-[(1-(2-nitrophenyl)ethoxy)carbonylamino-methyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (8α-A)

Clear oil. Rf: 0.34 (40% EtOAc/Hexane); IR (NaCl): ν 3355, 2967, 2253, 1723, 1526, 1510, 1251, 1179, 1033 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.10–1.18 (several d, 12H, Hv, J = 6.8 Hz), 1.60 (d, 3H, H11, J = 6.5 Hz), 1.72 (m, 1H, H2), 2.29 (m, 1H, H2), 2.50 (t, 2H, H, J = 6.0 Hz), 3.08 (m, 1H, H5), 3.24 (m, 3H, H5 + H6), 3.60 (m, 4H, H + Hw), 3.77 and 3.78 (2s, 6H, Me-DMT), 4.09 (m, 1H, H4), 4.19 (m, 1H, H1), 4.44 (m, 1H, H3), 6.15 (m, 1H, H10), 6.85 (m, 4H, Harom), 7.17–7.49 (several m, 10H, Harom), 7.70 (m, 2H, Harom), 7.93 (m, 1H, Harom) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.8 (d, C, J = 6.6 Hz), 22.5 (C11), 25.0 (d, 4Cv, J = 7.3 Hz), 38.0 (d, C2, J = 4.0 Hz), 38.1 (d, C2, J = 4.0 Hz), 44.3 (d, Cw, J = 12.2 Hz), 46.0 (C6), 46.2 (C6), 55.7 (2 O-CH3), 59.7 (d, C, J = 18.3 Hz), 65.1 (C5), 69.6 (C10), 75.9 (d, C3, J = 15.6 Hz), 76.0 (d, C3, J = 16.4 Hz), 79.0 (C1), 79.3 (C1), 85.7 (C4), 87.5 (C18), 114.1 (4Cg), 119.3 (CN), 125.2 (CHarom), 127.8 (Cd), 128.2 (CHarom), 128.3 (CHarom), 128.8 (2Cc), 129.4 (2Cb), 129.5 (CHarom), 131.4 (4Cf), 134.9 (CHarom), 137.2 (Ce), 137.3 (Ce), 137.4 (Ce), 140.0 (C12), 146.5 (Ca), 149.1 (C13), 158.0 (C=O), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ 148.0 ppm; HRMS (ESI+, m/z): calcd for C45H56N4O10P [M + H]+: 843.3729, found: 843.3726, calcd for C45H55N4NaO10P [M + Na]+: 865.3548, found: 865.3545, calcd for C45H55KN4O10P [M+K]+: 881.3287, found: 881.3300.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1α-[(1-(2-nitrophenyl)ethoxy)carbonylamino-methyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (8α-B)

Clear oil. Rf: 0.30 (40% EtOAc/Hexane); IR (NaCl): ν 3360, 2967, 2253, 1723, 1526, 1510, 1252, 1179, 1033 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.04 (d, 6H, Hv, J = 6.8 Hz), 1.15 (d, 6H, Hv, J = 6.8 Hz), 1.60 (d, 3H, H11, J = 6.6 Hz), 1.82 (m, 1H, H2), 2.31 (m, 1H, H2), 2.65 (t, 2H, H, J = 5.9 Hz), 3.06 (m, 1H, H5), 3.16 (m, 1H, H5), 3.24 (m, 2H, H6), 3.55 (m, 2H, Hw), 3.72 (m, 2H, H), 3.77 (s, 6H, Me-DMT), 4.07 (m, 1H, H4), 4.17 (m, 1H, H1), 4.42 (m, 1H, H3), 6.14 (m, 1H, H10), 6.83 (m, 4H, Harom), 7.16–7.49 (m, 10H, Harom), 7.70 (m, 2H, Harom), 7.93 (m, 1H, Harom) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 6.7 Hz), 22.5 (C11), 22.6 (C11), 24.9 (d, 2Cv, J = 7.7 Hz), 25.0 (d, 2Cv, J = 7.3 Hz), 38.2 (C2), 44.3 (d, Cw, J = 12.6 Hz), 46.2 (C6), 54.8 (2 O-CH3), 59.7 (d, C, J = 19.0 Hz), 65.2 (C5), 65.3 (C5), 69.6 (C10), 76.5 (d, C3, J = 16.2 Hz), 76.6 (d, C3, J = 17.7 Hz), 79.1 (C1), 79.3 (C1), 85.7 (d, C4, J = 5.7 Hz), 87.5 (C18), 114.1 (4Cg), 119.5 (CN), 125.2 (CHarom), 127.8 (Cd), 128.2 (CHarom), 128.3 (CHarom), 128.8 (2Cc), 129.3 (2Cb), 129.5 (CHarom), 131.3 (4Cf), 134.8 (CHarom), 137.2 (2Ce), 139.9 (C12), 146.4 (Ca), 149.1 (C13), 157.9 (C=O), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ 148.0 and 148.1 ppm; HRMS (ESI+, m/z): calcd for C45H56N4O10P [M + H]+: 843.3729, found: 843.3725, calcd for C45H55N4NaO10P [M + Na]+: 865.3548, found: 865.3550, calcd for C45H55KN4O10P [M+K]+: 881.3287, found: 881.3310.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1β-[(1-(2-nitrophenyl)ethoxy)carbonylamino-methyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (8β-A)

Clear oil. Rf: 0.41 (40% EtOAc/Hexane); IR (NaCl): ν 3356, 2932, 2253, 1725, 1526, 1510, 1251, 1179, 1033 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.13 (d, 6H, Hv, J = 6.8 Hz), 1.18 (d, 6H, Hv, J = 6.8 Hz), 1.49 and 1.52 (2d, 3H, H11, 6.5 Hz), 1.66–1.96 (several m, 2H, H2), 2.52 (t, 2H, H, J = 6.0 Hz), 3.07–3.29 (several m, 4H, H5 + H6), 3.48–3.71 (m, 4H, H + Hw), 3.77 and 3.78 (2s, 6H, Me-DMT), 4.03 (m, 1H, H4), 4.16 (m, 1H, H1), 4.40 (m, 1H, H3), 6.11 (m, 1H, H10), 6.85 (m, 4H, Harom), 7.11–7.64 (several m, 12H, Harom), 7.91 (m, 1H, Harom) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 6.8 Hz), 22.4 (C11), 22.5 (C11), 24.9 (d, 2Cv, J = 7.2 Hz), 24.9 (d, 2Cv, J = 7.3 Hz), 37.8 (C2), 38.2 (C2), 44.4 (d, Cw, J = 11.9 Hz), 45.0 (C6), 55.7 (2 O-CH3), 59.7 (d, C, J = 19.2 Hz), 65.2 (C5), 69.6 (C10), 77.6 (C3, cross-peak in HSQC), 78.9 (C1), 79.0 (C1), 87.0 (C4), 87.5 (C18), 114.1 (4Cg), 119.3 (CN), 125.2 (CHarom), 127.8 (Cd), 128.2 (CHarom), 128.8 (2Cc), 129.4 (2Cb), 129.5 (CHarom), 131.4 (4Cf), 134.8 (CHarom), 137.2 (Ce), 137.3 (Ce), 139.6 (C12), 146.4 (Ca), 149.1 (C13), 158.0 (C=O), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ 148.0 ppm; HRMS (ESI+, m/z): calcd for C45H56N4O10P [M + H]+: 843.3729, found: 843.3723, calcd for C45H55N4NaO10P [M + Na]+: 865.3548, found: 865.3541.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1β-[(1-(2-nitrophenyl)ethoxy)carbonylamino-methyl]-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (8β-B)

Clear oil. Rf: 0.34 (40% EtOAc/Hexane); IR (NaCl): ν 3363, 2933, 2253, 1729, 1509, 1250, 1179, 1034 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.08 (d, 6H, Hv, J = 6.8 Hz), 1.19 (d, 6H, Hv, J = 6.8 Hz), 1.50 and 1.53 (2d, 3H, H11, J = 6.4 Hz), 1.70–1.95 (several m, 2H, H2), 2.68 (t, 2H, H, J = 5.9 Hz), 3.08–3.41 (several m, 4H, H5 + H6), 3.60 (m, 2H, Hw), 3.78 and 3.79 (2s, 6H, Me-DMT), 3.78 (m, 2H, H), 4.01 (m, 1H, H4), 4.18 (m, 1H, H1), 4.41 (m, 1H, H3), 6.12 (m, 1H, H10), 6.85 (m, 4H, Harom), 7.18–7.63 (seveal m, 12H, Harom), 7.93 (m, 1H, Harom) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 6.6 Hz), 22.4 (C11), 22.5 (C11), 24.9 (d, 2Cv, J = 7.7 Hz), 25.0 (d, 2Cv, J = 7.3 Hz), 38.0 (C2), 38.2 (C2), 44.4 (d, Cw, J = 12.6 Hz), 45.0 (C6), 45.3 (C6), 55.7 (2 O-CH3), 59.7 (d, C, J = 19.1 Hz), 65.2 (C5),65.3 (C5), 69.6 (C10), 77.0 (C3, cross-peak in HSQC), 78.8 (C1), 78.9 (C1), 86.8 (d, C4, J = 5.5 Hz), 87.5 (C18),114.1 (4Cg), 119.5 (CN), 125.2 (CHarom), 127.8 (Cd), 128.1 (CHarom), 128.23 (CHarom), 128.8 (2Cc), 129.4 (2Cb), 129.5 (CHarom), 131.3 (4Cf), 134.8 (CHarom), 137.1 (Ce), 137.2 (Ce), 137.3 (Ce), 139.8 (C12), 146.4 (Ca), 149.0 (C13), 157.9 (C=O), 160.1 (2Ch) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ 147.6 ppm; HRMS (ESI+, m/z): calcd for C45H56N4O10P [M + H]+: 843.3729, found: 843.3732, calcd for C45H55N4NaO10P [M + Na]+: 865.3548, found: 865.3541.

Preparation of 1-Acetylmercaptomethyl-1,2-dideoxy-d-erythro-pentofuranose Phosphoramidites 16α and 16β

Synthesis of 9, 10, 11, and 12

Synthesis of 9α, 10α, 11α, and 12α was described previously by us.[19] A procedure analogous to that afforded 9β, 10β, 11β, and 12β. Yields are indicated in Scheme .

1,2-Dideoxy-1β-(methoxycarbonyl)-d-erythro-pentofuranose (9β)

Yellowish oil. Rf: 0.36 (10% MeOH/CH2Cl2); IR (NaCl): ν 3387, 2954, 1738 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 2.19 (m, 2H, H2), 3.57 (d, 2H, H5, J = 5.1 Hz), 3.75 (s, 3H, Me), 3.91 (dt, 1H, H4, J = 5.0, 2.8 Hz), 4.26 (dt, 1H, H3, J = 5.7, 2.9 Hz), 4.64 (dd, 1H, H1, J = 8.5, 7.4 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 39.7 (C2), 52.7 (O-CH3), 63.5 (C5), 73.2 (C3), 77.4 (C1), 89.5 (C4), 175.3 (C=O) ppm; HRMS (ESI+, m/z): calcd for C7H12NaO5 [M + Na]+: 199.0577, found: 199.0580.

3,5-Bis-O-(tert-butyldimethylsilyl)-1,2-dideoxy-1β-(methoxycarbonyl)-d-erythro-pentofuranose (10β)

Viscous liquid. Rf: 0.59 (20% EtOAc/Hexane); IR (NaCl): ν 2954, 2931, 2898, 2858, 1759, 1737 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 0.08 (s, 3H, Si-Me), 0.09 (s, 3H, Si-Me), 0.108 (s, 3H, Si-Me), 0.113 (s, 3H, Si-Me), 0.91 (s, 9H, Si-Bu), 0.92 (s, 9H, Si-Bu), 2.12 (m, 2H, H2), 3.51 (dd, 1H, H5, J = 10.9, 6.5 Hz), 3.68 (dd, 1H, H5, J = 10.8, 4.2 Hz), 3.73 (s, 3H, O-Me), 3.89 (ddd, 1H, H4, J = 6.3, 4.3, 1.8 Hz), 4.42 (m, 1H, H3), 4.61 (dd, 1H, H1, J = 8.8, 7.4 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ −5.32 (Si-CH3), −5.29 (Si-CH3), −4.53 (Si-CH3), −4.50 (Si-CH3), 18.7 (SiCMe3), 19.2 (SiCMe3), 26.3 (3 CH3-Bu), 26.4 (3 CH3-Bu), 39.8 (C2), 52.5 (O-CH3), 64.6 (C5), 75.0 (C3), 77.7 (C1), 90.0 (C4), 174.3 (C=O) ppm; HRMS (ESI+, m/z): calcd for C19H40NaO5Si2 [M + Na]+: 427.2306, found: 427.2308.

3,5-Bis-O-(tert-butyldimethylsilyl)-1,2-dideoxy-1β-(hydroxymethyl)-d-erythro-pentofuranose (11β)

Viscous liquid. Rf: 0.37 (20% EtOAc/Hexane); IR (NaCl): ν 3450, 2960, 2925, 1472, 1256 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 0.08 (s, 6H, Si-Me), 0.10 (s, 6H, Si-Me), 0.91 (s, 9H, Si-Bu), 0.92 (s, 9H, Si-Bu), 1.84 (m, 2H, H2), 3.50 (m, 2H, H6), 3.60 (dd, 1H, H5, J = 11.6, 4.0 Hz), 3.65 (dd, 1H, H5, J = 10.8, 4.2 Hz), 3.79 (ddd, 1H, H4, J = 6.3, 4.2, 2.5 Hz), 4.21 (m, 1H, H1), 4.36 (dt, 1H, H3, J = 5.1, 2.6 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ −5.3 (Si-CH3), −5.2 (Si-CH3), −4.5 (Si-CH3), −4.4 (Si-CH3), 18.9 (SiCMe3), 19.2 (SiCMe3), 26.3 (3 CH3-Bu), 26.5 (3 CH3-Bu), 38.0 (C2), 64.9 (C5), 65.4 (C6), 75.1 (C3), 80.6 (C1), 89.0 (C4) ppm; HRMS (ESI+, m/z): calcd for C18H40NaO4Si2 [M + Na]+: 399.2357, found: 399.2361.

3,5-Bis-O-(tert-butyldimethylsilyl)-1,2-dideoxy-1β-(tosyloxy)methyl-d-erythro-pentofuranose (12β)

Viscous liquid. Rf: 0.64 (20% EtOAc/Hexane); IR (NaCl): ν 2954, 2930, 2896, 2857, 1471, 1366, 1255 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 0.02 (s, 3H, Si-Me), 0.04 (s, 3H, Si-Me), 0.07 (s, 6H, Si-Me), 0.88 (s, 9H, Si-Bu), 0.89 (s, 9H, Si-Bu), 1.80 (m, 2H, H2), 2.46 (s, 3H, Ts-Me), 3.41 (dd, 1H, H5, J = 10.9, 5.9 Hz), 3.53 (dd, 1H, H5, J = 10.9, 4.1 Hz), 3.75 (ddd, 1H, H4, J = 6.2, 4.1, 2.3 Hz), 3.95 (dd, 1H, H6, J = 10.5, 5.6 Hz), 4.10 (dd, 1H, H6, J = 10.5, 3.4 Hz), 4.29 (m, 2H, H1 + H3), 7.44 (d, 2H, Harom, J = 8.5 Hz), 7.79 (d, 2H, Harom, J = 8.4 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ −5.3 (Si-CH3), −5.2 (Si-CH3), −4.6 (Si-CH3), −4.5 (Si-CH3), 18.8 (SiCMe3), 19.2 (SiCMe3), 21.6 (CH3-Ts), 26.3 (3 CH3-Bu), 26.5 (3 Bu-CH3), 37.7 (C2), 64.6 (C5), 72.8 (C6), 74.8 (C3), 77.1 (C1), 89.2 (C4), 129.1 (2 Carom), 131.1 (2 Carom), 134.4 (Cipso), 146.5 (Cipso) ppm; HRMS (ESI+, m/z): calcd for C25H47O6SSi2 [M + H]+: 531.2626, found: 531.2633.

Synthesis of 13α/13β

A solution of potassium thioacetate (1.7 equiv) in anhydrous DMF (0.5 M) was added dropwise to a solution of 12α/12β in anhydrous DMF (0.3 M). The reaction was stirred 6 h at 65 °C, and the mixture was dissolved in H2O and extracted with Et2O. The organic layer was dried, filtered, and evaporated to dryness. The residue was purified by column chromatography (gradient eluent 5–15% EtOAc/Hexane) to give 13α (70% yield) or 13β (75% yield).

1α-(Acetylmercaptomethyl)-3,5-bis-O-(tert-butyldimethylsilyl)-1,2-dideoxy-d-erythro-pentofuranose (13α)

Yellow oil. Rf: 0.63 (20% EtOAc/Hexane); IR (NaCl): ν 2954, 1697, 1257, 1109, 626 cm–1; 1H NMR (300.13 MHz, CDCl3): δ 0.03 (s, 3H, Si-Me), 0.04 (s, 3H, Si-Me), 0.06 (s, 6H, Si-Me), 0.87 (s, 18H, Si-Bu), 1.72 (dt, 1H, H2, J = 13.0, 4.2 Hz), 2.21 (ddd, 1H, H2, J = 13.2, 7.3, 6.0 Hz), 2.33 (s, 3H, CO-Me), 3.12 (dd, 1H, H6, J = 13.6, 5.6 Hz), 3.19 (dd, 1H, H6, J = 13.5, 7.4 Hz), 3.48 (dd, 1H, H5, J = 10.8, 5.6 Hz), 3.60 (dd, 1H, H5, J = 10.9, 3.8 Hz), 3.89 (m, 1H, H4), 4.15 (m, 1H, H1), 4.34 (dt, 1H, H3, J = 6.3, 3.4 Hz) ppm; 13C NMR (75.5 MHz, CDCl3): δ −5.3 (Si-CH3), −5.2 (Si-CH3), −4.7 (Si-CH3), −4.6 (Si-CH3), 18.1 (SiCMe3), 18.5 (SiCMe3), 25.9 (tBu-CH3), 26.1 (tBu-CH3), 30.7 (CO-CH3), 34.9 (C6), 39.9 (C2), 63.6 (C5), 73.7 (C3), 78.0 (C1), 87.2 (C4), 195.7 (C=O) ppm; HRMS (ESI+, m/z): calcd for C20H43O4SSi2 [M + H]+: 435.2415, found: 435.2413, calcd for C20H42NaO4SSi2 [M + Na]+: 457.2235, found: 457.2241, calcd for C20H42KO4SSi2 [M+K]+: 473.1974, found: 473.1971.

1β-(Acetylmercaptomethyl)-3,5-bis-O-(tert-butyldimethylsilyl)-1,2-dideoxy-d-erythro-pentofuranose (13β)

Yellow oil. Rf: 0.75 (20% EtOAc/Hexane); IR (NaCl): ν 2955, 1961, 1255, 1108, 626 cm–1; 1H NMR (300.13 MHz, CDCl3): δ 0.05 (s, 12H, Si-Me), 0.87 (s, 9H, Si-Bu), 0.89 (s, 9H, Si-Bu), 1.67 (ddd, 1H, H2, J = 12.6, 9.6, 5.6 Hz), 1.85 (ddd, 1H, H2, J = 12.6, 5.5, 2.2 Hz), 2.34 (s, 3H, CO-Me), 2.98 (dd, 1H, H6, J = 13.6, 6.5 Hz), 3.18 (dd, 1H, H6, J = 13.6, 4.8 Hz), 3.45 (dd, 1H, H5, J = 10.7, 6.1 Hz), 3.61 (dd, 1H, H5, J = 10.8, 4.0 Hz), 3.79 (m, 1H, H4), 4.28 (m, 2H, H1 + H3) ppm; 13C NMR (75.5 MHz, CDCl3): δ −5.3 (Si-CH3), −5.2 (Si-CH3), −4.6 (Si-CH3),), −4.5 (Si-CH3), 18.1 (SiCMe3), 18.5 (SiCMe3), 25.9 (tBu-CH3), 26.1 (tBu-CH3), 30.7 (CO-CH3), 33.8 (C6), 40.1 (C2), 63.7 (C5), 74.1 (C3), 77.1 (C1), 88.0 (C4), 195.6 (C=O) ppm; HRMS (ESI+, m/z): calcd for C20H43O4SSi2 [M + H]+: 435.2415, found: 435.2416, calcd for C20H42NaO4SSi2 [M + Na]+: 457.2235, found: 457.2235, calcd for C20H42KO4SSi2 [M+K]+: 473.1974, found: 473.1974.

Synthesis of 14α/14β

(−)-CSA (2 equiv) was added to a solution of 13α/13β in anhydrous MeOH (0.1 M) at 0 °C and the reaction was stirred at rt during 2 h. Solid NaHCO3 was then added and the mixture was stirred for a further 5 min. The solvent was evaporated, and the crude product was subjected to column chromatography (10% MeOH/CH2Cl2) to afford 14α (80% yield) or 14β (80% yield).

1α-(Acetylmercaptomethyl)-1,2-dideoxy-d-erythro-pentofuranose (14α)

Clear oil. Rf: 0.47 (10% MeOH/CH2Cl2); IR (NaCl): ν 3374, 2931, 1692, 629 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.71 (ddd, 1H, H2, J = 12.7, 6.7, 5.7 Hz), 2.30 (m, 1H, H2), 2.33 (s, 3H, CO-Me), 3.10 (dd, 1H, H6, J = 13.6, 5.9 Hz), 3.20 (dd, 1H, H6, J = 13.6, 6.4 Hz), 3.51 (dd, 1H, H5, J = 11.8, 5.1 Hz), 3.59 (dd, 1H, H5, J = 11.8, 3.9 Hz), 3.82 (q, 1H, H4, J = 4.0 Hz), 4.16 (m, 1H, H1), 4.21 (m, 1H, H3) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 30.4 (CO-CH3), 35.2 (C6), 40.4 (C2), 63.3 (C5), 73.3 (C3), 78.4 (C1), 87.4 (C4), 197.0 (C=O) ppm; HRMS (ESI+, m/z): calcd for C8H15O4S [M + H]+: 207.0686, found: 207.0688, calcd for C8H14NaO4S [M + Na]+: 229.0505, found: 229.0506, calcd for C8H14KO4S [M+K]+: 245.0244, found: 245.0245.

1β-(Acetylmercaptomethyl)-1,2-dideoxy-d-erythro-pentofuranose (14β)

Clear oil. Rf: 0.47 (10% MeOH/CH2Cl2); IR (NaCl): ν 3390, 2930, 1691, 630 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.76 (ddd, 1H, H2, J = 13.1, 9.6, 6.1 Hz), 1.92 (ddd, 1H, H2, J = 13.0, 5.6, 2.2 Hz), 2.33 (s, 3H, CO-Me), 3.10 (d, 2H, H6, J = 5.7 Hz), 3.52 (d, 2H, H5, J = 5.0 Hz), 3.77 (m, 1H, H4), 4.21 (m, 2H, H1 + H3) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 30.4 (CO-CH3), 34.2 (C6), 40.7 (C2), 63.9 (C5), 74.0 (C3), 78.6 (C1), 89.0 (C4), 196.9 (C=O) ppm; HRMS (ESI+, m/z): calcd for C8H15O4S [M + H]+: 207.0686, found: 207.0684, calcd for C8H14NaO4S [M + Na]+: 229.0505, found: 229.0502, calcd for C8H14KO4S [M+K]+: 245.0244, found: 245.0240.

Synthesis of 15α/15β

A procedure analogous to that described for the synthesis of 4α/4β, starting from 14α/14β, gave 15α (80% yield) or 15β (85% yield).

1α-(Acetylmercaptomethyl)-1,2-dideoxy-5-O-(4,4′-dimethoxytrityl)-d-erythro-pentofuranose (15α)

Clear oil. Rf: 0.32 (40% EtOAc/Hexane); IR (NaCl): ν 3413, 2929, 1692, 1508, 625 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.69 (ddd, 1H, H2, J = 12.6, 6.9, 5.4 Hz), 2.33 (m, 1H, H2), 2.34 (s, 3H, CO-Me), 3.12 (m, 4H, H5 + H6), 3.76 (s, 6H, Me-DMT), 3.98 (dt, 1H, H4, J = 5.1, 3.9 Hz), 4.23 (m, 2H, H1 + H3), 6.84 (d, 4H, Hg, J = 8.9 Hz), 7.24 (m, 3H, Hc + Hd), 7.31 (d, 4H, Hf, J = 8.9 Hz), 7.44 (d, 2H, Hb, J = 7.0 Hz)ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 30.5 (CO-CH3), 35.2 (C6), 40.7 (C2), 55.7 (2 O-CH3), 65.5 (C5), 74.2 (C3), 78.8 (C1), 86.6 (C4), 87.4 (C10), 114.0 (4Cg), 127.7 (Cd), 128.7 (2Cc), 129.3 (2Cb), 131.3 (4Cf), 137.3 (Ce), 137.4 (Ce), 146.5 (Ca), 160.0 (2Ch), 196.9 (C=O)ppm; HRMS (ESI+, m/z): calcd for C29H32NaO6S [M + Na]+: 531.1812, found: 531.1782, calcd for C29H32KO6S [M+K]+: 547.1551, found: 547.1520.

1β-(Acetylmercaptomethyl)-1,2-dideoxy-5-O-(4,4′-dimethoxytrityl)-d-erythro-pentofuranose (15β)

Clear oil. Rf: 0.29 (40% EtOAc/Hexane); IR (NaCl): ν 3402, 2929, 1693, 1508, 627 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.78 (ddd, 1H, H2, J = 13.0, 9.6, 5.8 Hz), 1.90 (ddd, 1H, H2, J = 13.1, 5.7, 2.2 Hz), 2.29 (s, 3H, CO-Me), 3.12 (m, 4H, H5+ H6), 3.76 (s, 6H, Me-DMT), 3.90 (m, 1H, H4), 4.22 (m, 1H, H3), 4.29 (dq, 1H, H1, J = 11.0, 5.6 Hz), 6.84 (d, 4H, Hg, J = 8.9 Hz), 7.22 (m, 3H, Hc + Hd), 7.33 (d, 4H, Hf, J = 8.9 Hz), 7.46 (d, 2H, Hb, J = 7.0 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 30.5 (CO-CH3), 34.3 (C6), 40.4 (C2), 55.7 (2 O-CH3), 65.6 (C5), 74.5 (C3), 78.5 (C1), 87.4 (C10), 87.8 (C4), 114.0 (4Cg), 127.2 (Cd), 128.7 (2Cc), 129.4 (2Cb), 131.3 (4Cf), 137.3 (Ce), 137.4 (Ce), 146.5 (Ca), 160.1 (2Ch), 196.7 (C=O) ppm; HRMS (ESI+, m/z): calcd for C29H32NaO6S [M + Na]+: 531.1812, found: 531.1808, calcd for C29H32KO6S [M+K]+: 547.1551, found: 547.1547.

Synthesis of 16α/16β

A procedure analogous to that described for the synthesis of 5α/5β, starting from 15α/15β, gave 16α (72% yield) or 16β (68% yield).

1α-(Acetylmercaptomethyl)-1,2-dideoxy-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (16α-A)

Yellowish oil. Rf: 0.48 (40% EtOAc/Hexane); IR (NaCl): ν 2965, 2226, 1961, 1509, 1179, 1034, 590 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ1.15 (d, 6H, Hv, J = 6.9 Hz), 1.18 (d, 6H, Hv, J = 6.9 Hz), 1.84 (m, 1H, H2), 2.34 (s, 3H, CO-Me), 2.37 (m, 1H, H2), 2.51 (t, 2H, H, J = 6.0 Hz), 3.04–3.26 (several m, 4H, H5 + H6), 3.62 (m, 4H, Hw + H), 3.78 (s, 6H, Me-DMT), 4.12 (m, 1H, H4), 4.27 (m, 1H, H1), 4.47 (m, 1H, H3), 6.86 (d, 4H, Hg, J = 8.9 Hz), 7.25 (m, 3H, Hc + Hd), 7.31 (d, 4H, Hf, J = 8.9 Hz), 7.44 (d, 2H, Hb, J = 7.0 Hz) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ 148.1 ppm; HRMS (ESI+, m/z): calcd for C38H50N2O7PS [M + H]+: 709.3071, found: 709.3063, calcd for C38H49N2NaO7PS [M + Na]+: 731.2890, found: 731.2884.

1α-(Acetylmercaptomethyl)-1,2-dideoxy-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (16α-A+B)

Yellowish oil. Rf: 0.48 and 0.44 (40% EtOAc/Hexane); IR (NaCl): ν 2967, 2231, 1970, 1509, 1178, 1033, 587 cm–1; 31P NMR (121.5 MHz, MeOH-d4): δ 148.0, 148.1 ppm.

1β-(Acetylmercaptomethyl)-1,2-dideoxy-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (16β-A)

Yellowish oil. Rf: 0.65 (40% EtOAc/Hexane); IR (NaCl): ν 2967, 2254, 1722,1509, 1178, 1033, 583 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.15 (d, 6H, Hv, J = 6.8 Hz), 1.18 (d, 6H, HvJ = 6.8 Hz), 1.87 (m, 1H, H2), 2.01 (m, 1H, H2), 2.30 (s, 3H, CO-Me), 2.52 (t, 2H, H, J = 5.9 Hz), 3.16 (m, 4H, H5 + H6), 3.62 (m, Hw + H), 3.78 (s, 6H, Me-DMT), 4.02 (m, 1H, H4), 4.30 (m, 1H, H1), 4.45 (m, 1H, H3), 6.86 (d, 4H, Hg, J = 8.9 Hz), 7.26 (m, 3H, Hc + Hd), 7.33 (d, 4H, Hf, J = 8.9 Hz), 7.46 (d, 2H, Hb, J = 7.0 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 7.2 Hz), 24.9 (d, Cv, J = 7.4 Hz), 25.0 (d, Cv, J = 7.4 Hz), 30.5 (CO-CH3), 34.1 (C6), 39.7 (d, C2, J = 4.3 Hz), 44.4 (d, 2Cw, J = 12.6 Hz), 55.7 (2 O-CH3), 59.7 (d, C, J = 18.4 Hz), 64.9 (C5), 76.3 (d, C3, J = 16.8 Hz), 78.7 (C1), 87.1 (d, C4, J = 4.6 Hz), 87.4 (C10), 114.1 (4Cg), 119.3 (CN), 127.8 (Cd), 128.8 (2Cc), 129.4 (2Cb), 131.4 (4Cf), 137.3 (Ce), 137.4 (Ce), 146.5 (Ca), 160.1 (2Ch), 196.7 (C=O) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ 148.0 ppm; HRMS (ESI+, m/z): calcd for C38H50N2O7PS [M + H]+: 709.3071, found: 709.3079, calcd for C38H49N2NaO7PS [M + Na]+: 731.2890, found: 731.2899, calcd for C38H49N2KO7PS [M+K]+: 747.2630, found: 747.2642.

1β-(Acetylmercaptomethyl)-1,2-dideoxy-d-erythro-pentofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (16β-B)

Yellowish oil. Rf: 0.58 (40% EtOAc/Hexane); IR (NaCl): ν 2966, 2253, 1963, 1509, 1178, 1035, 588 cm–1; 1H NMR (300.13 MHz, MeOH-d4): δ 1.08 (d, 6H, Hv, J = 6.8 Hz), 1.18 (d, 6H, HvJ = 6.8 Hz), 1.87 (m, 1H, H2, J = 13.1, 9.3, 5.9 Hz), 2.12 (m, 1H, H2, J = 12.0, 5.2, 2.0 Hz), 2.31 (s, 3H, CO-Me), 2.69 (t, 2H, H, J = 5.9 Hz), 3.16 (m, 4H, H5 + H6), 3.59 (m, 2H, Hw), 3.79 (s, 6H, Me-DMT), 3.80 (m, 2H, H),4.00 (m, 1H, H4), 4.31 (m, 1H, H1), 4.44 (m, 1H, H3), 6.87 (d, 4H, Hg, J = 8.9 Hz), 7.26 (m, 3H, Hc + Hd), 7.34 (d, 4H, Hf, J = 8.9 Hz), 7.47 (d, 2H, Hb, J = 7.0 Hz) ppm; 13C NMR (75.5 MHz, MeOH-d4): δ 20.9 (d, C, J = 6.9 Hz), 24.9 (d, Cv, J = 7.2 Hz), 25.0 (d, Cv, J = 7.0 Hz), 30.5 (CO-CH3), 34.1 (C6), 39.7 (d, C2, J = 3.1 Hz), 44.4 (d, 2Cw, J = 12.4 Hz), 55.7 (2 O-CH3), 59.8 (d, C, J = 19.1 Hz), 65.1 (C5), 76.7 (d, C3, J = 16.5 Hz), 78.6 (C1), 86.9 (d, C4, J = 5.4 Hz), 87.4 (C10), 114.1 (4Cg), 119.4 (CN), 127.8 (Cd), 128.7 (2Cc), 129.4 (2Cb), 131.4 (4Cf), 137.2 (Ce), 137.3 (Ce), 146.5 (Ca), 160.1 (2Ch), 196.8 (C=O) ppm; 31P NMR (121.5 MHz, MeOH-d4): δ 147.7 ppm; HRMS (ESI+, m/z): calcd for C38H50N2O7PS [M + H]+: 709.3071, found: 709.3085, calcd for C38H49N2NaO7PS [M + Na]+: 731.2890, found: 731.2905, calcd for C38H49N2KO7PS [M+K]+: 747.2630, found: 747.2661.

Synthesis of Solid Supports Functionalized with 1,2-Dideoxy-d-erythro-pentofuranose Derivatives

Preparation of the 3-O-Succinyl-1,2-dideoxy-d-erythro-pentofuranose Derivatives 17α, 17β, 18α, 18β, 19α, and 19β

5-O-DMT-monomers (4α, 4β, 7α, 7β, 15α, or 15β) were dried twice by evaporation with anhydrous CH2Cl2 and dissolved in anhydrous CH2Cl2 (0.02 M). Then, 1.5 equiv of succinic anhydride and 1.5 equiv of DMAP were added, and the reaction was stirred at rt overnight. After the addition of CH2Cl2, the mixture was washed with 0.1 M NaH2PO4 (pH 5). The organic layer was dried with Na2SO4, filtrated, and concentrated to dryness giving place to 3-O-succinate-2-deoxy-d-ribofuranose derivatives 17α, 17β, 18α, 18β, 19α, and 19β. The resulting succinates were used directly for the functionalization of the supports without further purification.

Incorporation of the 3-O-Succinates to an LCAA-CPG Solid Support

The 5-O-DMT-3-O-succinate derivatives (17α, 17β, 18α, 18β, 19α, or 19β) obtained in the previous step and 1 equiv of DMAP were dissolved in acetonitrile (0.1 M). Next, 1 equiv of 2,2′-dithio-bis(5-nitropyridine) dissolved in a mixture (0.3 M) of acetonitrile:CH2Cl2 (1:3) was added. Then, this solution was added to 1 equiv of Ph3P in acetonitrile 80 μL. This final solution was poured to a vial containing 0.5 equiv LCAA-CPG (70 μmol/g) that had been previously washed with acetonitrile. After 3 h of reaction, the resin was washed with CH2Cl2 and acetonitrile. Finally, a 1:1 mixture of acetic anhydride/Py/THF and methylimidazole/THF was added to the resin for 5 min. The solid support was washed with CH2Cl2 and acetonitrile and dried out. The degree of functionalization of all of the supports ranged around 20–25 μmol/g.

Synthesis of Pentafluorophenyl Fatty Acid Esters 25

Preparation of Pentafluorophenyl Oleate (25a)

Oleic acid 23a (1 mmol, 282.46 mg) was dissolved in CH2Cl2 (1 mL/mmol). Et3N (16 mmol, 2.25 mL) and pentafluorophenyl trifluoroacetate 24 (4 mmol, 0.67 mL) were added to the solution. Then, the reaction mixture was stirred at rt for 1 h. Afterward, the reaction mixture was diluted in CH2Cl2 (6 mL/mmol) and washed with aqueous saturated NaHCO3 solution (5 mL/mmol) and 1 M NaH2PO4 solution (5 mL/mmol). The organic layer was separated, dried out with Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography and eluted with CH2Cl2/Hexane (1:1, v/v) to yield the desired oleic ester 25a as a yellowish oil (420 mg, 93%). 1H NMR (CDCl3, 400.13 MHz): δ 0.86 (t, 3H, CH3, J = 7.0 Hz), 1.44–1.21 (m, 20H, (CH2)n), 1.70–1.82 (m, 2H,CH2CH2CO), 2.01 (m, 4H, CH2CH=CHCH2), 2.64 (t, 2H, CH2CO, J = 7.4 Hz), 5.30–5.38 (m, 2H, CH=CH) ppm; 13C NMR (CDCl3, 100.6 MHz): δ 14.0 (CH3), 22.6, 24.7, 27.1, 27.2, 28.8, 28.9, 29.0, 29.3, 29.5, 29.6, 29.7 (CH2), 31.9 (COCH2), 33.3 (CH2CH=CH), 129.6, 130.0 (CH=CH), 136.5, 138.0, 139.0, 139.8, 140.6 (Carom), 142.4 (CaromO), 169.5 (CO) ppm; 19F NMR (CDCl3, 376.5 MHz): δ −162.5–162.7 (m, 2F), −158.4 (t, 1F, J = 21.6 Hz), −152.8–153.1 (m, 2F) ppm.

Preparation of Pentafluorophenyl Palmitate (25b)

The palmitic acid ester 25b was synthesized similarly to what has been described above for the pentafluorophenyl oleate. In this case, palmitic acid 23b (1 mmol, 256.4 mg) was dissolved in 10 mL of CH2Cl2 due to solubility issues, and the reaction mixture was stirred at rt overnight. The isolation and purification steps were also mentioned in the preparation of the pentafluorophenyl oleate. The desired palmitic ester 25b was obtained as a white solid (407 mg, 96%). 1H NMR (CDCl3, 400.13 MHz): δ 0.86 (t, 3H, Me, J = 6.8 Hz), 1.24 (s, 24H, (CH2)n), 1.75 (m, 2H, CH2CH2CO), 2.64 (t, 2H, CH2CO, J = 7.4 Hz) ppm; 13C NMR (CDCl3, 100.6 MHz): δ 14.1 (CH3), 22.6, 24.7, 28.8, 29.1, 29.3, 29.4, 29.5, 29.6, 29.6, 29.6, 29.7, 31.9 (CH2), 33.3 (COCH2), 142.3–137.5 (6Carom), 169.6 (CO) ppm; 19F NMR (CDCl3, 376.5 MHz): δ −162.5–162.7 (m, 2F), −158.3 (t, 1F, J = 21.6 Hz), −152.8–152.9 (m, 2H) ppm.

Synthesis, Purification, and Characterization of Oligonucleotides Incorporating Monomers 4α, 4β, 7α, 7β, 15α, and 15β

Oligonucleotide Synthesis

Oligonucleotide sequences, shown in Table , were synthesized on several batches between 0.5 and 1 μmol scale. In all cases, the 0.5–1 μmol standard solid-phase phosphoramidite chemistry protocols were carried out using an automatic DNA synthesizer.[23] The 1,2-dideoxy-d-erythro-pentofuranose derivatives were site specifically inserted at 5′- and 3′-ends of the desired sequences. The solid supports of each one of them were used to introduce these modifications at the 3′-end of the sequence, and the corresponding phosphoramidites were incorporated at the 5′-end of the desired sequence. All the oligonucleotides were synthesized DMT-ON.

Oligonucleotide Deprotection and Purification

According to the derivatives introduced in the sequence, different deprotection procedures were used for its deprotection. The 5′-O-DMT group of the 3′-modified gapmer with each one of the six derivatives 4α, 4β, 7α, 7β, 15α, and 15β were removed with a solution of 3% TCA in CH2Cl2 on the solid support.

The solid support of the Gapmer containing the 15α and 15β nucleoside derivatives either at the 3′-end or at the 5′-end and RS15α were treated with a solution of 1% DBU in acetonitrile followed by a couple of washes with acetonitrile and followed with a wash with a solution of 1% Et3N/acetonitrile for 1 min.

All the gapmer sequences containing only one 4α, 4β, 7α, and 7β derivative in its 3′- or 5′-end and the sequences RS4α, RS7β, and 7αgapmer4α were treated with 32% aqueous ammonia solution at 55 °C overnight. The RS15α and the four gapmer15α, gapmer15β, 15αgapmer, and 15βgapmer were deprotected with the same ammonium solution with 0.1 M DTT. Then, the 5′-O-DMT group of the three RS sequences (4α, 7β, 15α) were removed by the direct addition of the ammonium solution over an OPC cartridge. Then, all the solutions of the RS sequences (4α, 7β, and 15α) and the 5′- and 3′-gapmers were desalted on a Sephadex G-25 using water as eluent.

The final products of RS4α, RS7β, RS15α, and the 3′-end-modified gapmers were HPLC analyzed with the DMT-OFF method with column D at a flow rate of 0.7 mL/min and an increasing gradient of acetonitrile (0% to 50%) over 0.1 M aqueous triethylammonium acetate, during 20 min.

The 5′-end gapmers (4α, 4β, 7α, 7β, 15α, and 15β) were HPLC purified with the DMT-ON method with the column B using a flow rate of 2 mL/min and an increasing gradient of acetonitrile (0% to 70%) over 0.1 M aqueous triethylammonium acetate, during 20 min. The product fractions were collected and concentrated. The resulting products were detritylated by treating them with 1 mL of 50% acetic acid solution for 30 min at rt followed with extraction with Et2O. The deprotected oligonucleotides were desalted in a Sephadex column and analyzed by HPLC.

The length and homogeneity of all the modified oligonucleotide sequences were verified by MALDI-TOF. The retention time for the oligonucleotide and the calculated and found mass are shown in Table .

Removal of the Photolabile Protecting Group of Oligonucleotides Modified with 7α and 7β Nucleoside Derivatives

The elimination of the photolabile protecting group NPEC from the oligonucleotide sequences was done directly on the solid support or in solution, after the release of the oligonucleotide from the support.

The oligonucleotides already detached from the solid support were exposed to irradiation at 340 nm (blacklight) for different periods of time (15, 30, 45, 60, and 120 min) in a solution of 100 μL H2O/acetonitrile (1:1, v/v). The samples of oligonucleotide still attached to the solid support were suspended in the same solvent conditions and placed under the UV–vis lamp for the 1, 2, and 6 h. Then, the oligonucleotides were deprotected and purified as explained in the previous section (section 5.2).

Oligonucleotides Conjugated with Fluorescein Isothiocyanate

The gapmer4α was left to react with fluorescein isothiocyanate (FITC) through its free amino group as follows. 52 nmol of gapmer4α was dissolved in 250 μL of an aqueous solution of 0.1 M NaHCO3 (pH 9) and 10 equiv of FITC (0.2 mg, 520 nmol) dissolved in 250 μL DMF was added and left to react at rt for 8 h. Then, 10 additional equiv of FITC was added and the mixture was left to react overnight at rt. The mixtures were concentrated to dryness and the residue resuspended in 1 mL of water. The solution was desalted by Sephadex G-25 and analyzed by HPLC.

Conjugation Reactions in Solution

Oligonucleotides containing 7α or 7β nucleoside derivatives were dissolved in 350 μL carbonate buffer solution (pH 9.0), DMF, and acetonitrile (1:4:2, v:v:v). After that, 20 μL Et3N and 10 equiv of pentafluorophenyl oleate or palmitate were added, and the reaction mixture was stirred overnight at rt. The solution was concentrated to dryness. Then, the products were redissolved in water and desalted in a Sephadex column and HPLC purified. The yield of the final products obtained in each conjugation is shown in Table .

Conjugation Reactions on the Solid Support

DMF (200 μL), Et3N (20 μL), and 10 equiv of oleoyl chloride or the corresponding ester were added to the oligonucleotides containing 7α or 7β nucleosides derivatives attached to either the 5′-end or the 3′-end and attached to the solid support. The reaction mixtures were left at rt for 2 h. Next, the excess of chemicals was washed off. The resulting solid supports were washed with acetonitrile and dried. Then, the solid supports were treated with ammonia for the removal of protecting groups and its release from the resin. The resulting oligonucleotide-conjugates were desalted and purified by HPLC. The yield of the final products obtained in each conjugation is shown in Table .

Oligonucleotide Double Conjugation with Fluorescein Isothiocyanate and Oleic Acid (FITC-7αgapmer4α-oleic)

The 7αgapmer4α first was photolyzed during 6 h, and then washed with acetonitrile and DMF. Then, it was left to react with fluorescein isothiocyanate (FITC) through its free amino group in the solid support as follows. 0.5 μmol of 7α-gapmer4α was suspended in 100 μL of DMF, and 20 equiv of TEA (2 μL 10 μmol) was added, and 20 equiv of FITC (4 mg, 10 μmol) dissolved in 250 μL DMF was added and left to react at rt for 2 h. The reaction was washed with acetonitrile and dried. Then, the solid support was treated with 32% aqueous ammonia solution at 55 °C overnight. The solution was desalted by Sephadex G-25 and dried. Next, the FITC-7α-gapmer4α oligonucleotide (24 nmol) was dissolved in 70 μL carbonate buffer solution (pH 9.0), DMF and acetonitrile (1:4:2, v:v:v). After that, 1 μL Et3N and 20 equiv of pentafluorophenyl oleate were added, and the reaction mixture was stirred overnight at rt. The solution was concentrated to dryness. Successively, the product was redissolved in water and desalted in a Sephadex column and HPLC purified. The yield of the final products obtained in each conjugation is shown in Table .