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

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Oligonucleotides Containing 1‑Aminomethyl or 1‑Mercaptomethyl-

2-deoxy‑^D‑ribofuranoses: Synthesis, Puriﬁcation, Characterization,

and Conjugation with Fluorophores and Lipids

̀

́

Virginia Mart í n-Nieves, Carme Fabrega, Marc Guasch, Susana Fernandez, Yogesh S. Sanghvi,

Miguel Ferrero,* and Ramon Eritja *

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ABSTRACT: 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.

thioureas.¹¹Although 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

INTRODUCTION

■

Therapeutic oligonucleotides have tremendous potential for

treating a variety of diseases if they can reach the target cells

selective introduction of ligands to oligonucleotides. Amino-

alkylalcohols, such as 6-aminohexanol^6,12or 5′-amino-2′,5′-

dideoxynucleoside¹³derivatives, are utilized for the introduc-

tion 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-propanediol¹⁴or 2-aminobutyl-1,3-propanediol de-

rivatives.¹⁵

On the other hand, thiol groups have a selective reactivity

with maleimide and haloacetamide derivatives to form

thioethers.¹¹The introduction of thiol groups in oligonucleo-

tides is usually done by preparing 3-mercaptopropanol and 6-

mercaptohexanol derivatives protected either by trityl¹⁶or

disulﬁde groups.^17,18

successfully upon administration. More recently, this task has

been accomplished by covalent conjugation of peptides, lipids,

and GalNAc to oligonucleotides.^1,2Often, preparation of these

conjugates requires the presence of a reactive group such as an

amino or thiol group within an oligonucleotide.³⁻⁵The

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.⁶⁻⁸Toward this end, research-

ers are developing nucleosidic and non-nucleosidic phosphor-

amidite derivatives that enable eﬃcient preparation of

oligonucleotide conjugates.^3,9Some 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.⁶Some 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

Received: December 31, 2020

Revised: January 22, 2021

Published: February 5, 2021

Amino groups react readily with carboxylic acid derivatives

via amide formation as well as with isothiocyanates to form

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We have recently described the synthesis of novel 1′-homo-

N-2′-deoxy-α-nucleosides¹⁹and 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.²⁰These

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 mercapto-

methyl 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 mercapto-

methyl 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.

Scheme 1. Synthesis of 1-Triﬂuoroacetylaminomethyl-1,2-

dideoxy-^D-erythro-pentofuranosyl-3-phosphoramidites

a

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,

ⁱPr₂NEt, CH₂Cl₂, rt, 1 h, 84% (5α) and 70% (5β).

Scheme 2. Synthesis of 1-Aminomethyl-1,2-dideoxy-D-

erythro-pentofuranosyl-3-phosphoramidites Bearing a

Photolabile Protecting Group at the Amino Function

a

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 1),

which is easily accessible to perform on a large scale.²¹

Treatment of the latter with LiAlH₄in 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.

a

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, ⁱPr₂NEt,

CH₂Cl₂, rt, 1 h, 78% (8α) and 72% (8β).

Preparation of phosphoramidites of 1α- and 1β-amino-

methyl-1,2-dideoxy-^D-erythro-pentofuranoses bearing a photo-

labile protecting group at the amino function is outlined in

Scheme 2. The amino diol 2 was reacted with 1-(2-

nitrophenyl)ethyl-N-succinimidyl carbonate²²to aﬀord carba-

mates 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.

The synthetic protocol for the 1-S-mercaptomethyl-1,2-

dideoxy-^D-erythro-pentofuranosyl-3-O-phosphoramidites 16α

or 16β is summarized in Scheme 3. 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

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with succinic anhydride yielding the corresponding succinate

derivatives 17α/17β, 18α/18β, and 19α/19β (Scheme 4).

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 3. Synthesis of 1-S-Acetylmercaptomethyl-1,2-

dideoxy-^D-erythro-pentofuranose Phosphoramidites

a

Scheme 4. Preparation of CPG Solid Supports

Functionalized with 1-Aminomethyl- or 1-Mercaptomethyl-

a

1,2-dideoxy-^D-erythro-pentofuranoses

a

Reagents and conditions: (a) KOH, MeOH, H₂O, 25 °C, 3 h, 85%

a

Reagents and conditions: (a) Succinic anhydride, DMAP, rt,

(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-diisopropyl-

chlorophosphoramidite, ⁱPr₂NEt, CH₂Cl₂, rt, 1 h, 72% (16α) and 68%

(16β).

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 1 were made on the automated

DNA synthesizer using standard protocols.²³The 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.

Next, the two RS oligonucleotides containing the amino-

methyl 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

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.

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

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a

Table 1. Sequence of Oligonucleotides and Its Characterization by MALDI-TOF

code

RS4α

RS7α

RS7β

RS15α

sequences (5′ → 3′)

CATTGTCCA-4α

CATTGTCCA-7α

CATTGTCCA-7β

CATTGTCCA-15α

MW (calcd)

MW (found)

2880.5

2880.3

b

c

b

2880.5/3072.5

2897.5/5792.1

5865

2880.5/3073.5

2897.2

5853.8

5854.5

5864.6

5855/6057

5880.8

5880.5

5864.5

Gapmer4α

Gapmer4β

Gapmer7α

Gapmer7β

Gapmer15α

Gapmer15β

4αGapmer

4βGapmer

7αGapmer

7βGapmer

15αGapmer

15βGapmer

7αGapmer4α

cguuTCCTTTGTTCugga-4α

cguuTCCTTTGTTCugga-4β

cguuTCCTTTGTTCugga-7α

cguuTCCTTTGTTCugga-7β

cguuTCCTTTGTTCugga-15α

cguuTCCTTTGTTCugga-15β

4α-cguuTCCTTTGTTCugga

4β-cguuTCCTTTGTTCugga

7α-cguuTCCTTTGTTCugga

7β-cguuTCCTTTGTTCugga

15α-cguuTCCTTTGTTCugga

15β-cguuTCCTTTGTTCugga

7β-cguuTCCTTTGTTCugga-4α

5865

5866/6061

b

5884

5883

5865

5866

5883

5866.8

5865/6042

5849/6057

5878

5880.8

6074.0/6267.6

6075/6268

a

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-

b

c

nucleotides. Expected MW with a photolabile protecting group. Expected MW of the dimer form with a disulﬁde bridge.

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.

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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.

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 1, and the MW are

shown in Table 1.

Table 2. Data from the Kinetic Studies for the Removal of

the NPEC of 7α and 7β Gapmers

on the CPG

a

b

photolysis

support

in solution phase

reaction time (min)

Gapmer7α (%)

Gapmer7β (%)

30

78

71

60

91

92

15

60

80

30

89

93

45

100

99

60

100

a

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

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 1. 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 2. 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.

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 2, 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

b

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

oligonucleotide.

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.

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 func-

tional 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^6,24−26with a 93% and 96% yield, respectively

(Scheme 5).

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

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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.

Scheme 5. Synthesis of Pentaﬂuorophenyl Esters of Fatty

Acids

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 6). For this purpose, the 7αgapmer4α modiﬁed at

Scheme 6. Synthesis of an Oligonucleotide Carrying Both a

Fluorophore (FITC) and a Lipid (Oleic)

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 3). However, conjugate gapmer4α−FITC

Table 3. Oligonucleotide Conjugates and Their

Characterization by MALDI-TOF

oligonucleotide-conjugates

yield (%)

MW (calc)

MW (found)

Gapmer4α-FITC

RS4α-Oleic

50

76

64

61

19

72

66

46

6

6256.1

3146

3121

6108

6463

6258

3145.6

3123.6

3124.0

6107.5

6465.2

6742.7

RS4α-Palmitic

a

RS7α-Palmitic

b

RS7α-Palmitic

a

Palmitic-7αGapmer

FITC-7αGapmer4α

b

c

FITC-7αGapmer4α-Oleic

20

6728

a

b

Both photolysis and conjugation in solution. Photolysis over the

c

solid support and conjugation in solution. Both photolysis and

conjugation on the solid support.

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 pentaﬂuor-

ophenyl oleate in solution. The ﬁnal product was HPLC

puriﬁed and characterized by mass spectrometry. The yield

obtained is shown in Table 3. These results are a step forward

to obtain multiple functionalized oligonucleotides for diag-

nostic and therapeutic applications.

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.

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 3.

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

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 phosphorami-

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dite 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 1).

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 3) 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-aminohexanol¹²or 6-mercaptohex-

anol.¹⁶5′-Amino¹³and 5′-mercapto²⁷dideoxynucleosides 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.²⁸⁻³⁰However, 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.

for the attachment of the reactive group allows easy

incorporation into an oligonucleotide using standard solid-

phase amidite chemistry.

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-propanol¹⁴and 2-butylamino-1,3-propanol¹⁵and may

produce diastereoisomeric mixtures. In addition, it has been

described that the 2-amino-1,3-propanol linker may produce

intramolecular side reactions.³¹Threoninol derivatives have

also been described for the preparation of thiolated

oligonucleotides.³²Amino-³³and mercapto-¹⁸functionalized

nucleosides at the nucleobases or at the 2′-position of a

ribonucleotide³⁴have 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.³⁵Furthermore, the cyano sugar ribose precursor could

be transformed to other interesting reactive groups such as

azide or alkyne groups for conjugation using cycloaddition

reactions.¹⁰The choice of using the 2-deoxyribose framework

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.

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Article

(gradient eluent MeOH−10% NH₃/MeOH) to aﬀord 2α or

2β (both contains traces of silica gel).

MATERIALS AND METHODS

■

1. General. 1.1. 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

(CDCl₃and MeOH-d₄) were obtained from reputable sources

and used as received. Thin-layer chromatography (TLC) was

carried out on aluminum-backed Silica-Gel 60 F₂₅₄plates. 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, Al-

drich) 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.

1α-Aminomethyl-1,2-dideoxy-^D-erythro-pentofuranose

(2α). Yellowish oil. R_f: 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. R_f: 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.

2.1.2. 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. 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).

1

Chemical shifts for H, ¹³C, ¹⁹F, and ³¹P NMR are given in

1,2-Dideoxy-1α-[N-(triﬂuoroacetyl)aminomethyl]-^D-eryth-

ro-pentofuranose (3α). Clear oil. R_f: 0.58 (20% MeOH/

CH₂Cl₂); IR (NaCl): ν 3404, 3302, 2940, 1713, 1216, 1191,

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 C₁₈2.5 μm (10 × 50 mm) and analytical

columns: column C: XbridgeTM OST C₁₈2.5 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.

2. Synthesis of 1-Functionalized 1,2-Dideoxy-^D-

erythro-pentofuranose Phosphoramidites. 2.1. Prepara-

tion of 1-Triﬂuoroacetylaminomethyl-1,2-dideoxy-^D-eryth-

ro-pentofuranose Phosphoramidites 5α and 5β. 2.1.1. Syn-

thesis 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

1

1160 cm⁻¹; H 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-eryth-

ro-pentofuranose (3β). Clear oil. R_f: 0.57 (20% MeOH/

CH₂Cl₂); IR (NaCl): ν 3395, 3315, 2945, 1712, 1192, 1162

1

cm⁻¹; H 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.

2.1.3. 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,

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saturated aqueous NaHCO₃was 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.

Clear oil. R_f: 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, H_v, J = 6.9

Hz), 1.18 (d, 6H, H_v, 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_y, 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+ H_w+ H_x), 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, H_g, J = 8.9 Hz), 7.24 (m, 3H, H_c+

H_d), 7.30 (d, 4H, H_f, J = 8.9 Hz), 7.45 (d, 2H, H_b, J = 7.0 Hz)

ppm; ¹³C NMR (75.5 MHz, MeOH-d₄): δ 20.9 (d, C_y, J = 7.3

Hz), 25.0 (d, 4C_v, J = 7.4 Hz), 38.2 (d, C₂, J = 4.0 Hz), 44.4

(d, 2C_w, J = 12.3 Hz), 45.2 (C₆), 55.7 (2 O-CH₃), 59.7 (d, C_x,

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 (4C_g), 117.5 (q,

CF₃, J = 286.8 Hz), 119.3 (CN), 127.8 (C_d), 128.8 (2C_c),

129.3 (2C_b), 131.3 (4C_f), 137.2 (C_e), 137.3 (C_e), 146.4 (C_a),

159.2 (q, CO, J = 37.6 Hz), 160.1 (2C_h) 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. R_f: 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, H_v, J

= 6.8 Hz), 1.17 (d, 6H, H_v, 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_y, 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+

H_w), 3.77 (s, 6H, Me-DMT), 3.78 (m, 2H, H_x), 4.12 (q, 1H,

H₄, J = 4.0 Hz), 4.33 (m, 1H, H₁), 4.46 (m, 1H, H₃), 6.85 (d,

4H, H_g, J = 8.9 Hz), 7.23 (m, 3H, H_c+ H_d), 7.30 (d, 4H, H_f, J

= 8.9 Hz), 7.43 (d, 2H, H_b, J = 7.0 Hz) ppm; ¹³C NMR (75.5

MHz, MeOH-d₄): δ 20.9 (d, C_y, J = 6.8 Hz), 23.6 (d, 2C_v, J =

7.1 Hz), 23.6 (d, 2C_v, J = 7.1 Hz), 38.3 (d, C₂, J = 2.8 Hz),

44.4 (d, 2C_w, J = 12.4 Hz), 45.3 (C₆), 55.7 (2 O-CH₃), 59.7

(d, C_x, 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 (4C_g), 117.5

(q, CF₃, J = 286.8 Hz), 119.5 (CN), 127.8 (C_d), 128.8 (2C_c),

129.3 (2C_b), 131.3 (4C_f), 137.1 (C_e), 137.2 (C_e), 146.4 (C_a),

159.2 (q, CO, J = 37.6 Hz), 160.1 (2C_h) 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)-1α-[N-

(triﬂuoroacetyl)aminomethyl]-^D-erythro-pentofuranose

(4α). Intense yellow oil. R_f: 0.22 (40% EtOAc/Hexane); IR

(NaCl): ν 3414, 3282, 2934, 1715, 1509, 1252, 1202, 1177

1

cm⁻¹; H 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, H_g, J = 8.9 Hz), 7.24 (m, 3H, H_c+

H_d), 7.31 (d, 4H, H_f, J = 8.9 Hz), 7.43 (d, 2H, H_b, 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 (4C_g), 117.6 (q, CF₃, J = 286.7 Hz),

127.7 (C_d), 128.7 (2C_c), 129.3 (2C_b), 131.3 (4C_f), 137.2 (C_e),

137.3 (C_e), 146.4 (C_a), 159.2 (q, CO, J = 37.0 Hz), 160.1

(2C_h) 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. R_f: 0.13 (40% EtOAc/Hexane); IR (NaCl):

1

ν 3424, 3331, 2934, 1721, 1510, 1251, 1216, 1177 cm⁻¹; H

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, H_g, J = 8.9 Hz), 7.21 (m, 3H, H_c+ H_d), 7.31

(d, 4H, H_f, J = 8.9 Hz), 7.44 (m, 2H, H_b) 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 (4C_g), 117.5 (q, CF₃, J = 286.8 Hz), 127.8 (C_d), 128.7

(2C_c), 129.3 (2C_b), 131.3 (4C_f), 137.2 (C_e), 137.3 (C_e), 146.4

(C_a), 159.2 (q, CO, J = 36.8 Hz), 160.1 (2C_h) 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.

2.1.4. 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

ⁱPr₂NEt (3 equiv) was added. The resulting solution was

cooled in an ice bath and 2-cyanoethyl N,N-diisopropylchlor-

ophosphoramidite (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)-1β-[N-

(triﬂuoroacetyl)aminomethyl]-^D-erythro-pentofuranosyl-3-

O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (5β-A).

Clear oil. R_f: 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, H_v, J

= 6.8 Hz), 1.19 (d, 6H, H_v, J = 6.8 Hz), 1.83 (m, 1H, H₂), 2.01

(m, 1H, H₂), 2.54 (t, 2H, H_y, J = 6.0 Hz), 3.15 (m, 2H, H₅),

3.47 (m, 2H, H₆), 3.65 (overlapped signal, 4H, H_x+ H_w), 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, H_g, J = 8.9 Hz), 7.22 (m, 3H, H_c+

H_d), 7.32 (d, 4H, H_f, J = 7.5 Hz), 7.45 (d, 2H, H_b, J = 7.3 Hz)

ppm; ¹³C NMR (75.5 MHz, MeOH-d₄): δ 20.9 (d, C_y, J = 6.8

Hz), 25.0 (d, 2C_v, J = 7.5 Hz), 25.0 (d, 2C_v, J = 7.5 Hz), 38.5

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).

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(d, C₂, J = 4.6 Hz), 44.4 (d, 2C_w, J = 12.0 Hz), 44.5 (C₆), 55.7

(2 O-CH₃), 59.7 (d, C_x, 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 (4C_g), 117.5 (q, CF₃, J = 286.8 Hz), 119.3 (CN), 127.8

(C_d), 128.7 (2C_c), 129.3 (2C_b), 131.3 (4C_f), 137.2 (C_e), 137.3

(C_e), 146.4 (C_a), 159.2 (q, CO, J = 36.8 Hz), 160.1 (2C_h)

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.

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. R_f: 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, H_arom), 7.72 (m, 2H, H_arom), 7.95 (d, 1H, H_arom, 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 (CH_arom), 128.2 (CH_arom),

129.6 (CH_arom), 134.8 (CH_arom), 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.

2.2.2. 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)-1β-[N-

(triﬂuoroacetyl)aminomethyl]-^D-erythro-pentofuranosyl-3-

O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (5β-B).

Clear oil. R_f: 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, H_v, J

= 6.8 Hz), 1.18 (d, 6H, H_v, 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_y, J = 6.0 Hz),

3.12 (d, 2H, H₅, J = 4.9 Hz), 3.46 (m, 2H, H₆), 3.59 (m, 2H,

H_w), 3.77 (m, 8H, Me-DMT + H_x), 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,

H_g, J = 8.9 Hz), 7.23 (m, 3H, H_c+ H_d), 7.31 (d, 4H, H_f, J = 8.9

Hz), 7.43 (d, 2H, H_b, J = 7.2 Hz) ppm; ¹³C NMR (75.5 MHz,

MeOH-d₄): δ 20.9 (d, C_y, J = 7.1 Hz), 25.0 (d, 2C_v, J = 7.0

Hz), 25.0 (d, 2C_v, J = 7.0 Hz), 38.5 (d, C₂, J = 3.6 Hz), 44.4

(d, 2C_w, J = 12.7 Hz), 44.5 (C₆), 55.7 (2 O-CH₃), 59.8 (d, C_x,

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 (4C_g), 117.5 (q,

CF₃, J = 286.8 Hz), 119.5 (CN), 127.8 (C_d), 128.8 (2C_c),

129.3 (2C_b), 131.3 (4C_f), 137.1 (C_e), 137.2 (C_e), 146.4 (C_a),

159.2 (q, CO, J = 37.6 Hz), 160.1 (2C_h) 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.

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1α-[(1-(2-

nitrophenyl)ethoxy)carbonylamino-methyl]-^D-erythro-pen-

tofuranose (7α). Yellowish oil. R_f: 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, H_arom), 7.14−7.36

(several m, 8H, H_arom), 7.44 (m, 2H, H_arom), 7.67 (m, 2H,

H

_arom), 7.91 (m, 1H, H_arom) ppm; ¹³C NMR (75.5 MHz,

2.2. Preparation of 1-NPEC-aminomethyl-1,2-dideoxy-^D-

erythro-pentofuranose Phosphoramidites 8α and 8β.

2.2.1. 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 carbonate²²

(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). Yel-

low oil. R_f: 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, H_arom), 7.72 (m, 2H, H_arom), 7.94 (d,

1H, H_arom, 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

(CH_arom), 128.3 (CH_arom), 129.5 (CH_arom), 134.8 (CH_arom),

134.9 (CH_arom), 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

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 (4C_g),

125.2 (CH_arom), 127.7 (C_d), 128.1 (CH_arom), 128.2 (CH_arom),

128.7 (2C_c), 129.4 (2C_b), 129.4 (CH_arom), 131.3 (4C_f), 134.9

(CH_arom), 137.3 (2C_e), 139.9 (C₁₂), 146.5 (C_a), 149.0 (C₁₃),

158.0 (CO), 160.0 (2C_h) 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-pen-

tofuranose (7β). Yellowish oil. R_f: 0.16 (50% EtOAc/Hexane);

1

IR (NaCl): ν 3424, 2931, 1722, 1525, 1510, 1252 cm⁻¹; H

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, H_arom), 7.17−7.65 (several m, 12H, H_arom), 7.93 (m, 1H,

H_arom) 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 (4C_g),

125.2 (CH_arom), 127.8 (C_d), 128.2 (CH_arom), 128.8 (2C_c),

129.4 (2C_b), 129.5 (CH_arom), 131.3 (4C_f), 134.8 (CH_arom),

137.3 (C_e), 137.4 (C_e), 139.8 (C₁₂), 146.5 (C_a), 149.0 (C₁₃),

157.9 (CO), 160.1 (2C_h) 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.

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2.2.3. 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).

phosphoramidite (8β-A). Clear oil. R_f: 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, H_v, J = 6.8 Hz), 1.18 (d, 6H, H_v, 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_y, J = 6.0 Hz), 3.07−3.29 (several m, 4H,

H₅+ H₆), 3.48−3.71 (m, 4H, H_x+ H_w), 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, H_arom), 7.11−7.64

(several m, 12H, H_arom), 7.91 (m, 1H, H_arom) ppm; ¹³C NMR

(75.5 MHz, MeOH-d₄): δ 20.9 (d, C_y, J = 6.8 Hz), 22.4 (C₁₁),

22.5 (C₁₁), 24.9 (d, 2C_v, J = 7.2 Hz), 24.9 (d, 2C_v, J = 7.3 Hz),

37.8 (C₂), 38.2 (C₂), 44.4 (d, C_w, J = 11.9 Hz), 45.0 (C₆), 55.7

(2 O-CH₃), 59.7 (d, C_x, 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 (4C_g), 119.3 (CN), 125.2 (CH_arom),

127.8 (C_d), 128.2 (CH_arom), 128.8 (2C_c), 129.4 (2C_b), 129.5

(CH_arom), 131.4 (4C_f), 134.8 (CH_arom), 137.2 (C_e), 137.3

(C_e), 139.6 (C₁₂), 146.4 (C_a), 149.1 (C₁₃), 158.0 (CO),

160.1 (2C_h) 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)-1α-[(1-(2-

nitrophenyl)ethoxy)carbonylamino-methyl]-^D-erythro-pen-

tofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

phosphoramidite (8α-A). Clear oil. R_f: 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, H_v, 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_y, J = 6.0 Hz), 3.08 (m, 1H, H₅), 3.24 (m, 3H, H₅+ H₆), 3.60

(m, 4H, H_x+ H_w), 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, H_arom), 7.17−7.49 (several m, 10H, H_arom),

7.70 (m, 2H, H_arom), 7.93 (m, 1H, H_arom) ppm; ¹³C NMR

(75.5 MHz, MeOH-d₄): δ 20.8 (d, C_y, J = 6.6 Hz), 22.5 (C₁₁),

25.0 (d, 4C_v, J = 7.3 Hz), 38.0 (d, C₂, J = 4.0 Hz), 38.1 (d, C₂,

J = 4.0 Hz), 44.3 (d, C_w, J = 12.2 Hz), 46.0 (C₆), 46.2 (C₆),

55.7 (2 O-CH₃), 59.7 (d, C_x, 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 (4C_g), 119.3

(CN), 125.2 (CH_arom), 127.8 (C_d), 128.2 (CH_arom), 128.3

(CH_arom), 128.8 (2C_c), 129.4 (2C_b), 129.5 (CH_arom), 131.4

(4C_f), 134.9 (CH_arom), 137.2 (C_e), 137.3 (C_e), 137.4 (C_e),

140.0 (C₁₂), 146.5 (C_a), 149.1 (C₁₃), 158.0 (CO), 160.1

(2C_h) 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-pen-

tofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

phosphoramidite (8α-B). Clear oil. R_f: 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, H_v, J = 6.8 Hz), 1.15 (d, 6H, H_v, 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_y, J = 5.9 Hz), 3.06 (m, 1H, H₅), 3.16 (m, 1H,

H₅), 3.24 (m, 2H, H₆), 3.55 (m, 2H, H_w), 3.72 (m, 2H, H_x),

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, H_arom),

7.16−7.49 (m, 10H, H_arom), 7.70 (m, 2H, H_arom), 7.93 (m, 1H,

H_arom) ppm; ¹³C NMR (75.5 MHz, MeOH-d₄): δ 20.9 (d, C_y, J

= 6.7 Hz), 22.5 (C₁₁), 22.6 (C₁₁), 24.9 (d, 2C_v, J = 7.7 Hz),

25.0 (d, 2C_v, J = 7.3 Hz), 38.2 (C₂), 44.3 (d, C_w, J = 12.6 Hz),

46.2 (C₆), 54.8 (2 O-CH₃), 59.7 (d, C_x, 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 (4C_g), 119.5 (CN), 125.2 (CH_arom), 127.8

(C_d), 128.2 (CH_arom), 128.3 (CH_arom), 128.8 (2C_c), 129.3

(2C_b), 129.5 (CH_arom), 131.3 (4C_f), 134.8 (CH_arom), 137.2

(2C_e), 139.9 (C₁₂), 146.4 (C_a), 149.1 (C₁₃), 157.9 (CO),

160.1 (2C_h) 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-pen-

tofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

phosphoramidite (8β-B). Clear oil. R_f: 0.34 (40% EtOAc/

Hexane); IR (NaCl): ν 3363, 2933, 2253, 1729, 1509, 1250,

1

1179, 1034 cm⁻¹; H NMR (300.13 MHz, MeOH-d₄): δ 1.08

(d, 6H, H_v, J = 6.8 Hz), 1.19 (d, 6H, H_v, 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_y, J = 5.9 Hz), 3.08−3.41 (several m, 4H, H₅+

H₆), 3.60 (m, 2H, H_w), 3.78 and 3.79 (2s, 6H, Me-DMT), 3.78

(m, 2H, H_x), 4.01 (m, 1H, H₄), 4.18 (m, 1H, H₁), 4.41 (m,

1H, H₃), 6.12 (m, 1H, H₁₀), 6.85 (m, 4H, H_arom), 7.18−7.63

(seveal m, 12H, H_arom), 7.93 (m, 1H, H_arom) ppm; ¹³C NMR

(75.5 MHz, MeOH-d₄): δ 20.9 (d, C_y, J = 6.6 Hz), 22.4 (C₁₁),

22.5 (C₁₁), 24.9 (d, 2C_v, J = 7.7 Hz), 25.0 (d, 2C_v, J = 7.3 Hz),

38.0 (C₂), 38.2 (C₂), 44.4 (d, C_w, J = 12.6 Hz), 45.0 (C₆), 45.3

(C₆), 55.7 (2 O-CH₃), 59.7 (d, C_x, 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

(4C_g), 119.5 (CN), 125.2 (CH_arom), 127.8 (C_d), 128.1

(CH_arom), 128.23 (CH_arom), 128.8 (2C_c), 129.4 (2C_b), 129.5

(CH_arom), 131.3 (4C_f), 134.8 (CH_arom), 137.1 (C_e), 137.2

(C_e), 137.3 (C_e), 139.8 (C₁₂), 146.4 (C_a), 149.0 (C₁₃), 157.9

(CO), 160.1 (2C_h) 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.

2.3. Preparation of 1-Acetylmercaptomethyl-1,2-dideoxy-

D-erythro-pentofuranose Phosphoramidites 16α and 16β.

2.3.1. Synthesis of 9, 10, 11, and 12. Synthesis of 9α, 10α,

11α, and 12α was described previously by us.¹⁹A procedure

analogous to that aﬀorded 9β, 10β, 11β, and 12β. Yields are

indicated in Scheme 3.

1,2-Dideoxy-1β-(methoxycarbonyl)-^D-erythro-pentofura-

nose (9β). Yellowish oil. R_f: 0.36 (10% MeOH/CH₂Cl₂); IR

1

(NaCl): ν 3387, 2954, 1738 cm⁻¹; H 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-

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-1β-[(1-(2-

nitrophenyl)ethoxy)carbonylamino-methyl]-D-erythro-pen-

tofuranosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

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1

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.

1257, 1109, 626 cm⁻¹; H 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-^tBu), 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.

3,5-Bis-O-(tert-butyldimethylsilyl)-1,2-dideoxy-1β-(me-

thoxycarbonyl)-^D-erythro-pentofuranose (10β). Viscous

liquid. R_f: 0.59 (20% EtOAc/Hexane); IR (NaCl): ν 2954,

1

2931, 2898, 2858, 1759, 1737 cm⁻¹; H 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-^tBu), 0.92

(s, 9H, Si-^tBu), 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-^tBu), 26.4 (3 CH₃-^tBu), 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-(hy-

droxymethyl)-^D-erythro-pentofuranose (11β). Viscous liquid.

R_f: 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-^tBu),

0.92 (s, 9H, Si-^tBu), 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-^tBu), 26.5 (3 CH₃-^tBu), 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.

1β-(Acetylmercaptomethyl)-3,5-bis-O-(tert-butyldime-

thylsilyl)-1,2-dideoxy-^D-erythro-pentofuranose (13β). Yellow

oil. R_f: 0.75 (20% EtOAc/Hexane); IR (NaCl): ν 2955, 1961,

1

1255, 1108, 626 cm⁻¹; H NMR (300.13 MHz, CDCl₃): δ

0.05 (s, 12H, Si-Me), 0.87 (s, 9H, Si-^tBu), 0.89 (s, 9H, Si-^tBu),

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.

3,5-Bis-O-(tert-butyldimethylsilyl)-1,2-dideoxy-1β-

(tosyloxy)methyl-^D-erythro-pentofuranose (12β). Viscous

liquid. R_f: 0.64 (20% EtOAc/Hexane); IR (NaCl): ν 2954,

2.3.3. 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).

1

2930, 2896, 2857, 1471, 1366, 1255 cm⁻¹; H 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-^tBu), 0.89 (s, 9H, Si-^tBu),

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, H_arom, J = 8.5 Hz), 7.79 (d, 2H, H_arom, 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-^tBu), 26.5 (3 ^tBu-

CH₃), 37.7 (C₂), 64.6 (C₅), 72.8 (C₆), 74.8 (C₃), 77.1 (C₁),

89.2 (C₄), 129.1 (2 C_arom), 131.1 (2 C_arom), 134.4 (C_ipso),

146.5 (C_ipso) ppm; HRMS (ESI⁺, m/z): calcd for

C₂₅H₄₇O₆SSi₂[M + H]⁺: 531.2626, found: 531.2633.

2.3.2. 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-butyldime-

thylsilyl)-1,2-dideoxy-^D-erythro-pentofuranose (13α). Yellow

oil. R_f: 0.63 (20% EtOAc/Hexane); IR (NaCl): ν 2954, 1697,

1α-(Acetylmercaptomethyl)-1,2-dideoxy-D-erythro-pento-

furanose (14α). Clear oil. R_f: 0.47 (10% MeOH/CH₂Cl₂); IR

1

(NaCl): ν 3374, 2931, 1692, 629 cm⁻¹; H 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-pento-

furanose (14β). Clear oil. R_f: 0.47 (10% MeOH/CH₂Cl₂); IR

1

(NaCl): ν 3390, 2930, 1691, 630 cm⁻¹; H 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),

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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.

2.3.4. 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).

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-pento-

furanosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

phosphoramidite (16β-A). Yellowish oil. R_f: 0.65 (40%

EtOAc/Hexane); IR (NaCl): ν 2967, 2254, 1722,1509,

1

1178, 1033, 583 cm⁻¹; H NMR (300.13 MHz, MeOH-d₄):

δ 1.15 (d, 6H, H_v, J = 6.8 Hz), 1.18 (d, 6H, H_vJ = 6.8 Hz),

1.87 (m, 1H, H₂), 2.01 (m, 1H, H₂), 2.30 (s, 3H, CO-Me),

2.52 (t, 2H, H_y, J = 5.9 Hz), 3.16 (m, 4H, H₅+ H₆), 3.62 (m,

H_w+ H_x), 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, H_g, J = 8.9 Hz), 7.26

(m, 3H, H_c+ H_d), 7.33 (d, 4H, H_f, J = 8.9 Hz), 7.46 (d, 2H,

H_b, J = 7.0 Hz) ppm; ¹³C NMR (75.5 MHz, MeOH-d₄): δ

20.9 (d, C_y, J = 7.2 Hz), 24.9 (d, C_v, J = 7.4 Hz), 25.0 (d, C_v, J

= 7.4 Hz), 30.5 (CO-CH₃), 34.1 (C₆), 39.7 (d, C₂, J = 4.3 Hz),

44.4 (d, 2C_w, J = 12.6 Hz), 55.7 (2 O-CH₃), 59.7 (d, C_x, 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 (4C_g), 119.3 (CN), 127.8

(C_d), 128.8 (2C_c), 129.4 (2C_b), 131.4 (4C_f), 137.3 (C_e), 137.4

(C_e), 146.5 (C_a), 160.1 (2C_h), 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.

1α-(Acetylmercaptomethyl)-1,2-dideoxy-5-O-(4,4′-dime-

thoxytrityl)-^D-erythro-pentofuranose (15α). Clear oil. R_f: 0.32

(40% EtOAc/Hexane); IR (NaCl): ν 3413, 2929, 1692, 1508,

1

625 cm⁻¹; H 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, H_g, J = 8.9 Hz), 7.24 (m, 3H, H_c+ H_d), 7.31 (d, 4H, H_f, J

= 8.9 Hz), 7.44 (d, 2H, H_b, 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 (4C_g), 127.7 (C_d), 128.7 (2C_c), 129.3 (2C_b),

131.3 (4C_f), 137.3 (C_e), 137.4 (C_e), 146.5 (C_a), 160.0 (2C_h),

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′-dime-

thoxytrityl)-^D-erythro-pentofuranose (15β). Clear oil. R_f: 0.29

(40% EtOAc/Hexane); IR (NaCl): ν 3402, 2929, 1693, 1508,

1β-(Acetylmercaptomethyl)-1,2-dideoxy-^D-erythro-pento-

furanosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

phosphoramidite (16β-B). Yellowish oil. R_f: 0.58 (40%

EtOAc/Hexane); IR (NaCl): ν 2966, 2253, 1963, 1509,

1

627 cm⁻¹; H 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, H_g, J = 8.9 Hz), 7.22

(m, 3H, H_c+ H_d), 7.33 (d, 4H, H_f, J = 8.9 Hz), 7.46 (d, 2H,

H_b, 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 (4C_g),

127.2 (C_d), 128.7 (2C_c), 129.4 (2C_b), 131.3 (4C_f), 137.3 (C_e),

137.4 (C_e), 146.5 (C_a), 160.1 (2C_h), 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.

1

1178, 1035, 588 cm⁻¹; H NMR (300.13 MHz, MeOH-d₄): δ

1.08 (d, 6H, H_v, J = 6.8 Hz), 1.18 (d, 6H, H_vJ = 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_y, J = 5.9 Hz),

3.16 (m, 4H, H₅+ H₆), 3.59 (m, 2H, H_w), 3.79 (s, 6H, Me-

DMT), 3.80 (m, 2H, H_x),4.00 (m, 1H, H₄), 4.31 (m, 1H, H₁),

4.44 (m, 1H, H₃), 6.87 (d, 4H, H_g, J = 8.9 Hz), 7.26 (m, 3H,

H_c+ H_d), 7.34 (d, 4H, H_f, J = 8.9 Hz), 7.47 (d, 2H, H_b, J = 7.0

Hz) ppm; ¹³C NMR (75.5 MHz, MeOH-d₄): δ 20.9 (d, C_y, J =

6.9 Hz), 24.9 (d, C_v, J = 7.2 Hz), 25.0 (d, C_v, J = 7.0 Hz), 30.5

(CO-CH₃), 34.1 (C₆), 39.7 (d, C₂, J = 3.1 Hz), 44.4 (d, 2C_w, J

= 12.4 Hz), 55.7 (2 O-CH₃), 59.8 (d, C_x, 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 (4C_g), 119.4 (CN), 127.8 (C_d), 128.7

(2C_c), 129.4 (2C_b), 131.4 (4C_f), 137.2 (C_e), 137.3 (C_e), 146.5

(C_a), 160.1 (2C_h), 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.

3. Synthesis of Solid Supports Functionalized with

1,2-Dideoxy-D-erythro-pentofuranose Derivatives.

3.1. 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 CH₂Cl₂and

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-

2.3.5. 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-pento-

furanosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

phosphoramidite (16α-A). Yellowish oil. R_f: 0.48 (40%

EtOAc/Hexane); IR (NaCl): ν 2965, 2226, 1961, 1509,

1

1179, 1034, 590 cm⁻¹; H NMR (300.13 MHz, MeOH-d₄):

δ1.15 (d, 6H, H_v, J = 6.9 Hz), 1.18 (d, 6H, H_v, 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_y, J = 6.0 Hz), 3.04−3.26 (several m, 4H, H₅+

H₆), 3.62 (m, 4H, H_w+ H_x), 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, H_g,

J = 8.9 Hz), 7.25 (m, 3H, H_c+ H_d), 7.31 (d, 4H, H_f, J = 8.9

Hz), 7.44 (d, 2H, H_b, 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-pento-

furanosyl-3-O-(2-cyanoethyl-N,N-diisopropyl)-

phosphoramidite (16α-A+B). Yellowish oil. R_f: 0.48 and 0.44

(40% EtOAc/Hexane); IR (NaCl): ν 2967, 2231, 1970, 1509,

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D-ribofuranose derivatives 17α, 17β, 18α, 18β, 19α, and 19β.

The resulting succinates were used directly for the function-

alization of the supports without further puriﬁcation.

7β, 15α, and 15β. 5.1. Oligonucleotide Synthesis.

Oligonucleotide sequences, shown in Table 1, were synthe-

sized 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.²³The 1,2-dideoxy-D-erythro-pentofuranose deriva-

tives 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.

5.2. Oligonucleotide Deprotection and Puriﬁcation.

According to the derivatives introduced in the sequence,

diﬀerent deprotection procedures were used for its depro-

tection. 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 CH₂Cl₂on the

solid support.

3.2. 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 CH₂Cl₂and 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 CH₂Cl₂and acetonitrile and dried out. The degree of

functionalization of all of the supports ranged around 20−25

μmol/g.

4. Synthesis of Pentaﬂuorophenyl Fatty Acid Esters

25. 4.1. 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 pentaﬂuor-

ophenyl 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 NaH₂PO₄solution (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,

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 triethylammo-

nium 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.

1

93%). H 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 (C_arom), 142.4 (C_aromO),

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.

4.2. 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 CH₂Cl₂due 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

1

was obtained as a white solid (407 mg, 96%). H NMR

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 1.

5.3. 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.

(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 (6C_arom),

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.

5. Synthesis, Puriﬁcation, and Characterization of

Oligonucleotides Incorporating Monomers 4α, 4β, 7α,

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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).

6. Preparation of Oligonucleotide Conjugates. 6.1. Oli-

gonucleotides 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.

6.2. 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 3.

6.3. 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 3.

6.4. Oligonucleotide Double Conjugation with Fluores-

cein 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 3.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.bioconjchem.0c00717.

■

sı

Level of purity is indicated by the inclusion of copies of

¹H, ¹³C, ³¹P, and DEPT NMR spectra; in addition, some

2D NMR experiments are shown, which were used to

assign the peaks (PDF)

AUTHOR INFORMATION

Corresponding Authors

■

Miguel Ferrero − Departamento de Química Orgánica e

Inorgánica, Universidad de Oviedo, 33006 Oviedo, Asturias,

Spain; orcid.org/0000-0002-9025-4535; Phone: +34

985 105 013; Email: mferrero@uniovi.es

Ramon Eritja − Department of Chemical and Biomolecular

Nanotechnology, Institute for Advanced Chemistry of

Catalonia (IQAC, CSIC), 08034 Barcelona, Spain; CIBER-

BBN Networking Centre on Bioengineering, Biomaterials and

Nanomedicine, 08034 Barcelona, Spain; orcid.org/0000-

0001-5383-9334; Phone: +34 934 006 145;

Email: recgma@cid.csic.es

Authors

Virginia Martín-Nieves − Departamento de Química

Orgánica e Inorgánica, Universidad de Oviedo, 33006

Oviedo, Asturias, Spain; orcid.org/0000-0003-0741-

3855

̀

Carme Fabrega − Department of Chemical and Biomolecular

Nanotechnology, Institute for Advanced Chemistry of

Catalonia (IQAC, CSIC), 08034 Barcelona, Spain; CIBER-

BBN Networking Centre on Bioengineering, Biomaterials and

Nanomedicine, 08034 Barcelona, Spain

Marc Guasch − Department of Chemical and Biomolecular

Nanotechnology, Institute for Advanced Chemistry of

Catalonia (IQAC, CSIC), 08034 Barcelona, Spain; CIBER-

BBN Networking Centre on Bioengineering, Biomaterials and

Nanomedicine, 08034 Barcelona, Spain

́

Susana Fernandez − Departamento de Química Orgánica e

Inorgánica, Universidad de Oviedo, 33006 Oviedo, Asturias,

Spain; orcid.org/0000-0002-2946-2108

Yogesh S. Sanghvi − Rasayan Inc., Encinitas, California

92024-6615, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.bioconjchem.0c00717

Funding

Financial support of this work by the Spanish Ministerio de

́

Ciencia e Innovacion (MICINN, Project CTQ2014-55015-P)

and Principado de Asturias (Project IDI/2018/000181) are

gratefully acknowledged. The authors acknowledge the

ﬁnancial support provided by CIBER-BBN an initiative funded

by the VI National R+D+I Plan 2008−2011, Iniciativa Ingenio

2010, Consolider Program and the Instituto de Salud Carlos

III with assistance from the European Regional Development

and ICTS NANBIOSIS (oligonucleotide synthesis platform

U29). The RE group is supported in part by the Spanish

́

Ministerio de Ciencia e Innovacion (CTQ2017-84415-R) and

364

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Article

dites; applications of 5 ′-amino-oligodeoxy-ribonucleotides. Nucleic

Acids Res. 15, 6181−6196.

(14) Nelson, P. S., Sherman-Gold, R., and Leon, R. (1989) A new

and versatile reagent for incorporating multiple primary aliphatic

amines into synthetic oligonucleotides. Nucleic Acids Res. 17, 7179−

7186.

Generalitat de Catalunya (2017SGR114). V. M.-N. thanks

FICYT (Asturias) for a predoctoral Severo Ochoa fellowship.

Notes

The authors declare no competing ﬁnancial interest.

DEDICATION

(15) Nelson, P. S., Kent, M., and Muthini, S. (1992) Oligonucleo-

tide labeling methods 3. Direct labeling of oligonucleotides employing

a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

Nucleic Acids Res. 20, 6253−6259.

■

This paper is dedicated to the memory of Prof. Enrique

Pedroso.

(16) Connolly, B. A., and Rider, P. (1985) Chemical synthesis of

oligonucleotides containing a free sulphydryl group and subsequent

attachment of thiol specific probes. Nucleic Acids Res. 13, 4485−4502.

ABBREVIATIONS

■

CPG, controlled pore glass; DBU, 1,8-diazabicyclo[5.4.0]-

undec-7-ene; DMAP, N,N-dimethylaminopyridine; DMF,

N,N-dimethylformamide; DMT, dimethoxytrityl; DMSO,

dimethylsulfoxide; DTT, dithiothreitol; FITC, ﬂuorescein

isothiocyanate; LCAA-CPG, long chain amino alkyl-controlled

pore glass; MALDI, matrix-assisted laser disorption/ionization;

NPEC, 1-(2-nitrophenyl)ethoxycarbonyl; OPC, oligonucleo-

tide puriﬁcation cartridge; Py, pyridine; RP-HPLC, reverse

phase high performance liquid chromatography; TCA,

trichloroacetic acid; TOF, time of ﬂight

̈

(17) Salo, H., Guzaev, A., and Lonnberg, H. (1998) Disulfide-

tethered solid supports for synthesis of photoluminescent oligonu-

cleotide conjugates: hydrolytic stability and labeling on the support.

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Article Doi

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

DOI: 10.1021/acs.bioconjchem.0c00717

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